title: L-theanine synthesis using γ-glutamyl transpeptidase from Bacillus licheniformis ER-15.
abstract: Recombinant γ-glutamyl transpeptidase (rBLGGT) from Bacillus licheniformis ER-15 was purified to homogeneity by ion-exchange chromatography. Molecular masses of large and small subunits were 42 and 22 kDa, respectively. The enzyme was optimally active at pH 9.0 and 60 °C and was alkali stable. K(m) and V(max) for γ-glutamyl-p-nitroanilide hydrochloride were 45 μM and 0.34 mM/min, respectively. L-Theanine synthesis was standardized using a one variable at a time approach followed by response surface methodology, which resulted in approximately 85-87% conversion of L-glutamine to L-theanine within 4 h. The standardized reaction contained 80 mM L-glutamine, 600 mM ethylamine, and 1.0 U/mL rBLGGTin 50 mM Tris-Cl (pH 9.0) at 37 °C. Similar conversions were also obtained with the enzyme immobilized in calcium alginate. Using immobilized enzyme, 35.2 g of L-theanine was obtained in three cycles of 1 L each. The product was purified by Dowex 50W X 8 hydrogen form resin and was confirmed by HPLC and proton NMR spectroscopy.
authors.affiliation: Department of Microbiology, University of Delhi , South Campus, New Delhi 110021, India.
authors.forename: Shruti
authors.initials: S
authors.lastname: Bindal
authors.forename: Rani
authors.initials: R
authors.lastname: Gupta
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D004800:Enzymes, Immobilized; D005021:Ethylamines / Q000378:metabolism; D005971:Glutamates / Q000096:biosynthesis*; D005973:Glutamine / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D005021:Ethylamines; D005971:Glutamates; D011994:Recombinant Proteins; D005973:Glutamine; C026166:theanine; D005723:gamma-Glutamyltransferase; C041564:ethylamine
keywords: Bacillus; RSM; immobilization; process optimization; theanine; γ-glutamyl transpeptidase (GGT)