BIORESOURCE TECHNOLOGY
·
2022
·
IF 11.889
Xiaoyu Chen,Zhe Dou,Tianwei Luo,Zewen Sun,Hongmin Ma,Guochao Xu,Ye Ni
Ancestral enzymes are promising for industrial biotechnology due to high stability and catalytic promiscuity. An effective protocol was developed for the directed resurrection of ancestral enzymes. Employing genome mining with diaryl alcohol dehydrogenase Kp ADH as the probe, descendant enzymes D10 and D11 were firstly identified. Then through ancestral sequence reconstruction, A64 was resurrected with a specific activity of 4.3 U·mg −1 . The optimum pH of A64 was 7.5, distinct from 5.5 of D10. The T15 50 and T m values of A64 were 57.5 °C and 61.7 °C, significantly higher than those of the descendant counterpart. Substrate spectrum of A64 was quantitively characterized with a Shannon-Wiener index of 2.38, more expanded than D10, especially, towards bulky ketones in Group A and B. A64 also exhibited higher enantioselectivity. This study provides an effective protocol for constructing of ancestral enzymes and an efficient ancestral enzyme of industrial relevance for asymmetric synthesis of chiral alcohols.
研究领域:
生物化学
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工业生物技术
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DOI
PubMed
高影响因子
TEXTILE RESEARCH JOURNAL
·
2020
·
IF 1.926
Yawei Wang,Yun Bai,Tong Shu,Pei Fan,Huashan Zhang,Ossi Turunen,Hairong Xiong,Longjiang Yu
A novel versatile glycoside hydrolase Cel5M was identified from Pectobacterium carotovorum and used in ramie degumming. The full-length Cel5M (GenBank: MH544226) containing the catalytic domain Cel5MD and its carbohydrate-binding module (CBM3) were expressed in E. coli. Both Cel5M and Cel5MD showed a broad substrate spectrum cleaving konjac glucomannan (KGM), carboxymethylcellulose (CMC) and oat spelt xylan. The optimal temperature of Cel5MD was 55oC with CMC and KGM, whereas the optimum temperature of Cel5M was 65oC with KGM and 60oC with CMC. The improved thermal activity with KGM was probably reflected also in the much lower Km with KGM than with CMC, indicating stronger binding of the full-length enzyme with KGM. Both enzymes were highly stable over a broad pH range from pH 5.0 to 9.0 with KGM. The use of the Cel5M enzyme together with the wild-type P. carotovorum HG-49 strain showed significant improvement when compared to using the mere strain HG-49 for application in ramie degumming. Moreover, the whole P. carotovorum Cel5M gene was over-expressed in P. carotovorum HG-49 itself to improve the ramie-degumming capacity. Altogether, this study indicates that in situ production of processing enzymes could be utilized in the textile industry.
研究领域:
生物技术
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酶学
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纺织科学
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产品关联
DOI
高影响因子
Molecular Catalysis
·
2024
·
IF 3.9
Yan-Fei Liang,Shuai Feng,Dan Wu,Meng Ma,Jin-Tao Ren,Qian Zhao,Wen-Yun Gao,Heng Li
1-Deoxy- D- xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in the 2-methyl- D- erythritol-4-phosphate (MEP) terpenoid biosynthetic pathway. To explore the active sites of the protein, we made a series of mutations to the key amino acid residues of Escherichia coli DXS including H49, R420, D427, and R478. The results show that several mutants including H49Q lose almost all their catalytic activity in DXP synthesis, but the double-point mutants H49QD427H and H49QD427 N recover 100 % activity of the wild type enzyme. The kinetic characterization displays that the affinity of the double-point mutants for pyruvate and D- glyceraldehyde-3-phosphate only slightly decreases. We also assessed impact of the mutation on the oxidative decarboxylation activity of all the mutants. Despite the fact that H49QD427H and H49QD427 N get back the full activity with regard to DXP synthesis, their oxidase activity remains unrestored. Furthermore, we assayed the acceptor substrate spectrum of the mutants and the results show that a large portion of them catalyzes ligation between pyruvate and nitrosobenzene to form N-phenyl-N-hydroxyacetamide, whereas R420D, R420F, and D427H exhibit activity in connection of pyruvate and benzaldehyde to afford phenylacetocarbinol (PAC), with the first two mutants yielding ( S )-PAC (yields 21 %-25 %, ee values 71–75 %) while D427H furnishing ( R )-PAC (yield 41 %, ee value 91 %), implying the key role of D427 for the acceptor substrate recognition and the stereoselectivity of the enzyme. It is worth noting that this is the first report of DXS mutants that can recognize benzaldehyde as an acceptor substrate and generate both enantiomers of PAC.
