Advances in Enzymes and Protein Engineering

Topic Information

Dear Colleagues,

Enzymes are proteins produced by living cells to catalyze a reaction—hence, they are often called biocatalysts. In recent decades, the utilization of enzymes for special synthetic purposes has become more and more popular in numerous fields of applied sciences. Biocatalysis, which refers to the use of microorganisms and enzymes in chemical reactions, has become increasingly popular, and is frequently applied in the industry, since enzymes are highly specific and display high catalytic efficiencies.

This Topic is focused on recent research and reviews about how different approaches have managed to improve enzymes for industrial applications and protein engineering. Research topics may include but are not limited to:

• Production of enzymes for industrial application;
• Application of enzymes in biotransformation of compounds;
• Examples of enhanced enzymes by directed evolution, enzyme engineering and metagenomics;
• Bioprocess scaling-up to industrial levels;
• Novel approaches to improve the stability and reutilization of the industrial enzymes;
• Multienzymatic systems development, including cofactor regeneration;
• Process intensification and successful examples of new industrial enzymatic applications;
• Protein engineering to create new enzymes with novel properties.

Prof. Dr. Yung-Chuan Liu
Prof. Dr. Jose M. Guisan
Prof. Dr. Antonio Zuorro
Topic Editors

Keywords

Participating Journals

Journal Name	Impact Factor	CiteScore	Launched Year	First Decision (median)	APC
Biomolecules biomolecules	4.8	9.4	2011	18.4 Days	CHF 2700
Catalysts catalysts	3.8	6.8	2011	13.9 Days	CHF 2200
International Journal of Molecular Sciences ijms	4.9	8.1	2000	16.8 Days	CHF 2900
Microorganisms microorganisms	4.1	7.4	2013	11.7 Days	CHF 2700
Molecules molecules	4.2	7.4	1996	15.1 Days	CHF 2700

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Published Papers (42 papers)

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18 pages, 3208 KiB  

Open AccessArticle

Research of Multicopper Oxidase and Its Degradation of Histamine in Lactiplantibacillus plantarum LPZN19

by Huijie Pei, Yilun Wang, Wei He, Lin Deng, Qinjie Lan, Yue Zhang, Lamei Yang, Kaidi Hu, Jianlong Li, Aiping Liu, Xiaolin Ao, Hui Teng, Shuliang Liu, Likou Zou, Ran Li and Yong Yang

Microorganisms 2023, 11(11), 2724; https://doi.org/10.3390/microorganisms11112724 - 8 Nov 2023

Cited by 6 | Viewed by 1741

Abstract

In order to explore the structural changes and products of histamine degradation by multicopper oxidase (MCO) in Lactiplantibacillus plantarum LPZN19, a 1500 bp MCO gene in L. plantarum LPZN19 was cloned, and the recombinant MCO was expressed in E. coli BL21 (DE3). After [...] Read more.

In order to explore the structural changes and products of histamine degradation by multicopper oxidase (MCO) in Lactiplantibacillus plantarum LPZN19, a 1500 bp MCO gene in L. plantarum LPZN19 was cloned, and the recombinant MCO was expressed in E. coli BL21 (DE3). After purification by Ni²⁺-NTA affinity chromatography, the obtained MCO has a molecular weight of 58 kDa, and it also has the highest enzyme activity at 50 °C and pH 3.5, with a relative enzyme activity of 100%, and it maintains 57.71% of the relative enzyme activity at 5% salt concentration. The secondary structure of MCO was determined by circular dichroism, in which the proportions of the α-helix, β-sheet, β-turn and random coil were 2.9%, 39.7%, 21.2% and 36.1%, respectively. The 6xj0.1.A with a credibility of 68.21% was selected as the template to predict the tertiary structure of MCO in L. plantarum LPZN19, and the results indicated that the main components of the tertiary structure of MCO were formed by the further coiling and folding of a random coil and β-sheet. Histamine could change the spatial structure of MCO by increasing the content of the α-helix and β-sheet. Finally, the LC-MS/MS identification results suggest that the histamine was degraded into imidazole acetaldehyde, hydrogen peroxide and ammonia. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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25 pages, 5284 KiB  

Open AccessReview

Ribonucleoside Hydrolases–Structure, Functions, Physiological Role and Practical Uses

by Leonid A. Shaposhnikov, Svyatoslav S. Savin, Vladimir I. Tishkov and Anastasia A. Pometun

Biomolecules 2023, 13(9), 1375; https://doi.org/10.3390/biom13091375 - 12 Sep 2023

Cited by 4 | Viewed by 2235

Abstract

Ribonucleoside hydrolases are enzymes that catalyze the cleavage of ribonucleosides to nitrogenous bases and ribose. These enzymes are found in many organisms: bacteria, archaea, protozoa, metazoans, yeasts, fungi and plants. Despite the simple reaction catalyzed by these enzymes, their physiological role in most [...] Read more.

Ribonucleoside hydrolases are enzymes that catalyze the cleavage of ribonucleosides to nitrogenous bases and ribose. These enzymes are found in many organisms: bacteria, archaea, protozoa, metazoans, yeasts, fungi and plants. Despite the simple reaction catalyzed by these enzymes, their physiological role in most organisms remains unclear. In this review, we compare the structure, kinetic parameters, physiological role, and potential applications of different types of ribonucleoside hydrolases discovered and isolated from different organisms. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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11 pages, 2433 KiB  

Open AccessArticle

Unraveling the Pivotal Roles of Various Metal Ion Centers in the Catalysis of Quercetin 2,4-Dioxygenases

by Xueyuan Yan, Han Xiao, Jinshuai Song and Chunsen Li

Molecules 2023, 28(17), 6238; https://doi.org/10.3390/molecules28176238 - 25 Aug 2023

Cited by 2 | Viewed by 1663

Abstract

Quercetin 2,4-dioxygenase (QueD) with various transition metal ion co-factors shows great differences, but the internal reasons have not been illustrated in detail. In order to explore the effects of metal ion centers on the catalytic reactivity of QueD, we calculated and compared the [...] Read more.

Quercetin 2,4-dioxygenase (QueD) with various transition metal ion co-factors shows great differences, but the internal reasons have not been illustrated in detail. In order to explore the effects of metal ion centers on the catalytic reactivity of QueD, we calculated and compared the minimum energy crossing point (MECP) of dioxygen from the relatively stable triplet state to the active singlet state under different conditions by using the DFT method. It was found that the metal ions play a more important role in the activation of dioxygen compared with the substrate and the protein environment. Simultaneously, the catalytic reactions of the bacterial QueDs containing six different transition metal ions were studied by the QM/MM approach, and we finally obtained the reactivity sequence of metal ions, Ni²⁺ > Co²⁺ > Zn²⁺ > Mn²⁺ > Fe²⁺ > Cu²⁺, which is basically consistent with the previous experimental results. Our calculation results indicate that metal ions act as Lewis acids in the reaction to stabilize the substrate anion and the subsequent superoxo and peroxo species in the reaction, and promote the proton coupled electron transfer (PCET) process. Furthermore, the coordination tendencies of transition metal ion centers also have important effects on the catalytic cycle. These findings have general implications on metalloenzymes, which can expand our understanding on how various metal ions play their key role in modulating catalytic reactivity. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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14 pages, 4925 KiB  

Open AccessArticle

Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum

by Jisub Hwang, Wanki Yoo, Seung Chul Shin, Kyeong Kyu Kim, Han-Woo Kim, Hackwon Do and Jun Hyuck Lee

Int. J. Mol. Sci. 2023, 24(15), 12022; https://doi.org/10.3390/ijms241512022 - 27 Jul 2023

Cited by 2 | Viewed by 2166

Abstract

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. Ea [...] Read more.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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14 pages, 1532 KiB  

Open AccessArticle

Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization

by Yeon-Jun Kim, Jun-Seop Shin, Kang Woo Lee, Hyo-Ji Eom, Byung Gwan Jo, Jin Woo Lee, Jun Hyoung Kim, So Yeon Kim, Jung Hoon Kang and Jae-Won Choi

Int. J. Mol. Sci. 2023, 24(13), 11083; https://doi.org/10.3390/ijms241311083 - 4 Jul 2023

Cited by 4 | Viewed by 3207

Abstract

Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, [...] Read more.

Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed Escherichia coli strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the PvLDH gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an E. coli strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this E. coli Rosetta(DE3), containing the original PvLDH gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by P. vivax. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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17 pages, 3034 KiB  

Open AccessArticle

RNA-Seq Analysis of Aboveground and Underground Parts of Biomass Sorghum Was Performed to Evaluate Its Suitability for Environmental Remediation

by Tao Zhou, Dingxun Ling, Qihao He, Ping Wang and Jian Zhu

Biomolecules 2023, 13(6), 925; https://doi.org/10.3390/biom13060925 - 31 May 2023

Cited by 1 | Viewed by 1966

Abstract

“Alto2” is a new biomass sorghum variety, which has the characteristics of fast growth, high growth, and strong cadmium (Cd) resistance, so it has the application prospect of soil remediation plants. In order to reveal the Cd resistance mechanism of this plant and [...] Read more.

