Substrate channel evolution of an esterase for the synthesis of cilastatin

2015 ◽  
Vol 5 (5) ◽  
pp. 2622-2629 ◽  
Author(s):  
Zheng-Jiao Luan ◽  
Fu-Long Li ◽  
Shuai Dou ◽  
Qi Chen ◽  
Xu-Dong Kong ◽  
...  
Keyword(s):  
Building Block ◽  
Site Directed Mutagenesis ◽  
Native Enzyme ◽  
Directed Mutagenesis ◽  
Whole Cell ◽  
Channel Evolution ◽  
Whole Cell Biotransformation

Error-prone PCR and site-directed mutagenesis around substrate channel were employed for improving an esterase (RhEst1) activity towards Cilastatin building block. RhEst1A147I/V148F/G254A showed 20 times higher activity than the native enzyme in whole cell biotransformation.

scholarly journals Mammalian inositol monophosphatase: the identification of residues important for the binding of Mg2+ and Li+ ions using fluorescence spectroscopy and site-directed mutagenesis

1993 ◽  
Vol 296 (3) ◽  
pp. 811-815 ◽  
Author(s):  
M G Gore ◽  
P Greasley ◽  
G McAllister ◽  
C I Ragan
Keyword(s):  
Fluorescence Spectroscopy ◽  
Specific Activity ◽  
Fluorescence Emission ◽  
Site Directed Mutagenesis ◽  
Native Enzyme ◽  
Mutant Enzyme ◽  
Directed Mutagenesis ◽  
Inositol Monophosphatase ◽  
Fluorescence Emission Intensity ◽  
Ph Dependent

The fluorescence properties of residue Trp-219 in inositol monophosphatase are sensitive to the ionization of neighbouring groups. The pH-dependent changes in the fluorescence emission intensity and wavelength of maximum emission appear to arise as the result of two separate ionizations in the proximity of Trp-219, namely due to the ionization of His-217 and Cys-218. By studying the curve of fluorescence intensity against pH, given by the mutants Cys-218→Ala or His-217→Gln, the pK of His-217 was determined to be 7.54 and the pK of Cys-218 was estimated to be about 8.2. These mutants have altered kinetic parameters for catalytic Mg2+ ions and inhibitory Mg2+ and Li+ ions. The Cys-218→Ala mutant enzyme is not subject to inhibition by concentrations of Mg2+ ions up to 400 mM and has a specific activity of 156% of the maximum obtainable activity of the native enzyme. The His-217→Gln mutant enzyme shows reduced sensitivity to inhibition by Mg2+ and Li+ ions, and has a specific activity of 110% of that obtainable for the native enzyme.

scholarly journals Engineering of CYP153A33 With Enhanced Ratio of Hydroxylation to Overoxidation Activity in Whole-Cell Biotransformation of Medium-Chain 1-Alkanols

2022 ◽  
Vol 9 ◽  
Author(s):  
Hyuna Park ◽  
Doyeong Bak ◽  
Wooyoung Jeon ◽  
Minjung Jang ◽  
Jung-Oh Ahn ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Dodecanoic Acid ◽  
P450 Monooxygenase ◽  
Oxidation Activity ◽  
Whole Cell ◽  
Structure Activity ◽  
Marinobacter Aquaeolei ◽  
Whole Cell Biotransformation ◽  
Dodecanedioic Acid ◽  
Hydroxylation Activity

α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.

Mutation Analysis of a Thermostable Exo-ß-D-Glucosaminidase from the Hyperthermophilic Archaeon Pyrococcus Horikoshii and Comparison with Two Mesophilic Exo-ß-D-Glucosaminidases

2013 ◽  
Vol 787 ◽  
pp. 172-176
Author(s):  
Hui Hui Shi ◽  
Yan Yan Gao ◽  
Tian Ming Liu ◽  
Bo Liu
Keyword(s):  
Hydrophobic Interactions ◽  
Ion Pairs ◽  
Structural Difference ◽  
Site Directed Mutagenesis ◽  
Native Enzyme ◽  
Directed Mutagenesis ◽  
Pyrococcus Horikoshii ◽  
Hyperthermophilic Archaeon ◽  
Amycolatopsis Orientalis ◽  
Amino Acids Composition

The mechanism of thermostabilization of the thermostable exo-ß-D-glucosaminidase (Bglph) from Pyrococcus horikoshii was investigated. Conserved domains analysis and C-terminal truncated mutation showed that the C-terminal region was indispensable for the enzymes thermostability. Two site directed mutagenesis proteins (C103A and C103AandC550A) were as active as the native enzyme and showed no structural difference with the native enzyme. By comparison with two mesophilic exo-ß-D-glucosaminidases (CsxA and CsxT) from actinomycetes Amycolatopsis orientalis and fungi Trichoderma reesei respectively, it was shown that the amino acids composition of Bglph was consistent with its high thermostability, hydrophobic interactions, homodimerization and ion pairs could play key roles. These results presented the overall properties of the thermostability of Bglph from diverse aspects.

The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

2013 ◽  
Vol 37 (3) ◽  
pp. 330
Author(s):  
Yanan WANG ◽  
Xudong LIU ◽  
Linlin MU ◽  
Zhipeng LIU ◽  
Chunmei LI ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Cynoglossus Semilaevis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Site-directed mutagenesis on alkaline phosphatasefrompseudomonas aeruginosapao1 for enhanced activity

2016 ◽  
Vol 7 (4) ◽  
Author(s):  
UMA SELVARAJ ◽  
THIRUMALAI MUTHUKUMARESAN ◽  
GAYATHRI VIJAYENDRAN ◽  
SENTHIL KUMAR DEVAN ◽  
VENU BABU P ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Enhanced Activity
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