Beta-galactosidase combination mutant with high transglycosylation, and preparation method and application thereof

A technology of galactosidase and high transglycosidase, which is applied in the field of β-galactosidase combination mutant and its preparation, can solve the problems of low yield, long reaction time and high production cost, and achieves high-efficiency transglycosidase activity, The effect of high transglycosidic activity

Active Publication Date: 2017-09-26
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

[0012] 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 combination mutant with high transglycosylation, and preparation method and application thereof
  • Beta-galactosidase combination mutant with high transglycosylation, and preparation method and application thereof
  • Beta-galactosidase combination mutant with high transglycosylation, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Prediction of tertiary structure and mutation site of β-galactosidase

[0036] 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.

[0037] The β-galactosidase gene laco', which removes its own signal peptide, was cloned from Aspergillus oryzae (Aspergillusoryze) in our laboratory, and it also consists of 2958 nucleotides. The specific sequence is shown in sequence 3. The gene encodes The protein of is also composed of 986 amino acids, and its 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 p...

Embodiment 2

[0042] Example 2: Construction of Pichia pastoris single point saturation mutant library

[0043] 1. Materials and methods

[0044] (1) Strain and carrier

[0045] 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, connected to the pPIC9 expression vector, and expressed in Pichia pastoris GS115; Escherichia coliTrans1-T1 competent cells were purchased from TransGen; pPIC9 expression vector, Pichia GS115 was purchased from Invitrogen.

[0046] (2) Preparation of medium and related solutions

[0047] For Pichia pastoris transformation, culture and screening conventional media and reagents, refer to the instructions of Invitrogen.

[0048] PTM trace salt: 0.6% CuSO 4 , 0.008% NaI 2 , 0.3% MnSO 4 , 0.02% Na 2 MoO 4 , 0.002%H...

Embodiment 3

[0078] Example 3: Screening of S219 Saturation Mutant Library and Synthesis of Oligosaccharides

[0079] 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 combination mutant with high transglycosylation. The beta-galactosidase combination mutant is obtained through site-saturation mutagenesis of two sites on the basis of removing an amino acid sequence of beta-galactosidase of aspergillus candidus and aspergillus oryzae of self-signal peptide, and the transglycosylation reaction rate of the mutant is improved by more than 100 percent compared with a wild type under the condition of keeping oligosaccharide forming amount unchanged. Meanwhile, the invention also discloses a DNA (Deoxyribonucleic Acid) molecule for coding the combination mutant, a recombinant expression vector containing the DNA molecule, and a host cell expressing the DNA molecule. In addition, the invention also provides a method for preparing the beta-galactosidase combination mutant with high transglycosylation by adopting the recombinant expression vector, and application of the combination mutant, the DNA molecule, the recombinant expression vector and the host cell in preparing the beta-galactosidase.

Description

[0001] This application is the application number of 201410514519.1, the application date is September 29, 2014, and the title is "a β-galactosidase combination mutant with high transglycosidic activity and its preparation method and application". Divisional application. technical field [0002] 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 [0003] 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 hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N15/56C12N15/81C12N1/19C12R1/84
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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