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DBAT mutation enzyme D166H and application thereof

A D166H, mutant enzyme technology, applied in the field of biomolecular catalysis, can solve problems such as enzyme instability and inactivation, and achieve the effects of reducing production cost, improving utilization rate and improving utilization rate

Active Publication Date: 2016-04-27
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the active center of the enzyme requires very precise conformational adjustments. It requires both flexibility to recognize the substrate and complete the catalytic reaction, and a certain rigidity to maintain a reasonable conformation to maintain good biological activity; the active center of the enzyme Maintaining a delicate balance between flexibility and rigidity, random mutations to the active center will lead to rapid destabilization or inactivation of the enzyme (Hilvert, 2013)

Method used

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  • DBAT mutation enzyme D166H and application thereof
  • DBAT mutation enzyme D166H and application thereof
  • DBAT mutation enzyme D166H and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Obtaining of wild-type DBAT

[0030] The gene sequence of the DBAT enzyme published on NCBI is SEQ ID NO: 1 (GenBank: JQ029678.1), GGAATTC (EcoRI restriction site) is added before the start codon ATG, and GCGGCCGCTTTA (NotⅠ) is added after the stop codon TGA Restriction site) to obtain the gene sequence of the DBAT enzyme with the restriction site.

[0031] Use restriction endonuclease EcoRI and NotI (Invitrogen Company) to carry out double enzyme digestion to the gene of DBAT enzyme; Simultaneously, use restriction endonuclease EcoRI and NotI to carry out double enzyme digestion to pET-32a (+) vector (Invitrogen Company) . The digested product was purified using a gel purification kit, and the above two digested products were ligated with T4 DNA ligase (Invitrogen). The ligation product was transformed into E.coliDH5α competent cells (Invitrogen Company), a small amount of plasmid extraction, enzyme digestion identification, PCR (primers are dbat-F and dbat...

Embodiment 2DB

[0034] The acquisition of embodiment 2DBAT mutant enzyme

[0035] (1) Construction of mutant enzyme vectors by using the whole plasmid PCR method

[0036] 1. A small amount of plasmid extraction of the recombinant plasmid pET-DBAT;

[0037] 2. Using the plasmid pET-DBAT as a template, combined with the mutation primers at both ends, using overlap extension PCR technology, amplified the dbat plasmid containing the mutation site. The PCR system is as follows:

[0038]

[0039] The Primer-F and Primer-R need to be changed according to different mutant enzymes, as shown in Table 1.

[0040] The primer pair D166AF and D166AR are used to obtain the DBAT mutant enzyme D166A, the amino acid sequence of which is SEQ ID NO: 4, and the coding nucleotide sequence is SEQ ID NO: 3.

[0041] The primer pair D166HF and D166HR are used to obtain DBAT mutant enzyme D166H or D166HR363H; the amino acid sequence of DBAT mutant enzyme D166H is SEQ ID NO: 6, and the encoding nucleotide sequence...

Embodiment 3

[0078] Example 3 Fermentation verification and enzyme activity analysis of mutant enzyme engineering strain TB21-D166H

[0079] (1) Fermentation, protein induction and purification of engineering strains

[0080] 1. Protein induced expression

[0081] The expression host E.coliBL21 was transformed with the recombinant plasmid to obtain the recombinant expression strain TBL21-D166H. Inoculate the positive clone into 100 mL of LB liquid medium (with 100 μg / mL of ampicillin added) according to the inoculum amount of 1%, cultivate at 37°C and 220 rpm until OD600=0.6-0.8, and add the final concentration of 1 mM inducer IPTG, 28 ℃ induction 3h.

[0082]The cells were collected by centrifugation at 8000 rpm for 10 min at 4°C, and resuspended by adding 20 mL of phosphate buffer (PBS, pH 7.4) per 100 mL of the original fermentation broth. The resuspended bacteria were crushed with an ultrasonic cell disruptor. The total ultrasonic time was 12 min, the working time was 6 s, the inter...

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Abstract

The invention discloses a DBAT mutation enzyme D166H and application thereof. The mutation enzyme D166H is obtained by changing a 166th amino acid of a DBAT enzyme with an amino acid sequence of SEQ ID NO: 2 from Asp to His. The amino acid sequence of the mutation enzyme D166H is SEQ ID NO: 6. The catalytic efficiency of utilizing acetyl coenzyme A of the mutation enzyme is improved by 11 times compared with that of a wild type enzyme, and the usage rate of acetyl coenzyme A and the yield of a product baccatin III are increased greatly. Meanwhile, in order to reduce the production cost, seven novel acyl donors are found, and acetic acid vinyl ester, amyl acetate and isopropenyl acetate can be utilized efficiently by the mutation enzyme. The utilization rate of acetic acid vinyl ester is improved by 74 times, and the mutation enzyme can replace an expensive acetyl coenzyme A substrate to be used for preparing baccatin III. So far, in research for baccatin III biosynthesis, research for using non-plant coenzymes as the acyl donors and achieving the high utilization rate is not found.

Description

technical field [0001] The invention belongs to the field of biomolecular catalysis, and in particular relates to a DBAT enzyme (10-deacetylbaccatin III-10β acetyltransferase) mutant enzyme D166H and its application. Background technique [0002] Customizing enzymes has long been a goal of biochemists. With the gradual maturity of molecular biology techniques, any modification of the protein sequence can decipher the catalytic mechanism of the enzyme and continue to rationally design the enzyme. Such rational design could be adding properties to existing enzymes, or even redesigning a protein. [0003] Enzymes with high catalytic activity are the key to exerting their important potential in the biological industry, and they are also the main direction of modern protein engineering research. The rational design of new catalysts will often expand our understanding of the biochemical properties of proteins, especially those rational protein modification processes with fewer e...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P17/02C12R1/19
CPCC12N9/1029C12P17/02C12Y203/01
Inventor 郭丽琼林俊芳尤琳烽黄佳俊叶志伟
Owner SOUTH CHINA AGRI UNIV
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