Mutation Breeding Of Streptomyces Mobaraensis With High-yield And Thermostable Transglutaminase

Transglutaminase (EC 2.3.2.13, Transglutaminase, TGase) is a kind of transferase which catalyze intramolecular or intermolecular cross-linking occurs via Na(y-glutamyl)lysine isopeptide bonds. The cross-linking reaction can significantly improve the gel strength, water binding capacity, thermostablility of protein, etc. It was widely used in food processing field, and has obtained the huge commercial value in domestic and abroad.Commercial TGase mainly derive from microorganisms. It is named microbial transglutaminase(MTG). In the process of production, the main reason of high production cost is the low-yield strain. And the poor thermostablility of MTG limits its application in food processing at a higher temperature. In order to obtain a Streptomyces mobaraensis strain with the high-yield of MTG, and at the same time improve the thermostability of MTG, the traditional mutagenesis technique was used to modify the Streptomyces mobaraensis which was preserved in ECNU laboratory. A high-yield strain A1 was successfully selected from nearly 15000 mutants by high-throughput screening method. And the MTG activity of the high-yield strain A1 was almost increased by 200% compared to the parent strain. In this paper, mutation and screening for high-yield strain, character of the high-yield strain A1, establishment of a rapid method of screening for thermostable transglutaminase from Streptomyces mobaraensis and the related mutation breeding were researched. Detailed results were as follows.Mutation and screening for high-yield strain. Optimum conditions of UV mutagenesis:The UV irradiation time was 30s, and the distance was 30cm, and then dark treatment time was 1h. The best NaNO2 mutagenesis conditions:concentration of NaNO2 was 2mg/ml, pH5.4, incubation for 30min. NTG mutagenesis conditions:the concentration of NTG was 2mg/ml, pH8.0, incubation for 60min. Three mutants selected from different mutation sources and preserved in ECNU laboratory were set as the parent strains. Using the above Optimum mutagenesis conditions, a high-yield strainA1 was successfully selected from 14280 mutants. The MTG activity of the high-yield strain Al reached to 6.08 U/mL, increased by 185.07% when compared to the parent strain, increased by 99.61% when compared to the pilot scale strain.The character of Streptomyces mobaraensis Al with high-yield MTG. Process of seed growth of the high-yield strain A1 was as follows:0 to 16h was the lag phase.16 to 32h was the logarithmic phase. Since the growth rate constant at the 24h was largest, the bacteria at logarithmic phase 24h were selected as the seeds for fermentation. Fermentation metabolic characteristics of the high-yield strain A1 were as follows:0 to 4 h was lag phase.4-12 h was the logarithmic phase.12-36 h was stationary phase.36-52 h was decline phase. Glycerin was quickly used during 0-28h, and was consumed completely at 32h. The amino nitrogen content significant decreased during 4-16h, then dropped to the 2.96g/L. There was an exponential increase in MTG activity during 20-32h. The maximum number of MTG activity was 6.25U/mL at 32h.The MTG activity of the high-yield strains A1 was increased by 216% compared to the parent strain, and the fermentation period was 4h shorter. The maximum biomass of Al in the fermentation process was close to that of parent strain. So the increase of MTG activity of A1 was the result of the increase of enzyme activity of individual cell.The high-yield strain A1 had good genetic stability. There was no significant difference between the average MTG activity of first generation and that of the seventh generation (P>0.05). Compared to the parent strain, the fermentation stability of A1 was improved. The relative enzyme activity of fifth generation from A1 and parent strain were 69.4% and 53.9% respectively. The pH stability interval of the high-yield strain A1 and parent strain were pH5-8. Tm values of the high-yield strain A1 and parent strain were 49.11 ℃ and 52.58℃ respectively. MTG from both strains had good thermostablility below 40 ℃, which activity could retain more than 80% within 50 minutes. The HPLC-MS analysis of purified MTG from A1 showed that the molecular weight was 37816.4Da, which was consistent with the theoretical molecular weight from the DNA sequencing. The amino acid sequence of pre-pro-MTG from A1 was the same as that from Sv. Ladakanum numbered AAO48277.1 in the NCBI database.In order to obtain Streptoverticillium mobaraense mutants producing thermostable MTG, a high-throughput screening method was established. The spores(107个/ml) were treated by NTG (2mg/ml, pH8.0) for 60 min, then the fermented supernatant were incubated at 70℃ for 7.5 min after fermentation in 96 well microtiters. Finally, MTG activity was determined at 37℃ air bath for 10 min.5 mutants with thermostable MTG were selected from 5200 mutants. The relative activity of the mutant 12-82 was 10%-20% higher than that of parent strain Al when incubated at 50℃,70℃and 80℃. When incubated at 50℃for 60min, the relative activity of 12-82 was 58.7%, which was almost 20% higher than that of parent strain Al. Tm value of MTG from mutant 12-82 and parent strain A1 were 55.98 ℃ and 49.17℃ respectively. The Tm value of 12-82 increased approximately 6℃.The MTG activity of the mutant 12-82 and parent strain A1 were 6.12U/mL and 5.46 U/mL. The mutant 12-82 was a Streptomyces mobaraensis with high-yield and thermostable MTG.