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A kind of thermophilic l-asparaginase mutant and its screening and fermentation method

A technology of asparaginase, mutant, applied in the fields of genetic engineering, enzyme engineering and fermentation engineering

Active Publication Date: 2020-08-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme activity yield and specific enzyme activity still need to be improved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029]Example 1: Construction, transformation and expression of recombinant plasmid pMA5-asnase

[0030] (1) The predicted L-asparaginase gene sequence of Pyrococcus yayanosii CH1 in NCBI, this sequence is 24.34% homologous to L-asparaginase derived from Escherichia coli, and L-asparagine derived from Bacillus subtilis The homology of the enzyme is 20.41%, and the homology with L-asparaginase derived from Thermococcus kodakarensis is 65.15%. According to the predicted L-asparaginase gene sequence (SEQ ID NO.1) of Pyrococcusyayanosii CH1, Shanghai Sangon Bioengineering (Shanghai) Co., Ltd. was commissioned to optimize the L-asparaginase gene and clone it into the vector pUCk. Using this as a template (SEQ ID NO.2), design primers for PCR amplification. The PCR product was purified and recovered using a gel recovery kit, and the concentration of the recovered product was checked by electrophoresis. The recovered product was stored in a 1.5ml centrifuge tube and stored in a -20...

Embodiment 2

[0033] Example 2: Screening construction and expression of mutants with high enzymatic activity

[0034] (1) Using pMA5-asnase as a template, use the GeneMorph II random mutagenesis kit to design primers for epPCR. According to the method described in step (2) of Example 1, connect the amplified product of epPCR to BamHI of pMA5 and MluI sites, and transformed into Bacillus subtilis 168. Coat the kanamycin-resistant plate of LB and culture for 12 hours.

[0035] (2) In an oven (without wind) at 95°C, preheat a 96 deep-well plate (covered) containing 0.5mL reaction solution (25mM L-asparagine, 50Mm Tris-HCl, pH=8) for 10 minute. Use an inoculation loop to pick a single colony of the mutant transformant from step (1) that has been cultured for 12 hours, inoculate it into a preheated 96 deep-well plate containing the reaction solution, and heat it in an oven (without wind) at 95°C for 10 minutes . Add 10uL Nessler reagent for color development, and after standing at room temp...

Embodiment 3

[0038] Embodiment 3: Construction of high-yielding L-asparaginase recombinant bacteria

[0039] (1) Using pMA5-S17G / A90S / R156S / K272A-2 as a template, design primers for PCR amplification, and connect the PCR product to the EcorV & HindIII restriction sites of pMA5 in the manner described in step (2) of Example 1, The recombinant plasmid pMA5-S17G / A90S / R156S / K272A was constructed. Store in -20°C refrigerator for later use.

[0040] (2) Extract the Bacillus subtilis 168 genome, use its genome as a template, design primers for PCR amplification, and connect the obtained products to pMA5-S17G / A90S / R156S / Between the EcoRI and EcoRV restriction sites of K272A, a recombinant plasmid pMA5-P containing different promoters was constructed 43 -S17G / A90S / R156S / K272A(P 43 The promoter sequence is shown in SEQ ID NO.10), pMA5-P groEs -S17G / A90S / R156S / K272A, pM A5-P sigX -S17G / A90S / R156S / K272A, pMA5-P trnQ -S17G / A90S / R156S / K272A, pMA5-P yxiE -S17G / A90S / R156S / K272A. These 5 kinds of ...

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Abstract

The invention discloses a thermophilic L-asparaginase mutant and a screening and fermentation method thereof, belonging to fields of gene engineering, enzyme engineering and fermentation engineering.In bacillus subtilis 168, an L-asparaginase coding gene from thermophilic bacterium Pyrococcus yayanosii CH1 is used as a template, a mutant library is built through an error-prone PCR technology, anda mutant strain with improved specific enzyme activity is screened out through a high-throughput screening method 'synchronized cell disruption and enzyme activity assay'. Mutant residues included ina positive mutant strain are analyzed, a composite mutant strain S17G / A90S / R156S / K272A with improved specific enzyme activity is built, wherein the specific enzyme activity reaches 3108U / mg. The expression level of the composite mutant in bacillus subtilis 168 is improved through a strong promoter P43 and an RBS optimization manner. Finally, the bacillus subtilis 168 containing the L-asparaginasecomposite mutant strain gene produces enzyme and is fermented in a 5L fermentation tank through medium optimization and a pH feeding coupling policy, and the enzyme activity yield of the L-asparaginase reaches 6453+ / -127U / mL.

Description

technical field [0001] The invention relates to a thermophilic L-asparaginase mutant and a screening and fermentation method thereof, belonging to the fields of genetic engineering, enzyme engineering and fermentation engineering. Background technique [0002] L-asparaginase (E.C.3.5.1.1) can catalyze the deamination of L-asparagine to generate L-aspartic acid and ammonia. L-asparaginase is mainly used in the pharmaceutical industry and food industry. In the pharmaceutical industry, L-asparaginase is used as one of the anticancer drugs because of its inhibitory effect on some tumors. E L-asparaginase II isolated and purified from .coli, Erwiniachrysanthemi and E.carotovora has been widely used in the treatment of acute lymphoblastic leukemia, lymphosarcoma and reticulocyte sarcoma; in the food industry, L-asparaginase II Amidase can reduce the content of L-asparagine, the precursor of carcinogen acrylamide in high-temperature processed foods such as frying and baking, there...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/82C12N15/55C12N15/70C12N15/75C12N15/81
CPCC12N9/82C12Y305/01001
Inventor 饶志明李谞张显徐书琴胡静宜徐美娟杨套伟
Owner JIANGNAN UNIV
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