The Biosynthesis Of Theanine: The Construction Of Recombinant Strain And Its Synthesis Constraints

Theanine is not only the main free amino acid of tea,but also the major component of peculiar flavour and refreshing fresh taste in tea soup.It already becomes the important symbol of the sign appraising high-grade green tea.Moreover,masses of researches and clinical trials show that theanine can promote the cerebral function and neurite extension, having anticancer function,reducing blood press and calming the nerves.For these reasons, its preparation studies become a hot spot on tea studies.It is very difficult and more expensive to extract a high-purity's theanine directly from tea leaves.The technics of chemosynthesis theanine handicraft is more complicated;The yield of theanine by tissue culture is low.So,the biosynthesis of theanine has important theory significance and practice value.The present paper applying the biological technology to the natural products preparation technology developed the project bacterium construction aspect to work.We constructed a recombinant strain by gene clone technology.The conditions of biosynthesis theanine were studied to improve output of theanine.Study results mainly were as follows:1,We got the gene ofγ-glutamyltranspeptidase by PCR.The recombinant plasmid pUC19-GGT was constructed by the gene ofγ-glutamyltranspeptidase and pUC19.The recombinant plasmid pUC19-GGT was transformed to E.coli DH5α.γ-Glutamyltranspepti-dase was produced with 0.1 mM IPTG in 32℃incubation.The activity of 1g fresh cells of recombinant strain was usually0.236 U/mL,it is about 2.6 times higher than the E.coli DH5α.The recombinant strain was incubated at 32℃for 6h,and then incubated at 32℃with 0.1 mM IPTG for 4h.In 1mL reaction system,when 0.35 M L-Gln was used,2.0 M ethylamine hydrochlorid,70 mg/mL fresh cells,Ph 9.5,and incubation at 30℃for 4 h were the optimum condition.The yield of theanine was 0.475 g/L.2,We got the gene of glutamine synthetase by PCR.The recombinant plasmid pET-GS was constructed by the gene of glutamine synthetase and pET32a.The recombinant plasmid pET-GS was transformed to E.coli BL21.Glutamine synthetase was produced with 0.1mM IPTG in 28℃incubation.The activity of fresh cells of recombinant strain was usually 41.79 U/mgprot,it is about 126.5 times higher than the E.coli BL21.3,The reaction condition for synthesis theanine of recombinant strain were investigated.The recombinant strain was incubated at 32℃for 6 h,and then incubated at 28℃with 0.1mM IPTG for 4h.In 1 mL reaction system,when 0.2 M L-sodium glutamate was used,1.2 M chlorhydric acid ethylamine,100 mM imidaxole,5 mM MnC12,30 mM ATP and 100 mg/mL frash cells,pH 9.5,and incubation at 30℃for 14 h were the optimum condition.The yield of theanine was 6.2 g/L.