Enzyme Catalysis, Biotransformation and Bioeconomy

A special issue of Catalysts (ISSN 2073-4344). This special issue belongs to the section "Biocatalysis".

Deadline for manuscript submissions: closed (20 May 2022) | Viewed by 25242

Special Issue Editors


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Guest Editor
Department of Chemistry, University of Lleida, Lleida, Spain
Interests: biocatalysis; organic synthesis; biomass conversion; bioeconomy and circular economy

E-Mail Website
Guest Editor
Department of Chemistry, University of Lleida, Lleida, Spain
Interests: biocatalysis; organic synthesis; bioeconomy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Currently, there are hundred different biocatalytic processes that have been implemented in various pharma, chemical, food, cosmetic and agro-based industries. Considering these facts, biocatalysis has become an essential tool in the synthesis of different chemicals with diverse industrial purposes. In this sense, the use of enzymes in many biotransformation processes has demonstrated that biocatalysts are a real alternative to traditional chemical processes. This is because biocatalysis can provide more sustainable, efficient, and less polluting methods than conventional methods. Likewise, methods based on the use of enzymes can further broaden their applicability and meet the criteria of sustainability if efficiently employed for the transformation of cheaply available agro-food wastes. They can give added value to by-products and waste and allow the development of valorization processes that are included within the bioeconomy and the circular economy.

The purpose of this Special Issue is to collect original research papers, reviews, and opinions focused on the latest developments in which various types of catalytic processes have been used to synthesize chemical compounds with industrial applications. Articles that present the latest research focused on developing strategies related to the biotransformation of industrial waste and by-products will be appreciated.

Dr. Edinson Yara-Varón
Prof. Dr. Ramon Canela-Garayoa
Guest Editors

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Keywords

  • enzymes
  • whole cells
  • biocatalysis
  • chemoenzymatic synthesis
  • biotransformation
  • novel biocatalytic processes
  • sustainable chemistry
  • renewable biomass
  • waste valorization

Published Papers (12 papers)