“Alto2” is a new biomass sorghum variety, which has the characteristics of fast growth, high growth, and strong cadmium (Cd) resistance, so it has the application prospect of soil remediation plants. In order to reveal the Cd resistance mechanism of this plant and pave the way for genetic breeding and cultivation of efficient remediation plants in the future, in this research, through the determination of Cd content in various tissues of sorghum under Cd stress and the physicochemical response combined with RNA-Seq analysis, the mechanism of Cd resistance of “Alto2” was initially revealed. The results show biomass sorghum “Alto2” was mainly connected with aboveground and underground parts through the MAPK signaling pathway and plant hormone signaling pathway, and transmit stress signal in response to Cd stress. Chelase and metal-binding proteins may be the functional genes mainly responsible for Cd enrichment and transport and regulated by stress signals. However, the expression of aboveground transporters was not significant. This may be because Cd in biomass sorghum is mainly concentrated in the underground part and is enriched by the chelation of secondary metabolites from plant roots by the cell wall leading to inhibition of aboveground transporter expression. The results of this study indicate that the biomass sorghum “Alto2” on Cd has high resistance, but the lack of the aboveground enrichment of transportability requires further research to improve the Cd transportability of this plant. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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18 pages, 1264 KiB  

Open AccessArticle

Mathematical and Machine Learning Approaches for Classification of Protein Secondary Structure Elements from Cα Coordinates

by Ali Sekmen, Kamal Al Nasr, Bahadir Bilgin, Ahmet Bugra Koku and Christopher Jones

Biomolecules 2023, 13(6), 923; https://doi.org/10.3390/biom13060923 - 31 May 2023

Cited by 1 | Viewed by 2004

Abstract

Determining Secondary Structure Elements (SSEs) for any protein is crucial as an intermediate step for experimental tertiary structure determination. SSEs are identified using popular tools such as DSSP and STRIDE. These tools use atomic information to locate hydrogen bonds to identify SSEs. When [...] Read more.

Determining Secondary Structure Elements (SSEs) for any protein is crucial as an intermediate step for experimental tertiary structure determination. SSEs are identified using popular tools such as DSSP and STRIDE. These tools use atomic information to locate hydrogen bonds to identify SSEs. When some spatial atomic details are missing, locating SSEs becomes a hinder. To address the problem, when some atomic information is missing, three approaches for classifying SSE types using $C\alpha$ atoms in protein chains were developed: (1) a mathematical approach, (2) a deep learning approach, and (3) an ensemble of five machine learning models. The proposed methods were compared against each other and with a state-of-the-art approach, PCASSO. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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15 pages, 1965 KiB  

Open AccessArticle

Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase

by Miesho Hadush Berhe, Xiangfei Song and Lishan Yao

Int. J. Mol. Sci. 2023, 24(10), 8963; https://doi.org/10.3390/ijms24108963 - 18 May 2023

Cited by 5 | Viewed by 3056

Abstract

Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect, we optimized the [...] Read more.

Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect, we optimized the protein sequence encoding for an LPMO from Bacillus amyloliquefaciens (BaLPMO10A) using the sequence consensus method. Enzyme activity was determined using the chromogenic substrate 2,6-Dimethoxyphenol (2,6-DMP). Compared with the wild type (WT), the variants exhibit up to a 93.7% increase in activity against 2,6-DMP. We also showed that BaLPMO10A can hydrolyze p-nitrophenyl-β-D-cellobioside (PNPC), carboxymethylcellulose (CMC), and phosphoric acid-swollen cellulose (PASC). In addition to this, we investigated the degradation potential of BaLPMO10A against various substrates such as PASC, filter paper (FP), and Avicel, in synergy with the commercial cellulase, and it showed up to 2.7-, 2.0- and 1.9-fold increases in production with the substrates PASC, FP, and Avicel, respectively, compared to cellulase alone. Moreover, we examined the thermostability of BaLPMO10A. The mutants exhibited enhanced thermostability with an apparent melting temperature increase of up to 7.5 °C compared to the WT. The engineered BaLPMO10A with higher activity and thermal stability provides a better tool for cellulose depolymerization. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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17 pages, 12622 KiB  

Open AccessArticle

Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein

by Yingqing Xu, Zijian Geng, Chengxi Yang, Hongwei Zhou, Yixing Wang, Buayisham Kuerban and Gang Luo

Molecules 2023, 28(7), 3041; https://doi.org/10.3390/molecules28073041 - 29 Mar 2023

Cited by 2 | Viewed by 2466

Abstract

Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein [...] Read more.

Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHβ secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing β-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHβ production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHβ. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHβ secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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13 pages, 2300 KiB  

Open AccessArticle

Study on the Interaction Mechanism of Methoxy Polyethylene Glycol Maleimide with Sweet Potato β-Amylase

by Xinhong Liang, Yaxin Kong, Huadi Sun, Ruixiang Zhao, Lingxia Jiao, Wanli Zhang and Bing Liu

Molecules 2023, 28(5), 2188; https://doi.org/10.3390/molecules28052188 - 27 Feb 2023

Cited by 3 | Viewed by 2291

Abstract

In this study, sweet potato β-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified β-amylase and the interaction mechanism between SPA and Mal-mPEG5000 was investigated. the changes in the functional groups of different amide [...] Read more.

In this study, sweet potato β-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified β-amylase and the interaction mechanism between SPA and Mal-mPEG5000 was investigated. the changes in the functional groups of different amide bands and modifications in the secondary structure of enzyme protein were analyzed using infrared spectroscopy and circular dichroism spectroscopy. The addition of Mal-mPEG5000 transformed the random curl in the SPA secondary structure into a helix structure, forming a folded structure. The Mal-mPEG5000 improved the thermal stability of SPA and protected the structure of the protein from breaking by the surrounding. The thermodynamic analysis further implied that the intermolecular forces between SPA and Mal-mPEG5000 were hydrophobic interactions and hydrogen bonds due to the positive values of ΔH^θ and ΔS^θ. Furthermore, the calorie titration data showed that the binding stoichiometry for the complexation of Mal-mPEG5000 to SPA was 1.26, and the binding constant was 1.256 × 10⁷ mol/L. The binding reaction resulted from negative enthalpy, indicating that the interaction of SPA and Mal-mPEG5000 was induced by the van der Waals force and hydrogen bonding. The UV results showed the formation of non-luminescent material during the interaction, the Fluorescence results confirmed that the mechanism between SPA and Mal-mPEG5000 was static quenching. According to the fluorescence quenching measurement, the binding constant (K_A) values were 4.65 × 10⁴ L·mol⁻¹ (298K), 5.56 × 10⁴ L·mol⁻¹ (308K), and 6.91 × 10⁴ L·mol⁻¹ (318K), respectively. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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18 pages, 4205 KiB  

Open AccessArticle

Computational Insights into the Allosteric Modulation of a Phthalate-Degrading Hydrolase by Distal Mutations

by Ran Xu, Yiqiong Bao, Mengrong Li, Yan Zhang, Lili Xi and Jingjing Guo

Biomolecules 2023, 13(3), 443; https://doi.org/10.3390/biom13030443 - 26 Feb 2023

Cited by 4 | Viewed by 3180

Abstract

Phthalate esters (PAEs) are a ubiquitous kind of environmental endocrine that disrupt chemicals, causing environmental and health issues. EstJ6 is an effective phthalate-degrading hydrolase, and its mutant with a combination of three non-conservative distal mutations has an improved activity against PAEs with unknown [...] Read more.

Phthalate esters (PAEs) are a ubiquitous kind of environmental endocrine that disrupt chemicals, causing environmental and health issues. EstJ6 is an effective phthalate-degrading hydrolase, and its mutant with a combination of three non-conservative distal mutations has an improved activity against PAEs with unknown molecular mechanisms. Herein, we attempt to fill the significant gap between distal mutations and the activity of this enzyme using computational approaches. We found that mutations resulted in a redistribution of the enzyme’s preexisting conformational states and dynamic changes of key functional regions, especially the lid over the active site. The outward motion of the lid upon the mutations made it easier for substrates or products to enter or exit. Additionally, a stronger substrate binding affinity and conformational rearrangements of catalytic reaction-associated residues in the mutant, accompanied by the strengthened communication within the protein, could synergistically contribute to the elevated catalytic efficiency. Finally, an attempt was made to improve the thermostability of EstJ6 upon introducing a distal disulfide bond between residues A23 and A29, and the simulation results were as expected. Together, our work explored the allosteric effects caused by distal mutations, which could provide insights into the rational design of esterases for industrial applications in the future. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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19 pages, 7471 KiB  

Open AccessReview

Possibilities of Using De Novo Design for Generating Diverse Functional Food Enzymes

by Xinglong Wang, Kangjie Xu, Yameng Tan, Song Liu and Jingwen Zhou

Int. J. Mol. Sci. 2023, 24(4), 3827; https://doi.org/10.3390/ijms24043827 - 14 Feb 2023

Cited by 8 | Viewed by 4360

Abstract

Food enzymes have an important role in the improvement of certain food characteristics, such as texture improvement, elimination of toxins and allergens, production of carbohydrates, enhancing flavor/appearance characteristics. Recently, along with the development of artificial meats, food enzymes have been employed to achieve [...] Read more.

