Abstract
The efficient industrial conversion of plant-derived cellulose to simple sugars and other value-added chemicals requires various highly stable and reactive enzymes. Industrial processes especially synchronous saccharification and fermentation (SSF)-based production of cellulosic bio-ethanol require enzymes that are active at lower temperatures. In this study, we have identified, characterized, and expressed the cold-adaptive endo-1,4-β-glucanase (BpEG) isolated from the Burkholderia pyrrocinia JK-SH007. The analysis of the predicted amino acid sequence indicated that BpEG belongs to GH family 8. The BpEG without the signal peptide was cloned into the expression vector pET32a and significantly expressed in Escherichia coli BL21 (DE3) competent cells. The SDS-PAGE and Western blot analysis of BpEG revealed that the recombinant BpEG was approximately 60 kDa. Purified recombinant BpEG exhibited hydrolytic activity against carboxymethyl cellulose (CMC) and phosphoric acid swollen cellulose (PASC), but not crystalline cellulose and xylan substrates. High performance, anion exchange, chromatography-pulsed amperometric detector (HPAEC-PAD) analysis of the enzymatic products obtained from depolymerization of 1,4-β-linked biopolymers of different lengths revealed an interesting cutting mechanism employed by endoglucanases. The recombinant BpEG exhibited 6.0 of optimum pH and 35°C of optimum temperature, when cultured with CMC substrate. The BpEG enzyme exhibited stable activity between pH 5.0 and 9.0 at 35°C. Interestingly, BpEG retained about 42% of its enzymatic activity at 10°C compared to its optimal temperature. This new cold-adaptive cellulase could potentially achieve synchronous saccharification and fermentation (SSF) making BpEG a promising candidate in the fields of biofuel, biorefining, food and pharmaceutical industries.
Highlights
Increasing global population and continuous depletion of fossil fuels worldwide have forced the search for renewable fuel alternatives (Zhao et al, 2011; Rafique and Rehman, 2017)
The BpEG amino acid sequence retrieved from the B. pyrrocinia JK-SH007 strain was aligned with the GH8 family proteins downloaded from the NCBI database using the ClustalW program
We have reported the isolation and characterization of the B. pyrrocinia JK-SH007 strain from the stems of Populus deltoids
Summary
Increasing global population and continuous depletion of fossil fuels worldwide have forced the search for renewable fuel alternatives (Zhao et al, 2011; Rafique and Rehman, 2017). The 1,4β-glucosidase (EC3.2.1.21) produces glucose from its cleavage of cello-oligosaccharides Among these enzymes, endo-β-1,4glucanase (EC 3.2.1.4) is extremely important as it cleaves the β-1,4-glycosidic bonds of cellulose and generates cellooligosaccharides of random length (Huang et al, 2016). The endo-β-1,4-glucanases active at lower temperatures could potentially be of higher importance industrially, offering low energy processes that are both cost and time-efficient. Similar studies conducted by Jin et al has reported an efficient cold-adapted lipase (LIPBA) isolated from the Burkholderia anthina NT15, which exhibited higher catalytic activities even at lower temperatures (Jin et al, 2012). We report the heterologous expression of a novel cold-adaptive endo-1,4-β-glucanase gene isolated from Burkholderia pyrrocinia
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