Gene cloning and expression of fungal lignocellulolytic enzymes from the rumen of gayal (Bos frontalis).

A total of 6,219 positive clones were obtained by constructing a BAC library of uncultured ruminal fungi of gayal, and two clones (xynF1 and eglF2) with lignocellulolytic enzyme activity were selected. The sequencing results showed that xynF1 and eglF2 had 903-bp, and 1,995-bp, open reading frames likely to encode β-xylanase (XynF1) and β-glucosidase (EglF2), respectively. The amino acid sequence of XynF1 had 99% coverage and 95% homology to the endo-β-1,4-xylanase encoded by the cellulase gene of Orpinomyces sp. LT-3 (GenBank accession No. AEO51791.1). The amino acid sequence of EglF2 had 99% coverage and 93% homology to the β-glucosidase encoded by the cellulase gene of Piromyces sp. E2 (GenBank accession No. CAC34952.1). Analysis using the SMART software showed that XynF1 contains a glycoside hydrolase family 11 functional module and a carbohydrate-binding module, while EglF2 contains a glycoside hydrolase family 1 functional module. XynF1 showed the highest relative enzymatic activity, up to 95%, at 45°C and pH 4.2, while EglF2 showed the highest relative enzymatic activity, up to 95%, at 55°C and pH 6.2. In this study, we achieved efficient expression of the xynF1 and eglF2 genes in Pichia pastoris, which laid a foundation for the practical application of the lignocellulolytic enzymes.