Food enzymes have an important role in the improvement of certain food characteristics, such as texture improvement, elimination of toxins and allergens, production of carbohydrates, enhancing flavor/appearance characteristics. Recently, along with the development of artificial meats, food enzymes have been employed to achieve more diverse functions, especially in converting non-edible biomass to delicious foods. Reported food enzyme modifications for specific applications have highlighted the significance of enzyme engineering. However, using direct evolution or rational design showed inherent limitations due to the mutation rates, which made it difficult to satisfy the stability or specific activity needs for certain applications. Generating functional enzymes using de novo design, which highly assembles naturally existing enzymes, provides potential solutions for screening desired enzymes. Here, we describe the functions and applications of food enzymes to introduce the need for food enzymes engineering. To illustrate the possibilities of using de novo design for generating diverse functional proteins, we reviewed protein modelling and de novo design methods and their implementations. The future directions for adding structural data for de novo design model training, acquiring diversified training data, and investigating the relationship between enzyme–substrate binding and activity were highlighted as challenges to overcome for the de novo design of food enzymes. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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18 pages, 3233 KiB  

Open AccessArticle

Molecular Insights into Substrate Binding of the Outer Membrane Enzyme OmpT

by Yubo Zhang and Marc Baaden

Catalysts 2023, 13(2), 214; https://doi.org/10.3390/catal13020214 - 17 Jan 2023

Cited by 2 | Viewed by 2816

Abstract

The enzyme OmpT of the outer membrane of Escherichia coli shows proteolytic activity and cleaves peptides and proteins. Using molecular dynamics simulations in a fully hydrated lipid bilayer on a time scale of hundreds of nanoseconds, we draw a detailed atomic picture of [...] Read more.

The enzyme OmpT of the outer membrane of Escherichia coli shows proteolytic activity and cleaves peptides and proteins. Using molecular dynamics simulations in a fully hydrated lipid bilayer on a time scale of hundreds of nanoseconds, we draw a detailed atomic picture of substrate recognition in the OmpT-holo enzyme complex. Hydrogen bonds and salt bridges are essential for maintaining the integrity of the active site and play a central role for OmpT in recognizing its substrate. Electrostatic interactions are critical at all stages from approaching the substrate to docking at the active site. Computational alanine scanning based on the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) approach confirms the importance of multiple residues in the active site that form salt bridges. The substrate fluctuates along the axis of the β-barrel, which is associated with oscillations of the binding cleft formed by the residue pairs D210-H212 and D83-D85. Principal component analysis suggests that substrate and protein movements are correlated. We observe the transient presence of putative catalytic water molecules near the active site, which may be involved in the nucleophilic attack on the cleavable peptide bond of the substrate. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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16 pages, 2650 KiB  

Open AccessArticle

Yeast Expressed Hybrid Peptide CLP Abridged Pro-Inflammatory Cytokine Levels by Endotoxin Neutralization

by Junhao Cheng, Baseer Ahmad, Muhammad Asif Raza, Henan Guo, Marhaba Ahmat, Xubiao Wei, Lulu Zhang, Zhongxuan Li, Qiang Cheng, Jing Zhang, Junyong Wang, Dayong Si, Yueping Zhang and Rijun Zhang

Microorganisms 2023, 11(1), 131; https://doi.org/10.3390/microorganisms11010131 - 4 Jan 2023

Cited by 6 | Viewed by 3413

Abstract

The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was [...] Read more.

The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was considered to have potent endotoxin-neutralizing activity with minimal cytotoxic and hemolytic activity. To achieve high secretion expression, an expression vector of pPICZαA-HSA-CLP was constructed by the golden gate cloning strategy before being transformed into Pichia pastoris and integrated into the genome. The recombinant CLP was purified through the Ni-NTA affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. The Limulus amebocyte lysate (LAL) test exhibited that the hybrid peptide CLP inhibited lipopolysaccharides (LPS) in a dose-dependent manner and was significantly (p < 0.05) more efficient compared to the parent peptides. In addition, it essentially diminished (p < 0.05) the levels of nitric oxide and pro-inflammatory cytokines (including TNF-α, IL6, and IL-1β) in LPS-induced mouse RAW264.7 macrophages. As an attendant to the control and the parental peptide LL37, the number of LPS-induced apoptotic cells was diminished compared to the control parental peptide LL37 (p < 0.05) with the treatment of CLP. Consequently, we concluded that the hybrid peptide CLP might be used as a therapeutic agent. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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15 pages, 2270 KiB  

Open AccessArticle

Construction and Validation of a New Naïve Sequestrin Library for Directed Evolution of Binders against Aggregation-Prone Peptides

by Linnea Charlotta Hjelm, Hanna Lindberg, Stefan Ståhl and John Löfblom

Int. J. Mol. Sci. 2023, 24(1), 836; https://doi.org/10.3390/ijms24010836 - 3 Jan 2023

Cited by 1 | Viewed by 2362

Abstract

Affibody molecules are small affinity proteins that have excellent properties for many different applications, ranging from biotechnology to diagnostics and therapy. The relatively flat binding surface is typically resulting in high affinity and specificity when developing binding reagents for globular target proteins. For [...] Read more.

Affibody molecules are small affinity proteins that have excellent properties for many different applications, ranging from biotechnology to diagnostics and therapy. The relatively flat binding surface is typically resulting in high affinity and specificity when developing binding reagents for globular target proteins. For smaller unstructured peptides, the paratope of affibody molecules makes it more challenging to achieve a sufficiently large binding surface for high-affinity interactions. Here, we describe the development of a new type of protein scaffold based on a dimeric form of affibodies with a secondary structure content and mode of binding that is distinct from conventional affibody molecules. The interaction is characterized by encapsulation of the target peptide in a tunnel-like cavity upon binding. The new scaffold was used for construction of a high-complexity phage-displayed library and selections from the library against the amyloid beta peptide resulted in identification of high-affinity binders that effectively inhibited amyloid aggregation. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)
(This article belongs to the Section Molecular Biology)

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13 pages, 3125 KiB  

Open AccessArticle

Development of Pectinase Based Nanocatalyst by Immobilization of Pectinase on Magnetic Iron Oxide Nanoparticles Using Glutaraldehyde as Crosslinking Agent

by Tayyaba Behram, Sidra Pervez, Muhammad Asif Nawaz, Shujaat Ahmad, Amin Ullah Jan, Haneef Ur Rehman, Shahbaz Ahmad, Nasir Mehmood Khan and Farman Ali Khan

Molecules 2023, 28(1), 404; https://doi.org/10.3390/molecules28010404 - 3 Jan 2023

Cited by 13 | Viewed by 2861

Abstract

To increase its operational stability and ongoing reusability, B. subtilis pectinase was immobilized on iron oxide nanocarrier. Through co-precipitation, magnetic iron oxide nanoparticles were synthesized. Scanning electron microscopy (SEM) and energy dispersive electron microscopy (EDEX) were used to analyze the nanoparticles. Pectinase was [...] Read more.

To increase its operational stability and ongoing reusability, B. subtilis pectinase was immobilized on iron oxide nanocarrier. Through co-precipitation, magnetic iron oxide nanoparticles were synthesized. Scanning electron microscopy (SEM) and energy dispersive electron microscopy (EDEX) were used to analyze the nanoparticles. Pectinase was immobilized using glutaraldehyde as a crosslinking agent on iron oxide nanocarrier. In comparison to free pectinase, immobilized pectinase demonstrated higher enzymatic activity at a variety of temperatures and pH levels. Immobilization also boosted pectinase’s catalytic stability. After 120 h of pre-incubation at 50 °C, immobilized pectinase maintained more than 90% of its initial activity due to the iron oxide nanocarrier, which improved the thermal stability of pectinase at various temperatures. Following 15 repetitions of enzymatic reactions, immobilized pectinase still exhibited 90% of its initial activity. According to the results, pectinase’s catalytic capabilities were enhanced by its immobilization on iron oxide nanocarrier, making it economically suitable for industrial use. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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24 pages, 7710 KiB  

Open AccessArticle

Construction of Bi-Enzyme Self-Assembly Clusters Based on SpyCatcher/SpyTag for the Efficient Biosynthesis of (R)-Ethyl 2-hydroxy-4-phenylbutyrate

by Jinmei Wang, Yuan Lu, Pengpeng Cheng, Chuyue Zhang, Lan Tang, Lihua Du, Jinghua Li and Zhimin Ou

Biomolecules 2023, 13(1), 91; https://doi.org/10.3390/biom13010091 - 1 Jan 2023

Cited by 6 | Viewed by 2911

Abstract

Cascade reactions catalyzed by multi-enzyme systems are important in science and industry and can be used to synthesize drugs and nutrients. In this study, two types of macromolecules of bi-enzyme self-assembly clusters (BESCs) consisting of carbonyl reductase (CpCR) and glucose dehydrogenase (GDH) were [...] Read more.

