Expression Of Kawachii Acid Protease In Non-spore Aspergillus Niger SH-2

Acid proteases are enzymes suitable for efficiently hydrolyzing proteins to amino acids and small peptides at pH 2-6.Since acid protease hydrolysis occurs in an acidic environment,corruption and deterioration due to bacterial growth are generally not present.Thus acid protease is widely used in the wine,feed,leather,pharmaceutical and other industries.At present,most studies on acidic proteases are focused on Aspergillus oryzae,Aspergillus niger,and Aspergillus usamii,while there have been few studies on the acidic proteases of Aspergillus kawachii.In this study,a strain of Aspergillus niger SH-2 with low protein background was used as the host,and Aspergillus kawachii was used as the gene donor of acidic proteases to construct a recombinant strain capable of expressing acid protease of Aspergillus kawachii.?1?Bioinformatics analysisAspergillus kawachi acid protease belongs to the aspartic protease family.This enzyme has a total of 394 amino acids.The first 20 amino acids are signal peptides.It was predicted that-NKT at the amino acid sequence 39 is an N-glycosylation site and this protein is hydrophilic protein.The model presented a flute-like structure with two active centers in the groove,three Zn binding sites and one disulfide bond.?2?Expression of acid protease of Aspergillus kawachiiAfter the recombinant strain was cultured in shake flask medium for 168 h,the crude enzyme liquid enzyme activity was 240 U/mL,and the enzyme activity was 9972 U/mL after fermentation in a 30 L fermentor for 240 h.The SDS-PAGE analysised showed that the molecular weight of Aspergillus kawachi acid protease was approximately 47 KDa.In the tank fermentation process of the recombinant strain 30 L,the feed rate and the ammonium chloride addition rate were restricted,and the stable physiological activity of the fermentation process can be maintained.The fermentation pH was controlled within the range of 4.7 to 4.8,and the bacterial concentration is maintained at 55%or more,which allowed stable and high yield of experimental fermentation.?3?Enzymatic propertiesThe optimum reaction temperature of Aspergillus kawachii acid protease was 35°C.When the temperature was lower than 25°C or higher than 50°C,the enzyme activity would be significantly reduced.The optimum reaction pH was 4.When the pH was lower or higher than 4,the enzyme activity was reduced.The pH value had a significant effect on the activity of Aspergillus kawachii acid protease,and a slight decrease in the pH value would caused a rapid decline in the enzyme activity.Mn²⁺and Cu²⁺had a significant activation effect on enzyme activity,and the enzyme activity was 3 and 2.5 times that of the control group.Zn²⁺and Mg²⁺had no obvious effect on the enzyme activity,while Cd²⁺had a significant inhibitory effect on the enzyme activity.Under this condition,the degree of hydrolysis of soy protein isolate reached 6.6%.?4?Effect on hydrolysis of soy proteinThe optimal temperature for the hydrolysis of soybean protein isolate by Aspergillus kawachii acid protease is about 60°C,the optimal pH is 5,the optimal hydrolysis time is 3h and the best E:S is 800:1.Under this condition,the degree of hydrolysis reaches 6.6%.Compared with the skewed acid protease,Aspergillus kawachi acid protease has stronger hydrolysis ability to the soy protein isolate.The effect of combined hydrolysis with other proteases showed that the combined effect of“Alkaline protease+Aspergillus kawachi acid protease”enhanced the effect of hydrolysis.