研究领域:
生物化学
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分子生物学
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产品关联
DOI
高影响因子
PROCESS BIOCHEMISTRY
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2022
·
IF 4.885
Wenhao Qu,Yajun Bai,Tai-Ping Fan,Xiaohui Zheng,Yujie Cai
Lactobacillus reuteri DSM20016 has several aldo-keto reductases (AKRs), and they were heterologously expressed in E. coli BL21 (DE3) in this study. Subsequently, these AKRs were purified, and their enzymatic properties were investigated. It was found that these enzymes used NADPH as coenzyme and did not depend on metal ions. The optimum pH of these AKRs was a slightly acidic (AKR-1 and AKR-6: 5.5; AKR-3 and AKR-8: 6; AKR-2 and AKR-4: 6.5). These enzymes had a broad substrate spectrum and could convert a variety of aldehydes to alcohols; however, they did not catalyze the reverse reactions. All results showed that Lactobacillus reuteri had the ability to produce various volatile compounds, which allowed Lactobacillus reuteri to impart complex aroma to something.
研究领域:
微生物学
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生物化学
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酶学
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食品科学
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DOI
高影响因子
JACS Au
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2024
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IF 8.5
Yaoyun Wu,Yaozhong Cui,Wei Song,Wanqing Wei,Zhizhen He,Jinyang Tao,Dejing Yin,Xiulai Chen,Cong Gao,Jia Liu,Liming Liu,Jing Wu
l-Amino acid oxidase (LAAO) is an important biocatalyst used for synthesizing α-keto acids. LAAO from Rhodococcus opacus (RoLAAO) has a broad substrate spectrum; however, its low total turnover number limits its industrial use. To overcome this, we aimed to employ crystal structure-guided density functional theory calculations and molecular dynamic simulations to investigate the catalytic mechanism. Two key steps were identified: S → [TS1] in step 1 and Int1 → [TS2] in step 2. We reprogrammed the transition states [TS1] and [TS2] to reduce the identified energy barrier and obtain a RoLAAO variant capable of catalyzing 19 kinds of l-amino acids to the corresponding high-value α-keto acids with a high total turnover number, yield (≥95.1 g/L), conversion rate (≥95%), and space-time yields ≥142.7 g/L/d in 12–24 h, in a 5 L reactor. Our results indicated the promising potential of the developed RoLAAO variant for use in the industrial production of α-keto acids while providing a potential catalytic-mechanism-guided protein design strategy to achieve the desired physical and catalytic properties of enzymes.
研究领域:
生物催化
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酶工程
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计算化学
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蛋白质工程
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2020
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IF 4.192
Yidong Zhou,Zhijian Ke,Hangting Ye,Mengting Hong,Yifei Xu,Mingliang Zhang,Wankui Jiang,Qing Hong
Carbaryl is a widely used carbamate pesticide in agriculture. The strain Rhizobium sp. X9 possesses the typical carbaryl degradation pathway in which carbaryl is mineralized via 1-naphthol, salicylate, and gentisate. In this study, we cloned a carbaryl hydrolase gene cehA and a novel two-component 1-naphthol hydroxylase gene cehC1C2. CehA mediates carbaryl hydrolysis to 1-naphthol and CehC1, an FMNH2 or FADH2-dependent monooxygenase belonging to the HpaB superfamily, and hydroxylates 1-naphthol in the presence of reduced nicotinamide-adenine dinucleotide (FMN)/flavin adenine dinucleotide (FAD), and the reductase CehC2. CehC1 has the highest amino acid similarity (58%) with the oxygenase component of a two-component 4-nitrophenol 2-monooxygenase, while CehC2 has the highest amino acid similarity (46%) with its reductase component. CehC1C2 could utilize both FAD and FMN as the cofactor during the hydroxylation, although higher catalytic activity was observed with FAD as the cofactor. The optimal molar ratio of CehC1 to CehC2 was 2:1. The Km and Kcat/Km values of CehC1 for 1-naphthol were 74.71 ± 16.07 μM and (8.29 ± 2.44) × 10–4 s–1·μM–1, respectively. Moreover, the enzyme activities and substrate spectrum between CehC1C2 and previously reported 1-naphthol hydroxylase McbC were compared. The results suggested that McbC had a higher 1-naphthol hydroxylation activity, while CehC1C2 had a broader substrate spectrum.