Cascade reactions catalyzed by multi-enzyme systems are important in science and industry and can be used to synthesize drugs and nutrients. In this study, two types of macromolecules of bi-enzyme self-assembly clusters (BESCs) consisting of carbonyl reductase (CpCR) and glucose dehydrogenase (GDH) were examined. Stereoselective CpCR and GDH were successfully fused with SpyCatcher and SpyTag, respectively, to obtain four enzyme modules, namely: SpyCatcher-CpCR, SpyCatcher-GDH, SpyTag-CpCR, and SpyTag-GDH, which were covalently coupled in vitro to form two types of hydrogel-like BESCs: CpCR-SpyCatcher-SpyTag-GDH and GDH-SpyCatcher-SpyTag-CpCR. CpCR-SpyCatcher-SpyTag-GDH showed a better activity and efficiently converted ethyl 2-oxo-4-phenylbutyrate (OPBE) to ethyl(R)2-hydroxy-4-phenylbutanoate ((R)-HPBE), while regenerating NADPH. At 30 °C and pH 7, the conversion rate of OPBE with CpCR-SpyCatcher-SpyTag-GDH as a catalyst reached 99.9%, with the ee% of (R)-HPBE reaching above 99.9%. This conversion rate was 2.4 times higher than that obtained with the free bi-enzyme. The pH tolerance and temperature stability of the BESCs were also improved compared with those of the free enzymes. In conclusion, bi-enzyme assemblies were docked using SpyCatcher/SpyTag to produce BESCs with a special structure and excellent catalytic activity, improving the catalytic efficiency of the enzyme. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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11 pages, 6138 KiB  

Open AccessArticle

Halogenation of Peptides and Proteins Using Engineered Tryptophan Halogenase Enzymes

by Barindra Sana, Ding Ke, Eunice Hui Yen Li, Timothy Ho, Jayasree Seayad, Hung A. Duong and Farid J. Ghadessy

Biomolecules 2022, 12(12), 1841; https://doi.org/10.3390/biom12121841 - 8 Dec 2022

Cited by 7 | Viewed by 3333

Abstract

Halogenation of bioactive peptides via incorporation of non-natural amino acid derivatives during chemical synthesis is a common strategy to enhance functionality. Bacterial tyrptophan halogenases efficiently catalyze regiospecific halogenation of the free amino acid tryptophan, both in vitro and in vivo. Expansion of their [...] Read more.

Halogenation of bioactive peptides via incorporation of non-natural amino acid derivatives during chemical synthesis is a common strategy to enhance functionality. Bacterial tyrptophan halogenases efficiently catalyze regiospecific halogenation of the free amino acid tryptophan, both in vitro and in vivo. Expansion of their substrate scope to peptides and proteins would facilitate highly-regulated post-synthesis/expression halogenation. Here, we demonstrate novel in vitro halogenation (chlorination and bromination) of peptides by select halogenase enzymes and identify the C-terminal (G/S)GW motif as a preferred substrate. In a first proof-of-principle experiment, we also demonstrate chemo-catalyzed derivatization of an enzymatically chlorinated peptide, albeit with low efficiency. We further rationally derive PyrH halogenase mutants showing improved halogenation of the (G/S)GW motif, both as a free peptide and when genetically fused to model proteins with efficiencies up to 90%. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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13 pages, 3541 KiB  

Open AccessFeature PaperArticle

Euterpe oleracea Mart (Açaizeiro) from the Brazilian Amazon: A Novel Font of Fungi for Lipase Production

by Iracirema S. Sena, Adriana M. Ferreira, Victor H. Marinho, Fabrício H. e Holanda, Swanny F. Borges, Agerdanio A. de Souza, Rosemary de Carvalho R. Koga, Adilson L. Lima, Alexandro C. Florentino and Irlon M. Ferreira

Microorganisms 2022, 10(12), 2394; https://doi.org/10.3390/microorganisms10122394 - 2 Dec 2022

Cited by 6 | Viewed by 2790

Abstract

Lipases (EC 3.1.1.3) are hydrolases that catalyze triglycerides hydrolysis in free fatty acids and glycerol. Among the microorganisms that produce lipolytic enzymes, the entophytic fungi stand out. We evaluated 32 fungi of different genera, Pestalotiopsis, Aspergillus, Trichoderma, Penicillium, Fusarium [...] Read more.

Lipases (EC 3.1.1.3) are hydrolases that catalyze triglycerides hydrolysis in free fatty acids and glycerol. Among the microorganisms that produce lipolytic enzymes, the entophytic fungi stand out. We evaluated 32 fungi of different genera, Pestalotiopsis, Aspergillus, Trichoderma, Penicillium, Fusarium, Colletotrichum, Chaetomium, Mucor, Botryodiplodia, Xylaria, Curvularia, Neocosmospora and Verticillium, isolated from Euterpe oleracea Mart. (Açaizeiro) from the Brazilian Amazon for lipase activity. The presence of lipase was evidenced by the deposition of calcium crystals. The endophytic Pestalotiopsis sp. (31) and Aspergillus sp. (24) with Pz 0.237 (++++) and 0.5 (++++), respectively, were the ones that showed the highest lipolytic activity in a solid medium. Lipase activity was rated in liquid medium, in a different range of temperatures (°C), pH and time (days). The values obtained in the production of lipase by the endophytic fungi were 94% for Pestalotiopsis sp. (31) and 93.87% for Aspergillus sp. (24). Therefore, it is emphasized that the endophytic fungus isolated the E. oleracea palm may be a potential candidate to produce enzymes of global commercial interest. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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25 pages, 6374 KiB  

Open AccessReview

Insights on Chemical Crosslinking Strategies for Proteins

by Brindha Jayachandran, Thansila N Parvin, M Mujahid Alam, Kaushik Chanda and Balamurali MM

Molecules 2022, 27(23), 8124; https://doi.org/10.3390/molecules27238124 - 22 Nov 2022

Cited by 56 | Viewed by 11123

Abstract

Crosslinking of proteins has gained immense significance in the fabrication of biomaterials for various health care applications. Various novel chemical-based strategies are being continuously developed for intra-/inter-molecular crosslinking of proteins to create a network/matrix with desired mechanical/functional properties without imparting toxicity to the [...] Read more.

Crosslinking of proteins has gained immense significance in the fabrication of biomaterials for various health care applications. Various novel chemical-based strategies are being continuously developed for intra-/inter-molecular crosslinking of proteins to create a network/matrix with desired mechanical/functional properties without imparting toxicity to the host system. Many materials that are used in biomedical and food packaging industries are prepared by chemical means of crosslinking the proteins, besides the physical or enzymatic means of crosslinking. Such chemical methods utilize the chemical compounds or crosslinkers available from natural sources or synthetically generated with the ability to form covalent/non-covalent bonds with proteins. Such linkages are possible with chemicals like carbodiimides/epoxides, while photo-induced novel chemical crosslinkers are also available. In this review, we have discussed different protein crosslinking strategies under chemical methods, along with the corresponding crosslinking reactions/conditions, material properties and significant applications. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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20 pages, 4928 KiB  

Open AccessArticle

Two Subgroups within the GH43_36 α-l-Arabinofuranosidase Subfamily Hydrolyze Arabinosyl from Either Mono-or Disubstituted Xylosyl Units in Wheat Arabinoxylan

by Kai P. Leschonski, Svend G. Kaasgaard, Nikolaj Spodsberg, Kristian B. R. M. Krogh and Mirjam A. Kabel

Int. J. Mol. Sci. 2022, 23(22), 13790; https://doi.org/10.3390/ijms232213790 - 9 Nov 2022

Cited by 7 | Viewed by 2327

Abstract

Fungal arabinofuranosidases (ABFs) catalyze the hydrolysis of arabinosyl substituents (Ara) and are key in the interplay with other glycosyl hydrolases to saccharify arabinoxylans (AXs). Most characterized ABFs belong to GH51 and GH62 and are known to hydrolyze the linkage of α-(1→2)-Ara and α-(1→3)-Ara [...] Read more.

