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Understanding and Utilization of Extreme Enzymes

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (30 June 2023) | Viewed by 19626

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Dr. Cheng Zhou

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Guest Editor

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
Interests: biochemistry; enzyme engineering; molecular biology; synthetic biology; metabiologic engineering; biological technology

Prof. Dr. Guimin Zhang

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Guest Editor

College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
Interests: protein engineering and high-efficiency expression of enzymes; mycotoxin degradation; biosensing of toxic pollutants in food, environment
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Dear Colleagues,

The extreme enzyme Extremozyme, mostly derived from extremophiles and extreme environment, has considerable economic potential for application to agriculture, food and beverages and feed, detergent, textile, leather, pulp and paper, pharmaceutical and biomining industries because of its unique properties and adaptation to extreme environments. Furthermore, the studies on extreme enzyme improve the understanding of the extremes of life. With the discovery of extreme enzyme combined with enzyme modification by directed evolution or rational engineering, the development of economical bioprocesses will accelerate and be enabled on an increasingly large scale. Although a number of biochemical, molecular and biotechnological application studies were done, further studies are still necessary for the full understanding and application extending of extreme enzymes. This Special Issue aims to present recent research advances on extreme enzymes, and the main emphasis is given, but not limited, to new extreme enzyme discovery, catalytic and adaptation mechanism, structural analysis. Manuscripts of original research, communications, and reviews are all welcome.

Dr. Zhou Cheng
Prof. Dr. Guimin Zhang
Guest Editors

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Keywords

extreme enzyme discovery
enzyme characterization and function
enzyme production and purification
enzyme structure
stability and adaptation mechanism
oligomeric state of extreme enzyme
extreme enzyme engineering
molecular evolution
biotechnological and industrial application

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Published Papers (7 papers)

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Research

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16 pages, 7924 KiB

Open AccessArticle

Construction of Fusion Protein with Carbohydrate-Binding Module and Leaf-Branch Compost Cutinase to Enhance the Degradation Efficiency of Polyethylene Terephthalate

by Yingxuan Chen, Shudi Zhang, Zhenyu Zhai, Shuo Zhang, Jun Ma, Xiao Liang and Quanshun Li

Int. J. Mol. Sci. 2023, 24(3), 2780; https://doi.org/10.3390/ijms24032780 - 1 Feb 2023

Cited by 13 | Viewed by 4585

Abstract

Poly(ethylene terephthalate) (PET) is a manufactured plastic broadly available, whereas improper disposal of PET waste has become a serious burden on the environment. Leaf-branch compost cutinase (LCC) is one of the most powerful and promising PET hydrolases, and its mutant LCC^ICCG shows high catalytic activity and excellent thermal stability. However, low binding affinity with PET has been found to dramatically limit its further industrial application. Herein, TrCBM and CfCBM were rationally selected from the CAZy database to construct fusion proteins with LCC^ICCG, and mechanistic studies revealed that these two domains could bind with PET favorably via polar amino acids. The optimal temperatures of LCC^ICCG-TrCBM and CfCBM-LCC^ICCG were measured to be 70 and 80 °C, respectively. Moreover, these two fusion proteins exhibited favorable thermal stability, maintaining 53.1% and 48.8% of initial activity after the incubation at 90 °C for 300 min. Compared with LCC^ICCG, the binding affinity of LCC^ICCG-TrCBM and CfCBM-LCC^ICCG for PET has been improved by 1.4- and 1.3-fold, respectively, and meanwhile their degradation efficiency on PET films was enhanced by 3.7% and 24.2%. Overall, this study demonstrated that the strategy of constructing fusion proteins is practical and prospective to facilitate the enzymatic PET degradation ability. Full article

(This article belongs to the Special Issue Understanding and Utilization of Extreme Enzymes)

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16 pages, 3097 KiB

Open AccessArticle

Structure of an Alkaline Pectate Lyase and Rational Engineering with Improved Thermo-Alkaline Stability for Efficient Ramie Degumming

by Cheng Zhou, Yuting Cao, Yanfen Xue, Weidong Liu, Jiansong Ju and Yanhe Ma

Int. J. Mol. Sci. 2023, 24(1), 538; https://doi.org/10.3390/ijms24010538 - 29 Dec 2022

