Research On The Neutral Protease From Bacillus Amyloliquefaciens BS5582

Neutral protease is widely used in brewing, food, textile, pharmaceutical, leather industry and so on. It takes up a large share of the total market of industrial enzyme in the world. Bacillus amyloliquefaciens BS5582 is a patented strain with high yield of neutral protease. In this paper, the neutral protease from BS5582 was studied in order to lay a good foundation for its further application.The quality of the wort is very important to the beer. We compared the effect of neutral protease from BS5582 and some commercial protease on the mashing of different malt both from home and abroad. The concentration ofα-amino-nitrogen from the malt which was treated by neutral protease from BS5582 was increased by 16.21% and 24.96%, respectively. And it was higher than the results from the commercial ones. Furthermore, its protein composition was more appropriate. The content of sensitive protein in the wort was decreased by 25.6% and 41.5%, respectively. Besides, the analysis of amino acid showed that the kind of amino acid which could be absorbed and used was increased.The neutral protease was purified with a four-step procedure including ammonium sulfate precipitation, ion-exchange, hydrophobic interaction, and gel filtration chromatography. A 5.6 fold increase in specific activity was achieved with the recovery of 3% and the specific activity of neutral protease reached 11278 U/mg. The molecular weight of the enzyme was estimated to be 37 kDa by SDS-PAGE. Further research showed that purified protease showed maximal activity at 50℃and lost its activity after being heated at 60℃for 10 min. It was found to be stable under different pH from 4 to 10 for 30-60 min with an optimum activity at pH 7.0. The enzyme activity was strongly inhibited by EDTA, indicating the enzyme was the metalloproteinase and markedly improved by Mn²⁺ for about 3 times when the concentration was 10 mmol. On the other hand, the activity was inhibited by Fe²⁺,Fe3+ or Ba²⁺. The impacts of Tween 20, Tween 80, Triton X-100, and SDS on neutral protease were also investigated. It was found that the protease activity was about 60% and 35% higher than the original one when treated by Tween 20 and Tween 80. The results of circular dichroism study showed that high temperature destroyed the secondary structure evidently, resulting in the lost of activity.The neutral protease gene was amplified from B. amyloliquefaciens BS5582 genomic DNA by PCR and then expressed in Pichia pastoris GS115. The activity of recombinant enzyme reached 761 U/mL after methanol induction during the fermentation process. The optimum activity of the recombinant protease was reached at pH7.0 and 50℃. It remained about 85% activity after being incubated at 40℃for 1 h, and showed excellent stability at pH4-9. What's more, Ca²⁺, Mg²⁺, and Mn²⁺ could activate the enzyme, whose activity was increased by 64%, 46%, and 18%.