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Research

11 pages, 1119 KiB  
Article
A Prolyl Endopeptidase from Flammulina velutipes Degrades Celiac Disease-Inducing Peptides in Grain Flour Samples
by Franziska Ersoy, Philine Beinhorn, Kathrin Schalk, Katharina A. Scherf, Ralf G. Berger and Ulrich Krings
Catalysts 2023, 13(1), 158; https://doi.org/10.3390/catal13010158 - 10 Jan 2023
Cited by 1 | Viewed by 2568
Abstract
Celiac disease (CD) is an inflammatory disorder of the small intestine. Gluten peptides are supposed to be responsible for the reaction, the best-researched of which is the so-called ‘33-mer’. Analogous peptides in secalins (rye) and hordeins (barley) have been described. This study presents [...] Read more.
Celiac disease (CD) is an inflammatory disorder of the small intestine. Gluten peptides are supposed to be responsible for the reaction, the best-researched of which is the so-called ‘33-mer’. Analogous peptides in secalins (rye) and hordeins (barley) have been described. This study presents the degradation of gliadins, glutenins, hordeins and secalins purified from the respective flours using a prolyl endopeptidase from the Basidiomycete Flammulina velutipes (FvpP). The flour fractions were incubated with the enzyme, and the cleavage sites were determined using high-resolution nLC-qTOF-MS/MS. For the wheat samples, eight cleavage sites in the 33-mer peptide were shown, and all of the six described epitopes were successfully cleaved. For the commercially available prolyl-specific endopeptidase from Aspergillus niger (An-Pep), which was used as a control, only two cleavage sites that cleaved three of the six epitopes were identified. For the secalins, four prolyl-specific cleavage sites in the CD-active peptide QPFPQPQQPIPQ were found for the FvpP but none for the An-Pep. The CD-active peptide QPFPQPEQPFPW in C-hordein was cleaved at three prolyl-specific positions by the FvpP. The study proves the usability of FvpP to degrade CD-inducing peptides in real-grain flour samples and indicates its higher effectiveness compared with An-Pep. A clinical study would be required to assess the therapeutic or preventive potential of FvpP for CD. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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12 pages, 2758 KiB  
Article
Immobilization of a Bienzymatic System via Crosslinking to a Metal-Organic Framework
by Raneem Ahmad, Sydnie Rizaldo, Sarah E. Shaner, Daniel S. Kissel and Kari L. Stone
Catalysts 2022, 12(9), 969; https://doi.org/10.3390/catal12090969 - 29 Aug 2022
Cited by 8 | Viewed by 1992
Abstract
A leading biotechnological advancement in the field of biocatalysis is the immobilization of enzymes on solid supports to create more stable and recyclable systems. Metal-organic frameworks (MOFs) are porous materials that have been explored as solid supports for enzyme immobilization. Composed of organic [...] Read more.
A leading biotechnological advancement in the field of biocatalysis is the immobilization of enzymes on solid supports to create more stable and recyclable systems. Metal-organic frameworks (MOFs) are porous materials that have been explored as solid supports for enzyme immobilization. Composed of organic linkers and inorganic nodes, MOFs feature empty void space with large surface areas and have the ability to be modified post-synthesis. Our target enzyme system for immobilization is glucose oxidase (GOx) and chloroperoxidase (CPO). Glucose oxidase catalyzes the oxidation of glucose and is used for many applications in biosensing, biofuel cells, and food production. Chloroperoxidase is a fungal heme enzyme that catalyzes peroxide-dependent halogenation, oxidation, and hydroxylation. These two enzymes work sequentially in this enzyme system by GOx producing peroxide, which activates CPO that reacts with a suitable substrate. This study focuses on using a zirconium-based MOF, UiO-66-NH2, to immobilize the enzyme system via crosslinking with the MOF’s amine group on the surface of the MOF. This study investigates two different crosslinkers: disuccinimidyl glutarate (DSG) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinidimide (NHS), providing stable crosslinking of the MOF to the enzymes. The two crosslinkers are used to covalently bond CPO and GOx onto UiO-66-NH2, and a comparison of the recyclability and enzymatic activity of the single immobilization of CPO and the doubly immobilized CPO and GOx is discussed through assays and characterization analyses. The DSG-crosslinked composites displayed enhanced activity relative to the free enzyme, and all crosslinked enzyme/MOF composites demonstrated recyclability, with at least 30% of the activity being retained after four catalytic cycles. The results of this report will aid researchers in utilizing CPO as a biocatalyst that is more active and has greater recyclability. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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22 pages, 2013 KiB  
