Enhancing Catalytic Efficiency of an Actinoplanes utahensis Echinocandin B Deacylase through Random Mutagenesis and Site-Directed Mutagenesis

2019 ◽  
Vol 190 (4) ◽  
pp. 1257-1270
Author(s):  
Ying-Nan Cheng ◽  
Shuai Qiu ◽  
Feng Cheng ◽  
Chun-Yue Weng ◽  
Ya-Jun Wang ◽  
...  
Keyword(s):  
Random Mutagenesis ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Echinocandin B

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals Replacement of Tyrosine 181 by Phenylalanine in Gentisate 1,2-Dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 Enhances Catalytic Activities

2005 ◽  
Vol 187 (21) ◽  
pp. 7543-7545 ◽  
Author(s):  
Chew Ling Tan ◽  
Chew Chieng Yeo ◽  
Hoon Eng Khoo ◽  
Chit Laa Poh
Keyword(s):  
Catalytic Efficiency ◽  
Relative Activity ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Specific Mutation ◽  
Catalytic Activities ◽  
Pseudomonas Alcaligenes

ABSTRACT xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k cat/Km ) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

2013 ◽  
Vol 79 (13) ◽  
pp. 4072-4077 ◽  
Author(s):  
Xuguo Duan ◽  
Jian Chen ◽  
Jing Wu
Keyword(s):  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Optimum ◽  
Widespread Application ◽  
Content Type ◽  
Major Factors

ABSTRACTPullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase fromBacillus deramificanswere selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that theKmvalues for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and theKcat/Kmvalues increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

scholarly journals Enhancing The Photocatalytic Conversion Of Pt(IV) Substrates By Flavoprotein Engineering

2021 ◽  
Author(s):  
Juan Gurruchaga-Pereda ◽  
Virginia Martínez-Martínez ◽  
Elena Formoso ◽  
Oksana Azpitarte ◽  
Elixabete Rezabal ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Triplet Excited State ◽  
Excited State Lifetime ◽  
Redox Activation ◽  
Oxygen Generator ◽  
Singlet Oxygen Generator ◽  
First Time ◽  
Catalytic Capacity

Our recent work demonstrates that certain flavoproteins can catalyze the redox activation of Pt(IV) prodrug complexes under light irradiation. Herein, we used site directed mutagenesis on the mini Singlet Oxygen Generator (mSOG) to modulate the photocatalytic activity of this flavoprotein towards two model Pt(IV) substrates. Among the prepared mutants, Q103V mSOG displayed enhanced catalytic efficiency as a result of its longer triplet excited state lifetime. This study shows, for the first time, that protein engineering can improve the catalytic capacity of a protein towards metal-containing substrate.

scholarly journals Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase

2005 ◽  
Vol 71 (9) ◽  
pp. 5433-5439 ◽  
Author(s):  
Michiyo Yanase ◽  
Hiroki Takata ◽  
Kazutoshi Fujii ◽  
Takeshi Takaha ◽  
Takashi Kuriki
Keyword(s):  
Heat Treatment ◽  
Random Mutagenesis ◽  
Cumulative Effect ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Triple Mutant ◽  
Original Activity ◽  
Glucan Phosphorylase

ABSTRACT The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.

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