Beta-galactosidase two-site mutant with high transglycosylation and low hydrolytic activity and preparation method thereof

Abstract

The invention provides a beta-galactosidase two-site mutant with the high transglycosylation and the low hydrolytic activity. The mutant is obtained through the steps that site-specific mutagenesis on an E303 site and an F341 site of beta-galactosidase, wherein the E303 site is mutagenized into cysteine (Cys) from original glutamic acid (Glu), and the F341 site is mutagenized into serin (Ser) from original phenylalanine (Phe); a two-site mutant is subjected to KI oxidation treatment. The invention further relates to a construction method and application of the two-site mutant. According to the beta-galactosidase two-site mutant, a bacillus stearothermophilus source beta-galactosidase (BgaB) is taken as a function renovation objective, a mixture of oligosaccharides larger than a disaccharide can be obtained by catalyzing substrate lactose through the obtained two-site mutant, the synthetic amount reaches 9.5%, and the degradation capacity of the two-site mutant to the synthetic product is reduced. In addition, the mutant is suitable for catalyzing lactose hydrolysis and galactooligosaccharide synthesis under the neutral and high-temperature conditions.

Description

【Technical field】 [0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a double-point mutant of β-galactosidase with high transglycosidic activity and low hydrolytic activity, and also relates to a double-point mutant of β-galactosidase with high transglycosidic activity and low hydrolytic activity method of preparation. 【Background technique】 [0002] The industrial preparation of galacto-oligosaccharides (GOS) mainly uses lactose as a substrate and is synthesized through the catalysis of β-galactosidase transglycosidase. The full name of β-galactosidase (β-galactosidase) is β-D-galactoside galactoside hydrolase (β-D-galactoside hydrolase, EC 3.2.1.23), which has two functions of catalyzing lactose hydrolysis and transglycosidation. However, due to the low transglycosidic activity of β-galactosidase and the catalytic characteristics of substrate hydrolysis, it has become the main technical bottleneck probl...

AI Technical Summary

Problems solved by technology

Since the β-galactosidase derived from Aspergillus leucobacter or Aspergillus oryzae is an acidic β-galactosidase, its optimum pH is between pH 2.5 and 5.5. This catalytic characteristic limits its use as a catalyst at high temperature and to a certain extent. Synthesis of galacto-oligosaccharides under neutral conditions

[0004] On the other hand, the successful acquisition of the mutants of CN 201410148999 needs to rely on the accuracy of bioinformatics and prediction algorithms, and at the same time needs to cooperate with high-throughput screening methods with high sensitivity and high throughput. The operation is complicated and the success rate is relatively low

When this method is applied to the transformation of transglycosidic activity, due to the unidirectional nature of the screening, it cannot effectively take into account the influence of hydrolytic activity on the catalytic efficiency of transglycosidic transglycosidation, resulting in the phenomenon that the hydrolytic activity of the mutants with improved transglycosidic activity improved at the same time.

Images