| As a kind of transferase,the microbial transglutaminase(MTG)can catalyze the cross-linking reaction between the same or different proteins to make them become the polymer composites.Because of deriving from microorganisms,it is convenient for industrial production and has been widely used in the pharmaceutical industry,especially in the field of food industry.However,the current commercial MTG has been faced with the problem of low thermal stability and it is restricted in the field of high-temperature food.In order to achieve a MTG which has great thermal stability,this paper took eight wild type streptomyces strains which could produce MTG as research objects,and finally seeked out one strain named MJ-7 from them through the way of bioinformatics and half-life test.MJ-7 could produce MTG named MTG-MJ7,which have the better thermal stability than commercial MTG.Then this paper optimized fermentation condition and purification method for achieving high purity MTG-MJ7,in order to analyze its enzymatic properties.The optimized formulation and immobilization method were found,which could improve the thermal stability of MTG-MJ7,and then it was taken into application in high temperature spray drying and food study.The main conclusion of this paper are as follows:1.This paper took eight wild type streptomyces strains which could produce MTG as research objects.With the help of Whole genome analysis,this paper picked out three strains,the theoretical amino acid sequence of whose products were different from the commercial MTG.And their products were named MTG-MJ5,MTG-MJ6 and MTG-MJ7,respectively.The structures of MTG-MJ5,MTG-MJ6,MTG-MJ7 and commercial MTG were analyzed by bioinformatics softwares,and it was proved that MTG-MJ7 had the best thermal stability of them through the half-life test.These point,taken together,lead this paper to conclude that the thermal stability of enzyme is related to the alpha-helix and hydrogen bond,the thermal stability is in directly proportion to the number of alpha-helix and hydrogen bond.The theoretical amino acid sequence of MTG-MJ7 was verified through the PCR technology and the sequencing of PCR production;2.Then the fermentation conditions of MJ-7 and purification methods of MTG-MJ7 were optimized in order to obtain an industrialized process of MTG-MJ7.The optimum carbon and nitrogen sources were determined to be glycerol and yeast extract powder FM802,respectively.The enzyme activity of MJ-7 strain reached 0.91 U/m L under this optimal conditions,it was increased by 82%,compared with the enzyme activity under non-optimized conditions.Then the effect of temperature and p H on the enzyme production were studied through the lab-scale fermentation production process.Finally,the optimal conditions were determined to be 25?and p H 6.5,and the enzyme activity of MJ-7 strain reached 0.95 U/m L with it,it was increased by 10.5%,compared with that of non-optimized conditions(25?and p H 6.5 in fermenter).After industrial purification methods which include ultrafiltration concentration and ethanol precipitation,the enzyme activity was increased from 0.95 U/m L to 16.97 U/m L,the recovery rate was 71.45%;And the specific activity was increased from 0.16 U/mg to0.59 U/mg,the purification multiple was 3.69.The feasibility of industrial purification was verified;3.The further purification was studied to improve the purity of MTG-MJ7,in order to analyze its enzymatic property and explore whether the impurities have an effect on the thermal stability.The method of anion exchange chromatography was determined in the further purification,the optimal conditions were p H 8.0,phosphate buffer with ion concentration of 10 mmol/L and sample loading amount of 10 mg.Under these conditions,the enzyme activity recovery was up to 76.25%and the specific activity was increased from 0.59 U/mg to 4.33 U/mg,the purification multiple was 7.34.Finally,combined with gel chromatography,MTG-MJ7 reached 98.7%electrophoretic purity.The half-life of MTG-MJ7 was increased by 50%under 80?after purification,it indicated that the reduction of impurities would contribute to the improvement of thermal stability;4.The enzymatic properties of the purified MTG-MJ7 were analyzed.Firstly,the molecular weight of the purified MTG-MJ7 was determined to be 37836.39 Da by high resolution mass spectrometry,which was very close to the molecular weight of the theoretical sequence,so the authenticity of the theoretical sequence was verified.The optimum substrate was found by substrate specificity test.Its optimum temperature was between 54?and 56?,which was higher than the commercial MTG,and its half-life under medium-high temperature was also longer than that of the commercial MTG,which could verify that the excellent thermal stability of MTG-MJ7.Its optimum p H and p H stability were consistent with commercial MTG;5.The thermal stability of MTG-MJ7 was increased after purification,but it was still far away from commercialization.In order to further improve the thermal stability of MTG-MJ7,the formulation and immobilization method were studied in this paper.In terms of formulation,the?KTA-96-well plate high-efficiency screening method was pioneered to screen out the concentration range of glycerol and trehalose.Through the orthogonal experimental design,and antibacterial,metal ions were taken into account,the optimum formulation was determined to be 35%glycerin,21%trehalose,0.003%H2O2 and 100 mmol/L potassium chloride,it could increase the thermal stability of MTG-MJ7,its half-life under 80?was extended by 33.3%than that of purified MTG-MJ7.After immobilized by sodium alginate(the optimal conditions are 2%sodium alginate,2%calcium chloride and 30 min gelation time),its half-life under 80?was further extended by 116.7%than that of purified MTG-MJ7;6.The above results were applied to the production and food study under high temperature.It was suitable for the production method of high temperature spray drying.After high temperature spray drying,the recovery rate was 86.2%.Under the temperature of 60?,it could increase gel strength of gelatin and egg white protein products more significantly compared with commercial MTG.And the feasibility of one-step method for making meatballs was proved.The hardness of meatball which was cooked by one-step method was 408.5±24.2 g,and its elasticity was 47.4±2.6%,the results were very close to that of meatball which was cooked by two-step method.Their cross section were both smooth under the scanning electron microscope,there were no holes and gullies exist.In conclusion,this paper studied the properties of MTG-MJ7 from the aspects of fermentation,purification,property identification and production application,analyzed its excellent thermal stability,and also found the way to increase the thermal stability artificially.All the results laid the foundation of realizing the commercialization of MTG-MJ7. |
PDF Full Text Request