JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
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2015
·
IF 2.128
Hui Fei,Gang Xu,Jian-Ping Wu,Li-Rong Yang
DERA SEP (2-deoxy- d -ribose-5-phosphate aldolase cloned from Staphylococcus epidermidis ) shows higher catalytic activity toward 2-deoxy- d -ribose-5-phosphate (DRP) than that of DERA ECO (2-deoxy-ribose aldolase cloned from Escherichia coli ). However, the acetaldehyde tolerance of DERA SEP is still at low level, which limits its use in large-scale applications. Although the mechanism responsible for enzyme inactivation in the presence of solvents is unclear, earlier work indicated the positive correlation between the tolerance of an enzyme for organic solvents and the rigidity of its structure. The acetaldehyde tolerance of DERA SEP was improved by site-directed mutagenesis on the basis of enhancing the rigidity of the protein structure. After calculating the virtual mutation energy (stability) of the enzyme and experimental verification, variant 10 mutant DERA SEP with three amino acid substitutions (T120C, G174I, and G213C) was harvested after three rounds of site-directed mutagenesis. It showed surprisingly high tolerance to acetaldehyde, with almost 70.5% residual catalytic activity after exposure to 300 mM acetaldehyde for 2 h, while the wild-type DERA SEP retained only 11.3% activity under the same condition. Because the rigidity of the protein was increased after incorporating the mutations, the specific activity of the variant 10 was only half that of DERA SEP . Thus, the acetaldehyde tolerance of DERA SEP was efficiently increased by enhancing the rigidity of the structure. These results suggested that the inactivation of DERA by exposure to acetaldehyde may result from protein structural damage.
研究领域:
生物化学
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酶工程
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DOI
高影响因子
Biomolecules
·
2024
·Vol. 14
·期 9
·1188
·
IF 4.8
Jiao Ma,Zhongke Sun,Zifu Ni,Yanli Qi,Qianhui Sun,Yuansen Hu,Chengwei Li
This study identified a salt-tolerant GH11 xylanase, Xyn st , which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xyn st and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xyn st in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan ( K m = 0.30 mg mL −1 for Xyn st and 0.18 mg mL −1 for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency ( K cat / K m = 15483.33 mL mg −1 s −1 ). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xyn st did. The conversion efficiencies of Xyn st and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.
研究领域:
生物化学
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分子生物学
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DOI
PubMed
高影响因子
Systems Microbiology and Biomanufacturing
·
2021
·Vol. 2
·期 3
·473-486
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IF 0
Wu Aiai,Bai Yajun,Fan TaiPing,Zheng Xiaohui,Cai Yujie
l -Lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum. l -Lactate dehydrogenase gene (LF- l -LDH0845) with reducing activity towards 3,4-dihydroxyphenylpyruvate and phenylpyruvate was obtained from Lactobacillus fermentum JN248. To change the substrate specificity of LDH0845 and improve its catalytic activity towards large substrates, site-directed mutation of Tyr221 was performed by analyzing the amino acids in the active center. Kinetic parameters show that the k cat values of Y221F mutant on 3,4-dihydroxyphenylpyruvate, 4-methyl-2-oxopentanoate, and glyoxylate are 1.21 s −1 , 1.35 s −1 , and 0.72 s −1 , respectively, which are 420%, 150% and 130% of the wild-type LDH0845. This study shows that the mutations of Y221 can significantly change the substrate specificity of LDH0845, making it become a potential tool enzyme for the reduction of alpha-keto carboxylic acids with large functional groups.
