ENZYME AND MICROBIAL TECHNOLOGY
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2021
·
IF 3.493
Miao Tian,Shaowei Huang,Zhiyuan Wang,Junying Fu,Pengmei Lv,Changlin Miao,Tao Liu,Lingmei Yang,Wen Luo
The propeptide is a short sequence that facilitates protein folding. In this study, four highly active Rhizomucor miehei lipase (RML) mutants were obtained through saturation mutagenesis at three propeptide positions: Ser8, Pro35, and Pro47. The enzyme activities of mutants P35 N, P47 G, P47 N, and S8E/P35S/P47A observed at 40 °C, and pH 8.0 were 10.19, 7.53, 6.15, and 8.24 times of that wild-type RML, respectively. The S8E/P35S/P47A mutant showed good thermostability. After incubation at 40 °C for 1 h, 98.98 % of its initial activity remained, whereas wild-type RML retained only 78.76 %. This result indicated that the enhancement of hydrophilicity of 35- and 47- amino-acid residues could promote the interaction between the propeptide and the mature peptide and the enzyme activity and expression level. Highly conserved sites had a more significant impact on enzyme performance than did other sites, similar to the Pro35 and Pro47 mutants showed in this study. This study provides a new idea for protein modification: enzyme performance can be improved through propeptide regulation.
研究领域:
生物化学
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酶学
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蛋白质工程
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DOI
PubMed
高影响因子
Molecular Catalysis
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2022
·
IF 5.089
Yajiao Zhang,Minjie Liu,Huijing Wang,Juan Lin,Fener Chen
Non-catalytic sites are commonly present in many enzymes, but how to classify and modulate such sites for activity improvement is not well-explored. Herein a non-catalytic cavity in the 3-ketosteroid-Δ 1 -dehydrogenase (Δ 1 -KstD) from Mycobacterium smegmatis ( Ms KstD1), which could competitively bind to substrate to form non-catalytic complex, was predicted next to substrate tunnel by the CAVER/molecular dynamics. Based on that, a rational design was performed by Focused Site-directed Iterative Saturation Mutagenesis (FSISM) for blocking the non-catalytic cavity to enhance substrate enter active site to improve the Δ 1 -dehydrogenation activity of Ms KstD1. We obtained the best quadruple mutant (H132M\L113F\V419W\M51L) with 10-fold higher specific activity toward hydrocortisone than that of wild type enzyme. The Δ 1 -dehydrogenation space time yield toward hydrocortisone reached 36.0 g/L/h to produce prednisolone. This work displays how to combine focused mutagenesis with computational analysis to effectively identify and modify non-catalytic cavity to enhance enzyme activity for efficient production of Δ 1 -3-ketosteroid.
研究领域:
生物化学
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酶工程
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计算生物学
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高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2012
·Vol. 97
·期 8
·3355-3362
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IF 3.425
Guo Fei,Xu Haoming,Xu Hongna,Yu Hongwei
Despite directed evolution being a practical and efficient method of improving the properties of enzymes, a trade-off between the targeted property and other essential properties often exists which hinders the efficiency of directed evolution. In our previous work, mutant CVH of an esterase from Rhodobacter sphaeroides with high enantioselectivity was obtained by directed evolution, unfortunately its activity cannot catch another mutant YH. To compensate the trade-off of mutant CVH, site-directed saturation mutagenesis was conducted on four residues, three (Asn62, Met121, and Leu145) were hot spots determined from directed evolution, and one (Tyr27) was introduced to make up the large distance between a mutation (Asn62) and the substrate. A new mutant (HMVY) with high enantioselectivity and comparable activity to YH was obtained. According to the kinetic analysis and molecular dynamics simulations, it was understood that the high enantioselectivity and poor activity of mutant CVH was caused by different decrement of efficiency constants to two isomers, ( R )-, ( S )-methyl mandelate, and the high enantioselectivity and activity of mutant HWVY was caused by improved activity towards the preferred substrate (( S )-methyl mandelate), which provided the interpretation of the trade-off compensation. This work could provide a way to compensate the trade-off of enantioselectivity and activity in the process of enzyme evolution.