Fungal arabinofuranosidases (ABFs) catalyze the hydrolysis of arabinosyl substituents (Ara) and are key in the interplay with other glycosyl hydrolases to saccharify arabinoxylans (AXs). Most characterized ABFs belong to GH51 and GH62 and are known to hydrolyze the linkage of α-(1→2)-Ara and α-(1→3)-Ara in monosubstituted xylosyl residues (Xyl) (ABF-m2,3). Nevertheless, in AX a substantial number of Xyls have two Aras (i.e., disubstituted), which are unaffected by ABFs from GH51 and GH62. To date, only two fungal enzymes have been identified (in GH43_36) that specifically release the α-(1→3)-Ara from disubstituted Xyls (ABF-d3). In our research, phylogenetic analysis of available GH43_36 sequences revealed two major clades (GH43_36a and GH43_36b) with an expected substrate specificity difference. The characterized fungal ABF-d3 enzymes aligned with GH43_36a, including the GH43_36 from Humicola insolens (HiABF43_36a). Hereto, the first fungal GH43_36b (from Talaromyces pinophilus) was cloned, purified, and characterized (TpABF43_36b). Surprisingly, TpABF43_36b was found to be active as ABF-m2,3, albeit with a relatively low rate compared to other ABFs tested, and showed minor xylanase activity. Novel specificities were also discovered for the HiABF43_36a, as it also released α-(1→2)-Ara from a disubstitution on the non-reducing end of an arabinoxylooligosaccharide (AXOS), and it was active to a lesser extent as an ABF-m2,3 towards AXOS when the Ara was on the second xylosyl from the non-reducing end. In essence, this work adds new insights into the biorefinery of agricultural residues. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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14 pages, 4164 KiB  

Open AccessArticle

A Programmable, DNA-Exclusively-Guided Argonaute DNase and Its Higher Cleavage Specificity Achieved by 5′-Hydroxylated Guide

by Shichao Sun, Dejin Xu, Lin Zhu, Bei Hu and Zhen Huang

Biomolecules 2022, 12(10), 1340; https://doi.org/10.3390/biom12101340 - 21 Sep 2022

Cited by 7 | Viewed by 3015

Abstract

Argonaute proteins exist widely in eukaryotes and prokaryotes, and they are of great potential for molecular cloning, nucleic acid detection, DNA assembly, and gene editing. However, their overall properties are not satisfactory and hinder their broad applications. Herein, we investigated a prokaryotic Argonaute [...] Read more.

Argonaute proteins exist widely in eukaryotes and prokaryotes, and they are of great potential for molecular cloning, nucleic acid detection, DNA assembly, and gene editing. However, their overall properties are not satisfactory and hinder their broad applications. Herein, we investigated a prokaryotic Argonaute nuclease from a mesophilic bacterium Clostridium disporicum (CdAgo) and explored its overall properties, especially with 5′-hydroxylated (5′-OH) guides. We found that CdAgo can exclusively use single-stranded DNA (ssDNA) as guide to cleave ssDNA and plasmid targets. Further, we found the length of the efficient guide is narrower for the 5′-OH guide (17–20 nt) than for the 5′-phosphorylated guide (5′-P, 14–21 nt). Furthermore, we discovered that the 5′-OH guides can generally offer stronger mismatch discrimination than the 5′-P ones. The 5′-OH guides offer the narrower length range, higher mismatch discrimination and more accurate cleavage than the 5′-P guides. Therefore, 5′-OH-guide-directed CdAgo has great potential in biological and biomedical applications. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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11 pages, 2652 KiB  

Open AccessArticle

Discovery of the Key Mutation Site Influencing the Thermostability of Thermomyces lanuginosus Lipase by Rosetta Design Programs

by Enheng Zhu, Xia Xiang, Sidi Wan, Huabiao Miao, Nanyu Han and Zunxi Huang

Int. J. Mol. Sci. 2022, 23(16), 8963; https://doi.org/10.3390/ijms23168963 - 11 Aug 2022

Cited by 6 | Viewed by 2201

Abstract

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, [...] Read more.

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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9 pages, 4323 KiB  

Open AccessCommunication

Antibodies Regulate Dual-Function Enzyme IYD to Induce Functional Synergy between Metabolism and Thermogenesis

by Sunghyun Kang, Hwan-Woo Park and Kyung Ho Han

Int. J. Mol. Sci. 2022, 23(14), 7834; https://doi.org/10.3390/ijms23147834 - 15 Jul 2022

Cited by 5 | Viewed by 2234

Abstract

Iodotyrosine deiodinase (IYD) is a type of deiodinase enzyme that scavenges iodide from the thyroid gland. Previously, we showed that H3 Ab acts as an agonist on IYD to induce migration of cells to the heart and differentiate human stem cells into brown [...] Read more.

Iodotyrosine deiodinase (IYD) is a type of deiodinase enzyme that scavenges iodide from the thyroid gland. Previously, we showed that H3 Ab acts as an agonist on IYD to induce migration of cells to the heart and differentiate human stem cells into brown adipocyte-like cells. To continue this study, we investigated the dual function of IYD in hypothyroidism by blocking IYD and in thermogenesis by looking at the induction of brown adipocyte-like cells by treatment with H3 Ab in a mouse model. Surprisingly, our results suggest H3 Ab acts on IYD as both an antagonist and agonist to reduce T4 and increase core body temperature in the mouse model. Taken together, the data suggest IYD has a dual function that can regulate physiological metabolism and enhance thermogenesis. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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16 pages, 30374 KiB  

Open AccessArticle

A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation

by Yinping Pan, Shijin Tang, Minghai Zhou, Fanglin Ao, Zhuozhou Tang, Liancai Zhu, Deshuai Lou, Jun Tan and Bochu Wang

Catalysts 2022, 12(7), 781; https://doi.org/10.3390/catal12070781 - 14 Jul 2022

Cited by 3 | Viewed by 2438

Abstract

7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays an important role in the biosynthesis of tauroursodeoxycholic acid (TUDCA) using complex substrate chicken bile powder as raw material. However, chicken bile powder contains 4.74% taurocholic acid (TCA), and a new by-product tauroursocholic acid (TUCA) will be produced, having [...] Read more.

7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays an important role in the biosynthesis of tauroursodeoxycholic acid (TUDCA) using complex substrate chicken bile powder as raw material. However, chicken bile powder contains 4.74% taurocholic acid (TCA), and a new by-product tauroursocholic acid (TUCA) will be produced, having the risk of causing colorectal cancer. Here, we obtained a novel NADP(H)-dependent 7α-HSDH with good thermostability from Ursus thibetanus gut microbiota (named St-2-2). St-2-2 could catalyze taurochenodeoxycholic acid (TCDCA) and TCA with the catalytic activity of 128.13 and 269.39 U/mg, respectively. Interestingly, by a structure-based C-terminal truncation strategy, St-2-2△C10 only remained catalytic activity on TCDCA (14.19 U/mg) and had no activity on TCA. As a result, it can selectively catalyze TCDCA in waste chicken bile powder. MD simulation and structural analysis indicated that enhanced surface hydrophilicity and improved C-terminal rigidity affected the entry and exit of substrates. Hydrogen bond interactions between different subunits and interaction changes in Phe249 of the C-terminal loop inverted the substrate catalytic activity. This is the first report on substrate selectivity of 7α-HSDH by C-terminal truncation strategy and it can be extended to other 7α-HSDHs (J-1-1, S1-a-1). Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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15 pages, 2870 KiB  

Open AccessArticle

Cloning, Expression, Purification, and Characterization of β-Galactosidase from Bifidobacterium longum and Bifidobacterium pseudocatenulatum

by Mingzhu Du, Shuanghong Yang, Tong Jiang, Tingting Liang, Ying Li, Shuzhen Cai, Qingping Wu, Jumei Zhang, Wei Chen and Xinqiang Xie

Molecules 2022, 27(14), 4497; https://doi.org/10.3390/molecules27144497 - 14 Jul 2022

Cited by 7 | Viewed by 3706

Abstract

Expression and purification of β-galactosidases derived from Bifidobacterium provide a new resource for efficient lactose hydrolysis and lactose intolerance alleviation. Here, we cloned and expressed two β-galactosidases derived from Bifidobacterium. The optimal pH for BLGLB1 was 5.5, and the optimal temperature was [...] Read more.

