BIOTECHNOLOGY LETTERS
·
2016
·Vol. 38
·期 6
·1009-1013
·
IF 1.639
Kang Le,Bai Yajun,Cai Yujie,Zheng Xiaohui
Objectives To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3. Results The gene encoding BioH was cloned and overexpressed in E. coli . The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p -coumarate substrates were: K
m
values of 0.48, 6.3, and 1.9 mM, respectively, and k
cat
/ K
m
values of 9.3, 3.8, and 3.8 mM −1 s −1 , respectively. Conclusions Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.
研究领域:
生物化学
·
微生物学
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DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2013
·
IF 2.579
Lidan Ye,Hongna Xu,Hongwei Yu
Esterase BioH is a critical enzyme for Biotin synthesis in Escherichia coli , which has been previously found to be active in the acylation of secondary alcohols and amines. Directed evolution towards improved acylation activity requires a high-throughput screening method. The aim of this study is to explore whether the acylation activity of BioH can be improved by directed evolution of its hydrolysis activity. A colorimetric method based on p -nitrophenyl butyrate hydrolysis was adopted in the high-throughput determination of hydrolysis activity. The wild-type BioH showed a hydrolysis activity of 18 U/mg, and the specific activities for α-phenylethanol and α-phenylethylamine acylation were 12.8 U/mg and 3.5 U/mg, respectively. After two rounds of directed evolution, seven mutants with improved hydrolysis activity were obtained, among which, K213E, Q70L/M170T and M197L/K213E also showed improvement in acylation activity. To further improve the acylation activity, site mutations were generated in different combinations at the four hot spots Q70L, M170T, M197L and K213E. Among the resulting mutants, Q70L/M197L/K213E showed the highest activity in α-phenylethylamine acylation with a 120% improvement, while Q70L/K213E had the highest α-phenylethanol acylation activity, which was improved by 70%. The results demonstrated that directed evolution of the hydrolysis activity might be a possible approach to improve the acylation activity of the esterase BioH.
研究领域:
生物化学
·
分子生物学
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DOI
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2010
·
IF 2.193
Gangfeng Ren,Hongwei Yu
After analyzing protein surface structure, the opposite side of the activity site of esterase BioH was observed to be hydrophilic. Hence subsequently, a hydrophilic-modified solid support was selected to immobilize enzyme through oriented adsorption. The immobilized enzyme remained 86% of its original activity under the acylation of 1-phenylethanol, much more than those immobilized on other solid supports with low hydrophilicity (20–35.6%), as the mode of the immobilization could facilitate enzyme to expose its active site to the substrate. This demonstrated that the strategy to select solid support based on the analysis of protein surface structure was successful. It was also noticed that the substrate of the reaction was obviously adsorbed onto the support, therefore the adsorption effect could not be ignored and it was necessary to obtain the adsorption isotherm of the substrate for a more authentic result. The parameter K m shifted from 884.56 to 296.09 mM after immobilization. This was possibly mainly caused by the increment of substrate concentration around the enzyme due to the substrate adsorbing on the support. It could be another explanation for why those parameters were changed after enzyme immobilization in addition to some existing proposed reasons such as stabilizing conformation, facilitating dispersion of enzyme, etc.
研究领域:
生物技术
·
酶学
·
蛋白质工程
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DOI
高影响因子
PROCESS BIOCHEMISTRY
·
2014
·
IF 2.524
Ling Jiang,Bo Wang,Rong-rong Li,Sa Shen,Hong-wei Yu,Li-dan Ye
Enzymatic catalytic promiscuity has received increasing attention in the past decade. In this research, ten enzymes were investigated for the promiscuous activity in catalysis of the Michael addition-cyclization cascade reaction of p -nitrobenzalacetone with 1,3-cyclohexanedione to prepare 2-hydroxy-2-methyl-4-(4-nitrophenyl)-3,4,7,8-tetrahydro-2 H -chromen-5(6 H )-one in anhydrous media, and control experiments were conducted to exclude false positive results. The highest yield (46.1%) was observed with Escherichia coli BioH esterase and the optimal reaction condition was: 1 mmol α,β-unsaturated ketone, 1 mmol 1,3-dicarbonyl compound, 20 mg E. coli BioH esterase, 20 ml N,N-dimethylformamide at 37 °C for 120 h. To preliminarily investigate the mechanism, site-directed mutagenesis was performed on the hydrolysis catalytic triad of BioH, and the results indicated “alternate-site enzyme promiscuity”. When a series of substituted benzalacetones and 1,3-cyclic diketones were used as the reactants, yields of up to 76.3% were achieved. These results imply the potential industrial application of E. coli BioH in the preparation of dihydropyran derivatives.