研究领域:
生物化学
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分子生物学
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酶工程
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DOI
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
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2015
·Vol. 16
·期 4
·7273-7288
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IF 2.862
Dongming Lan,Qian Wang,Jinxin Xu,Pengfei Zhou,Bo Yang,Yonghua Wang
Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase fromMalassezia globosais a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273–278 and the 3104 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.Keywords:mono- and diacylglycerol lipase;thermostability;substrate selectivity;site-directed mutagenesis
研究领域:
生物化学
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蛋白质工程
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
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2018
·Vol. 19
·期 3
·683
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IF 3.687
Quan Gan,Xin Li,Xinwei Zhang,Lanlan Wu,Chongjun Ye,Ying Wang,Junshan Gao,Yan Meng
β-fructofuranosidase (β-FFase) belongs to the glycosyl-hydrolase family 32 (GH32), which can catalyze both the release of β-fructose from β-d-fructofuranoside substrates to hydrolyze sucrose and the synthesis of short-chain fructooligosaccharide (FOS).BmSuc1has been cloned and identified from the silkwormBombyx morias a first animal type of β-FFase encoding gene. It was hypothesized that BmSUC1 plays an important role in the silkworm-mulberry adaptation system. However, there is little information about the enzymatic core sites of BmSUC1. In this study, we mutated three amino acid residues (D63, D181, and E234) that represent important conserved motifs for β-FFase activity in GH32 to alanine respectively by using site-directed mutagenesis. Recombinant proteins of three mutants and wild type BmSUC1 were obtained by using a Bac-to-Bac/BmNPV expression system and BmN cells. Enzymatic activity, kinetic properties, and substrate specificity of the four proteins were analyzed. High Performance Liquid Chromatography (HPLC) was used to compare the hydrolyzing and transfructosylating activities between D181A and wtBmSUC1. Our results revealed that the D63A and E234A mutations lost activity, suggesting that D63 and E234 are key amino acid residues for BmSUC1 to function as an enzyme. The D181A mutation significantly enhanced both hydrolyzing and transfructosylating activities of BmSUC1, indicating that D181 may not be directly involved in catalyzation. The results provide insight into the chemical catalyzation mechanism of BmSUC1 inB. mori. Up-regulated transfructosylating activity of BmSUC1 could provide new ideas for usingB. moriβ-FFase to produce functional FOS.Keywords:Bombyx mori;β-fructofuranosidase;site-directed mutagenesis;hydrolysis;transfructosylation
研究领域:
酶学
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分子生物学
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生物化学
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PubMed
高影响因子
Molecular Catalysis
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2022
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IF 5.089
Xiaowen Wu,Yingying Jiang,Zengyu Wang,Xiaobin Yu,Zhoutong Sun,Wei Luo
An NAD + -dependent formate dehydrogenase from Lodderomyces elongisporus ( Le FDH) was expressed in Escherichia coli and then purified by nickel affinity chromatography. The analysis of enzymatic properties showed that the specific activity of Le FDH was much high (16.26 U·mg –1 ), while the thermal stability may be insufficient for industrial application. To further enhance the thermal stability by semi-rational design, sequences alignment, homologous modeling and molecular dynamic simulations were conducted. As a result, seven non-conserved amino acid residues far from the active center were selected for site-directed mutagenesis. Several mutants as well as a binary mutant displayed improved thermal stability and unreduced specific activity. Then temperature and pH stability, kinetic parameters and specific activity, and structural characteristics of wild-type and mutant Le FDH were compared and evaluated. The variant K21P/K22R exhibited an increased half-life by 211% at 60 °C and a 4.5 °C higher T m than those of wild type, and good stability in alkaline environment. This binary mutant enhances the operational stability of Le FDH in the catalytic reaction and thus improves its potential for application in coenzyme regenerations.
研究领域:
生物化学
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分子生物学
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DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2022
·Vol. 106
·期 7
·2493-2501
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IF 5.56
Zhang Feng,Cheng Feng,Jia Dong-Xu,Liu Qian,Liu Zhi-Qiang,Zheng Yu-Guo
Obtaining a sucrose isomerase (SIase) with high catalytic performance is of great importance in industrial production of isomaltulose (a reducing sugar). In order to obtain such SIase mutant, a high-throughput screening system in microtiter plate format was developed based on a widely used 2,4-dinitrosalicylic acid (DNS) method for determination of reducing sugar. An SIase from Erwinia sp. Ejp617 ( Er SIase) was selected to improve its catalytic efficiency. After screening of ~ 8000 mutants from a random mutagenesis library, Q209 and R456 were identified as beneficial positions. Saturation mutagenesis of the two positions resulted in a double-site mutant Er SIase_Q209S-R456H that showed the highest catalytic efficiency, and its specific activity reached 684 U/mg that is 17.5-fold higher than that of the wild-type Er SIase. By employing the lyophilized Escherichia coli ( E. coli ) cells harboring Er SIase_Q209S-R456H, a high space–time yield (STY = 3.9 kg/(L·d)) was achieved toward 600 g/L sucrose. Furthermore, the in silico analysis suggested that the hydrogen bond network was improved and steric hindrance was reduced due to the beneficial substitutions.