Expression and purification of β-galactosidases derived from Bifidobacterium provide a new resource for efficient lactose hydrolysis and lactose intolerance alleviation. Here, we cloned and expressed two β-galactosidases derived from Bifidobacterium. The optimal pH for BLGLB1 was 5.5, and the optimal temperature was 45 °C, at which the enzyme activity of BLGLB1 was higher than that of commercial enzyme E (300 ± 3.6 U/mg) under its optimal conditions, reaching 2200 ± 15 U/mg. The optimal pH and temperature for BPGLB1 were 6.0 and 45 °C, respectively, and the enzyme activity (0.58 ± 0.03 U/mg) under optimum conditions was significantly lower than that of BLGLB1. The structures of the two β-galactosidase were similar, with all known key sites conserved. When o-nitrophenyl-β-D-galactoside (oNPG) was used as an enzyme reaction substrate, the maximum reaction velocity (V_max) for BLGLB1 and BPGLB1 was 3700 ± 100 U/mg and 1.1 ± 0.1 U/mg, respectively. The kinetic constant (K_m) of BLGLB1 and BPGLB1 was 1.9 ± 0.1 and 1.3 ± 0.3 mmol/L, respectively. The respective catalytic constant (k_cat) of BLGLB1 and BPGLB1 was 1700 ± 40 s⁻¹ and 0.5 ± 0.02 s⁻¹, respectively; the respective k_cat/K_m value of BLGLB1 and BPGLB1 was 870 L/(mmol∙s) and 0.36 L/(mmol∙s), respectively. The K_m, k_cat and V_max values of BLGLB1 were superior to those of earlier reported β-galactosidase derived from Bifidobacterium. Overall, BLGLB1 has potential application in the food industry. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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21 pages, 2228 KiB  

Open AccessReview

Biotechnology, Bioengineering and Applications of Bacillus Nattokinase

by Li Yuan, Chen Liangqi, Tang Xiyu and Li Jinyao

Biomolecules 2022, 12(7), 980; https://doi.org/10.3390/biom12070980 - 13 Jul 2022

Cited by 42 | Viewed by 9681

Abstract

Thrombosis has threatened human health in past decades. Bacillus nattokinase is a potential low-cost thrombolytic drug without side-effects and has been introduced into the consumer market as a functional food or dietary supplement. This review firstly summarizes the biodiversity of sources and the [...] Read more.

Thrombosis has threatened human health in past decades. Bacillus nattokinase is a potential low-cost thrombolytic drug without side-effects and has been introduced into the consumer market as a functional food or dietary supplement. This review firstly summarizes the biodiversity of sources and the fermentation process of nattokinase, and systematically elucidates the structure, catalytic mechanism and enzymatic properties of nattokinase. In view of the problems of low fermentation yield, insufficient activity and stability of nattokinase, this review discusses the heterologous expression of nattokinase in different microbial hosts and summarizes the protein and genetic engineering progress of nattokinase-producing strains. Finally, this review summarizes the clinical applications of nattokinase. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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20 pages, 4045 KiB  

Open AccessArticle

Molecular Recognition of Surface Trans-Sialidases in Extracellular Vesicles of the Parasite Trypanosoma cruzi Using Atomic Force Microscopy (AFM)

by Alexa Prescilla-Ledezma, Fátima Linares, Mariano Ortega-Muñoz, Lissette Retana Moreira, Ana Belén Jódar-Reyes, Fernando Hernandez-Mateo, Francisco Santoyo-Gonzalez and Antonio Osuna

Int. J. Mol. Sci. 2022, 23(13), 7193; https://doi.org/10.3390/ijms23137193 - 28 Jun 2022

Cited by 10 | Viewed by 3382

Abstract

Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease. These enzymes have been related to infectivity, escape from immune [...] Read more.

Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease. These enzymes have been related to infectivity, escape from immune surveillance and pathogenesis exhibited by this protozoan parasite. In this work, atomic force microscopy (AFM)-based single molecule-force spectroscopy is implemented as a suitable technique for the detection and location of functional TS on the surface of extracellular vesicles (EVs) released by tissue-culture cell-derived trypomastigotes (Ex-TcT). For that purpose, AFM cantilevers with functionalized tips bearing the anti-TS monoclonal antibody mAb 39 as a sense biomolecule are engineered using a covalent chemical ligation based on vinyl sulfonate click chemistry; a reliable, simple and efficient methodology for the molecular recognition of TS using the antibody-antigen interaction. Measurements of the breakdown forces between anti-TS mAb 39 antibodies and EVs performed to elucidate adhesion and forces involved in the recognition events demonstrate that EVs isolated from tissue-culture cell-derived trypomastigotes of T. cruzi are enriched in TS. Additionally, a mapping of the TS binding sites with submicrometer-scale resolution is provided. This work represents the first AFM-based molecular recognition study of Ex-TcT using an antibody-tethered AFM probe. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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15 pages, 3710 KiB  

Open AccessArticle

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

by Jiafeng Niu, Ruxue Yan, Juan Shen, Xiaoyu Zhu, Fanqiang Meng, Zhaoxin Lu and Fengxia Lu

Int. J. Mol. Sci. 2022, 23(12), 6588; https://doi.org/10.3390/ijms23126588 - 13 Jun 2022

Cited by 10 | Viewed by 3250

Abstract

Type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) was efficiently produced and secreted in Bacillus subtilis RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in B. [...] Read more.

Type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) was efficiently produced and secreted in Bacillus subtilis RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in B. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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23 pages, 2737 KiB  

Open AccessArticle

Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA

by Isabella M. Acquistapace, Emma J. Thompson, Imke Kühn, Mike R. Bedford, Charles A. Brearley and Andrew M. Hemmings

Int. J. Mol. Sci. 2022, 23(11), 6346; https://doi.org/10.3390/ijms23116346 - 6 Jun 2022

Cited by 6 | Viewed by 3803

Abstract

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P₄ and [...] Read more.

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P₄ and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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13 pages, 2537 KiB  

Open AccessArticle

Structural Basis for the Regiospecificity of a Lipase from Streptomyces sp. W007

by Zexin Zhao, Siyue Chen, Long Xu, Jun Cai, Jia Wang and Yonghua Wang

Int. J. Mol. Sci. 2022, 23(10), 5822; https://doi.org/10.3390/ijms23105822 - 22 May 2022

Cited by 12 | Viewed by 2779

Abstract

The efficiency and accuracy of the synthesis of structural lipids are closely related to the regiospecificity of lipases. Understanding the structural mechanism of their regiospecificity contributes to the regiospecific redesign of lipases for meeting the technological innovation needs. Here, we used a thermostable [...] Read more.

The efficiency and accuracy of the synthesis of structural lipids are closely related to the regiospecificity of lipases. Understanding the structural mechanism of their regiospecificity contributes to the regiospecific redesign of lipases for meeting the technological innovation needs. Here, we used a thermostable lipase from Streptomyces sp. W007 (MAS1), which has been recently reported to show great potential in industry, to gain an insight into the structural basis of its regiospecificity by molecular modelling and mutagenesis experiments. The results indicated that increasing the steric hindrance of the site for binding a non-reactive carbonyl group of TAGs could transform the non-specific MAS1 to a α-specific lipase, such as the mutants G40E, G40F, G40Q, G40R, G40W, G40Y, N45Y, H108W and T237Y (PSI > 80). In addition, altering the local polarity of the site as well as the conformational stability of its composing residues could also impact the regiospecificity. Our present study could not only aid the rational design of the regiospecificity of lipases, but open avenues of exploration for further industrial applications of lipases. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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24 pages, 8633 KiB  

Open AccessArticle

Fluorescent Imaging of Extracellular Fungal Enzymes Bound onto Plant Cell Walls

by Neus Gacias-Amengual, Lena Wohlschlager, Florian Csarman and Roland Ludwig

Int. J. Mol. Sci. 2022, 23(9), 5216; https://doi.org/10.3390/ijms23095216 - 6 May 2022

Cited by 1 | Viewed by 3087

Abstract

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and [...] Read more.

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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18 pages, 2473 KiB  

Open AccessArticle

The Nitrogen Atom of Vitamin B₆ Is Essential for the Catalysis of Radical Aminomutases

by Amarendra Nath Maity, Jun-Ru Chen, Quan-Yuan Li and Shyue-Chu Ke

Int. J. Mol. Sci. 2022, 23(9), 5210; https://doi.org/10.3390/ijms23095210 - 6 May 2022

Viewed by 2725

Abstract

Radical aminomutases are pyridoxal 5′-phosphate (PLP, a B₆ vitamer)-dependent enzymes that require the generation of a 5′-deoxyadenosyl radical to initiate the catalytic cycle, to perform a 1,2 amino group shift reaction. The role of the nitrogen atom of PLP in radical aminomutases [...] Read more.

