Beta-galactosidase combined mutant with high transglycosylation activity as well as preparation method and application of beta-galactosidase combined mutant

A technology of galactosidase and high transglycosidase is applied in the field of β-galactosidase combination mutants and their preparation, and can solve the problems of low yield, long reaction time, low galacto-oligosaccharide synthesis yield and the like, and achieves the High-efficiency transglycosidic activity, effect of high transglycosidic activity

Active Publication Date: 2015-01-07
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] So far, whether it is the screening and separation of natural enzymes, process optimization or genetic engineering to improve the expression and properties of β-galactosidase, it has not changed the status quo of low transglycosidic activity and low yield of β-galactosidase , resulting in low synthesis yield of galactooligosaccharides, long reaction time, and high production costs, which seriously restrict the cheap production and application of galactooligosaccharides

Method used

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  • Beta-galactosidase combined mutant with high transglycosylation activity as well as preparation method and application of beta-galactosidase combined mutant
  • Beta-galactosidase combined mutant with high transglycosylation activity as well as preparation method and application of beta-galactosidase combined mutant
  • Beta-galactosidase combined mutant with high transglycosylation activity as well as preparation method and application of beta-galactosidase combined mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Prediction of tertiary structure and mutation site of β-galactosidase

[0035] The β-galactosidase gene lacb' that removes its own signal peptide is cloned from Aspergillus candidus in our laboratory. The gene that removes its own signal peptide sequence consists of 2958 nucleotides. The specific sequence is as follows: As shown in 1, the protein encoded by the gene consists of 986 amino acids, and the specific sequence is shown in sequence 2.

[0036] The β-galactosidase gene laco', which removes its own signal peptide, was cloned from Aspergillus oryze in our laboratory, and it also consists of 2958 nucleotides. The specific sequence is shown in sequence 3. The gene The encoded protein also consists of 986 amino acids, and the specific sequence is shown in SEQ ID NO:4. There are only three amino acid differences between its amino acid sequence and the protein encoded by the lacbˊ gene: at position 231: lacbˊ(Gly), lacoˊ(Ser); at position 401: lacbˊ(Met), l...

Embodiment 2

[0041] Example 2: Construction of Pichia pastoris single point saturation mutant library

[0042] 1. Materials and methods

[0043] (1) Strain and carrier

[0044] The wild-type gene is derived from the β-galactosidase gene lacb' of Aspergillus leucobacter that removed its own signal peptide, and the β-galactosidase gene laco' from Aspergillus oryzae, which were cloned in the previous stage of our laboratory. The specific sequence is as follows: 1 and Sequence 3, linked to pPIC9 expression vector, and expressed in Pichia pastoris GS115; Escherichia coli Trans1-T1 competent cells were purchased from TransGen; pPIC9 expression vector, Pichia GS115 was purchased from Invitrogen.

[0045] (2) Preparation of medium and related solutions

[0046] For Pichia pastoris transformation, culture and screening conventional media and reagents, refer to the instructions of Invitrogen.

[0047] PTM trace salt: 0.6% CuSO 4 , 0.008% NaI 2 , 0.3% MnSO 4 , 0.02% Na 2 MoO 4 , 0.002%H 3 BO...

Embodiment 3

[0077] Example 3: Screening of S219 Saturation Mutant Library and Synthesis of Oligosaccharides

[0078] 200 positive Pichia pastoris clones from the S219 mutant library were selected to measure their transglycoside activity (oligosaccharide production), and their nucleotide sequences were determined. Sequencing showed that these mutants were mutated to 8 different amino acids, all of which could improve the transglycosidic activity of the mutant enzyme (see Table 3), especially amino acids with smaller side chains such as Gly, Ala, Val and negatively charged The polar amino acid Glu is more prominent, and the mutation to Gly is the most obvious (see attached Figure 4 ), the production of oligosaccharides can be increased by 26.6%. After mutation to small side chain and negatively charged Glu, the production of oligosaccharides increased by 25.7%. When S219 was mutated to Ala and Val, the production of oligosaccharides increased by 15.0% and 15.5%, respectively. Mutated to A...

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Abstract

The invention belongs to the fields of gene engineering and genetic engineering, and discloses a beta-galactosidase combined mutant with high transglycosylation activity. On the basis of amino acid sequences of beta-galactosidase of aspergillus candidus and aspergillus oryzae, from which signal peptides are removed, a beta-galactosidase combined mutant is obtained through fixed-site saturation mutagenesis of double sites; under the condition of keeping the oligosaccharide production amount invariable, the transglycosylation reaction rate of the mutant is improved by over 100% in comparison with a wild mutant; and meanwhile, the invention also discloses a DNA molecule of encoding the combined mutant, a recombinant expression vector containing the DNA molecule, and a host cell of expressing the DNA molecule. In addition, the invention also provides a method for preparing the beta-galactosidase with high transglycosylation activity by using the recombinant expression vector, and applications of the combined mutant, the DNA molecule, the recombinant expression vector and the host cell in preparation of the beta-galactosidase.

Description

technical field [0001] The invention relates to the field of genetic engineering and genetic engineering, in particular to a combined mutant of beta-galactosidase with high transglycosidic activity and its preparation method and application. Background technique [0002] Galactooligosaccharides (GOS) are a kind of oligosaccharides with special biological functions that cannot be digested and absorbed in the human gastrointestinal tract, but directly enter the large intestine and are well utilized by various bifidobacteria. It can improve the micro-ecological environment in the human body, facilitate the proliferation of bifidobacteria and other beneficial bacteria, and improve the immune function of the human body. At the same time, GOS produces organic acids through metabolism to lower the pH value of the intestines, inhibit the growth of Salmonella and spoilage bacteria in the intestines, reduce the production of toxic fermentation products and harmful bacterial enzymes, r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N15/56C12N15/81C12N1/19
CPCC12N9/2471C12N15/815C12Y302/01023
Inventor 张伟张宇宏刘波孙宁张佳琳
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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