关键词: 米曲霉, 重组碱性蛋白酶, 毕赤酵母, 克隆, 表达, 水解特性

Abstract:

Alkaline proteases from Aspergillus oryzae are of efficient hydrolysis ability and preferable debittering effect to plant protein. In order to obtain a large amount of alkaline proteases for industrial applications,the alkaline protease gene from A. oryzae was cloned by using RT-PCR technique,and was transformed into Pichia pastoris KM71 strain,thus being successfully expressed. Then,the protease was used in the hydrolysis experiments of bovine insulin B-chain,soy protein and peanut protein. The results show that ( 1) when the protease is induced to express in a 10-L fermentor,the protease activity of the fermentation broth is up to 4100U/mL; ( 2) the optimum pH value and temperature of the protease are respectively 8.5~9.5 and 50℃,and the protease is stable at a pH value of 6.0 to 10.0 below 40℃; ( 3) the protease is of extensive peptide-bond selectivity; and ( 4) the protease displays higher hydrolysis activity to soy and peanut protein than some commercial proteases. The above-mentioned results indicate that the recombinant protease is of potential application prospects.

Key words: Aspergillus oryzae, recombinant alkaline protease, Pichia pastoris, cloning, expression, hydrolysis characteristic