Elsevier

Bioresource Technology

Volume 170, October 2014, Pages 513-521
Bioresource Technology

A highly active beta-glucanase from a new strain of rumen fungus Orpinomyces sp.Y102 exhibits cellobiohydrolase and cellotriohydrolase activities

https://doi.org/10.1016/j.biortech.2014.08.016Get rights and content

Highlights

  • A new strain of rumen fungus was isolated and designated Orpinomyces sp.Y102.

  • CelC7 from the cDNA library of Orpinomyces sp.Y102 encodes an exocellulase.

  • The N-terminus of CelC7 may be a dockerin-containing domain.

  • The N-terminal truncated forms of CelC7 have relatively high activities.

  • Truncated CelC7 has cellobiohydrolase and cellotriohydrolase activities.

Abstract

A new strain of rumen fungus was isolated from Bos taurus, identified and designated Orpinomyces sp.Y102. A clone, celC7, isolated from the cDNA library of Orpinomyces sp.Y102, was predicted to encode a protein containing a signal peptide (Residues 1–17), an N-terminal dockerin-containing domain, and a C-terminal cellobiohydrolase catalytic domain of glycoside hydrolase family 6. CelC7 was insoluble when expressed in Escherichia coli. Deletion of 17 or 105 residues from the N-terminus significantly improved its solubility. The resulting enzymes, CelC7(-17) and CelC7(-105), were highly active to β-glucan substrates and were stable between pH 5.0 and 11.0. CelC7(-105) worked as an exocellulase releasing cellobiose and cellotriose from acid-swollen Avicel and cellooligosaccharides, and displayed a Vmax of 6321.64 μmole/min/mg and a Km of 2.18 mg/ml to barley β-glucan. Further, the crude extract of CelC7(-105) facilitated ethanol fermentation from cellulose. Thus, CelC7(-105) is a good candidate for industrial applications such as biofuel production.

Introduction

Cellulose, a polymer of d-glucose connected by β-1,4 linkage, is the major component of plant cell wall. To release glucose from lignocellulose as feedstock to produce ethanol is the idea for the second generation bioethanol (Hasunuma et al., 2013, Zhang et al., 2006), in which the efficient decomposition of cellulose is a key determining factor for the feasibility of this strategy. Therefore, highly effective cellulases, enzymes catalyzing the hydrolysis of cellulose, are required. Cellulase is also applied in several other industries, such as textile, detergent, and pulp/paper industries (Ko et al., 2010, Miao et al., 2014, Zhao et al., 2012). The utilization of enzymes in these industries to replace the traditional chemical processing is friendlier to the environment. Thus, the market of cellulase is expected to grow (Cherry and Fidantsef, 2003, Zhang et al., 2006).

At least three types of cellulases are required to work in synergy to convert cellulose into glucose monomer. Endocellulase or endoglucanase (endo-β-1,4-glucanase; EC 3.2.1.4) randomly cleaves the internal β-1,4 bonds of cellulose, generating shorter chains or oligosaccharides. Exocellulase or exoglucanase is usually referred to enzymes that hydrolyze cellulose by releasing cellobiose successively from the reducing end (EC 3.2.1.176; cellobiohydrolase I) or non-reducing end (EC 3.2.1.91; cellobiohydrolase II) of cellulose. Meanwhile, exocellulases that liberate glucose successively from the termini of cellulose are also reported (EC 3.2.1.74; glucan 1,4-β-glucosidase or exo-1,4-β-glucosidase). Finally, β-glucosidase (EC 3.2.1.21) dissects cellobiose or oligoglucosaccharides into glucose (Zhang et al., 2006). The work of endocellulase and exocellulase is considered the rate limiting step for the degradation of cellulose (Zhang et al., 2006). In the classification of glycoside hydrolase (EC 3.2.1.-) (Henrissat and Bairoch, 1996), endocellulases are found in 17 families; type I cellobiohydrolases are classified in families 7, 9, and 48, whereas type II cellobiohydrolases are in families 5, 6, 7, and 9.

The most common sources for exploring cellulases were bacteria and fungi (Kumar et al., 2008), and the available cellulases in the market were mostly cloned from Trichoderma (e.g. Trichoderma reesei, Trichoderma viride) and Aspergillus (Zhang et al., 2006). Evidence suggested that rumen fungi contain cellulases with much higher activities than those from Trichoderma (Chen et al., 2003, Harhangi et al., 2003, Nagy et al., 2007, Qiu et al., 2000). Thus, rumen fungi are regarded as a potential source for seeking highly efficient cellulases. Since cultivation of rumen fungi on a large scale is not easy due to their unique growth conditions, the number of cellulases cloned from rumen fungi is limited.

In this study, a new strain of the fungus Orpinomyces was isolated from the rumen of cattle (Bos taurus), and a cDNA library was constructed from its total RNA. The library was subjected to screening for β-glucan- or carboxymethyl cellulose (CMC)-degrading activity. The biochemical properties of the enzyme encoded by one of the positive clones, designated celC7, were investigated.

Section snippets

Bacterial strains and chemicals

Escherichia coli BL21(DE3) (Novagen, Madison, WI, USA) was used for protein expression and purification and E. coli DH5α (Yeastern Biotech, Taipei, Taiwan) for plasmid propagation and isolation. Oat spelt xylan, CMC, glucose, cellobiose, cellotriose, cellooligosaccharides, p-nitrophenyl-β-d-cellobioside, p-nitrophenyl-β-d-galactopyranoside, and chemicals for buffer preparations were purchased from Sigma–Aldrich (St. Louis, MO, USA); Avicel from Asahi Chemical (Tokyo, Japan); CBH I, barley

Identification of Isolate Y102 and sequence analysis of celC7

The fungi of the genus Orpinomyces feature polycentric thalli, filamentous rhizoids, and polyflagellate zoospores (Ho and Barr, 1995). These morphological characteristics were clearly observed during the growth of Isolate Y102. The size of mature thalli of Y102 was 1–15 mm in diameter. These results validly indicated that Isolate Y102 belongs to the genus Orpinomyces.

The ITS1 and partial rDNA sequence of Orpinomyces sp.Y102 were sequenced and registered in National Center for Biotechnology

Conclusions

CelC7 isolated from a new strain of rumen fungus Orpinomyces sp.Y102 was found to be a cellobiohydrolase and cellotriohydrolase. Its N-terminus is dispensable for the catalytic activity and may be a dockerin-containing domain. The N-terminal truncated form CelC7(-105) has a relatively high specific activity to β-glucan substrates, and is capable of facilitating ethanol production from cellulose. Thus, it is a good candidate for applications requiring cellulases such as biofuel production. Our

Acknowledgements

The financial support of the National Science Council Taiwan to H.L. Cheng (Grant No. NSC 99-ET-E-020-001-ET and NSC 100-ET-E-020-001-ET), and to Y.C. Chen (Grant No. NSC 102-2628-B-020-001-MY3) is greatly appreciated.

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