Article
Rendered-Protein Hydrolysates as a Low-Cost Nitrogen Source for the Fungal Biotransformation of 5-Hydroxymethylfurfural
by Diana Cosovanu, Alberto Millán Acosta, Pau Cabañeros López, Krist V. Gernaey, Qian Li, Rene Lametsch, Ramon Canela-Garayoa, Jordi Eras and Gemma Villorbina
Catalysts 2022, 12(8), 839; https://doi.org/10.3390/catal12080839 - 30 Jul 2022
Cited by 1 | Viewed by 1677
Abstract
5-hydroxymethylfurfural (HMF) is a platform chemical that can be converted into a wide range of high-value derivatives. Industrially, HMF-based derivatives are synthesized via chemical catalysis. However, biocatalytic transformation has emerged as an attractive alternative. Significant advances have been made in the last years [...] Read more.
5-hydroxymethylfurfural (HMF) is a platform chemical that can be converted into a wide range of high-value derivatives. Industrially, HMF-based derivatives are synthesized via chemical catalysis. However, biocatalytic transformation has emerged as an attractive alternative. Significant advances have been made in the last years using isolated enzymes and whole-cell biocatalysts in HMF biotransformation. Nonetheless, one of the major bottlenecks is the cost of the process, mainly due to the microorganism growth substrate. In this work, biotransformation studies to transform HMF into 2,5-di(hydroxymethyl)furan (DHMF) were carried out with the fungus Fusarium striatum using low-cost protein hydrolysates. The protein hydrolysates were obtained from fines, an unexploited material produced during the rendering process of meat industry waste residues. Given the high content in the protein of fines, of around 46%, protein hydrolysis was optimized using two commercially available proteases, Alcalase 2.4 L and Neutrase 0.8 L. The maximum degree of hydrolysis (DH) achieved with Alcalase 2.4 L was 21.4% under optimal conditions of 5% E/S ratio, pH 8, 55 °C, and 24 h. On the other hand, Neutrase 0.8 L exhibited lower efficiency, and therefore, lower protein recovery. After optimization of the Neutrase 0.8 L process using the response surface methodology (RSM), the maximum DH achieved was 7.2% with the variables set at 15% E/S ratio, initial pH 8, 40 °C, and 10.5 h. Using these hydrolysates as a nitrogen source allowed higher sporulation of the fungus and, therefore, the use of a lower volume of inoculum (three-fold), obtaining a DHMF yield > 90%, 50% higher than the yield obtained when using commercial peptones. The presented process allows the transformation of animal co- and by-products into low-cost nitrogen sources, which greatly impacts the industrial feasibility of HMF biotransformation. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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14 pages, 3976 KiB  
Article
PTCL1-EstA from Paenarthrobacter aurescens TC1, a Candidate for Industrial Application Belonging to the VIII Esterase Family
by Qinyu Li, Xiaojia Chen, Xiangcen Liu, Zheng Chen, Yang Han, Peng Zhou, Jiping Shi and Zhijun Zhao
Catalysts 2022, 12(5), 473; https://doi.org/10.3390/catal12050473 - 23 Apr 2022
Cited by 1 | Viewed by 1820
Abstract
The esterase PTCL1-EstA from Paenarthrobacter aurescens TC1 was expressed in Escherichia coli and characterized. An 1152 bp open reading frame encoding a 383 amino acid polypeptide was successfully expressed, the C-terminally His6-tagged PTCL1-EstA enzyme was purified, and the predicted molecular mass of the [...] Read more.
