The invention discloses to further optimization of a process for generating L-
theanine from L-
glutamine and
ethylamine catalyzed by gamma-glutamyl transpeptidase secreted from recombinant pMA5-ggt / Bacillus. subtilis by using a method that double substrates are added in different batches, and belongs to the field of
fermentation engineering and
enzyme engineering. The process comprises the following steps: culturing the strain of
bacillus subtilis which is established in previous operation and is used for secreting gamma-glutamyl transpeptidase in a
fermentation tank; concentrating supernate crude
enzyme liquid for conversion of
theanine; on the basis, adding the substrates in different batches so as to further regulate and control the conversion process, that is, in a
reaction system with 60U / mL of the
enzyme liquid, adding substrates
glutamine and
ethylamine into the
system every other two hours, wherein the addition amount of
glutamine is fixed to be 20 mM, the ratio of a donor to a
receptor is controlled to be 1:12, the pH value is adjusted to be 10, 164.2 mM of
theanine can be obtained after 18 hours, and the conversion rate of glutamine can be up to 91.2%. The method has the advantages that the operation is simple, the cost is low, the yield of theanine is high, the substrate conversion rate is high, and the like, and is beneficial for industrial large-scale production.