Key points
A sucrose isomerase mutant with high catalytic efficiency was obtained . The highest space–time yield was achieved toward high-concentration sucrose . The optimized H-bond network contributed to the enhanced catalytic efficiency .
研究领域:
生物化学
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工业酶学
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DOI
PubMed
高影响因子
Bioengineered
·
2019
·
IF 1.544
Jingyuan Weng,Liangliang Chen,Yinchu Cheng,Yan Li,Honghua Jia,Hua Zhou,Ping Wei
Quercetin-4′-O-glucoside is one of the major quercetin derivatives in the mature red onion bulb. It has an adjuvant effect on allergies, asthma, arthritis, and cancer. The present study aimed to use uridine diphosphate glycosyltransferase 88A1 (UGT88A1) from Arabidopsis thaliana to achieve the enzymatic synthesis of quercetin-4′-O-glucoside from quercetin. The results showed that UGT88A1 was most active at pH 9.0. The optimum temperature of UGT88A1 for synthesizing quercetin-4′-O-glucoside was 45°C, which was a little lower than that for synthesizing quercetin-3-O-glucoside (50°C). One mutant, V18R, of UGT88A1 was obtained by site-directed mutation and showed a greater affinity (Km 0.20 mM) and twice the enzyme activity (552.3 mU/mg) towards quercetin compared with the wild-type enzyme (0.36 mM and 227.6 mU/mg, respectively). The possible reason could be attributed to the distance change between the 18th amino-acid residue of UGT88A1 and the substrate quercetin, as deduced by molecular simulation.
研究领域:
植物学
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生物化学
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PubMed
高影响因子
MOLECULES
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2023
·Vol. 28
·期 18
·6738
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IF 4.6
Shuanghu Fan,Jingjing Guo,Shaoyan Han,Haina Du,Zimeng Wang,Yajuan Fu,Hui Han,Xiaoqiang Hou,Weixuan Wang
Phthalic acid esters (PAEs), which are widespread environmental contaminants, can be efficiently biodegraded, mediated by enzymes such as hydrolases. Despite great advances in the characterization of PAE hydrolases, which are the most important enzymes in the process of PAE degradation, their molecular catalytic mechanism has rarely been systematically investigated.Acinetobactersp. LUNF3, which was isolated from contaminated soil in this study, demonstrated excellent PAE degradation at 30 °C and pH 5.0–11.0. After sequencing and annotating the complete genome, the genedphAN1, encoding a novel putative PAE hydrolase, was identified with the conserved motifs catalytic triad (Ser201-Asp295-His325) and oxyanion hole (H127GGG130). DphAN1 can hydrolyze DEP (diethyl phthalate), DBP (dibutyl phthalate) and BBP (benzyl butyl phthalate). The high activity of DphAN1 was observed under a wide range of temperature (10–40 °C) and pH (6.0–9.0). Moreover, the metal ions (Fe2+, Mn2+, Cr2+and Fe3+) and surfactant TritonX-100 significantly activated DphAN1, indicating a high adaptability and tolerance of DphAN1 to these chemicals. Molecular docking revealed the catalytic triad, oxyanion hole and other residues involved in binding DBP. The mutation of these residues reduced the activity of DphAN1, confirming their interaction with DBP. These results shed light on the catalytic mechanism of DphAN1 and may contribute to protein structural modification to improve catalytic efficiency in environment remediation.Keywords:Acinetobacter;biodegradation;genome sequencing;HSL family hydrolase;site-directed mutagenesis
研究领域:
环境科学
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微生物学
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分子生物学
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DOI
PubMed
高影响因子
Biomolecules
·
2023
·Vol. 13
·期 2
·300
·
IF 5.5
Huanyu Wang,Qianru Lin,Mingwang Liu,Wen Ding,Nanhai Weng,Hao Ni,Jing Lu,Mingsheng Lyu,Shujun Wang