The esterase PTCL1-EstA from Paenarthrobacter aurescens TC1 was expressed in Escherichia coli and characterized. An 1152 bp open reading frame encoding a 383 amino acid polypeptide was successfully expressed, the C-terminally His6-tagged PTCL1-EstA enzyme was purified, and the predicted molecular mass of the purified PTCL1-EstA was 40.6 kDa. The EstA family serine hydrolase PTCL1-EstA belongs to the esterase family VIII, contains esterase-labeled S-C-S-K sequences, and homologous class C beta-lactamase sequences. PTCL1-EstA favored p-nitrophenyl esters with C2-C6 chain lengths, but it was also able to hydrolyze long-chain p-nitrophenyl esters. Homology modelling and substrate docking predicted that Ser59 was an active site residue in PTCL1-EstA, as well as Tyr148, Ala325, and Asp323, which are critical in catalyzing the enzymatic reaction of p-nitrophenyl esters. PTCL1-EstA reached the highest specific activity against p-nitrophenyl butyrate (C4) at pH 7.0 and 45 °C but revealed better thermal stability at 40 °C and maintained high relative enzymatic activity and stability at pH 5.0–9.0. Fermentation medium optimization for PTCL1-EstA increased the enzyme activity to 510.76 U/mL, tapping the potential of PTCL1-EstA for industrial production. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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14 pages, 3283 KiB  
Article
Purification and Structural Characterization of the Auxiliary Activity 9 Native Lytic Polysaccharide Monooxygenase from Thermoascus aurantiacus and Identification of Its C1- and C4-Oxidized Reaction Products
by Weishuai Yu, Imran Mohsin, Anastassios C. Papageorgiou and Duochuan Li
Catalysts 2022, 12(2), 139; https://doi.org/10.3390/catal12020139 - 23 Jan 2022
Cited by 2 | Viewed by 2691
Abstract
Auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are copper-dependent oxidoreductases that use O2 or H2O2 to perform oxidative cleavage of cellulose in the presence of an electron donor. Combined with cellulases, they can assist in a more efficient [...] Read more.
Auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are copper-dependent oxidoreductases that use O2 or H2O2 to perform oxidative cleavage of cellulose in the presence of an electron donor. Combined with cellulases, they can assist in a more efficient cleavage of cellulose. AA9 LPMOs have therefore attracted considerable attention in recent years for use in biotechnological applications. Here, a native AA9 LPMO (nTaAA9A) from the thermophilic fungus Thermoascus aurantiacus was purified and characterized. The enzyme was shown to be active and able to cleave cellulose and xylan to produce C1- and C4-oxidized products. It was also found to retain about 84.3, 63.7, and 35.3% of its activity after incubation for 30 min at 60, 70, and 80 °C, respectively, using quantitative activity determination. The structure was determined to 1.36 Å resolution and compared with that of the recombinant enzyme expressed in Aspergillus oryzae. Structural differences in the glycosylated Asn138 and in solvent-exposed loops were identified. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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14 pages, 3646 KiB  
Article
Mutagenesis of the l-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides
by Sven Bordewick, Ralf G. Berger and Franziska Ersoy
Catalysts 2021, 11(11), 1385; https://doi.org/10.3390/catal11111385 - 17 Nov 2021
Cited by 2 | Viewed by 1666
Abstract
The l-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed [...] Read more.
The l-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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12 pages, 2750 KiB  
Article
Sesquiterpene Cyclases from the Basidiomycete Cerrena unicolor
by Nils Püth, Franziska Ersoy, Ulrich Krings and Ralf G. Berger
Catalysts 2021, 11(11), 1361; https://doi.org/10.3390/catal11111361 - 12 Nov 2021
Cited by 4 | Viewed by 1946
Abstract
Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these [...] Read more.
Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these fungi. The basidiomycete Cerrena unicolor, a known sesquiterpene producer, was in silico screened for sesquiterpene cyclases via homology Basic Local Alignment Search Tool searches. Cyclase genes identified were cloned and heterologously expressed in Escherichia coli Bl21 using pCOLD I as the expression vector. Ten cyclases were successfully produced and purified, and their identity was confirmed using amino acid sequencing of tryptic peptides by nano-liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry. Gas chromatography/mass spectrometry analysis was applied to characterize these cyclases according to the formation of sesquiterpene hydrocarbons and oxidized terpenoids. Bioinformatic characterization and phylogenetic determination allowed for the classification of these diverse fungal enzymes. A representative single and a multi-product STS, respectively, were further analyzed for their dependency from divalent metal cations as a cofactor for the catalytic activity. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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9 pages, 1504 KiB  
