Continuous endoglucanase mutant based on co-evolution analysis and application thereof

An endoglucanase and mutant technology, applied in the field of genetic engineering, can solve the problems of lack and difficulty in satisfying the directional design and transformation of persistent endoglucanase

Active Publication Date: 2022-08-05
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is still a lack of overall and systematic theoretical understanding of the persistence mechanism of persistent endoglucanases, and it is difficult to meet the needs of the directional design and transformation of persistent endoglucanases. Therefore, co-evolutionary analysis based on large-scale sequence alignment The method not only provides theoretical guidance for protein engineering technology to modify persistent endoglucanase to improve its catalytic activity, but also helps to elucidate the special catalytic mechanism of this type of enzyme

Method used

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  • Continuous endoglucanase mutant based on co-evolution analysis and application thereof
  • Continuous endoglucanase mutant based on co-evolution analysis and application thereof
  • Continuous endoglucanase mutant based on co-evolution analysis and application thereof

Examples

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Effect test

Embodiment 1

[0024] Example 1 EVcoupling software analysis and screening of co-evolutionary amino acid residue pairs

[0025] Through the NCBI database, the known GH5 family persistent endoglucanase sequences were subjected to protein sequence alignment to find and obtain its homologous protein family (Pfam:PF00150). The EVfold bioinformatics tool was used to align and analyze the homologous sequences of persistent endoglucanase, calculate the degree of correlation between amino acid residues, and map the generated protein co-evolution network to the reference sequence with high similarity. Using PLM as the sole scoring criterion, pairs of key amino acid residues likely to have co-evolutionary relationships were identified and selected. In this example, based on the PLM algorithm, amino acid residue pairs with scores greater than 0.8 were selected for further experiments, namely K51 / L93, K84 / L126, K47 / A89, and V61 / V260.

Embodiment 2

[0026] Example 2 Construction of Persistent Endoglucanase Mutants and Screening Procedures for Optimal Mutants

[0027] Iterative saturation mutation was performed on the key amino acid residue pairs determined in Example 1, and the method disclosed by BinWu (Biotechnology for Biofuels, 2018, 11:20) was used to construct a recombinant plasmid containing SEQ ID NO: 2, with SEQ ID NO: 2 The sequence shown is a template. With reference to Vazyme biological products and operation manuals, the corresponding mutation primers designed as follows are used to amplify the site-directed mutation sequence from the whole plasmid. The primers used are as follows:

[0028]

[0029] The codons of nnn corresponding to different amino acids are as follows: TGT(Cys), GAT(Asp), GAA(Glu), TTT(Phe), GGC(Gly), CAT(His), ATT(Ile), AAA(Lys), CTG (Leu), ATG(Met), AAT(Asn), CCG(Pro), CAG(Gln), CGT(Arg), TCA(Ser), ACA(Thr), GTT(Val), TGG(Trp), TAT (Tyr).

[0030]Design mutants: first round of mutati...

Embodiment 3

[0051] Example 3 Analysis of enzymatic properties of persistent endoglucanase mutants

[0052] Determination of hydrolytic activity of persistent endoglucanase mutants

[0053] Definition of enzyme activity unit: One unit of enzyme activity is the amount of enzyme required to produce 1 mmol of reducing sugar from the substrate at 60°C and pH 6.0 per minute.

[0054] (1) Endo-glucanase: Accurately weigh 1 g of sodium carboxymethyl cellulose (CMC-Na) and dissolve it in 100 mL of Na 2 HPO 4 -KH 2 PO 4 Buffer (50mM, pH 6.0), stir and mix well, accurately pipette 1.5mL into the test tube as the enzyme reaction substrate, preheat at 60°C for 5min, add 0.5mL of properly diluted protease solution, put it in a water bath shaker at 60°C for reaction For 10 min, add 3 mL of DNS reagent, react in a boiling water bath for 5 min and then quickly cool to room temperature, and measure the absorbance value at a wavelength of 540 nm with the inactivated enzyme reaction solution as a control...

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Abstract

The invention relates to a persistent endoglucanase mutant with remarkably improved enzyme activity and application of the persistent endoglucanase mutant. The mutant comprises 51st and / or 93rd amino acid mutation of an amino acid sequence, and the amino acid sequence of the endo-cellulase is as shown in SEQ ID NO: 1. An iterative saturated mutation technology is adopted, based on evolutionary coupling analysis, the molecular structure of the persistent endoglucanase is optimized by selecting an amino acid residue pair with high coupling strength as a key mutation site, and the enzyme activity is improved. Compared with a wild enzyme, the continuous endoglucanase mutant has the advantages that the exonuclease activity and the endonuclease activity of the continuous endoglucanase mutant are obviously enhanced, and the continuous endoglucanase mutant has important significance on efficient degradation of a cellulose substrate and reduction of the production cost.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a class of persistent endoglucanase mutants with improved enzyme activity and applications thereof. Background technique [0002] Cellulose is the most widely distributed and abundant renewable resource on earth, and its efficient biotransformation is of great significance to achieve my country's "carbon neutrality" vision, solve the energy crisis and promote sustainable social development. However, hydrolysis The high cost of enzymes required in the process is still an important bottleneck restricting the commercialization of cellulose conversion technology. [0003] Processive endoglucanase, as a kind of bifunctional cellulose hydrolase, can efficiently degrade cellulose to generate small molecular oligosaccharides. Problems such as poor performance have seriously affected the application of this enzyme in the field of low-cost and high-efficiency bioconversion of cellulose. ...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12P19/02C12P19/14C12R1/19
CPCC12N9/2405C12N15/70C12P19/02C12P19/14Y02E50/10
Inventor吴斌吕科旻吴穆俊琦高振储建林何冰芳
OwnerNANJING UNIV OF TECH