The high-degree polymerization of isomaltooligosaccharide (IMO) not only effectively promotes the growth and reproduction of Bifidobacterium in the human body but also renders it resistant to rapid degradation by gastric acid and can stimulate insulin secretion. In this study, we chose the engineered strain expressed dextranase (PsDex1711) as the research model and used the AutoDock vina molecular docking technique to dock IMO4, IMO5, and IMO6 with it to obtain mutation sites, and then studied the potential effect of key amino acids in this enzyme on its hydrolysate composition and enzymatic properties by site-directed mutagenesis method. It was found that the yield of IMO4 increased significantly to 62.32% by the mutant enzyme H373A. Saturation mutation depicted that the yield of IMO4 increased to 69.81% by the mutant enzyme H373R, and its neighboring site S374R IMO4 yield was augmented to 64.31%. Analysis of the enzymatic properties of the mutant enzyme revealed that the optimum temperature of H373R decreased from 30 °C to 20 °C, and more than 70% of the enzyme activity was maintained under alkaline conditions. The double-site saturation mutation results showed that the mutant enzyme H373R/N445Y IMO4 yield increased to 68.57%. The results suggest that the 373 sites with basic non-polar amino acids, such as arginine and histidine, affect the catalytic properties of the enzyme. The findings provide an important theoretical basis for the future marketable production of IMO4 and analysis of the structure of dextranase.Keywords:molecular docking;site-directed mutagenesis;isomaltooligosaccharide;high degree of polymerization;GH49 family dextranase
研究领域:
生物化学
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分子生物学
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PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2024
·
IF 3.9
Jia Xu,Tao Li,Wei E. Huang,Ning-Yi Zhou
Rieske non-heme dioxygenase family enzymes play an important role in the aerobic biodegradation of nitroaromatic pollutants, but no active dioxygenases are available in nature for initial reactions in the degradation of many refractory pollutants like 2,4-dichloronitrobenzene (24DCNB). Here, we report the engineering of hotspots in 2,3-dichloronitrobenzene dioxygenase from Diaphorobacter sp. strain JS3051, achieved through molecular dynamic simulation analysis and site-directed mutagenesis, with the aim of enhancing its catalytic activity toward 24DCNB. The computationally predicted activity scores were largely consistent with the detected activities in wet experiments. Among them, the two most beneficial mutations (E204M and M248I) were obtained, and the combined mutant reached up to a 62-fold increase in activity toward 24DCNB, generating a single product, 3,5-dichlorocatechol, which is a naturally occurring compound. In silico analysis confirmed that residue 204 affected the substrate preference for meta-substituted nitroarenes, while residue 248 may influence substrate preference by interaction with residue 295. Overall, this study provides a framework for manipulating nitroarene dioxygenases using computational methods to address various nitroarene contamination problems.
研究领域:
环境生物学
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酶工程
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计算生物学
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DOI
PubMed
高影响因子
FOOD AND BIOPRODUCTS PROCESSING
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2021
·
IF 4.481
Peizhou Yang,Shuhua Lu,Wei Xiao,Zhi Zheng,Suwei Jiang,Shaotong Jiang,Shuying Jiang,Jieshun Cheng,Danfeng Zhang
Aflatoxin B 1 (AFB 1 ) is highly carcinogenic and teratogenic as a secondary metabolite of Aspergillus flavus and Aspergillus parasiticus . Trametes versicolor aflatoxin B 1 -degrading enzyme (TV-AFB 1 D) has capability of AFB 1 degradation. In this study, in order to improve the catalytic performance of TV-AFB 1 D, the molecular docking and site-directed mutagenesis techniques were applied to modify the key amino acid sites. Three mutation sites of Y305A, E436A, and H554A were selected for the directed mutation of TV-AFB 1 D. Four TV-AFB 1 D combinations of mutant H554A, Y305A/H554A, E436A/H554A, Y305A/E436A/H554A had potential to modify the catalytic performance according to the value of de-folding free energy. The TV-AFB 1 D mutations were expressed in engineering E. coli BL21 (DE3) with the size of approximately 77 kDa. The optimal temperature and pH value of TV-AFB 1 D were 32 °C and pH 7, respectively. E436A/H554A mutant represented the highest enzyme activities among these four mutants. The specific activity of E436A/H554A mutant (36 U/mg) was 1.84-fold in comparison with wild-type TV-AFB 1 D (19.6 U/mg) under the conditions of pH 7 and 32 °C. This study would contribute to the activity increase of TV-AFB 1 D by the performance improvement of AFB 1 degradation by site-directed mutagenesis approach.
研究领域:
酶学
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分子生物学
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食品安全
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微生物学
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DOI
高影响因子
危险品化学品经营许可证（带存储）