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
Molecular Catalysis
·
2023
·
IF 4.6
Bi-Han Guan,Wu-Tong Yin,Bo-Han Cao,Chen Zhao,Jiang Pan,Jian-He Xu
L-Carnosine (L-Car) is a biologically active dipeptide with antioxidant and anti-aging effects, formed by condensation of β-alanine (β-Ala) with L-histidine (L-His), which is used widely in the pharmaceutical and cosmetic industries. Here we report a dipeptide hydrolase originating from Bacillus megaterium , designated as Bm PepD, whose synthetic activity reached 6.5 times that of Sm PepD, which is the highest active dipeptidase reported so far for L-Car biosynthesis in vitro . By site-directed saturation mutagenesis, we obtained a significantly improved variant (T171N) of Bm PepD. It showed a specific activity of 22.9 U mg −1 protein , reaching 1.4 times that of wild-type Bm PepD or 9.1 times that of Sm PepD, which is the highest ever reported among this enzyme class. After optimizing the reaction conditions of variant Bm PepD T171N , we performed the synthetic reaction of L-Car simply from the two free amino acids (0.2 M L-His and 6.5 M β-Ala), achieving a titer of 31.3 mM L-Car with a specific productivity of 283 g Car · g catalyst −1 . This work provides a highly active and substrate (β-alanine)-tolerant dipeptide hydrolase for the facile synthesis of dipeptide L-Car via reversed hydrolysis.
研究领域:
生物化学
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酶工程
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合成生物学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
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2022
·Vol. 23
·期 17
·9663
·
IF 6.208
Yawen Zhou,Linshu Jiao,Juan Shen,Huibing Chi,Zhaoxin Lu,Huawei Liu,Fengxia Lu,Ping Zhu
Low catalytic activity is a key factor limiting the widespread application of type II L-asparaginase (ASNase) in the food and pharmaceutical industries. In this study, smart libraries were constructed by semi-rational design to improve the catalytic activity of type II ASNase fromBacillus licheniformis. Mutants with greatly enhanced catalytic efficiency were screened by saturation mutations and combinatorial mutations. A quintuple mutant ILRAC was ultimately obtained with specific activity of 841.62 IU/mg andkcat/Kmof 537.15 min−1·mM−1, which were 4.24-fold and 6.32-fold more than those of wild-type ASNase. The highest specific activity andkcat/Kmwere firstly reported in type II ASNase fromBacillus licheniformis. Additionally, enhanced pH stability and superior thermostability were both achieved in mutant ILRAC. Meanwhile, structural alignment and molecular dynamic simulation demonstrated that high structure stability and strong substrate binding were beneficial for the improved thermal stability and enzymatic activity of mutant ILRAC. This is the first time that enzymatic activity of type II ASNase fromBacillus licheniformishas been enhanced by the semi-rational approach, and results provide new insights into enzymatic modification of L-asparaginase for industrial applications.Keywords:L-asparaginase;protein engineering;saturation mutagenesis;catalytic efficiency;molecular dynamics simulation
研究领域:
生物技术
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酶学
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蛋白质工程
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DOI
PubMed
高影响因子
BMC BIOTECHNOLOGY
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2021
·Vol. 21
·期 1
·1-9
·
IF 2.563
Zhang Zhiwei,Liu Yang,Zhao Jing,Li Wenqiang,Hu Ruiwen,Li Xia,Li Aitao,Wang Yaping,Ma Lixin
Background The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering.Results Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g.Conclusion This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.
研究领域:
生物化学
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蛋白质工程
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酶学
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DOI
PubMed
高影响因子
CHEMOSPHERE
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2023
·
IF 8.8
Yan-Yan Liu,Yi-Xin Zhang,Hua-Mei Wen,Xiao-Long Liu,Xin-Jiong Fan
Phthalate esters (PAEs) and parabens are environmental pollutants that can be toxic to human health. Herein, a cold-adapted esterase from the Mao-tofu metagenome named Est1260 was screened for its PAE-hydrolyzing potential in cold temperatures. The results showed that purified Est1260 could degrade a variety of PAEs and parabens at temperatures as low as 0 °C. After careful analysis of the structural information and molecular docking, site-saturation mutation was conducted at the identified hotspots. Protein expression of variant A1B6 doubled, and its thermal stability significantly improved (24 times) without sacrificing activity at low temperatures. In addition, Est1260 and its variants were activated by NaCl and demonstrated resistance to high concentrations of saline (up to 5 M), making it a potential biocatalyst for bioremediation of PAE and paraben-polluted environments.
