Mutation Breeding Of Chryseobacterium Proteolyticum For Increasing Protein Glutaminase-production

Protein-Glutaminase (Protein-Glutaminase, PG, EC3.5.1.44) is a new type of glutaminase, it plays a role on deamidating proteins、short peptides or peptides’side chain. What’s more, it has an obvious effect on improving protein’s functional properties such as solubility、foaming ability and so on, which makes it have a great application prospect in food industry.However, the strains producing PG has low enzymatic activity and low yield, which become a limit of the strains’ application. In order to solve the problem, this paper has established the mutation method as well as high throughput screening and assaying method, laid a foundation for the PG industrialization.The important results were as follow:1、The method of fermentation in different systemsThe system of primary screen:The strains was fermented by 48-deep wells’with 1mL fermentation broth per well, and enzymatic activity was assayed after 14h fermentation, the variable coefficient of enzymatic activity is 14.7%.The second screening system:The strains was fermented by tube system with 3mL fermentation broth, and enzymatic activity was assayed after 12h fermentation. The validation system is 250mL flask system with 20mL fermentation broth, and enzymatic activity was assayed after 12h fermentation,and the best strain was chosed for the next round of mutation.2、The method of scale upThe correlation coefficient between tubes of 3mL liquid volume and 48-deep wells of 1mL liquid volume is 0.73, between 20mL/250mL flasks and 1mL/4.5mL 48-deep wells is 0.78, between tubes of 3mL liquid volume and 20mL/250mL flasks is over 0.9.The system of scale up is convenient and stable.3、The establishment of the method of mutationUV mutagenesis methods:strains Y8, irradiation distance:30cm, mutagenesis time:10s, fatality rate:99.31%, the positive mutation rate:33%, the enzyme activity was most increased by 150% in 650 strains’ primary screening; strain Y8, irradiation distance:45 cm, mutagenesis time:10s, fatality rate:80%, the positive mutation rate: 35%;enzyme activity was most increased by 145% in 557 strains’primary screening.NIT mutagenesis methods:strains Q9, best conditions:concentration of NIT:0.6 mg/mL, the fatality rate:99%, the positive mutation rate:30.4%; enzymatic activity was most increased by 88%in 400 strains’ primary screening.4、The method of high throughput screening and assayingMethylamine pressure screening:To inhibit the growth of Chryseobacterium protoelyticum,0.04% methylamine was added to the solid LB culture or liquid LB culture, and the accuracy of this method in applying for primary screening is 65%.High throughput assaying method:microplate reader was used to measure the AmaxV of fermentation broth’s dynamic coloration reaction. The AmaxV’s variation coefficient of PG’standard sample in a 96 well plate system is 10% and the fermented liquid is 18%, and for standard samples, the enzymatic activity of PG was significantly positive correlated with the ΔmaxV (R2=0.999).In addition, Using the method in nitrite sodium (nitrite, NIT) mutation (0.6mg/ml NIT treat 109CFU/mL bacteria for 20min), the R2 between induced strains’enzymatic activity and the Amax is 0.89.5、The mutation and the identification of high-yield strainsThrough three rounds of UV mutation, the strain Y8-UV3-80438 improved 125% was gotten, whose average enzymatic activity among 8 passages is 0.9U/mL, the strain Y8-UV3-80412 improved 133% was gotten, whose average enzymatic activity among 8 passages is 0.93U/mL. Through three rounds of NIT mutation, the strain Q9-NIT3-121412 improved 63% was gotten, whose enzymatic activity’variation coefficient among 8 passages is 8%.The result of PCR and sequencing says that there is no change among the nucleotide sequences and amino acids, suggesting the mutation site might be in the non-coding regions.