Expression of high-temperature resistant β-glucosidase in Pichia pastoris

Abstract

β-Glucosidase is involved in the degradation of carbohydrates, phenolic compounds, some natural products and antibiotics, and the decomposition of cellulose, lignin and other polysaccharides in the plant cell wall, which is important for the release and recycling of nutrients, and is widely used in the fields of food, pharmaceuticals, skincare and cosmetics, etc. Thermal stability of β-glucosidase is one of the key factors determining the cost-effectiveness of biorefinery. Utilizing microbial molecular biology technology, microbial technology, and enzyme engineering technology to create high-temperature-resistant β-glucosidase is of great scientific significance and great market application prospect. In this study, the β-glucosidase gene was cloned from a strain of Pyrococcus furiosus and molecularly modified to obtain the β-glucosidase mutant gene 371-β-glucosidase (T371A). The mutant gene was transformed into Pichia pastoris yeast strain GS115 by polyethylene glycol-mediated transformation, and SDS-PAGE was used to demonstrate that the T371A gene had been integrated into the Pichia pastoris yeast genome and was successfully expressed, which provided a reliable basis for the subsequent evaluation of the high-temperature tolerance of β-glucosidase and the preparation of its enzyme preparations. The results showed that the mutant β-glucosidase exhibited significant thermophilicity and thermal stability with increasing temperatures, a feature consistent with the Pyrococcus furiosus strain from which the gene was derived. The data showed that the enzyme activities of T371A β-glucosidase and enzyme powder were increased by 44.30% to 257.17% and 40.93% to 161.27%, respectively, compared with those of the wild-type β-glucosidase under high temperature conditions. By putting the transgenic Picrasinol yeast into a 50L fermenter for scale-up culture and high-temperature spray-drying treatment to become enzyme powder, the enzyme powder with a yield of 4.17 g/L and a water content of 9.9% was obtained, and its enzyme activity was only lost by 17.23%, which possessed the same catalytic properties as that of T371A, and the T371A enzyme powder also showed the advantages of a longer storage period and a higher quality of storage. It was further found that the enzyme activity of T371A and its enzyme powder continued to increase with the increase of reaction time under the high temperature condition of 100 ℃, which indicated that the enzyme did not reach the maximum reaction rate at 100 ℃ and the optimal temperature was much higher than 100 ℃, which demonstrated significant heat resistance and thermal stability. In summary, β-glucosidase, as an enzyme with a wide range of application prospects, its heat resistance is of great significance and application prospects in industrial and research fields. This study lays the foundation for expanding its application in a wider temperature range by exploring its heat resistance.