Breeding Of Protein Glutaminase Strain And Its Application In Enzymatic Hydrolysis Of Soybean Protein

Protein Glutaminase（PGase or PG for short）is a kind of hydrolytic enzyme that can hydrolyze glutamine residues on the side chain of proteins and polypeptides.It does not catalyze free glutamine and side chain asparagine residues.It has strong substrate specificity.Its deamidation can significantly improve the functional properties of plant proteins（solubility,gelation,emulsification,foaming,etc）,thus improving the processing properties of plant proteins.Many studies have successfully applied PG to the modification of proteins,thus improving the functional properties of proteins,such as soybean protein,coconut protein,wheat gluten,rice protein,pea protein,etc.At present,PG are mainly obtained by microbial fermentation.However,at present,there are few strains with high production of PG in domestic research,and there are few strains with high production of protein glutaminase,It is urgent to breed high-yield PG strains by artificial mutagenesis to meet the requirements of industrialization.In order to obtain the high yield strain,this paper used ultraviolet（UV）mutagenesis and atmospheric and room temperature plasma（ARTP）mutagenesis techniques to mutagenize on the laboratory preserved Chryseobacterium proteolyticum 1003,and then the enzymatic properties of the high yield mutant and the starting strain were studied.The effects of mutant PG enzyme solution on deamidation degree and functional properties of soybean protein isolate（SPI）were also studied.The main research results are as follows:1.Mutation breeding of protein glutaminase strainsIn this study,the optimal treatment time of the UV mutagenesis and ARTP mutagenesis was explored.UV mutagenesis parameters were fixed:the treatment power was 11 W,and the strain was placed under UV lamp for 30 cm.When the mutagenesis time was 30 s,the mortality rate of the strain was 88%,and the positive mutation rate was about12%,the mutagenesis effect was the best.The mutagenesis parameters of ARTP were fixed:the treatment power was 125 W,the mutagenesis gas flow was 10 splm,and it was placed 2mm below the ion source.When the mutagenesis time was 30 s,the fatality rate was about81%,and the positive mutation rate was about 13%,and the mutagenesis effect was the best.In this paper,96 deep-well plate primary screening and flask re-screening were used,and enzyme activity/OD₆₀₀was used as the screening index.Finally,a mutant strain UUA-3D8with stable heredity and 20%increase in enzyme activity was screened out from 3288mutant strains,and the enzyme activity reached 2.16 U/m L.2.Effect of protein glutaminase on functional properties of soybean protein isolateThe mutant UUA-3D8 was fermented,the supernatant was obtained by centrifugation,ethanol precipitation and ultrafiltration to obtain PG enzyme solution.The purified PG enzyme solution was added into 1%SPI solution,and the enzyme activities of the prepared100 m L SPI solution were 0 U,0.5 U,10 U and 100 U,respectively.After reaction at 37°C and 130 rpm for 1 h,SPI were dialyzed,freeze-dried and stored in a dryer.The effect of different PG supplemental levels on the functional properties of SPI was determined.The results showed that the deamidation effect of SPI was more significant with the increase of PG supplemental levels.When the enzyme activity was 100 U,the deamidation degree of soybean protein isolate was 22%,SPI aggregates decreased,solubility increased from21.5%to 58%,and thermal stability decreased slightly.When the enzyme activity was 10 U,the functional properties of deaminated SPI were significantly improved,and the emulsifying activity was 26.97 m²/g,which was 85.62%higher than that of undeaminated SPI;The emulsifying stability was improved by 53.21%;The foaming stability increased from 22.83%to 25.60%.When the enzyme activity was 0.5 U,The foaming ability was also increased from 34.60%to 46.13%.In this study,the mutant library of protein glutaminase was successfully established by UV and ARTP mutagenesis.Finally,a mutant enzyme with stable activity increased by 20%was screened out and used to modify SPI.This study is beneficial to increase the enzyme activity of industrial strains and provide some reference for improving the processing characteristics of SPI.