Cloning of two cellulase genes from endophytic Paenibacillus polymyxa GS01 and comparison with cel 44C‐man 26A

Abstract

Endophytic bacteria are acknowledged as a new source of genes, proteins and other biochemical compounds, which are often used in biochemical processes. In this study, Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). Two cellulase genes, cel 5A and cel 5B, were cloned from GS01, and encode 334 aa and 573 aa proteins, respectively. Cel5A and Cel5B each contain a glycosyl hydrolase family 5 (GH5) catalytic domain. The molecular mass of Cel5A and Cel5B were estimated to be 33 kDa and 61 kDa, respectively, by CMC-SDS-PAGE. When purified from Escherichia coli Cel5A and Cel5B both displayed cellulase activity with pH optima of 7.0 and 6.0, respectively and shared a temperature optimum of 50 degrees C. Neither enzyme had detectable xylanase, lichenase, or mannase activity, in contrast to the multifunctional Cel44C-Man26A enzyme of P. polymyxa which displays cellulase, xylanase, lichenase and mannanase activities. However, Cel5A and Cel5B exhibited higher specific cellulase activity than Cel44C-Man26A (120% and 140%, respectively). Cel5A and Cel5B mutants with alanine substitutions at a conserved glutamic acid in the GH5 domain (Glu 179 of Cel5A and Glu184 of Cel5B) lacked cellulase activity, suggesting that this residue is important for GH5 domain function.

Isolation and characterization of endophytic bacteria fromPlectranthus tenuiflorusmedicinal plant in Saudi Arabia desert and their antimicrobial activities

“…Generally, in presence study, Gram-positive bacteria displayed broad hydrolase potential than Gram-negative bacteria (SanchezPorro et al 2003;Rhoban et al 2009). Cho et al (2008) isolated two cellulose hydrolase genes (cel5A and cel5B) from endophytic Paenibacillus polymyxa GS01 of ginseng roots. In general, the hydrolytic enzymes of endophytes appear to be important for the colonization of plant roots Reinhold-Hurek and Hurek 1998;Sakiyama et al 2001).…”

Enhancing the Enzymatic Activity of the Multifunctional β-Glycosyl Hydrolase (Cel44C-Man26AP558) from Paenibacillus polymyxa GS01 Using DNA Shuffling

“…In many cases, the correlation between hydrophobic interactions and protein stability has been demonstrated. In particular, the side chains of alanine, valine, leucine, and isoleucine tend to cluster together within proteins, stabilizing protein structure by means of hydrophobic interactions (Tang et al, 2006;Cho et al, 2008a). In the case of the P438A substitution, it is proposed that the increased hydrophobicity of the environment after the substitutions contributed to the enhanced enzyme activity of the trifunctional β-glycosyl hydrolase.…”

“…The cellulase, xylanase, and lichenase activities of the recombinant E. coli clones were determined by measuring the amount of reducing sugars produced during the incubation with the substrate. One unit of enzyme activities for cellulase, xyalnase and lichenase was defined as described elsewhere (Cho et al, 2008a).…”

“…At present, 29 amino acid sequences of GH44 family members have been determined (Warner et al, 2010). Several researchers reported that GH44 enzymes exclusively cleave β-1, 4 bonds between glycosyl and xylosyl residues and that they have different abilities to attack the bonds of xylan, lichenan, and different cellulose forms, such as Avicel, acid-swollen cellulose, and carboxymethylcellulose (CMC) (Crameri et al, 1998;Cho et al, 2006Cho et al, , 2007Cho et al, , 2008aCho et al, , 2008bWarner et al, 2010). The development of directed-evolution techniques was stimulated by the advent of DNA shuffling (Crameri et al, 1998;Kataeva et al, 2002), a PCR-like process that introduces and recombines random point mutations within a particular gene (Ash et al, 1993).…”

“…This strain was shown to produce a wide variety of secondary metabolites, such as plant growth-regulating substances, antibiotic compounds, and plant cell wall-degrading enzymes (Cho et al, 2006(Cho et al, , 2007(Cho et al, , 2008a(Cho et al, , 2008b. In addition, we previously reported that the truncated Cel44C-Man26AP558 protein was a trifunctional β -glycosyl hydrolase, demonstrating cellulase, xylanase, and lichenase activities (Cho et al, 2006).…”