Cloning, purification and study of recombinant GH3 family β-glucosidase from Penicillium verruculosum

Volkov, P.V.; Rozhkova, A.M.; Zorov, I.N.; Sinitsyn, A.P.

Авторы: Volkov Pavel V., Rozhkova Alexandra M., Zorov Ivan N., Sinitsyn Arkady P.
Журнал: Biochimie
Том: 168
Год издания: 2020
Издательство: Elsevier BV
Местоположение издательства: Netherlands
Первая страница: 231
Последняя страница: 240
DOI: 10.1016/j.biochi.2019.11.009
Аннотация: A novel bgl1 gene, encoding GH3 family β-glucosidase from Penicillium verruculosum (PvBGL), was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain under the control of the strong xylA gene promoter. The recombinant rPvBGL was purified and their properties were studied in comparison with those of rAnBGL from Aspergillus niger expressed previously in the same fungal host. The rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data) and displayed the enzyme maximum activity at pH 4.6 and 65 °C. The enzyme half-life time at 60 °C was found to be 87 min. Unlike the rAnBGL, the rPvBGL was not adsorbed on microcrystalline cellulose, which gives the latter enzyme an advantage in cellulose conversion with a longer time of hydrolysis. Conclusions A novel gene bgl1, encoding a β-glucosidase from P. verruculosum was cloned and successfully expressed in the recipient fungal strain of P. canescens under the control of the strong xylA gene promoter. The recombinant rPvBGL was isolated in a homogeneous form. The enzyme exhibited a high activity against pNPG, cellobiose and other cellooligosaccharides. Surprisingly, it also demonstrated a remarkable ability to hydrolyze laminarin and a mixed β −1,3-1,4-glucan from barley. rPvBGL converted both polymeric substrates to glucose much quicker than the recombinant BGL from A. niger (rAnBGL). However, because of the higher inhibition by the reaction product (glucose), the rPvBGL was inferior to the rAnBGL when added as an accessory activity to the P. verruculosum multienzyme cellulase preparation in hydrolysis of MCC. However, in the hydrolysis of milled aspen wood, representing a real lignocellulosic feedstock, both BGLs under study demonstrated similar boosting effects, leading to a complete conversion of aspen wood cellulosic fraction after 48 h of the reaction, which is more interesting in terms of biotechnological application of the rPvBGL under study. Thus, this study has shown the possibility of using rPvBGL for the construction of complex and balanced cellulase enzyme preparations based on the fungus P.verruculosum.
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Cloning, purification and study of recombinant GH3 family β-glucosidase from Penicillium verruculosumстатья

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