Article
Recombinant Production of Arginyl Dipeptides by l-Amino Acid Ligase RizA Coupled with ATP Regeneration
by Sven Bordewick, Tim A. Mast, Ralf G. Berger and Franziska Ersoy
Catalysts 2021, 11(11), 1290; https://doi.org/10.3390/catal11111290 - 27 Oct 2021
Cited by 3 | Viewed by 2140
Abstract
Arginyl dipeptides like Arg-Ser, Arg-Ala, and Arg-Gly are salt-taste enhancers and can potentially be used to reduce the salt content of food. The l-amino acid ligase RizA from B. subtilis selectively synthesizes arginyl dipeptides. However, industrial application is prevented by the high [...] Read more.
Arginyl dipeptides like Arg-Ser, Arg-Ala, and Arg-Gly are salt-taste enhancers and can potentially be used to reduce the salt content of food. The l-amino acid ligase RizA from B. subtilis selectively synthesizes arginyl dipeptides. However, industrial application is prevented by the high cost of the cofactor adenosine triphosphate (ATP). Thus, a coupled reaction system was created consisting of RizA and acetate kinase (AckA) from E. coli providing ATP regeneration from acetyl phosphate. Both enzymes were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were varied and analyzed by RP-HPLC with fluorescence detection. Under optimal conditions the system produced up to 5.9 g/L Arg-Ser corresponding to an ATP efficiency of 23 g Arg-Ser per gram ATP. Using similar conditions with alanine or glycine as second amino acid, 2.6 g/L Arg-Ala or 2.4 g/L Arg Gly were produced. The RizA/AckA system selectively produced substantial amounts of arginyl dipeptides while minimizing the usage of the expensive ATP. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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13 pages, 2222 KiB  
Article
Characterization of a Novel Lutein Cleavage Dioxygenase, EhLCD, from Enterobacter hormaechei YT-3 for the Enzymatic Synthesis of 3-Hydroxy-β-ionone from Lutein
by Zhangde Long, Naixin Duan, Yun Xue, Min Wang, Jigang Li, Zan Su, Qibin Liu, Duobin Mao and Tao Wei
Catalysts 2021, 11(11), 1257; https://doi.org/10.3390/catal11111257 - 20 Oct 2021
Cited by 2 | Viewed by 1635
Abstract
3-Hydroxy-β-ionone, a flavor and fragrance compound with fruity violet-like characteristics, is widely applied in foodstuff and beverages, and is currently produced using synthetic chemistry. In this study, a novel lutein cleavage enzyme (EhLCD) was purified and characterized from Enterobacter hormaechei YT-3 [...] Read more.
3-Hydroxy-β-ionone, a flavor and fragrance compound with fruity violet-like characteristics, is widely applied in foodstuff and beverages, and is currently produced using synthetic chemistry. In this study, a novel lutein cleavage enzyme (EhLCD) was purified and characterized from Enterobacter hormaechei YT-3 to convert lutein to 3-hydroxy-β-ionone. Enzyme EhLCD was purified to homogeneity by ammonium sulfate precipitation, Q-Sepharose, phenyl-Sepharose, and Superdex 200 chromatography. The molecular mass of purified EhLCD, obtained by SDS-PAGE, was approximately 50 kDa. The enzyme exhibited the highest activity toward lutein, followed by zeaxanthin, β-cryptoxanthin, and β-carotene, suggesting that EhLCD exhibited higher catalytic efficiency for carotenoid substrates bearing 3-hydroxy-ionone rings. Isotope-labeling experiments showed that EhLCD incorporated oxygen from O2 into 3-hydroxy-β-ionone and followed a dioxygenase reaction mechanism for different carotenoid substrates. These results indicated that EhLCD is the first characterized bacterial lutein cleavage dioxygenase. Active EhLCD was also confirmed to be a Fe2+-dependent protein with 1 molar equivalent of non-haem Fe2+. The purified enzyme displayed optimal activity at 45 °C and pH 8.0. The optimum concentrations of the substrate, enzyme, and Tween 40 for 3-hydroxy-β-ionone production were 60 μM lutein/L, 1.5 U/mL, and 2% (w/v), respectively. Under optimum conditions, EhLCD produced 3-hydroxy-β-ionone (637.2 mg/L) in 60 min with a conversion of 87.0% (w/w), indicating that this enzyme is a potential candidate for the enzymatic synthesis of 3-hydroxy-β-ionone in biotechnological applications. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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11 pages, 1876 KiB  
Article
Hot Spots of Phytoene Desaturase from Rhodobacter sphaeroides Influencing the Desaturation of Phytoene
by Bo Hyun Choi, Sung Hui Kim and Pyung Cheon Lee
Catalysts 2021, 11(10), 1248; https://doi.org/10.3390/catal11101248 - 18 Oct 2021
Cited by 1 | Viewed by 2379
Abstract
