Identification and characterization of an Endo-glucanase secreted from cellulolytic Escherichia coli ZH-4

Abstract

Background In the previous study, the cellulolytic Escherichia coli ZH-4 isolated from bovine rumen was found to show extracellular cellulase activity and could degrade cellulose in the culture. The goal of this work was to identify and characterize the secreted cellulase of E. coli ZH-4. It will be helpful to re-understand E. coli and extend its application in industry. Results A secreted cellulase was confirmed to be endo-glucanase BcsZ which was encoded by bcsZ gene and located in the cellulose synthase operon bcsABZC in cellulolytic E. coli ZH-4 by western blotting. Characterization of BcsZ indicated that a broad range of pH and temperature tolerance with optima at pH 6.0 and 50 °C, respectively. The apparent Michaelis–Menten constant (K m ) and maximal reaction rate (V max ) for BcsZ were 8.86 mg/mL and 0.3 μM/min·mg, respectively. Enzyme activity of BcsZ was enhanced by Mg 2+ and inhibited by Zn 2+ , Cu 2+ and Fe 3+ . BcsZ could hydrolyze carboxymethylcellulose (CMC) to produce cello-oligosaccharides, cellotriose, cellobiose and glucose. Conclusions It is confirmed that extracellular cellulolytic capability of E. coli ZH-4 was attributed to BcsZ, which explained why E. coli ZH-4 can grow on cellulose. The endo-glucanase BcsZ from E. coli -ZH4 has some new characteristics which will extend the understanding of endo-glucanase. Analysis of the secretion characteristics of BcsZ provided a great reference for applying E. coli in multiple industrial fields.

Systematic Analysis of Escherichia coli Isolates from Sheep and Cattle Suggests Adaption to the Rumen Niche

“…Besides, genome analysis showed that bcsZ was the sole cellulase gene in these rumen-derived E. coli isolates, indicating that it is responsible for the observed extracellular endoglucanase activity. There were two amino acid differences in the BcsZ of rumen E. coli compared with E. coli MG1655 and W3110, Ser63 to Phe (Ser-Phe) and Ala71 to Val (Ala-Val), which was in agreement with previous results (21) and with the phylogenetic tree of BcsZ shown in Fig. S2 in the supplemental material.…”

“…Notably, this strain was able to degrade cellulose, as well as to produce ethanol and hydrogen (20). The responsible cellulose-degrading enzyme was later confirmed by Western blot and sequence analysis to be an endoglucanase (encoded by the bcsZ gene in the bcs gene cluster) (21). Genome analyses revealed that bcsZ was the sole cellulase gene in the E. coli genome and is responsible for extracellular endoglucanase activity.…”

“…Protein concentrations were measured using the Bradford method with bovine serum albumin as the standard (50). Western blot analysis was performed as described in our previous publication (21).…”

Succession of Gut Microbial Structure in Twin Giant Pandas During the Dietary Change Stage and Its Role in Polysaccharide Metabolism

“…Actually, as generally considered, the E. coli cannot degrade cellulose, and more than 75% of the endoglucanases from GH8 produced by bacterial taxa such as E. coli at Proteobacteria are as a component of the bacterial cellulose synthesis (bcs) system (Berlemont and Martiny, 2013), but interestingly, most of them are potentially required to correct packing of cellulose microfibrils (Mazur and Zimmer, 2011); that is, GH8 may retain its activity of endoglucanase to cello-oligosaccharides in the bcs system (Scapin et al, 2017). Under the product enrichment conditions, such as microfibril-like structured cellulose, GH8 will be secreted extracellularly, and out of the bcs system, it can hydrolyze soluble cellulose and cello-oligosaccharides in the environment (Pang et al, 2019), and structure-specific GH8 subfamily can directly hydrolyze amorphous CMC and crystalline cellulose (Attigani et al, 2016). In this special environment with large amount of bamboo fiber and complex microbial interaction, how the bacterial endoglucanases from GH8 participate in cellulose metabolism is attractive for further research.…”