Rational and random mutagenesis of GDEst-95 carboxylesterase: New functionality insights

被引：1

作者：

Malunavicius, Vilius ^{[1
]}

Vaskevicius, Laurynas ^{[1
]}

Gusaite, Ausrine ^{[1
]}

Gudiukaite, Renata ^{[1
]}

机构：

[1] Vilnius Univ, Inst Biosci, Life Sci Ctr, Sauletekis Ave 7, LT-10257 Vilnius, Lithuania

来源：

INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES | 2024年 / 256卷

关键词：

Geobacillus carboxylesterase; Site-directed mutagenesis; Random mutagenesis; Error-prone PCR; Protein engineering; GEOBACILLUS-THERMOLEOVORANS YN; THERMOSTABLE ESTERASE; MOLECULAR-CLONING; LIPOLYTIC ENZYMES; CRYSTAL-STRUCTURE; STEAROTHERMOPHILUS; CLASSIFICATION; PROTEINS; GD-95RM; LIPASES;

D O I：

10.1016/j.ijbiomac.2023.128331

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Lipolytic enzymes are important contributors in industrial processes from lipid hydrolysis to biofuel production or even polyester biodegradation. While these enzymes can be used in numerous applications, the genotypephenotype space of certain promising enzymes is still poorly explored. This limits the effective application of such biocatalysts. In this work the genotype space of a 55 kDa carboxylesterase GDEst-95 from Geobacillus sp. 95 was explored using site-directed mutagenesis and directed evolution methods. In this study four site-directed mutants (Gly108Arg, Ala410Arg, Leu226Arg, Leu411Ala) were created based on previous analysis of GDEst95 carboxylesterase. Error-prone PCR resulted three mutants: two of them with distal mutations: GDEst-RM1 (Arg75Gln), GDEst-RM2 (Gly20Ser Arg75Gln) and the third, GDEst-RM3, with a distal (Ser210Gly) and Tyr317Ala (amino acid position near to the active site) mutation. Mutants with Ala substitution displayed approximately twofold higher specific activity. Arg mutations lead a reduced specific activity, retaining 2.86 % (Gly108Arg), 10.95 % (Ala410Arg), and 44.23 % (Leu226Arg) of lipolytic activity. All three random mutants displayed increased specific activity as well as improved catalytic properties. This research provides the first deeper insights into the functionality of understudied Geobacillus spp. carboxylesterases with 55 kDa in size.

引用

页数：12