Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis

A technology for galactosidase and lactose hydrolysis, which is applied to beta-galactosidase GaLT1 and its application in lactose hydrolysis, and can solve problems such as poor stability

Pending Publication Date: 2022-03-08
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are various design schemes for parental short peptides, and there are many successful reports, but there are also many ineffective or even worse reports.
For example, in the Chinese invention patent 201810069643.X, the effect of 7 different amphipathic short peptides on the temperature stability of a glucose oxidase was tried, and it was found that one amphipathic short peptide would make the enzyme stability worse, and the rest had no effect on the enzyme's temperature stability. The stability has been improved to varying degrees, but the highest one is only incubated at 60°C for 30min

Method used

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  • Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis
  • Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis
  • Artificially modified beta-galactosidase GaLT1 and application thereof in lactose hydrolysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Sequence design and synthesis of artificially modified β-galactosidase GaLT1

[0024] (1) Amphiphilic short peptide design

[0025] The laboratory designed a total of 8 parental short peptides:

[0026] ①AEAEAKAKAEAEAKAKAEAEAKAK;

[0027] ②AEAEAKAKAEAEAKAK;

[0028] ③LELELKLKLELELKLK;

[0029] ④ADADAKAKADADAKAKA;

[0030] ⑤ADADARARADADARAR;

[0031] ⑥AEAEAHAHAEAEAHAH;

[0032] ⑦HNANARARHNANARARHNANARARHNANARAR;

[0033] ⑧ANANARARANANARAR.

[0034] (2) The fusion of the amphipathic short peptide and the wild-type β-galactosidase (named GaLTO) derived from T. scotoductus. The amino acid sequence of the wild-type β-galactosidase GaLT0 is derived from the whole genome sequencing of T. scotoductus strain, the number in the NCBI database is WP_015716994.1, and it is annotated as α-amylase (alpha-amylase). There is no literature report on the related research of this protein. Entrusted Shanghai Sangon Biological Co., Ltd. to fuse and connect the designed 8...

Embodiment 2

[0035] Example 2: Recombinant expression and protein purification of artificially modified β-galactosidase GaLT1

[0036] 1. The genes of β-galactosidases fused with different parental short peptides were respectively cloned into pET22b, and then transferred into Escherichia coli BL21(DE3) strains to induce expression according to the standard procedure of molecular cloning. The obtained bacterial cells are homogeneously crushed under high pressure, and the centrifuged supernatant is crude enzyme liquid.

[0037] 2. The crude enzyme solution is purified by anion exchange chromatography to obtain artificially modified β-galactosidase recombinant protein. Protein purity was checked by SDS-PAGE. After routine enzyme activity testing, GaLT1 with the best comprehensive catalytic ability was screened out from 8 artificially modified β-galactosidases. The recombinant expression and protein purification of the enzyme are as follows: figure 1shown. M is a high molecular weight stan...

Embodiment 3

[0038] Embodiment 3: the optimal reaction temperature of artificially transformed β-galactosidase GaLT1 and wild-type enzyme GaLT0

[0039] The enzyme reaction system was 500 μL, including 100 μL of 10 mM oNPG, 5 μL of appropriately diluted pure enzyme solution, and 395 μL of 50 mM phosphate buffer (pH 7.0). Carry out the reaction at 30-70°C for 10 minutes, and use the same volume of 1M Na at the end of the reaction 2 CO 3 The reaction was terminated, and the absorbance was measured at 405 nm. Such as figure 2 As shown, the optimum reaction temperature of the wild-type β-galactosidase GaLT0 is 50°C, and the optimum reaction temperature of the artificially modified β-galactosidase GaLT1 is increased to 55°C.

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Abstract

The invention discloses an artificially modified beta-galactosidase GaLT1 and an application of the artificially modified beta-galactosidase GaLT1 in hydrolysis of lactose, the beta-galactosidase GaLT1 is formed by connecting an artificially modified amphiphilic oligopeptide sequence to an N-terminal amino acid sequence of wild type beta-galactosidase GaLT0 derived from T.cotoductus, and the amino acid sequence of the beta-galactosidase GaLT1 is as shown in SEQ ID NO: 1. Compared with wild type beta-galactosidase GaLT0, the beta-galactosidase GaLT1 disclosed by the invention has the advantages that the temperature stability is obviously improved, and the half-life period at 55 DEG C is prolonged by 10 times; besides, the milk lactose degradation rate of the beta-galactosidase GaLT1 is also obviously improved, 95% of lactose in milk can be degraded within 2 hours at 55 DEG C, and the national standard of lactose-free milk products is met.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an artificially modified β-galactosidase GaLT1 and its application in hydrolyzing lactose. Background technique [0002] Lactose is a disaccharide consisting of glucose and galactose. Milk is rich in lactose. In some people, the secretion of lactase in the small intestinal mucosa is insufficient or even not secreted, resulting in the inability to effectively hydrolyze lactose. This symptom is called lactose intolerance. Lactose-free milk products (lactose content <0.5%, w / w, Weijianfa [2007] No. 300) have appeared on the market for this consumer group. A common method for preparing these lactose-free milk products is to use lactase (ie β-galactosidase, β-gaLactosidase, EC: 3.2.1.23) to degrade lactose in milk. [0003] β-galactosidase is widely distributed in animals, plants and microorganisms. At present, there are two commercially available β-galactosidases, but the price is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N15/56C12N15/70C12N1/21C12P19/14C12R1/19
CPCC12N9/2471C12N15/70C12Y302/01023C12P19/14
Inventor 彭惠孔慧慧李艺冰王煜吴海芳高毅
Owner ANHUI UNIVERSITY
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