In-Vitro Refolding and Characterization of Recombinant Laccase (CotA) From Bacillus pumilus MK001 and Its Potential for Phenolics Degradation

Abstract

Among lignocellulolytic enzymes, laccases are the most versatile, broadly specific, and largely studied enzyme with a wide range of biotechnological potential. Putative laccase (CotA) from Bacillus pumilus MK001 was cloned and expressed in E. coli. In addition to soluble bioactive fraction, inactive inclusion body fraction was also harvested and refolded under optimized conditions resulting in 64 % of refolding efficiency. The enzyme was found to be thermostable exhibiting a half-life of 60 min at 80 °C. UV thermal CD spectra also supported the observation as about 9 % increase in β-sheets was recorded after thermal induction. The 3D CotA structure was constructed through homology modeling and the best selected model was verified through PROCHECK, ERRAT, Verify 3D, and PROSA servers. Final 3D model showed potential binding affinities with ferulic acid, caffeic acid, and vanillin. Results of the docking studies were further validated by HPLC analysis which signified the efficient bioconversion ability of CotA.

Degradation of Aflatoxin B1 and Zearalenone by Bacterial and Fungal Laccases in Presence of Structurally Defined Chemicals and Complex Natural Mediators

“…Bs CotA, the B. subtilis spore coat laccase, is responsible for the biosynthesis of the brown spore pigment [26]. The specific activity of recombinant Bs CotA (6.7 U/mg) was similar to the laccase from Klebsiella pneumoniae (7.1 U/mg) [36], but lower than those of Bacillus pumilus MK001 (73 U/mg) [37] and B. pumilus DSM 27 (200 U/mg) [38]. ABTS, DMP, and SGZ are all substrates of Bs CotA, a feature shared by Bs CotA and other laccases [39].…”

Manganese Stress Adaptation Mechanisms of Bacillus safensis Strain ST7 From Mine Soil

“…It was ascertained that approximately six genes were hemicellulases such as xylanase, according to the hierarchies of KAAS enrichment analysis. Furthermore, two genes coding for manganese catalase and multicopper oxidase domain-containing protein (MCO) might participate in lignin degradation, which was the case for manganese peroxidase and bacterial laccase based on a homology blast with Bacillus pumilus ( Kumar et al, 2016a ; Ogonda et al, 2021 ).…”

“…The biodegradation of lignin requires oxidase and peroxidase, such as manganese peroxidase and laccase ( Ma et al, 2020 ). Although no Bacillus members can ferment lignocellulosic material without prior pre-treatment of biomass saccharification, genes from ST7 transcriptome coding for cellulases, hemicellulases, and enzymes to decompose lignin were expressed at a high level according to the hierarchies of the KAAS enrichment analysis, and homology blasts with other Bacillus species ( Kumar et al, 2016a ; Ogonda et al, 2021 ). The other genes were related to the production of high-value chemicals such as ethanol, acetate, and lactate from cellulose.…”

Structural analysis and biochemical properties of laccase enzymes from two Pediococcus species

“…Pp4816 and Pa5930 contain the four strictly conserved copper ligand motifs characteristics of MCO enzymes (Fig. 1) including consensus His residues, a typical trait of classical laccases (Fernandes et al, 2014;Kumar et al, 2016). Pp4816 and Pa5930 have a relatively high sequence identity (59%); however, Pp4816 is closer related to the L. plantarum J16 laccase (66% identity) described by Callej on et al published, since its gene sequence was not included in the report (nor could it be deduced from the primers described) (Xu and Fang, 2019).…”