Efficient Expression Of γ-glutamyltranspeptidase By Recombinant Corynebacterium Crenatum And Its Application For L-theanine Production

L-theanine, a natural amino acid found almost exclusively in tea plants, has many physiological and pharmacological benefits for human body.Thus it was widely used as an additive in food, beverage and drug industries. Enzymatic synthesis is the overriding method for L-theanine production as it is energy-efficient and environment-friendly.Among those enzymes exhibiting catalytic ability, γ-glutamyltranspeptidase has attracted most attention by its unique superiority. In this work the γ-glutamyltranspeptidase(GGT) from B. subtilis 168 was expressed in C. crenatum SYPA5-5,which was screened after traditional mutagenesis earlier in our lab,to obtain the reconstructed strains. Then to achieve higher GGT activity and higher theanine production through series of gene operations and condition optimization.The detailed research contents were as follows.(1) Two genes ggt and ?sp ggt(without signal peptide fragment)from B. subtilis were cloned and expressed in C. crenatum SYPA5-5, respectively, thus generating two engineered strains C. crenatum SYPA5-5/pXMJ19-?sp ggt(without signal peptide) and C. crenatum SYPA5-5/pXMJ19-ggt(with signal peptide). In C. crenatum SYPA5-5/pXMJ19-?sp ggt, GGT was only detected intracellularly with activity of 0.90 U?m L-1, whereas in C. crenatum SYPA5-5/pXMJ19-ggt, most of GGT was secreted to extracellular space. The intracellular and extracellular GGT activities in the above strain C. crenatum SYPA5-5/pXMJ19-ggt were 0.27 U?m L-1 and 3.24 U?mL-1 respectively.The total activity was 4-folds of that in the former strain. This finding suggested that the signal peptide of GGT was responsible for the secretion and the higher GGT activity and could work in C. crenatum strain system. Then GGT in the culture supernatant of C. crenatum SYPA5-5/pXMJ19-ggt was adopted for theanine transformation and considerable amount of theanine was detected.(2) ggt gene was polyadenylated at the 3’end by introducing poly(A/T) tails in the forms of AAA, AAAAAA, TTTAAA, AAATTTAAA, TTTAAATTTAAA, TTTAAAAAAAAA, respectively. The resulting six genes were cloned and expressed in C. crenatum SYPA5-5 by pXMJ19 vector, constructing recombinant strains(R1-R6 for short).The effect of the poly(A/T) tails on GGT expression in recombinant strains was analysed from the level of transcription and translation. Results showed that R3 exhibited the highest transcriptional level and enzyme activity, 2.5-folds and 1.6-folds of the ones in the control strain C. crenatum SYPA5-5/ pXMJ19-ggt.The rest of the strains showed either the similar level(R1 and R4, R6) or the dramatically decrease(R2, R5).(3) The sequence of the extended-10 region(GCTCGTATAAT) of the tac promoter in pXMJ19 was replaced with TGTGGTACCAT by overlap PCR, constructing a new recombinant plasmid pXMJ19-tacM.Then the new plasmid was used to express the ggtR3 gene, which carried TTTAAA at the 3’end, constructing recombinant strain C. crenatum SYPA5-5/pXMJ19-tacM-ggtR3. It was observed that the extracellular GGT enzyme activity of the recombinant strain was 2-folds of the control strain C. crenatum SYPA5-5/pXMJ19-tac-ggtR3.The fermentation optimization of C. crenatum SYPA5-5/pXMJ19-tacM-ggt R3 was carried out and the results showed that the highest GGT activity was 11.96 U mL-1 in the supernatant, 15% increase over the beginning under the concentration of glucose at 10%; adding 0.8 mmol L-1 of IPTG at 0 h.(4) The conditions catalyzed by GGT for L-theanine production were investigated. The optimal ratio of L-glutamine to ethylamine was 1:3, corresponding optimal enzyme amount was 0.06 U mL-1.Fedding-transformation was conducted under the following conditions: GGT 0.15 U m L-1, pH 10, 37°C, and 20 mmol L-1 L-glutamine and 60 mmol L-1 ethylamine were fed every two hours. Theanine at 75.48 mmol?L-1 was generated after 10 h. GGT was immobilized by mesoporous CaCO3–alginate composite gel to detect enzyme activity in the transformation process. The immobilized enzyme showed higher stability with the recovery rate of 75%. To sustain the GGT activity of 0.06 U m L-1, 0.45 U at 2 h, 0.46 U at 4 h, 0.49 U at 6 h, 0.49 U at 8 h should be added. Supplementation was ceased after 10 h and the L-theanine production reached 89.76 mmol L-1 with the conversion rate of 89.76%.