研究领域:
环境微生物学
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生物化学
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农药生物降解
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产品关联
DOI
PubMed
高影响因子
Catalysis Science & Technology
·
2019
·Vol. 9
·期 21
·5943-5952
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IF 5.726
Lian Xu,Li-Chao Wang,Xin-Qi Xu,Juan Lin
L-Threonine transaldolase (LTTA) is a putative serine hydroxymethyltransferase (SHMT) that can catalyze the trans-aldehyde reaction of L-threonine and aldehyde to produce L-threo-β-hydroxy-α-amino acids with excellent stereoselectivity. In the present study, an L-threonine transaldolase from Pseudomonas sp. (PsLTTA) was mined and expressed in Escherichia coli BL21 (DE3). A substrate spectrum assay indicated that PsLTTA only consumed L-threonine as the donor substrate and could accept a wide range of aromatic aldehydes as acceptor substrates. Among these substrates, PsLTTA could catalyze p-methylsulfonyl benzaldehyde and L-threonine to produce L-threo-p-methylsulfonylphenylserine with a high conversion rate (74.4%) and a high de value (79.9%). The conversion and stereoselectivity of PsLTTA were found to be dramatically influenced by the concentration of the whole cell, the co-solvent and the reaction temperature. Through conditional optimization, L-threo-p-methylsulfonylphenylserine was obtained with 67.1% conversion and a near-perfect de value (94.5%), the highest stereoselectivity for an L-threo-β-hydroxy-α-amino acid so far reported by enzymatic synthesis. Finally, synthesis of L-threo-p-methylsulfonylphenylserine at a 100 mL scale by whole-cell biocatalysis was conducted. This is the first systematic report of L-threonine transaldolase as a robust biocatalyst for preparation of β-hydroxy-α-amino acids, which can provide new insights for β-hydroxy-α-amino acids synthesis.
研究领域:
生物化学
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生物催化
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有机合成
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产品关联
DOI
高影响因子
ACS Catalysis
·
2018
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IF 11.384
Guo-Chao Xu,Yue Wang,Ming-Hui Tang,Jie-Yu Zhou,Jing Zhao,Rui-Zhi Han,Ye Ni
A hydroclassified combinatorial saturation mutagenesis (HCSM) strategy was proposed for reshaping the substrate binding pocket by dividing 20 amino acids into four groups based on their hydrophobicity and size. These smart HCSM libraries could significantly reduce screening effort especially for the simultaneous mutagenesis of three or more residues and lacking high throughput screening methods. Employing HCSM strategy, the stereoselectivity of KpADH, an alcohol dehydrogenase from Kluyveromyces polysporus, was efficiently improved to 99.4% ee. (4-Chlorophenyl)(pyridin-2-yl)methanone (CPMK), generally regarded as a “hard-to-reduce” ketone, was used as a model substrate, and its corresponding chiral alcohol products could be utilized as antihistamine precursors. The best variant 50C10 displayed higher binding affinity and catalytic efficiency toward CPMK with KM/kcat of 59.3 s–1·mM–1, 3.51-fold that of KpADH. Based on MD simulations, increased difference between two binding pockets, enhanced hydrophobicity, and π–π and halogen–alkyl interactions were proposed to favor the enantioselective recognition and substrate binding in 50C10. Substrate spectrum analysis revealed that 50C10 exhibited improved enantioselectivity toward diaryl ketones especially with halo- or other electron-withdrawing groups. As much as 500 mM CPMK could be asymmetrically reduced into chiral diaryl alcohols with ee of 99.4% and a space–time yield of 194 g·L–1·d–1 without addition of external NADP+. This study provides an effective mutagenesis strategy for the protein engineering of substrate specificity and enantioselectivity.
研究领域:
蛋白质工程
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生物催化
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酶催化
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手性合成
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药物前体
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分子动力学模拟
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生物化学
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）