Radical aminomutases are pyridoxal 5′-phosphate (PLP, a B₆ vitamer)-dependent enzymes that require the generation of a 5′-deoxyadenosyl radical to initiate the catalytic cycle, to perform a 1,2 amino group shift reaction. The role of the nitrogen atom of PLP in radical aminomutases has not been investigated extensively yet. We report an alternative synthetic procedure to provide easy access to 1-deazaPLP (dAPLP), an isosteric analog of PLP which acts as a probe for studying the role of the nitrogen atom. Our results revealed that lysine 5,6-aminomutase (5,6-LAM), a radical aminomutase, reconstituted with dAPLP cannot turn over a substrate, demonstrating that the nitrogen atom is essential for radical aminomutases. In contrast, biochemical and spectroscopic studies on the S238A variant reconstituted with PLP revealed a minuscule loss of activity. This apparent anomaly can be explained by a water-mediated rescue of activity in S238A, as if mimicking the active site of lysine 2,3-aminomutase. This study leads to a better comprehension of how enzymes harness the optimum capability of PLP to realize catalysis. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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9 pages, 1668 KiB  

Open AccessArticle

Investigating the Functional Role of the Cysteine Residue in Dehydrin from the Arctic Mouse-Ear Chickweed Cerastium arcticum

by Il-Sup Kim, Woong Choi, Ae Kyung Park, Hyun Kim, Jonghyeon Son, Jun Hyuck Lee, Seung Chul Shin, T. Doohun Kim and Han-Woo Kim

Molecules 2022, 27(9), 2934; https://doi.org/10.3390/molecules27092934 - 4 May 2022

Viewed by 1894

Abstract

The stress-responsive, SK₅ subclass, dehydrin gene, CaDHN, has been identified from the Arctic mouse-ear chickweed Cerastium arcticum. CaDHN contains an unusual single cysteine residue (Cys143), which can form intermolecular disulfide bonds. Mutational analysis and a redox experiment confirmed that the [...] Read more.

The stress-responsive, SK₅ subclass, dehydrin gene, CaDHN, has been identified from the Arctic mouse-ear chickweed Cerastium arcticum. CaDHN contains an unusual single cysteine residue (Cys143), which can form intermolecular disulfide bonds. Mutational analysis and a redox experiment confirmed that the dimerization of CaDHN was the result of an intermolecular disulfide bond between the cysteine residues. The biochemical and physiological functions of the mutant C143A were also investigated by in vitro and in vivo assays using yeast cells, where it enhanced the scavenging of reactive oxygen species (ROS) by neutralizing hydrogen peroxide. Our results show that the cysteine residue in CaDHN helps to enhance C. arcticum tolerance to abiotic stress by regulating the dimerization of the intrinsically disordered CaDHN protein, which acts as a defense mechanism against extreme polar environments. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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12 pages, 3041 KiB  

Open AccessArticle

YsHyl8A, an Alkalophilic Cold-Adapted Glycosaminoglycan Lyase Cloned from Pathogenic Yersinia sp. 298

by Shilong Zhang, Yujiao Li, Feng Han and Wengong Yu

Molecules 2022, 27(9), 2897; https://doi.org/10.3390/molecules27092897 - 2 May 2022

Cited by 8 | Viewed by 2218

Abstract

A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) [...] Read more.

A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) was cloned from the genome of Yersinia sp. 298 and subjected to recombinant expression. The specific activity of the recombinase YsHyl8A was 11.19 U/mg, with an optimal reaction temperature of 40 °C and 50% of its specific activity remaining after thermal incubation at 30 °C for 1 h. In addition, its optimal reaction pH was 7.5, and while it was most stable at pH 6.0 in Na₂HPO₄-citric acid buffer, it remained highly stable at pH 6.0–11.0. Further, its enzymatic activity was increased five-fold with 0.1 M NaCl. YsHyl8A, as an endo-lyase, can degrade both HA and CS, producing disaccharide end-products. These properties suggested that YsHyl8A possessed both significant alkalophilic and cold-adapted features while being dependent on NaCl, likely resulting from its marine source. Yersinia is a typical fish pathogen, with glycosaminoglycan lyase (GAG lyase) as a potential pathogenic factor, exhibiting strong hyaluronidase and chondroitinase activity. Further research on the pathogenic mechanism of GAG lyase may benefit the prevention and treatment of related diseases. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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14 pages, 3221 KiB  

Open AccessArticle

Characterization of a Thermostable and Surfactant-Tolerant Chondroitinase B from a Marine Bacterium Microbulbifer sp. ALW1

by Mingjing Mou, Qingsong Hu, Hebin Li, Liufei Long, Zhipeng Li, Xiping Du, Zedong Jiang, Hui Ni and Yanbing Zhu

Int. J. Mol. Sci. 2022, 23(9), 5008; https://doi.org/10.3390/ijms23095008 - 30 Apr 2022

Cited by 6 | Viewed by 2314

Abstract

Chondroitinase plays an important role in structural and functional studies of chondroitin sulfate (CS). In this study, a new member of chondroitinase B of PL6 family, namely ChSase B6, was cloned from marine bacterium Microbulbifer sp. ALW1 and subjected to enzymatic and structural [...] Read more.

Chondroitinase plays an important role in structural and functional studies of chondroitin sulfate (CS). In this study, a new member of chondroitinase B of PL6 family, namely ChSase B6, was cloned from marine bacterium Microbulbifer sp. ALW1 and subjected to enzymatic and structural characterization. The recombinant ChSase B6 showed optimum activity at 40 °C and pH 8.0, with enzyme kinetic parameters of K_m and V_max against chondroitin sulfate B (CSB) to be 7.85 µg/mL and 1.21 U/mg, respectively. ChSase B6 demonstrated thermostability under 60 °C for 2 h with about 50% residual activity and good pH stability under 4.0–10.0 for 1 h with above 60% residual activity. In addition, ChSase B6 displayed excellent stability against the surfactants including Tween-20, Tween-80, Trion X-100, and CTAB. The degradation products of ChSase B6-treated CSB exhibited improved antioxidant ability as a hydroxyl radical scavenger. Structural analysis and site-directed mutagenesis suggested that the conserved residues Lys248 and Arg269 were important for the activity of ChSase B6. Characterization, structure, and molecular dynamics simulation of ChSase B6 provided a guide for further tailoring for its industrial application for chondroitin sulfate bioresource development. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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12 pages, 3045 KiB  

Open AccessArticle

Structure-Guided Engineering of a Family IV Cold-Adapted Esterase Expands Its Substrate Range

by Nehad Noby, Rachel L. Johnson, Jonathan D. Tyzack, Amira M. Embaby, Hesham Saeed, Ahmed Hussein, Sherine N. Khattab, Pierre J. Rizkallah and D. Dafydd Jones

Int. J. Mol. Sci. 2022, 23(9), 4703; https://doi.org/10.3390/ijms23094703 - 24 Apr 2022

Cited by 3 | Viewed by 2487

Abstract

Cold active esterases have gained great interest in several industries. The recently determined structure of a family IV cold active esterase (EstN7) from Bacillus cohnii strain N1 was used to expand its substrate range and to probe its commercially valuable substrates. Database mining [...] Read more.

Cold active esterases have gained great interest in several industries. The recently determined structure of a family IV cold active esterase (EstN7) from Bacillus cohnii strain N1 was used to expand its substrate range and to probe its commercially valuable substrates. Database mining suggested that triacetin was a potential commercially valuable substrate for EstN7, which was subsequently proved experimentally with the final product being a single isomeric product, 1,2-glyceryl diacetate. Enzyme kinetics revealed that EstN7’s activity is restricted to C2 and C4 substrates due to a plug at the end of the acyl binding pocket that blocks access to a buried water-filled cavity. Residues M187, N211 and W206 were identified as key plug forming residues. N211A stabilised EstN7 allowing incorporation of the destabilising M187A mutation. The M187A-N211A double mutant had the broadest substrate range, capable of hydrolysing a C8 substrate. W206A did not appear to have any significant effect on substrate range either alone or when combined with the double mutant. Thus, the enzyme kinetics and engineering together with a recently determined structure of EstN7 provide new insights into substrate specificity and the role of acyl binding pocket plug residues in determining family IV esterase stability and substrate range. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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19 pages, 5307 KiB  

Open AccessArticle

A Novel β-Hairpin Peptide Z-d14CFR Enhances Multidrug-Resistant Bacterial Clearance in a Murine Model of Mastitis

by Xue Wang, Shuxian Li, Mengze Du, Ning Liu, Qiang Shan, Yunjing Zou, Jiufeng Wang and Yaohong Zhu

Int. J. Mol. Sci. 2022, 23(9), 4617; https://doi.org/10.3390/ijms23094617 - 21 Apr 2022

Cited by 9 | Viewed by 2481

Abstract

The widespread prevalence of antimicrobial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) have gained comprehensive attention as one of the major alternatives to antibiotics. However, low antibacterial activity and high-cost production have limited the applications of natural AMPs. [...] Read more.

