| Transglutaminase (TGase) can catalyze protein’s crosslinking by acyl transferring between or within the proteins, it can change the function properties of protein. Thus, transglutaminase has great potential applications in food medicine, textile and other industries. At present, commercial transglutaminase is produced by fermentation of Streptoverticillium mobaraense. The industrial costs is very high because of low enzyme production.In order to improve the production and decrease costs, we bred high-yield transglutaminase strains by protoplasts fusion in this study.The main research of this paper includes that, established and optimized the methods and conditions of protoplasts preparation, researched the difference of protoplasts preparation and regeneration between different strains, optimized the protoplasts fusion condition, screened high-yield transglutaminase-producing strains through several rounds of protoplasts fusion, made a preliminary studies of carbon source and nitrogen source on high-yield strains, and carried on fermentation tank test. The main conclusions were as follows:1) Protoplasts preparation:Incubated 108 spores to YEME medium and cultured 36-45 h with 30℃ 200 r/min, the concentration of lysozyme was 8 mg/mL, enzymolysis temperature was 30℃, centrifuged protoplasts suspension with 15 min to 500 r/min after 90-180 min of digestion, we got protoplasts of high purity and concentration. The colonies changed little after 8 days under the condition of 4 or-20℃, when those were preserved in P buffer or P buffer containing 20% glycerol. The diameter of protoplasts colony was smaller than the colony of mycelium, those enzyme were different significantly after fermentation though 96-well plates. Protoplasts’size and uniformity had significant difference between different strains in microphotograph.2) Researched the different of protoplasts preparation and regeneration from different strains for the best fusion. The different strains’optimal enzymolysis time were different when enzymolysis temperature was 30℃ and enzyme concentration was 6 mg/mL. Strain 266,5591 and D were 1 h, Strains A and B were 2 h. Under the condition, protoplasts purity of the four strains were 99.9%,98.9%,98.9%,73.6%. And the regeneration rate were 699.44%,351.96%, 1885.39% and 10.31%. There was obvious difference in colony morphology of them after protoplasts regeneration.3) The smaller molecular weight PEG had less influence on the colony number of protoplast regeneration. Protoplasts’enzyme activity after PEG treatment had no significant difference, but compared with the mycelium enzyme activity.The protoplasts lethality rate could reach 100% after 180 s by treatment in 60℃. Protoplasts were illuminated with 30 W ultraviolet and the distance is 15 cm, lethality rate was 100% after 120 s. There had four PEG of difference molecular weight, and PEG 2000 is best, the fusion rate could reach 1.47 ‰, the optimal fusion time was 30 min. It could be observed that a large number of protoplasts aggregation in the protoplasts fusion under contrast microscope, and formed the fusion larger diameter cell than protoplasts.There had two different 96-well plates fermentation in preliminary screening, included fusants fermentation directly with 96-well plates and fermentation with 96-well plates after fusants expanding culture. The first method, the accuracy was low, and it was purposeful, had a small work-loads. The second method was high accuracy and had a big workload. Two methods had different advantages and disadvantages, we screened high-yield strains with two different methods. In strain 847 and A, there was a positive correlation between enzyme activity and the color depth of fermentation liquor. This feature could be used as a standard for screening high-yield strains to improve the screening efficiency and reduce the workload.4) The parent strains for fusion had different time to reach best condition of protoplasts preparation and regeneration under 6 mg/mL enzyme concentration. Strain poA was 30 min, the regeneration rate was 47.43%, protoplasts purity was 91.08%; poB was 90 min, the concentration of protoplasts of 2.235×105 per milliliter, regeneration rate reached 591.22%, and it is 12.47 times to poA; poC was 15 min, regeneration rate reached 24.47%, the purity of protoplasm was 97.74%; poD was 45 min, regeneration rate was 158.32%, the purity was 96.44%.In the fusion of multiple rounds, we screened the quality microbial strains by 96-well plate preliminary screening, test tube fermentation and shake flask fermentation. We studied 6 strains of high-yield strains in curve of transglutaminase production. Enzyme production curve of strain 102302 was only consistent with the starting strain poD,64.04% improved than the starting strain poD; Strain 112528 was different with all starting strains, its enzyme improved 25.38% than poA,23.84% higher than poC,84.01% higher than poD,16.34% higher than poE.5) Did a preliminary optimization of carbon source and nitrogen source on 3 strains high-yield transglutaminase strains. Glucose and glycerol were the best carbon source, peptone and peptone from fish were optimal nitrogen source. Researched the metabolic characteristics of strain 112528 in 3 L fermentation tank. MTGase was accumulated in 16 h quickly, the highest enzyme activity was 9.28 U/mL in 40 h. Fermentation, dissolved oxygen decreased rapidly, it dropped to 3.3% in 12 h. pH decreased firstly, and it reached the lowest,6.24 in 24 h. Then, pH increased until it decreased again in 48 h. Glycerol was consumed within 24 h and biomass increased to 0.033 g/mL in 34 h. Amino nitrogen increased after fall firstly, the minimum is 0.68 g/L in 24 h. |
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