Secretory expression of a β-xylosidase gene fromThermomyces lanuginosusinEscherichia coliand characterization of its recombinant enzyme

Abstract

Aims: To characterize a β‐xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results: A gene (designated TlXyl43) encoding β‐xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017‐bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β‐xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6‐fold to homogeneity with an estimated mass of 51·6 kDa by SDS‐PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a Ki value of 63 mmol l−1. Conclusions: In this study, a GH family 43 β‐xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study: This is the first report of the cloning and functional expression of a β‐xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.

Production and Characteristics of a Novel Xylose- and Alkali-tolerant GH 43 β-xylosidase from Penicillium oxalicum for Promoting Hemicellulose Degradation

“…Based on the Lineweaver-Burk plots, the calculated Km and Vmax of the Xyl43 using the p NPX as substrate were 4.05 mM and 88.82 μmol min −1 mg −1 , respectively. Table 1 showed the Xyl43 had significantly lower Km values than the β-xylosidases from Humicola insolens (12.2 mM) 6 , Paecilomyces thermophile (8 mM) 17 , Neurospora crassa (8.9 mM) 24 , but similar to the β-xylosidases reported from Thermomyces lanuginosus and Paecilomyces thermophile 13 , 20 . Moreover, this recombinant Xyl43 exhibited higher Vmax toward p NPX than that of some β-xylosidases reported.…”

“…1 ) were described and characterized now. Most of the known fungal β-xylosidases from GH 43 were greatly tolerant to xylose, for example, Xyl43A and Xyl43B with the Ki values of 79 mM and 292 mM from Humicola insolens respectively 6 , TlXyl43 with the Ki value of 63 mM from Thermomyces lanuginosus 13 , PtXyl43 with the Ki value of 139 mM from Paecilomyces thermophile 14 . These β-xylosidases without predicting signal peptides were expressed in Escherichia coli ( E .…”

Secretory Expression and Characterization of Two Hemicellulases, Xylanase, and β-Xylosidase, Isolated from Bacillus Subtilis M015

“…Previous studies have shown that some β-xylosidases experience end-product inhibition by xylose present in the reaction mix [ 15 , 25 ]. We have observed a similar effect on XynB where its relative activity quickly dropped to 55 % in the presence of 20 mM of xylose and continued decreasing with increasing xylose concentration in the reaction mix (Fig.…”

Molecular cloning and comparative sequence analysis of fungal β-Xylosidases

“…Bacterial enzymes are mostly expressed heterologously in E. coli (Teng et al 2011) but fungal enzymes are generally purified from their original cultures (Ohta et al 2010) or produced in different high-level expression systems such as Aspergillus (Kitamoto et al 1999) or P. pastoris (Chen et al 2012b). With methanol induction a β-Xylosidase (Xyl3A) from Humicola insolens Y1 was successfully overexpressed in P. pastoris GS115 without signal peptide (Xia et al 2015) and the secretion of enzyme was facilitated by using the signal peptide of the yeast α-factor into the culture supernatant.…”