The widespread prevalence of antimicrobial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) have gained comprehensive attention as one of the major alternatives to antibiotics. However, low antibacterial activity and high-cost production have limited the applications of natural AMPs. In this study, we successfully expressed recombinant Zophobas atratus (Z. atratus) defensin for the first time. In order to increase the antimicrobial activity of peptide, we designed 5 analogues derived from Z. atratus defensin, Z-d13, Z-d14C, Z-d14CF, Z-d14CR and Z-d14CFR. Our results showed that Z-d14CFR (RGCRCNSKSFCVCR-NH₂) exhibited a broad-spectrum antimicrobial activity to both Gram-positive bacteria and Gram-negative bacteria, including multidrug-resistant bacteria. It possessed less than 5% hemolysis and 10% cytotoxicity, even at a high concentration of 1 mg/mL. Antimicrobial mechanism studies indicated that Z-d14CFR performed antimicrobial effect via inhibiting biofilm formation, disrupting bacterial membrane integrity and inducing cellular contents release. Furthermore, Z-d14CFR showed a great therapeutic effect on the treatment of multidrug-resistant Escherichia coli (E. coli) infection by enhancing bacterial clearance, decreasing neutrophils infiltration and the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in a murine model of mastitis. Our findings suggest that Z-d14CFR could be a promising candidate against multidrug-resistant bacteria. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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19 pages, 4476 KiB  

Open AccessArticle

Whole-Genome Sequence and Comparative Analysis of Trichoderma asperellum ND-1 Reveal Its Unique Enzymatic System for Efficient Biomass Degradation

by Fengzhen Zheng, Tianshuo Han, Abdul Basit, Junquan Liu, Ting Miao and Wei Jiang

Catalysts 2022, 12(4), 437; https://doi.org/10.3390/catal12040437 - 13 Apr 2022

Cited by 11 | Viewed by 3577

Abstract

The lignocellulosic enzymes of Trichoderma asperellum have been intensely investigated toward efficient conversion of biomass into high-value chemicals/industrial products. However, lack of genome data is a remarkable hurdle for hydrolase systems studies. The secretory enzymes of newly isolated T. asperellum ND-1 during lignocellulose [...] Read more.

The lignocellulosic enzymes of Trichoderma asperellum have been intensely investigated toward efficient conversion of biomass into high-value chemicals/industrial products. However, lack of genome data is a remarkable hurdle for hydrolase systems studies. The secretory enzymes of newly isolated T. asperellum ND-1 during lignocellulose degradation are currently poorly known. Herein, a high-quality genomic sequence of ND-1, obtained by both Illumina HiSeq 2000 sequencing platforms and PacBio single-molecule real-time, has an assembly size of 35.75 Mb comprising 10,541 predicted genes. Secretome analysis showed that 895 proteins were detected, with 211 proteins associated with carbohydrate-active enzymes (CAZymes) responsible for biomass hydrolysis. Additionally, T. asperellum ND-1, T. atroviride IMI 206040, and T. virens Gv-298 shared 801 orthologues that were not identified in T. reesei QM6a, indicating that ND-1 may play critical roles in biological-control. In-depth analysis suggested that, compared with QM6a, the genome of ND-1 encoded a unique enzymatic system, especially hemicellulases and chitinases. Moreover, after comparative analysis of lignocellulase activities of ND-1 and other fungi, we found that ND-1 displayed higher hemicellulases (particularly xylanases) and comparable cellulases activities. Our analysis, combined with the whole-genome sequence information, offers a platform for designing advanced T. asperellum ND-1 strains for industrial utilizations, such as bioenergy production. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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15 pages, 3429 KiB  

Open AccessArticle

Charged Residue Implantation Improves the Affinity of a Cross-Reactive Dengue Virus Antibody

by Huiling Wei, Jie Tan, Bingjie Zhou, Xiaotong Guan, Qiaoxian Zhong and Jiaqi Wang

Int. J. Mol. Sci. 2022, 23(8), 4197; https://doi.org/10.3390/ijms23084197 - 11 Apr 2022

Cited by 1 | Viewed by 2951

Abstract

Dengue virus (DENV) has four serotypes that complicate vaccine development. Envelope protein domain III (EDIII) of DENV is a promising target for therapeutic antibody development. One EDIII-specific antibody, dubbed 1A1D-2, cross-reacts with DENV 1, 2, and 3 but not 4. To improve the [...] Read more.

Dengue virus (DENV) has four serotypes that complicate vaccine development. Envelope protein domain III (EDIII) of DENV is a promising target for therapeutic antibody development. One EDIII-specific antibody, dubbed 1A1D-2, cross-reacts with DENV 1, 2, and 3 but not 4. To improve the affinity of 1A1D-2, in this study, we analyzed the previously solved structure of 1A1D-2-DENV2 EDIII complex. Mutations were designed, including A54E and Y105R in the heavy chain, with charges complementary to the epitope. Molecular dynamics simulation was then used to validate the formation of predicted salt bridges. Interestingly, a surface plasmon resonance experiment showed that both mutations increased affinities of 1A1D-2 toward EDIII of DENV1, 2, and 3 regardless of their sequence variation. Results also revealed that A54E improved affinities through both a faster association and slower dissociation, whereas Y105R improved affinities through a slower dissociation. Further simulation suggested that the same mutants interacted with different residues in different serotypes. Remarkably, combination of the two mutations additively improved 1A1D-2 affinity by 8, 36, and 13-fold toward DENV1, 2, and 3, respectively. In summary, this study demonstrated the utility of tweaking antibody-antigen charge complementarity for affinity maturation and emphasized the complexity of improving antibody affinity toward multiple antigens. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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11 pages, 2804 KiB  

Open AccessArticle

Efficient Synthesis of cis-4-Propylcyclohexanol Using a Mutant Alcohol Dehydrogenase Coupled with Glucose Dehydrogenase

by Licheng Wu, Zhiwei Wang, Xiaolei Guo, Pengfu Liu, Ziduo Liu and Gaobing Wu

Catalysts 2022, 12(4), 406; https://doi.org/10.3390/catal12040406 - 6 Apr 2022

Cited by 2 | Viewed by 2393

Abstract

cis-4-Propylcyclohexanol is an important intermediate for synthesizing trans-2-(4-propylcyclohexyl)-1,3-propanediol, which is widely used in the manufacture of liquid crystal displays. In this study, cis-4-propylcyclohexanol was prepared using a mutant alcohol dehydrogenase from Lactobacillus kefir (LK-TADH, A94T/F147L/L199H/A202L) coupled with glucose dehydrogenase. Using [...] Read more.

cis-4-Propylcyclohexanol is an important intermediate for synthesizing trans-2-(4-propylcyclohexyl)-1,3-propanediol, which is widely used in the manufacture of liquid crystal displays. In this study, cis-4-propylcyclohexanol was prepared using a mutant alcohol dehydrogenase from Lactobacillus kefir (LK-TADH, A94T/F147L/L199H/A202L) coupled with glucose dehydrogenase. Using the optimal catalytic conditions, 125 g/L (250 g) of 4-propylcyclohexanone was completely transformed after 5 h, and 225.8 g of cis-4-propylcyclohexanol (cis/trans ratio of 99.5:0.5) was obtained through extraction and rotary evaporation at a yield of 90.32%. This study reports a potential method for the green production of cis-4-propylcyclohexanol as the key intermediate of trans-2-(4-propylcyclohexyl)-1,3-propanediol at an industrial level. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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25 pages, 16452 KiB  

Open AccessReview

Substrate-Specific Engineering of Amino Acid Dehydrogenase Superfamily for Synthesis of a Variety of Chiral Amines and Amino Acids

by Feng Zhou, Yan Xu, Yao Nie and Xiaoqing Mu

Catalysts 2022, 12(4), 380; https://doi.org/10.3390/catal12040380 - 29 Mar 2022

Cited by 13 | Viewed by 4637

Abstract

Amino acid dehydrogenases (AADHs) are a group of enzymes that catalyze the reversible reductive amination of keto acids with ammonia to produce chiral amino acids using either nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺) as cofactors. [...] Read more.

Amino acid dehydrogenases (AADHs) are a group of enzymes that catalyze the reversible reductive amination of keto acids with ammonia to produce chiral amino acids using either nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺) as cofactors. Among them, glutamate dehydrogenase, valine dehydrogenase, leucine dehydrogenase, phenylalanine dehydrogenase, and tryptophan dehydrogenase have been classified as a superfamily of amino acid dehydrogenases (s-AADHs) by previous researchers because of their conserved structures and catalytic mechanisms. Owing to their excellent stereoselectivity, high atom economy, and low environmental impact of the reaction pathway, these enzymes have been extensively engineered to break strict substrate specificities for the synthesis of high value-added chiral compounds (chiral amino acids, chiral amines, and chiral amino alcohols). Substrate specificity engineering of s-AADHs mainly focuses on recognition engineering of the substrate side chain R group and substrate backbone carboxyl group. This review summarizes the reported studies on substrate specificity engineering of s-AADHs and reports that this superfamily of enzymes shares substrate specificity engineering hotspots (the inside of the pocket, substrate backbone carboxyl anchor sites, substrate entrance tunnel, and hinge region), which sheds light on the substrate-specific tailoring of these enzymes. Full article

(This article belongs to the Topic Advances in Enzymes and Protein Engineering)

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