Phytoene desaturase (CrtI, E.C. 1.3.99.31) shows variable desaturation activity, thereby introducing different numbers of conjugated double bonds (CDB) into the substrate phytoene. In particular, Rhodobacter sphaeroides CrtI is known to introduce additional 6 CDBs into the phytoene with 3 CDBs, generating neurosporene with [...] Read more.
Phytoene desaturase (CrtI, E.C. 1.3.99.31) shows variable desaturation activity, thereby introducing different numbers of conjugated double bonds (CDB) into the substrate phytoene. In particular, Rhodobacter sphaeroides CrtI is known to introduce additional 6 CDBs into the phytoene with 3 CDBs, generating neurosporene with 9 CDBs. Although in-depth studies have been conducted on the function and phylogenetic evolution of CrtI, little information exists on its range of CDB-introducing capabilities. We investigated the relationship between the structure and CDB-introducing capability of CrtI. CrtI of R. sphaeroides KCTC 12085 was randomly mutagenized to produce carotenoids of different CDBs (neurosporene for 9 CDBs, lycopene for 11 CDBs, and 3,4-didehydrolycopene for 13 CDBs). From six CrtI mutants producing different ratios of neurosporene/lycopene/3,4-didehydrolycopene, three amino acids (Leu163, Ala171, and Ile454) were identified that significantly determined carotenoid profiles. While the L163P mutation was responsible for producing neurosporene as a major carotenoid, A171P and I454T produced lycopene as the major product. Finally, according to the in silico model, the mutated amino acids are gathered in the membrane-binding domain of CrtI, which could distantly influence the FAD binding region and consequently the degree of desaturation in phytoene. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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10 pages, 2228 KiB  
Article
Screening of Acetyl Donors and the Robust Enzymatic Synthesis of Acetyl-CoA by 10-Deacetylbaccatin III-10-β-O-acetyltransferase
by Bo-Yong Zhang, Hao Wang, Ting Gong, Tian-Jiao Chen, Jing-Jing Chen, Jin-Ling Yang and Ping Zhu
Catalysts 2021, 11(10), 1240; https://doi.org/10.3390/catal11101240 - 15 Oct 2021
Viewed by 1585
Abstract
Acetyl-CoA is the precursor of many bio-manufacturing products and is also the hub of the cellular metabolism of energy and substances. However, acetyl-CoA is not a bulk commodity and its application is hindered due to its high cost and low yield. In this [...] Read more.
Acetyl-CoA is the precursor of many bio-manufacturing products and is also the hub of the cellular metabolism of energy and substances. However, acetyl-CoA is not a bulk commodity and its application is hindered due to its high cost and low yield. In this study, we screened acetyl donor candidates and utilized 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) in the synthesis of acetyl-CoA with CoASH as the acetyl acceptor. Among the tested candidates, acetylsalicylic acid methyl ester was identified to be the best acetyl donor, followed by acetyl-trans-resveratrol, acetylsalicylic acid ethyl ester, acetylsalicylsalicylic acid, and 4-acetoxyacetanilide. The enzymatic reaction conditions were optimized and the maximum yield of acetyl-CoA reached 14.82 mg/mL, which is the highest yield among all reported approaches to date. Meanwhile, 4.22 mg/mL of the by-product salicylic acid methyl ester, which is another industrial material, was produced. Additionally, a preliminary purification process for acetyl-CoA was established, in which 40 mg acetyl-CoA (HPLC purity > 98%) was acquired from the finished 20 mL reaction system (feeding 46 mg CoASH and 34 mg ASME) with a recovery rate of 86%. Our study lays the foundation for the large-scale production of acetyl-CoA by an enzymatic approach and will promote its application in different fields. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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9 pages, 2364 KiB  
Article
Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity
by Jing-Jing Chen, Xiao Liang, Tian-Jiao Chen, Jin-Ling Yang and Ping Zhu
Catalysts 2021, 11(9), 1042; https://doi.org/10.3390/catal11091042 - 28 Aug 2021
Viewed by 1568
Abstract
The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular [...] Read more.
The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr268 and Ser466 were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2. Full article
(This article belongs to the Special Issue Enzyme Catalysis, Biotransformation and Bioeconomy)
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