Study On The Fermentation And Heterologous Expression Of The Microbial Transglutaminase

As a very important enzyme in agricultural products processing industry,microbial transglutaminase?MTG?is widely applied in the processing and production of dairy products,seafood products,meat products,bakery products,soybean products,edible films,etc.Nowadays,MTG could be produced by fermentation of natural strains or heterologous expressed by bacteria or yeast.Active enzymes can be obtained directly through the fermentation of natural strains,which is accompanied by long fermentation period and complex process.Recombinant strains give short fermentation period and simple composition,nevertheless appropriate expression strategies are needed,otherwise inclusion bodies or inactive enzymes maybe form.To solve above prolems,actionomycetes strain107.3 and 109.5,which were isolated and confirmed as MTG-producing strains previously,were studied.The mutagenesis of strain,the downstream processing exploration of MTG,the cloning and heterologous expression of MTG gene,the analysis of MTG characters,the modification of MTG molecular,etc.were carried out in this study.The main results are as follows:Phylogenetic analysis of MTG-producing strains.Based on the sequencing of 16S rDNA and construction of phylogenetic trees,strain 107.3 was definited as the similar species of Streptomyces hygroscopicus;and strain 109.5 was similar to Amycolatopsis keratinophila.Streptomyces hygroscopicus is known as one of the best high-production species of MTG in the world.Meanwhile,it's the first time that the production of MTG from strain of Amycolatopsis was reported.Mutagenesis of strain 109.5 and extraction of MTG.The optimum seed medium of strain 109.5 was defined as:glucose 10.0 g/L,peptone 10.0 g/L,yeast extract 10.0 g/L,MgSO₄·7H₂O 0.5 g/L,K₂HPO₄ 0.7 g/L,KH₂PO₄ 0.3 g/L,at pH7.0.Thirty hours after inoculation in the seed medium at 30°C,200r/min,the cultures were transferred at 0.5%?v/v?ratio into the fermentation medium.After fermentation for 70 h,the enzyme activity reached the climax.Ultraviolet mutagenesis,hydroxylamine mutagenesis and multifunctional plasma mutagenesis system?MPMS?were applied on strain 109.5 successively,and the mutant PM-66 was obtained.The optimum medium of mutant PM-66 was difined as glucose 25g/L,peptone 35 g/L,yeast extract 1 g/L,NaCl 2 g/L,at pH 7.2 through the orthogonal test.Under this culture condition,the MTG activity of PM-66 reached 1.421±0.009 U/mL,which was 3.55-fold higher than the previous wild type strain.The method of semi-quantitative high throughput screening of mutants on solid medium was constructed;based on the image grayscale obtaind by the software Image J.This screening method increased the speed of mutants screening and provided new train of thought for other strains'screening which are not suitable for liquid colorimetry.Extraction of MTG from the fermentation broth of mutant PM-66 was achived by aqueous two phase extracton?ATPE?.The optimum ATPS?aqueous two phase system?for MTG from PM-66 consisted of 26%w/w PEG 1000,19%w/w ammonium sulfate and 5%w/w NaCl,at pH 6.0.In this ATPS,MTG was partitioned into the PEG-rich phase with a maximum yield of 86.51%and SA was 0.83 U/mg.After ATPE,9%w/w ammonium sulfate powder was added to the PEG phase and the precipitate of MTG could be collected.This is the first report about concentration and preliminary extraction of MTG from fermentation broth of Amycolatopsis sp.Gene cloning,expression and modification of MTG from strain 107.3.Firstly,the MTG gene of strain 107.3 was cloned and transferred into Escherichia coli.With IPTG?isopropyl?-D-thiogalactoside?inducing,the MTG activity was 0.372±0.008 U/mL in the supernatant of cell disruption liquid of recombinant E.coli.The properties of MTG were analyzed after purification by Ni²⁺-affinity chromatography.The optimal temperature was35?and the optimal pH was 7.The MTG activity could be increased slightly by Na⁺and K⁺,but inhibited by Zn²⁺,Cu²⁺,Mg²⁺,Fe²⁺and Mn²⁺at different extent;while almost not influenced by Ca²⁺.Secondly,fourteen mutants of MTG from strain 107.3 with different residues missing or substituting were constructed and expressed in E.coli.Many of them gave higher specific activities or better thermal stabilities than the wild-type MTG.Another eight residue-substitution mutants on the Y133 site were constructed and expressed in E.coli.The specific activities of six of them were better than wild-type enzyme.The results illustrated that the positive mutation sites mainly located at the N-terminal of the MTG and around the active site cleft.And Y133 was a positive site of MTG molecular.Lastly,to study the function of pro-peptide during the secretion of mature TGase,the pro-peptide of S.hygroscopicus?Hs?and S.mobaraensis?Sm?,the mature TGase of them two,were transferred into Pichia pastoris X33,respectively.The pro-TGase of these two streptomycetes were also transferred into X33.With 1.5%v/v methanol inducing,MTG activity of 0.200±0.005 U/mL was detected in the supernant of fermentation broth of recombinant yeast strain 6711,which contains Hs pro-TGase,after cultured for 96 h at30?.The results showed that the active expression of MTG could be achived only when the pro-peptide was cut off correctly from pro-TGase,and then the mature TGase was folded into correct conformation.The seperate expression of pro-peptide and mature TGase could not give active MTG production.