Cited by 4 | Viewed by 2945

Abstract

Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed β-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca²⁺-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔT_m values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T₅₀¹⁵ value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T₅₀¹⁵ value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions (4 h, 60 °C, pH 10.0), compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases. Full article

(This article belongs to the Special Issue Understanding and Utilization of Extreme Enzymes)

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13 pages, 2025 KiB

Open AccessArticle

Thermophilic Inorganic Pyrophosphatase Ton1914 from Thermococcus onnurineus NA1 Removes the Inhibitory Effect of Pyrophosphate

by Yajing Li, Xue Yang and Renjun Gao

Int. J. Mol. Sci. 2022, 23(21), 12735; https://doi.org/10.3390/ijms232112735 - 22 Oct 2022

Cited by 4 | Viewed by 2301

Abstract

Pyrophosphate (PP_i) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PP_i can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from Thermococcus onnurineus NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg²⁺ at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32–41%, while that of short-chain DNA was only improved by 9.5–15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications. Full article

(This article belongs to the Special Issue Understanding and Utilization of Extreme Enzymes)

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19 pages, 3030 KiB

Open AccessArticle

Characterization of a Novel Fe²⁺ Activated Non-Blue Laccase from Methylobacterium extorquens

by Abidan Ainiwaer, Yue Liang, Xiao Ye and Renjun Gao

Int. J. Mol. Sci. 2022, 23(17), 9804; https://doi.org/10.3390/ijms23179804 - 29 Aug 2022

Cited by 12 | Viewed by 2036

Abstract

Herein, a novel laccase gene, Melac13220, was amplified from Methylobacterium extorquens and successfully expressed in Escherichia coli with a molecular weight of approximately 50 kDa. The purified Melac13220 had no absorption peak at 610 nm and remained silent within electron paramagnetic resonance spectra, suggesting that Melac13220 belongs to the non-blue laccase group. Both inductively coupled plasma spectroscopy/optical emission spectrometry (ICP-OES) and isothermal titration calorimetry (ITC) indicated that one molecule of Melac13220 can interact with two iron ions. Furthermore, the optimal temperature of Melac13220 was 65 °C. It also showed a high thermolability, and its half-life at 65 °C was 80 min. Melac13220 showed a very good acid environment tolerance; its optimal pH was 1.5. Cu²⁺ and Co²⁺ can slightly increase enzyme activity, whereas Fe²⁺ could increase Melac13220′s activity five-fold. Differential scanning calorimetry (DSC) indicated that Fe²⁺ could also stabilize Melac13220. Unlike most laccases, Melac13220 can efficiently decolorize Congo Red and Indigo Carmine dyes even in the absence of a redox mediator. Thus, the non-blue laccase from Methylobacterium extorquens shows potential application value and may be valuable for environmental protection, especially in the degradation of dyes at low pH. Full article

(This article belongs to the Special Issue Understanding and Utilization of Extreme Enzymes)

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20 pages, 4761 KiB

Open AccessArticle

Identification of a Novel Lipase with AHSMG Pentapeptide in Hypocreales and Glomerellales Filamentous Fungi

by Denise Esther Gutiérrez-Domínguez, Bartolomé Chí-Manzanero, María Mercedes Rodríguez-Argüello, Jewel Nicole Anna Todd, Ignacio Islas-Flores, Miguel Ángel Canseco-Pérez and Blondy Canto-Canché

Int. J. Mol. Sci. 2022, 23(16), 9367; https://doi.org/10.3390/ijms23169367 - 19 Aug 2022

