Studies on the cyclodextrin glycosyltransferase from bacillus and studies on the α-amylase from aureobasidium pullulans

Abstract

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_{c...