Comparative Study On Alkaline Protease Produced By Pichia Pastoris And Bacillus Subtilis

Alkaline protease is a kind of biocatalyst with high catalytic performance and eco-friendliness.It can quickly and efficiently decompose proteinaceous materials into peptides and amino acids.And it is widely used in washing products,leather processing and food processing industries.Alkaline protease can be overexpressed by a heterologous expression host in the fermentation process of alkaline protease.The process of microbial fermentation of alkaline protease has the advantages of short cycle,high yield,low cost and easy availability of nutrients,and is suitable for industrial large-scale cultivation.In the fermentation production and application research of alkaline protease,most people only focus on one strain that produces alkaline protease,and lack of understanding of recombinant expression vector construction,fermentation conditions,ultrafiltration purification and washing performance differentiation of different strains.In this paper,the differences of the recombinant expression vector construction,fermentation conditions,ultrafiltration purification and washing performance between Pichia pastoris and Bacillus subtilis are compared and discussed.Firstly,new alkaline protease heterologous expression host(Pichia pastoris p PICZ?-AP and Bacillus subtilis p BE2R-AP)are successfully constructed and expressed.Subsequently,two strains are subjected to fermentation of the target product,and it is found that the activity of alkaline protease obtained by the B.subtilis is higher than that of the Pichia pastoris.Secondly,the optimal fermentation conditions for the alkaline protease production by Pichia pastoris and Bacillus subtilis are compared.It is found that the optimum enzyme activity of Pichia pastoris takes longer to obtain than that of Bacillus subtilis.In terms of the utilization types of nutrients,Pichia pastoris prefers to use methanol,glycerol and YPD;Bacillus subtilis is preferable for glucose and yeast extract instead of methanol and glycerol.In terms of the ratio of nutrients,Pichia pastoris has a lower single demand for carbon source than that of Bacillus subtilis,but it is higher than Bacillus subtilis in terms of total carbon source addition.About the physical factors of the culture process,the difference of optimum enzyme yield p H for the two strains is not much different;however,in terms of culture temperature,the lower temperature is more conducive for the expression of Pichia pastoris,and Pichia pastoris requires relatively more oxygen in the fermentation process.Thirdly,both the Pichia pastoris and the Bacillus subtilis fermentation broth were effectively concentrated by ultrafiltration,and the activity of alkaline protease in the fermentation broth was improved.Compared with Bacillus subtilis,ultrafiltration process of Pichia pastoris broth has a lower membrane flux and a longer ultrafiltration time,which may be caused by the larger solute particle size of the fermentation broth of Pichia pastoris.In the ultrafiltration process of Pichia pastoris and Bacillus subtilis,the particle size and Zeta potential of solute are dynamically changed.The change in the particle size and the absolute value of the Zeta potential is the same,and both increase with the ultrafiltration time,which is related to the aggregation of the solute in the fermentation broth.Finally,the washing performance of alkaline proteases derived from different strains before and after ultrafiltration is compared under the same total enzyme activity.After ultrafiltration of the two strains fermentation broths,the alkaline protease washing performance is slightly decreased.The washing performance of the fermentation broth are ranked as follows: detergent added with Pichia pastoris fermentation broth > detergent added with Bacillus subtilis fermentation broth > detergent without added alkaline protease;the washing performance of Pichia pastoris,Bacillus subtilis and commercially available alkaline are compared under the different total enzyme activity.The washing results of the proteases are ranked as follows: detergent added with Pichia pastoris alkaline protease > commercially available alkaline protease ? detergent added with Bacillus subtilis alkaline protease,and the washing performance increases with the increase of enzyme activity.From the ART-FTIR spectrum before and after washing of the Chinese national standard protein-contaminated swatches JB02,it can be seen that alkaline proteases derived from Pichia pastoris and Bacillus subtilis both can effectively destroy the peptide bonds of protein stains and the secondary structure of proteins during the washing process.The protein stains can be decomposed and detached from Chinese national standard protein-contaminated swatches JB02,thus the enzyme-added detergent has better washing performance.