Breeding And Fermenting Of Transglutaminase Producing Strain Streptomyces Mobaraensis

Transglutaminase (abbreviated TGase, EC2.3.2.13) crosslinks the protein molecules, changing various functional properties and nutritional value of protein. TGase has been widely used in food, cosmetics and medical industry. TGase widely exists in plants, animals, microorganisms and the human body. Among them, TGase producted from the microorganism fermentation (MTG) has been widely used in large-scale industrial production because of some specialties such as the small molecular weight, the calcium ion indepence, and low price.HS47 (preservation number:CGMCCNo.10804) is a new wild strain obtained by natural selection, security of MTG produced by it was certificated by the Food and Drug Administration (abbreviated FDA), so MTG produced by it can be safely used in Food. However, the enzyme production ability of the strain is too low to realize the industrialization of the MTG production.Therefore, this article adopts the traditional physical and chemical mutagenesis method supplemented 96 well-plates high-throughput screening system to process S. mobaraensis breeding so as to achieve the aim of improving MTG activity.At the same time, through optimization of fermentation medium components and improvement of culture conditions to improve’the level of the MTG fermentation so as to lay a foundation for the realization of industrialized production of MTG.The main results were as follows:(1)The basic characteristics and screening methods of strain HS47. HS47 is strict aerobic bacteria.Its mycelium gathered on the tablet just as thin layers. Seen through the microscope, mycelium was slender and branch, gathered to form a smooth dough. The fermentation course of HS47 was:cultivate seeds under 30℃ for 24 h, put 10% of the seed liquid to the fermentation medium, culture under the same temperature for about 40h. Growth characteristics of HS47 was as follows:before 36h, bacteria grew exponentially, then biomass basically remain unchanged, after maximum biomass of 31.8 g/L was achieved in 44h, bacteria biomass decreased. MTG production process of HS47 was:MTG activity began to produce when fermented 8h, improved rapidly from 12h, achieved 2.5U/mL in 36h, then enzyme activity began to decline. The carbon source metabolic process of HS47 was: between 8h and 20h, glycerol consumption rate was the largest, then glycerol consumption rate decreased, glycerol was cost completely after 30h. The whole change trend of ammonia nitrogen metabolism of HS47 was rise-fell to a steady-rise. The pH change for HS47:0～ 20 h, the pH fell sharply to 6.5, late in 32～48 h of fermentation, the pH increased to 8.1. Add Ampicillin or chloramphenicol to the medium can improve the MTG activity of screening strains. The original 96 well-plates high-throughput screening method of laboratory was improved. The screening speed is therefore increased further, the whole time of a cycle shortened from 31 d to 25 d, and the variation coefficient of screening strains was smaller and screening result was more accurate at the same time.(2)Mutation breeding of HS47 and the basic characteristics of high yield strain. The best UV mutagenesis time to HS47 was 60s, distance was 30 cm, dark processing time was 60 min.The best NTG mutagenesis conditions for HS47:concentration was 2 mg/mL, action time was 60 min, pH was 9.0. NIT mutagenic concentration was 2 mg/mL, pH was 4.5, action time was 30 min. The best ARTP mutagenic time was 30s, processing power was 120 w, dealing distance was 2 mm, gas flow rate was 10SLM. Using the above mutagenesis method to breeding strain HS47 for four rounds,1 high-yield strain was screened from 36780 strains whose MTG activity achieved 7.7 U/mL,2.08 times higher than the wild-type strain HS47. At the time of the highest enzyme activity, biomass of high-yield strain was slightly higher than the wild-type strain. The enzyme production time and fermentation period of high-yield strain was delayed for 4h compared to wild-type strain. The glycerol and amino nitrogen consumption of high-yield strain and wild-type strain have no significant difference during fermentation time.High-yield strain has a better stability on genetic than the wild-type strain. The best fermentation carbon source of high-yield strain was glycerol, the best fermentation nitrogen source was fish peptone.(3) ptimization of the fermentation medium of HS47 mutagenic strain.With the aid of the Design Expert software, using the method of response surface experiment design steps, based on the MTG activity of 40h during fermentation, the fermentation medium of HS47 mutagenic strain was optimized. Plackett -Burman experiment determined three main influence factors to MTG activity of HS47 mutagenic strain:glycerol, fish peptone and Twain 80. The steepest uphill experment determined the center of the response surface analysis, namely 17.90 g/L glycerol,19.60 g/L fish peptone and 0.60 g/L Twain 80. Central composite test determined the optimized fermentation medium formulas were 17.72 g/L glycerin,19.56 g/L fish peptone and 0.65 g/L Twain 80. After the optimization of fermentation medium, MTG activity was increased by 24.7%.