研究领域:
环境科学
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生物化学
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生物技术
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DOI
PubMed
高影响因子
Catalysts
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2020
·Vol. 10
·期 12
·1470
·
IF 3.52
Fei Liu,Junping Zhou,Meijuan Xu,Taowei Yang,Minglong Shao,Xian Zhang,Zhiming Rao
Unusual α-amino acids (UAAs) are important fundamental building blocks and play a key role in medicinal chemistry. Here, we constructed a hydrogen-borrowing dual-enzyme cascade for efficient synthesis of UAAs from α-hydroxy acids (α-HAs). D-mandelate dehydrogenase fromLactobacillus brevis(LbMDH) was screened for the catalysis of α-HAs to α-keto acids but with low activity towards aliphatic α-HAs. Therefore, we rational engineeredLbMDH to improve its activity towards aliphatic α-HAs. The substitution of residue Leu243 located in the substrate entrance channel with nonpolar amino acids like Met, Trp, and Ile significantly influenced the enzyme activity towards different α-HAs. Compared with wild type (WT), variant L243W showed 103 U/mg activity towards D-α-hydroxybutyric acid, 1.7 times of the WT’s 60.2 U/mg, while its activity towards D-mandelic acid decreased. Variant L243M showed 2.3 times activity towards D-mandelic acid compared to WT, and its half-life at 40 °C increased to 150.2 h comparing with 98.5 h of WT. By combiningLbMDH with L-leucine dehydrogenase fromBacillus cereus, the synthesis of structurally diverse range of UAAs from α-HAs was constructed. We achieved 90.7% conversion for L-phenylglycine production and 66.7% conversion for L-α-aminobutyric acid production. This redox self-sufficient cascade provided high catalytic efficiency and generated pure products.Keywords:D-mandelate dehydrogenase;unusual α-amino acids;rational engineering;site-saturation mutagenesis;hydrogen-borrowing cascade
研究领域:
生物催化
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酶工程
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药物化学
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DOI
高影响因子
ADVANCED SYNTHESIS & CATALYSIS
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2023
·Vol. 365
·期 7
·1048-1057
·
IF 5.4
Xiao-Ling Tang,Ning Li,Yan-Lai Liu,Jing-Peng Li,Kai-Xuan Zhao,Zhi-Qiang Liu,Yu-Guo Zheng
L-Methionine is the unique sulfur-containing amino acid essential for humans and animals, which is widely used in pharmaceutical, food and feed industries. Its green and efficient production has attracted a great deal of attention. Fermentation-enzymatic coupling route is regarded to be promising for L-methionine biosynthesis, while O- succinyl-L-homoserine mercaptotransferase (MetZ) is the key biocatalyst. Exploring and engineering of MetZ with ideal catalytic properties is highly desired. Herein, the MetZ from Chromobacterium violaceum ( Cv MetZ) was screened and through in silico analyses, potential beneficial amino acid residues both at the access channel and around the oxygen anion holes were anchored for site-saturation mutagenesis. The positive mutants were obtained with improved activity for L-methionine biosynthesis using O -succinyl-L-homoserine (OSH) and methyl mercaptan as substrate. The best mutant was further applied for L-methionine production and supply of methyl mercaptan was optimized in a fed-batch approach. The conversion of 100 g/L OSH reached 92% with L-methionine yield of 62.6 g/L, which was well coupled with the OSH fermentation level.