研究领域:
生物化学
·
酶工程
·
有机合成
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DOI
高影响因子
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
·
2014
·Vol. 61
·期 5
·603-610
·
IF 1.322
Rongrong Li,Ling Jiang,Lidan Ye,Jie Lu,Hongwei Yu
The esterase BioH from Escherichia coli was covalently immobilized onto the surface of the functional magnetic nanosupport in an oriented manner. The surface of the Fe 3 O 4 nanosupport was modified with acyl azide groups or both acyl azide groups and hydroxyl groups. The protein loading of the support was increased from 55 to 99 mg/g by an improvement in hydrophilicity, and the activity retention of the immobilized esterase on the nanosupport was improved by 40% after hydrophilic modification (30% and 70% of the free BioH, respectively). For the BioH immobilized on the hydrophilic-modified nanosupport, the recovery activity remained 80% of the original activity after 10 times of recycling. The catalytic kinetics and thermo-/pH-stability of the immobilized esterase BioH were also determined and compared with those of the free enzyme. The comparatively high activity retention, improved thermo-/pH-stability, and good reusability of the immobilized enzyme indicate that oriented covalent immobilization is an efficient method for immobilizing esterase BioH.
研究领域:
纳米生物技术
·
酶工程
·
生物催化
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应用微生物学
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DOI
高影响因子
BIOTECHNOLOGY AND BIOENGINEERING
·
2014
·Vol. 111
·期 9
·1729-1739
·
IF 4.164
Fei Guo,Stefan Franzen,Lidan Ye,Jiali Gu,Hongwei Yu
Aromatic interactions specific to aryl radicals were introduced into two esterases, BioH from Escherichia coli and RspE from Rhodobacter sphaeroides to control their enantioselectivity in the asymmetric hydrolysis of prochiral aryl glutaric acid diesters. As a result, the enantiomeric excess (ee) of the S- product of dimethyl 3-phenylglutarate was increased from 25% (BioH wild type) to 96% (B_L83F/L86F) and from 13% (RspE wild type) to >99% (R_Y27R), respectively, while another variant of RspE R_M121F gave a reversed ee of 50% ( R -product). Similar enhancement or reversion of enantioselectivity were also observed in the hydrolysis of three other prochiral aryl diesters (dimethyl 3-(4-flouro)-phenylglutarate, dimethyl 3-(4-cholo)-phenylglutarate and dimethyl 3-(3,4-dicholo)-phenylglutarate). Especially, the mutant R_Y27R was shown to be an excellent S -selective hydrolase for prochiral aryl diesters, with ee of all S -products >99%. In the mutants with altered enantioselectivity, the successful introduction of designed aromatic interactions was confirmed by molecular dynamics simulations and binding free energy analysis. These results demonstrate that aromatic interaction is one of the origins of enzyme enantioselectivity, the tuning of which leads to dramatic change in enantioselectivity. Besides, the successful engineering of the enantioselectivity in two different proteins toward four different substrates suggests that the introduction of aromatic interactions is a generally applicable strategy in the control of enantioselectivity toward aryl substrates. Biotechnol. Bioeng. 2014;111: 1729–1739. © 2014 Wiley Periodicals, Inc.
研究领域:
生物技术
·
生物化学
·
酶工程
·
有机化学
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DOI
PubMed
高影响因子
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