Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

Abstract

Background: Cellulases played an important role in the production of bioenergy and bio-products. Cellulases from bacteria with some special characteristics drew great attention due to its fast growth speed, wide adaption to harsh environment, and production of multi-function cellulases.Results: An endoglucanase gene egls from Bacillus velezensis A4 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme Egls was partially purified using aqueous two-phase system. The highest recovery rate of the enzyme was 90.39% at PEG 4000 (25% w/w), phosphate buffer 8.08% (w/w) (pH 6.0), and NaCl (5% w/w). The enzyme molecular weight was 55 KD estimated by zymogram. The optimal pH and temperature of recombinant enzyme Egls were pH 6.0 and 55 °C, respectively. The enzyme was stable at pH range of 5.0-7.0 at 55 °C for 60 min. The enzyme exhibited K m , V max , K cat values as 63.38 mg/ml, 55.6 mg/min, and 3.93 × 10 3 /S, respectively. The addition of 10 mM of Mg 2+ , Mn 2+ , or 5% (w/w) of Triton-X 100 in the reaction system enhanced the enzyme activity significantly. The enzyme showed both endoglucanase and exoglucanase activity. Conclusions:An endoglucanase gene egls from B. velezensis A4 was cloned and expressed in E. coli BL21 (DE3). The recombinant enzyme Egls was purified by aqueous two-phase system and characterized. The enzyme can be applied for the efficient pretreatment of lignocellulosic biomass for bioenergy and bio-products production.

Production of a halotolerant endo-1,4-β-glucanase by a newly isolated Bacillus velezensis H1 on olive mill wastes without pretreatment: purification and characterization of the enzyme

“…6a). This value is higher than the optimum pH for the previously published β-1,3-1,4-D-glucanase from B. velezensis ZJ20 (pH 5.0) (Xu et al 2016), and slightly higher than the endoglucanase from B. velezensis A4 (pH 5.0-6.0) (Liu et al 2018). Activity was still relatively high at pH 5.5 (over 85% of the optimum) as well as at pH 7.0 (70%) and 7.5 (50%).…”

“…Egl-257 from B. circulans KSM-N257 was also able to hydrolyze lichenan and CMC and was classified as a true endo-1,4-glucanase, despite showing 76.3% similarity to a lichenase from B. circulans WL-12 (Hakamada et al 2002). The substrate specificity of the purified H1 endoglucanase differs from that of the 55-kDa endoglucanase from B. velezensis A4 (activity only on CMC, filter paper, and avicel) (Liu et al 2018), and the 54-kDa endoglucanase from B. amyoliquefaciens DL-3, which was able to degrade all substrates tested in our study, but showed higher activity on CMC (Lee et al 2008).…”

“…Endoglucanases with molecular masses ranging from 25 to 30 kDa have been purified from B. velezensis ZJ20 (27.3 kDa) (Xu et al 2016), B. halodurans C-125 (26.7 kDa) (Akita et al 2005) and B. amyloliquefaciens ATCC 23,350 (26 kDa) (Sun et al 2012), as well as from fungi such as Gloeophyllum trabeum (26.2 kDa) (Miotto et al 2014) and Trichoderma koningii ZJU-T (26.5 kDa) (Wang et al 2007). Other endoglucanases secreted by Bacillus velezensis strains, have higher molecular masses and are classified as glycosyl hydrolases family 5, such as endoglucanase from B. velezensis A4 (55 kDa) (Liu et al 2018) and B. velezensis Y1 (52.31 kDa) (Khalid et al 2021).…”

Soluble expression of recombinant active cellulase in E.coli using B.subtilis (natto strain) cellulase gene

“…Variations in the usage of solubility tags/signal peptide are also reported by some researchers. Amore et al [17] and Liu et al [29] used signal peptide of source organism whereas Kim et al [30] expressed cellulase on the outer membrane of E. coli in fusion with ice nucleation protein (membrane protein of P. syringae).…”

Research advance of Bacillus velezensis: bioinformatics, characteristics, and applications

“…The study of genomes allows the targeted cloning of the desired gene to produce an enzyme with good stability, high activity, and a high yield [28] . The significance of cloning and expression of the enzyme genes is shown in Fig.…”