Abstract
ABSTRACT Bacterial CotA-laccases exhibit higher activity in alkaline pH and salt concentration conditions compared to laccases from white-rot fungi. They are considered as green catalysts in decolorizing of industrial dyes. However, CotA-laccases are limited due to the low yield and catalytic efficiency as the spore-bound nature of CotA. A DNA shuffling strategy was applied to generate a random mutation library. To improve laccase activities, a mutant (T232P/Q367R 5E29) with two amino acid substitutions was identified. The catalytic efficiency of mutant 5E29 was 1.21 fold higher compared with that of the wild-type. The Km and kcat values of 5E29 for SGZ were of 20.3 ± 1.3 µM and 7.6 ± 2.7 s−1. The thermal stability was a slight enhancement. Indigo Carmine and Congo red were efficiently decolorized by using this mutant at pH 9.0. These results provide that 5E29 CotA-laccase is a good candidate for biotechnology applications under alkaline condition, with an effective decolorization capability.
Highlights
Laccases belong to the multi-copper oxidase family and catalyze the reduction of molecular oxygen to water by the oxidation of substrate molecules, such as polyphenols, polyamines and certain inorganic ions[1]
The coat protein A (CotA)-laccases orthologs from different Bacillus species such as B.subtiils [8], B.pumilus [9], B.amyloliquefaciens [4], B.licheniformis [10], Bacillus clausii [11], Bacillus vallismortis [12] and Bacillus sphaericus [13], have been expressed heterologously in E. coli
The open reading frame of B. subtilis LS03 and B. amyloliquefaciens LS05 CotA-laccase genes wascloned, and the sequence was deposited in the GenBank database under accession No.GU972588 and No.GU972590
Summary
Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) belong to the multi-copper oxidase family and catalyze the reduction of molecular oxygen to water by the oxidation of substrate molecules, such as polyphenols, polyamines and certain inorganic ions[1]. The CotA-laccases orthologs from different Bacillus species such as B.subtiils [8], B.pumilus [9], B.amyloliquefaciens [4], B.licheniformis [10], Bacillus clausii [11], Bacillus vallismortis [12] and Bacillus sphaericus [13], have been expressed heterologously in E. coli. This expression pattern often results in inactive inclusion bodies and low catalytic effiency. The dye decolorization capacity of this mutant was evaluated
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