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

一种半乳糖苷酶、高转糖苷的技术，应用在β-半乳糖苷酶突变体及其制备领域，能够解决产量低、生产成本高、β-半乳糖苷酶转糖苷活性低等问题

Active Publication Date: 2014-06-25
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

[0010] However, so far, no matter whether it is screening and isolation of natural enzymes, process optimization, or improving the expression and properties of β-galactosidase through genetic engineering, the low transglycosidase activity and low yield of β-galactosidase have not been changed. The status quo of galactooligosaccharides, thus resulting in low synthesis yield of galactooligosaccharides and high production costs, which seriously restrict the cheap production and application of galactooligosaccharides

Method used

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  • Beta-galactosidase mutant with high transglycosylation activity and preparation method and application thereof
  • Beta-galactosidase mutant with high transglycosylation activity and preparation method and application thereof
  • Beta-galactosidase mutant with high transglycosylation activity and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Prediction of tertiary structure and mutation site of β-galactosidase

[0033] 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.

[0034] The β-galactosidase gene laco', which removes its own signal peptide, was cloned from Aspergillus oryze in our laboratory. 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), lacoˊ...

Embodiment 2

[0039] Example 2: Construction of Pichia pastoris single point saturation mutant library

[0040] 1. Materials and methods

[0041] (1) Strain and carrier

[0042] 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.

[0043] (2) Preparation of medium and related solutions

[0044] For Pichia pastoris transformation, culture and screening conventional media and reagents, refer to the instructions of Invitrogen.

[0045] 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 3...

Embodiment 3

[0075] Example 3: Screening of S219 Saturation Mutant Library and Synthesis of Oligosaccharides

[0076] 200 positive Pichia pastoris clones from the S219 mutant library were selected to determine their transglycosidic activity and their nucleotide sequences. 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%. The amount of oligosaccharides increased by 25.7% after mutation to a small side chain and negatively charged Glu, when S219 was mutated to Ala and Val, the amount of oligosaccharides increased by 15.0% and 15.5%, respectively, and the mutations to Asp, Arg, and Leu The amount of sugar pr...

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Abstract

The invention belongs to the field of genetic engineering, and discloses a beta-galactosidase mutant with high transglycosylation activity. On the basis of removal of an amino acid sequence of beta-galactosidase of aspergillus candidus and aspergillus oryzae of a signal peptide, the beta-galactosidase mutant is obtained through fixed point saturated mutation of a unit point. The transglycosylation activity of the mutant is improved by above 15 percent in comparison of the transglycosylation activity of a wild type mutant. Meanwhile, the invention further discloses a DNA molecule for coding the mutant, a recombinant expression vector containing the DNA molecule and a host cell for expressing the DNA molecule. In addition, the invention further provides a method for preparing the beta-galactosidase mutant with high transglycosylation activity by adopting the recombinant expression vector, and application of the mutant, the DNA molecule, the recombinant expression vector and the host cell to the 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 beta-galactosidase mutant 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, regulate gast...

Claims

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Application Information

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