The invention discloses a process for producing recombinant alpha-
cyclodextrin glucosyltransferase in a fermenting way by culturing
escherichia coli in
high density through a temperature two-stage control strategy and a constant
oxygen-dissolved and feed-supplemented batch technology, belonging to the technical field of
fermentation engineering. The method comprises the following steps of: inoculating
recombinant escherichia coli BL21(DE3) as a production strain at the
inoculation quantity of 5-10 percent and fermenting in batch; starting adding a supplementing liquid in a flowing mode when dissolved
oxygen rises to about 80-100 percent, ensuring that a strain body exponentially grows, controlling the temperature of the growth
phrase of the strain body to be 33-37DEG C and maintaining the dissolved
oxygen to 20-30 percent; adding 0.75-1.5 percent (
mass volume percent) of
glycine when the strain body OD600 reaches about 15-30; reducing the temperature to be 23-27DEG C and continuously supplementing 0.2-0.4g.l<-1>.h<-1> when the strain body OD600 reaches 45-60 and supplementing the supplementing liquid by adopting a gradient diminishing mode at the same time; and enabling the enzymeactivity of the
extracellular alpha-
cyclodextrin glucosyltransferase to reach 200-280U / ml by fermenting culture for 30-35 hours. By adopting the strategy to ferment, the invention realizes the high-efficiency
extracellular expression of the alpha-
cyclodextrin glucosyltransferase and greatly improves the production intensity. The invention has the advantages of wide source of raw materials, simple and feasible process and suitability for large-scale production; with effective
extracellular expression, the
pollution on host
bacteria hybrid proteins can be greatly reduced and the purification efficiency of the
protein can be improved. The invention lays a foundation for large-scale production of the alpha-cyclodextrin glucosyltransferase.