Biocatalytic Preparation Of L-theanine And Fermentation Process

Theanine is a kind of non-protein amino acids with important physiological functions in tea plants.At present,it has been found that natural L-theanine is only found in tea trees and mushrooms,and the content is low.L-theanine is synthesized in roots,transported to buds and leaves and accumulated.L-theanine has a variety of medicinal values,and has important functions in immune regulation,anti-tumor,improving the activity of anti-tumor drugs,anti-oxidation,reducing blood pressure,regulating brain nerve and so on.In this study,the production mode of L-theanine is biocatalytic synthesis,including enzyme catalysis and fermentation.In this experiment,a γ-glutamylmethylamide synthetase(γ-glutamylmethylamidesynthetase)from Methylophagy bacteria(Methylovorusmays No.9)was used to catalyze the synthesis of L-theanine,which was coupled with polyphosphate kinase of Rhodo-bacter sphaeroides to construct a genetic engineering strain.Using sodium L-glutamate and ethylamine hydrochloride as substrates,a method for producing L-theanine was established.The main contents are as follows:(1)The γ-glutamylmethylamide synthetase gene gmas from Methylophagy bacteria and the polyphosphate kinase gene ppk from Rhodo-bacter sphaeroides were both obtained by total gene synthesis and constructed into the expression vector p ET-21a(+)by genetic engineering.The recombinant plasmid p ET21a-ppk+gmas was obtained successfully.Using E.coli BL21(DE3)as the host strain,the genetic engineering strain BL21/p ET21a-ppk+gmas was constructed and the soluble expression of GMAS and PPK proteins in the host bacteria was optimized by induction.(2)After the optimization of cell crushing power and crushing time,the analysis of variance of the data showed that there was a very significant difference.It was confirmed that90 W pill 25 min was the best crushing condition.After optimizing the induction temperature,IPTG concentration and induction time,the analysis of variance of the data showed that there was a very significant difference.It was confirmed that 28℃,1 mmol/L IPTG and 8 h were the best induction conditions,the specific activity of intracellular crude enzyme was increased,and the soluble expression accounted for more than 90% of the total expression.Using the purified enzyme reaction,the yield of L-theanine reached 39.7 g/L,and the yield reached92.2%.Using the purified enzyme powder reaction,the yield of L-theanine reached 38.1 g/L,and the yield reached 88.7%.(3)The genetically engineered strain E.coli BL21/p ET21a-ppk+gmas was used as the catalyst for whole-cell transformation,and the conditions such as substrate molar ratio and wet cell weight were optimized.The data were analyzed by variance analysis.The yield of L-theanine reached 28.7 g/L and the conversion rate was 62%.(4)The production of L-theanine was increased by adding ethylamine and glucose to the fermentation of genetically engineered strain E.coli BL21/p ET21a-ppk+gmas.The yield of L-theanine reached 40.6 g/L.