Cyclodextrin glycosyltransferase(CGTase; EC 2.4.1.19) catalyzes the formation of cyclodextrins from starch and related α-1, 4-glucans. CGTases from different bacterial sources produce a mixture of three type of cyclodextrins, α-, β-, and γ-cyclodextrins. It has been proposed that the variations in substrate specificity and products ascribed to the relationships between their similar catalytic centers and different subsite structures. But what determines the product specificity is not clear yet. In order to clarify this, two types of amino acids were selected and mutated by site-directed mutagenesis, which place inside bottom of active site pocket and which place upper outside of the active site pocket. Tyr 100 and Trp 101 place inside and Arg 47, Tyr 89, Asn 94 place outside of the pocket.
Traditional megaprimer PCR mutagenesis methods had some drawbacks, self -annealing of the megaprimer and low frequency of mutation were the problems. A new method of PCR mutagenesis was developed. Two 5' mismatching primers and one nonmatching primer which had restriction site were used in this method. After second PCR reaction, selective cloning of the mutated gene were possible. Among 29 tested clones, 28 were mutants. Using this method developed, twelve mutants were made and expressed successfully by using E.coli Top10F' carrying pBR322 derivatives containing mutated gene. These mutated protein were purified and characterized. Among 12 mutants, N94S, Y100F, and W101D showed remarkable changes in producing product specificity. The wild type CGTase from Bacillus sp. I-5 produced mixtures of α-, β-, and γ-cyclodextrins in a ratio of 15 : 68 : 17.
When the inside amino acids of the pocket were changed, α-CD formation were strongly decreased and the ratio of β-, γ- CDs were increased. Smaller amino acids suffered more severe effect. Ratio of β- CD of Y100F and W101D mutant were about 80% and ratio of α- CD of these were less than 5%. They retained about 50% of the $k_{cat}$ values for β- CD forming activity, but the $k_{cat}$ values for coupling between cyclodextrins and maltose were reduced to about 7%. These mutants retained cyclization activities but lost coupling activities between cyclodextrins and maltose. Tyr-100 is located at near the subsite S1 and replacement of this amino acid maybe changed this structure. Coupling reaction starts by the binding of cyclodextin, subsequent cleavage of cyclodextrin between subsites S1 and S1'. And the non-reducing end of acceptor maltooligosaccharides binds to S1', and couples to the reducing end of hydrolyzed cyclodextrins.
When the outside amino acids of the pocket were changed, α-CD fraction of the N94S mutant CGTase was increased upto 33% of the total CDs. In this case, the $k_{cat}$ values for coupling between β-CD and maltose were reduced to 81% of wild-type, but the $k_{cat}$ values for coupling between α-CD and maltose were slightly increased. This can be explained by that formation of β-CD needs more subsite than formation α-CD. Asn-94 is located near subsite S4. Replacing Asn to small size Ser maybe caused slight shrinkage of active site pocket. Coupling activity between β-CD and maltose of Y89S and Y89N were increased about 2 times. And other mutants little affect on the production ratio of CDs.
In order to optimize the CGTase production conditions, chemostat studies were done. The specific CGTase activity was decreased with increase in ]dilution rate. And the concentration and productivity of CGTase were increased 3- and 9- fold respectively, compared with batch cultures. When nitrogen was limited, the CGTase was very low, but in case of oxygen limitation the CGTase activity was retained constant level. This is indicating that the energy status of the cell is important for CGTase production.
A new α-amylase from Aureobasidium pullulans GM21 were isolated, purified, and characterized. Molecular weight was 67,000, the optimum pH and temperature were 5.0 and 40℃. This enzyme reduced the molecular weight of pullulan by cleaving the rare maltotetraose sites of pullulan.