Cited by 5 | Viewed by 2003

Abstract

Lipases are enzymes that hydrolyze triglycerides to fatty acids and glycerol. A typical element in lipases is a conserved motif of five amino acids (the pentapeptide), most commonly G-X-S-X-G. Lipases with the pentapeptide A-X-S-X-G are present in species of Bacillus, Paucimonas lemoignei, and the yeast Trichosporon asahii; they are usually thermotolerant and solvent resistant. Recently, while searching for true lipases in the Trichoderma harzianum genome, one lipase containing the pentapeptide AHSMG was identified. In this study, we cloned from T. harzianum strain B13-1 the lipase ID135964, renamed here as ThaL, which is 97.65% identical with the reference. We found that ThaL is a lid-containing true lipase of cluster III that belongs to a large family comprising highly conserved proteins in filamentous fungi in the orders Hypocreales and Glomerellales, in which predominantly pathogenic fungi are found. ThaL was expressed in conidia, as well as in T. harzianum mycelium, where it was cultured in liquid minimal medium. These results—together with the amino acid composition, absence of a signal peptide, mitochondrial sorting prediction, disordered regions in the protein, and lineage-specific phylogenetic distribution of its homologs—suggest that ThaL is a non-canonical effector. In summary, AHSMG-lipase is a novel lipase family in filamentous fungi, and is probably involved in pathogenicity. Full article

(This article belongs to the Special Issue Understanding and Utilization of Extreme Enzymes)

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16 pages, 3694 KiB

Open AccessArticle

Discovery of a New Microbial Origin Cold-Active Neopullulanase Capable for Effective Conversion of Pullulan to Panose

by Meixing Wang, Huizhen Hu, Buyu Zhang, Yang Zheng, Pan Wu, Zhenghui Lu and Guimin Zhang

Int. J. Mol. Sci. 2022, 23(13), 6928; https://doi.org/10.3390/ijms23136928 - 22 Jun 2022

Cited by 8 | Viewed by 2407

Abstract

Panose is a type of functional sugar with diverse bioactivities. The enzymatic conversion bioprocess to produce high purity panose with high efficiency has become increasingly important. Here, a new neopullulanase (NPase), Amy117 from B. pseudofirmus 703, was identified and characterized. Amy117 presented the optimal activity at pH 7.0 and 30 °C, its activity is over 40% at 10 °C and over 80% at 20 °C, which is cold-active. The enzyme cleaved α-1, 4-glycosidic linkages of pullulan to generate panose as the only hydrolysis product, and degraded cyclodextrins (CDs) and starch to glucose and maltose, with an apparent preference for CDs. Furthermore, Amy117 can produce 72.7 mg/mL panose with a conversion yield of 91% (w/w) based on 80 mg/mL pullulan. The sequence and structure analysis showed that the low proportion of Arg, high proportion of Asn and Gln, and high α-helix levels in Amy117 may contribute to its cold-active properties. Root mean square deviation (RMSD) analysis also showed that Amy117 is more flexible than two mesophilic homologues. Hence, we discovered a new high-efficiency panose-producing NPase, which so far achieves the highest panose production and would be an ideal candidate in the food industry. Full article

(This article belongs to the Special Issue Understanding and Utilization of Extreme Enzymes)

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Review

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12 pages, 1652 KiB

Open AccessReview

Exploring Oxidoreductases from Extremophiles for Biosynthesis in a Non-Aqueous System

by Shizhen Wang, Hangbin Lei and Zhehui Ji

Int. J. Mol. Sci. 2023, 24(7), 6396; https://doi.org/10.3390/ijms24076396 - 29 Mar 2023

Cited by 7 | Viewed by 2609

Abstract

Organic solvent tolerant oxidoreductases are significant for both scientific research and biomanufacturing. However, it is really challenging to obtain oxidoreductases due to the shortages of natural resources and the difficulty to obtained it via protein modification. This review summarizes the recent advances in gene mining and structure-functional study of oxidoreductases from extremophiles for non-aqueous reaction systems. First, new strategies combining genome mining with bioinformatics provide new insights to the discovery and identification of novel extreme oxidoreductases. Second, analysis from the perspectives of amino acid interaction networks explain the organic solvent tolerant mechanism, which regulate the discrete structure-functional properties of extreme oxidoreductases. Third, further study by conservation and co-evolution analysis of extreme oxidoreductases provides new perspectives and strategies for designing robust enzymes for an organic media reaction system. Furthermore, the challenges and opportunities in designing biocatalysis non-aqueous systems are highlighted. Full article

(This article belongs to the Special Issue Understanding and Utilization of Extreme Enzymes)

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