研究领域:
生物化学
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合成生物学
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DOI
高影响因子
Molecular Catalysis
·
2024
·
IF 3.9
Yuqinxin Xie,Dongzhi Wei,Jinping Lin
(4 S )-3-[(5 S )-5-(4-fluorophenyl)-5‑hydroxy-valyl]-4-phenyl-1,3-oxazacyclopentane-2-one (( S )-Fop alcohol) is a key chiral intermediate for the synthesis of ezetimibe, and could be synthesized via asymmetric reduction of ( S )-4-phenyl-3-[5-(4-fluorophenyl)-5-oxopentanoyl]-2-oxazolidione (Fop dione). However, discovering and engineering of ketoreductases toward bulky-bulky (diaryl) ketones is still challenging. Previously, we identified an alcohol dehydrogenase Gox0525 from Gluconobacter oxydans DSM2343 which possessed strict diastereoselectivity ( d.e. value > 99%) but low activity toward Fop dione. In this study, a semi-rational design based on the focused rational iterative site-specific mutagenesis (FRISM) based on site-directed saturation mutagenesis was performed to improve the catalytic efficiency of Gox0525. The variant M4 (Y92G/P93M/Y94P/L151V) shows a 64-fold enhanced catalytic efficiency ( K cat / K m ) and 47-fold in specific activity compared with the wild type Gox0525. Engineered Escherichia coli cells co-expressing the variant M4 and glucose dehydrogenase from Bacillus subtilis (BsGDH) for NADPH regeneration were employed as biocatalysts for gram-scale reaction of Fop dione. As a result,95 mM (33.76 g/L) Fop dione was completely transformed within 4 h, affording ( S )-Fop alcohol with > 99% d.e. value, the yield of 96%, and the space-time yield of 195.6 g/L/d.
研究领域:
生物技术
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生物化学
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药物化学
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酶学
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化工
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产品关联
DOI
高影响因子
JOURNAL OF BIOTECHNOLOGY
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2015
·
IF 2.871
Yue-De Cao,Yu-Cai He,Hao Li,Guo-Yin Kai,Jian-He Xu,Hui-Lei Yu
Hyoscyamine 6 β -hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (−)-hyoscyamine and the subsequent epoxidation of 6 β -hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus ( Aa H6H). A double mutant, Aa H6H M1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34 h cultivation of Escherichia coli cells harboring Aa h6h m1 in a 5-L bioreactor with a working volume of 3 L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500 mg L −1 (−)-hyoscyamine in 97% conversion, and 1.068 g of the product were isolated, corresponding to a space-time yield of 251 mg L −1 d −1 . This study shows that the protein engineering of some key enzymes is a promising and effective way for improving the production of rare natural products such as scopolamine.
研究领域:
生物化学
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生物技术
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微生物学
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酶学
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产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2020
·
IF 3.448
Zelong Li,Runtao Zhu,Yongqi Liu,Jiaqi Li,Haofeng Gao,Nan Hu
L-theanine, a unique amino acid in green tea with health benefits, can be enzymatically synthesized by γ-glutamyltranspeptidase (γ-GT; EC 2.3.2.2). Here, a salt-tolerant γ-glutamyltranspeptidase from a marine bacterium Bacillus amyloliquefaciens was expressed in Escherichia. coli BL21 (DE3) and was shown to be optimally active at 55 °C, pH 8.5 and alkali stable. A mutant, with higher transpeptidation activity, was obtained following two rounds of directed evolution using error-prone PCR and site-saturation mutagenesis. The mutation increased the ratio of transpeptidation to hydrolysis from 1.6 to 35.6. Additionally, Kinetic analysis exhibited 17.5% decrease of K m , 13.0-fold increase of K cat , and 16.3-fold increase of K cat / K m in mutant V319A/S437 G versus the wild-type. The 3-D modelling analysis revealed a tighter binding pocket in mutant V319A/S437 G. The frequency of hydrogen bond between donor substrate and two residues in the catalytic pocket (Gly437 and Thr375) was enhanced, which stabilized the ligand binding and thus improved the catalytic efficiency. The optimal conditions for the biocatalytic synthesis were determined as pH 10.0, 20 μg mL -1 Ba GT, 200 mM L-glutamine, 2 M ethylamine, and a reaction time of 5 h. The V319A/S437 G mutant was shown to increase the percentage yield of L-theanine from 58% to 83%. These results indicate the great potential of V319A/S437 G in L-theanine production after further study.
研究领域:
生物化学
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生物技术
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酶学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）