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Beta-glucosidases are present in all domains of living organisms and catalyse various biological functions. They hydrolyse β-1,4 glycosidic linkages and synthesise glucosides through transglycosylation or reverse hydrolysis. β-glucosidases are an important class of enzymes having significant prospects in industrial biotechnology. However, cellulolytic microorganisms produces this enzyme in insufficient amounts. This presents a great obstacle in large-scale biotechnology applications. Therefore, the search for novel β-glucosidases is ongoing to counteract this issue. Genome sequencing of Thermomyces lanuginosus SSBP revealed multiple β-glucosidase genes. This study was aimed at characterising five T. lanuginosus SSBP β-glucosidases (Bgls) expressed in Pichia pastoris. Minimal methanol medium (MM) is commonly used for induction of expression in P. pastoris. In this medium, only Bgl2 was expressed after 144 hours. An activity of 71.9 U/ml was obtained whereas Bgl1, Bgl3, Bgl4 and Bgl5 were not detectable. Yeast extract, peptone and methanol (YPM) was then used as an alternative medium. In YPM, all the enzymes were produced after 168 hours of induction of expression. Bgl1, Bgl2, Bgl3, Bgl4 and Bgl5 activities were 1.5 U/ml, 310.8 U/ml, 0.9 U/ml, 1.8 U/ml and 0.9 U/ml, respectively. The sizes of Bgls were determined using nucleotide sequences. Bgl1, Bgl2, Bgl3, Bgl4 and Bgl5 sizes were 99.9 kDa, 46.5 kDa, 46.8 kDa, 68.9 kDa and 54.3 kDa, respectively. After partial purification, the specific activities of 50.4 U/mg for Bgl1, 553.7 U/mg for Bgl2, 72.0 U/mg for Bgl3, 111.6 U/mg for Bgl4 and 44.0 U/mg for Bgl5 were obtained. The Bgls performed optimally at pH 6.0 and temperature of 50-60℃. Bgl2 and Bgl4 hydrolysed all the tested natural substrates of different linkages, indicating broad substrate specificity. Bgl1, Bgl3 and Bgl5 selectively hydrolysed β-1,3/6-linked substrates (gentiobiose and laminarin). Bgl2 was the dominating recombinant enzyme and it showed the ability to work in synergy with a commercial cellulase to hydrolyse microcrystalline (MCC) and carboxymethyl cellulose (CMC). Upon supplementation of Bgl2, a 58% and 91% increase in glucose production was achieved from MCC and CMC, respectively. Therefore, this enzyme has potential to be used in cellulose degradation for valorisation of waste lignocellulosic biomass.

Characterisation of recombinant beta-glucosidases from Thermomyces lanuginosus SSBP and investigation of their synergistic potential in cellulose hydrolysis