A Novel Cold-Adaptive Endo-1,4-β-Glucanase From Burkholderia pyrrocinia JK-SH007: Gene Expression and Characterization of the Enzyme and Mode of Action

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.

Knockout of a highly GC‐rich gene in Burkholderia pyrrocinia by recombineering with freeze‐thawing transformation

“…Genome sequencing analysis of B. pyrrocinia revealed that the genomic DNA was rich in GC regions (Kwak & Shin, 2015). The endo‐1,4‐β‐glucanase gene ( BpEG ) (74.71% GC) was chosen as the target gene, which can cleave carboxymethyl cellulose (CMC) into glucose, as we previously reported (Chen, Ye, Sista Kameshwar et al, 2020). The three‐fragment cassette was constructed by overlap‐extension PCR combined with touchdown PCR instead of the classic digestion‐ligation system (Figure 4a).…”

“…B. pyrrocinia JK‐SH007, B. multivorans WS‐FJ9, and B. cenocepacia NSM‐05 were cultured in LB medium at 30 °C. The CMC medium (Chen, Ye, Susta Kameshwar et al, 2020) consisted of 1.5% carboxymethyl cellulose, 0.05% MgSO4.7H 2 O, 0.1% NaNO 3 , 0.1% KH 2 PO 4 , and 0.1% yeast extract.…”

“…The three‐fragment cassette for HR was constructed using overlap‐extension PCR combined with touchdown PCR technology, as described in the Supporting Information. First, both the upstream and downstream fragments of BpEG (Chen, Ye, Susta Kameshwar et al, 2020) were amplified by PCR (Table 3). These processes were named PCR1 (primer pair B) and PCR3 (primer pair C).…”

“…In particular, the GC content of B. pyrrocinia is more than 65% (Kwak & Shin, 2015). The proportion of GC in some genes of B. pyrrocinia is as high as 75% (Chen, Ye, Sista Kameshwar et al, 2020). This high‐GC content DNA leads to difficulties in the amplification of large PCR products and the construction of the mutagenesis cassette (Hube et al., 2005; Kang et al., 2011).…”

“…cellulose (CMC) into glucose, as we previously reported(Chen, Ye, Sista Kameshwar et al, 2020). The three-fragment cassette was constructed by overlap-extension PCR combined with touchdown PCR instead of the classic digestion-ligation system (Figure4a).…”