(191u) Homologous Constitutive Secretory Expression of Halo, Acid and Thermo-Tolerant ?-Glucosidase in Marine Aspergillus niger

β-glucosidase, a key component of cellulase, plays an important role in the degradation of cellulose. Besides, it is widely used in food and beverage industry for hydrolyzing the flavor precursors of fruit, vegetable and tea to rich full flavors. However, the application of β- glucosidase is often limited by different environmental factors, such as high salt, high temperature and extreme pH, which lead to reduced enzyme activity.

A halo-tolerant marine Aspergillus niger, isolated from the East China Sea, can produce β-glucosidases with high enzyme activity. It was found that the β-glucosidases had good properties of halo, acid and thermo-tolerance. In order to further study the characteristics of the enzyme accurately, a part of one β-glucosidase gene was amplified from the genome of marine A. niger by using degenerate primers, which were designed according to the conserved sequences of β-glucosidases within A. niger. And then the complete gene, which includes introns, was obtained by hiTAIL-PCR. The target gene contains 7 exons and 6 introns by comparing with its mRNA, and the length of complete gene and coding region gene are 2603 bp and 2298 bp, respectively. Subsequently, the complete gene was placed under the constitutive promoter PgpdA and then transformed into its own host with the help of Agrobacterium tumefaciens. Finally, a recombinant which constitutively expresses β-glucosidase was obtained, and the expression quantity was about 1 g·L^-1 in the supernatant determined by SEC-HPLC after 7 days of fermentation in the medium containing 0.5% yeast extract, 1% peptone, 3% NaCl, 2% glucose and 0.4% KH₂PO₄. Moreover, the enzyme accounted for more than 90% of the total proteins in the supernatant and its apparent molecular weight was 110 kDa. At the end, the characteristics of the enzyme were studied after removing the pigments, salt ions and other small molecular weight impurities by ultrafiltration. It was found that the optimum salinity, pH and temperature were 0.3-0.5 mol·L^-1 NaCl, 2.2-3.8 and 60-70 °C, respectively. Significantly, the enzyme activity remained 60% at the condition of 4 mol·L^-1NaCl, pH 1.0 or 80 °C.

Filamentous fungi have a strong ability of protein secretion. In this study, marine A. niger was used as the expression host for constitutive secretory expression of its own β-glucosidase. The expression level in the recombinant strain fully demonstrates the superiority of filamentous fungi in secretory expression of macromolecular protein which is more than 100 kDa. What’s more, β-glucosidase is the dominant component in the supernatant at the non-induced condition since most of the self-secreted proteins are inducible proteins in A. niger. Thus, its enzymatic characteristics can be determined after a simple ultrafiltration of fermentation supernatant. Furthermore, the β-glucosidase, which has strong halo, acid and thermo-tolerance, shows a great application prospect in the chemical and food industry.

Keywords: β-glucosidase; marine Aspergillus niger; enzymatic characteristics; tolerance

Supported by the National Natural Science Foundation of China and Scientific Program of Zhejiang Province (2015C33219); Corresponding author : Shan-Jing Yao, E-mail: yaosj@zju.edu.cn