Breeding Of Protein Glutaminase High-producing Strains By Protoplast Fusion

Protein-glutaminase is a novel protein-deamidating enzyme and is capable of hydrolyzing side chain amido groups of protein to release ammonia.It catalyzes the deamidation of intact protein without transglutaminase and protease activities.The targets of this enzyme are glutaminyl residues in short peptides or proteins,but not asparaginyl residues or free glutamines.The secondary structures of protein changed after deamidated by protein-glutaminase,the compositions of flexible structures increased thus the solubility of protein and other functions improved,such as emulsifiability,foamability.Protein-glutaminase can further expand the application ranges of proteins,so it has great potential for industrial applications as an effective method to modify proteins.There are many reports about protein-glutaminase,but most studies focused more on its application than the producing strains at home and abroad.Microbiology Lab of East China Normal University spended 5 years in screening microorganisms from soils and successfully isolated 45 strains that produced the target enzyme and 33 of the strains are Chryseobacterium proteolyticum.Protein-glutaminase production of wild type strains are too low,it is necessary to increase productions by mutation before applied the strains into industrial production.In this study,two wild type strains were selected as parent strains and a new strain with high protein-glutaminase production had been screened after four rounds of protoplast fusion.The main results were as follows:1.Determination of parents strains for protoplast fusionChryseobacterium proteolyticum bank of 33 strains was constructed for selecting parent strains.33 Chryseobacterium proteolyticum strains were divided into three groups according to automatic ribotyping clustering analysis.Among those strains,enzyme activity of L11 was the highest,and Y8 was the most stable one.So,Y8 and L11 were selected as parent strains by taking the results of automated ribotyping,the soil samples and protein-glutaminase production into account.2.Determination of conditions for protoplast fusionIn this study,protoplast fusion was applied to the breeding of protein glutaminase high-producing strains for the first time.The determined examination conditions were as follows:the cells were collected and then diluted to OD600 was 2.00,0.1 mol/L EDTA and 0.1%SDS were added until the final concentrations were 0.008 mol/L EDTA and 0.002%SDS as pretreatment.The cells were then resuspended in SMM buffer containing 25 mg/mL lysozyme.It was suitable for protoplasts to receive 3 min radiation treatments under 15 W UV-light in the distance of 30 cm and be treated in 60? for 2 min as inactivating conditions.The conditions of fusion were:the fusion temperature was 20?;the fusion time was 40min,the shaking rate was 200 r/min.Under the optimal conditions,the formation rate was 91.19%+±3.56%,regeneration rate was 66.53%+2.71%,purity was 85.87%±1.18%and fusion rate was 1.52%±0.22%.3.Breeding of protein glutaminase high-producing strains by four rounds of protoplast fusionY8 and L11 were selected as parents for protoplast fusion in the first round,11233 strains were screened in 48-well plate.Then after 48-well plates,test tube and shake-flask system three kinds of secondary screening,protein glutaminase production of high-producing strains was 31.51%,19.12%,18.96%and 21.32%higher than their parent stains,respectively.PF-Y0448 was obtained with protein-glutaminase production was 175%and 143%higher than protein glutaminase production of Y8 and L11.PF-Y0448 was subcultured for 10 times,the average protein glutaminase production was 1.14 ± 0:09 U/mL,the coefficient of variation was 8.17%.4.Characteristic analysis of protein glutaminase high-producing strainThe growth and enzyme production curve showed that the logarithmic phase of L11 and PF-Y0448 was 4 h-12 h.Protein glutaminase began to product at 8 h and reached the highest point at 16 h and 19 h for L11 and PF-Y0448,respectively.The results showed that protein glutaminase production of different carbon sources and nitrogen sources are different,with 5 g/L of lactose as carbon source,15 g/L poly peptone as the nitrogen source reached the highest production.Adding 5 g/L lactose after glucose depleted is helpful to increase protein glutamines production while 0.3 g/L glucose as the carbon source.5.Re-sequencing analysis of Lll and PF-Y0448To find out the mechanism of high protein glutaminase production,this study uses the third-generation sequencing technology to analysis whole genome sequencing of L11.Gene of protein-glutaminase located in orf03588.Orf03589 and orf03588 belong to one operon and are regulated by one promoter,but the fuction of orf03589 are not confirmed.With L11 as the reference sequence using the second-generation sequencing technology to analysis genome sequencing of PF-Y0448.The functional annotation based on the distribution of SNPs in PF-Y0448 was mainly alssified as carbohydrate transport and metabolism,inorganic ion transport and metabolism,amino acid transport and metabolism,nucleotide metabolism and amino acid metabolism pathway.NR functional annotation based on the distribution of InDels in PF-Y0448 was hypothetical protein.Lacking of deep analysis of the sequencing results,the results can not be associated with the enzyme activity,so it is impossible to obtain the possible reasons for high production.