WO2017031839A1 - Mutant de la l-asparaginase dote d'une activité enzymatique améliorée et son procédé de construction - Google Patents

Mutant de la l-asparaginase dote d'une activité enzymatique améliorée et son procédé de construction Download PDF

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Publication number
WO2017031839A1
WO2017031839A1 PCT/CN2015/094554 CN2015094554W WO2017031839A1 WO 2017031839 A1 WO2017031839 A1 WO 2017031839A1 CN 2015094554 W CN2015094554 W CN 2015094554W WO 2017031839 A1 WO2017031839 A1 WO 2017031839A1
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Prior art keywords
mutant
recombinant
asparaginase
seq
gene
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PCT/CN2015/094554
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English (en)
Chinese (zh)
Inventor
饶志明
龙水清
张显
杨套伟
徐美娟
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江南大学
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Publication of WO2017031839A1 publication Critical patent/WO2017031839A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/10—General methods of cooking foods, e.g. by roasting or frying
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to an L-asparaginase mutant with improved enzyme activity and a construction method thereof, and belongs to the technical field of genetic engineering.
  • L-asparaginase amidohydrolase (E.C. 3.5.1.1) is capable of hydrolyzing deamination of L-asparagine to form L-aspartic acid and ammonia.
  • L-asparaginase has anti-tumor activity and has been used in the treatment of acute lymphoblastic leukemia and Hodgkin's disease. In recent years, it has been found that L-asparaginase can also reduce the formation of acrylamide in fried foods.
  • the size, structure and nature of L-asparaginase vary from source to source. L-asparaginase is widely available, and L-asparaginase is found in guinea pig serum, plants and microorganisms.
  • L-asparaginase The problem of heterologous expression of L-asparaginase is that the protein expression is low and the L-asparaginase enzyme activity is low. Therefore, site-directed mutagenesis of L-asparaginase and enhancement of extracellular enzyme activity are of great significance for improving the industrial application prospect of L-asparaginase.
  • the present invention first provides an L-asparaginase mutant having improved enzymatic activity, the amino acid sequence of which is the sequence shown in SEQ ID NO.
  • the nucleotide sequence encoding the mutant is the sequence shown in SEQ ID NO.
  • the mutant is based on an amino acid such as the amino acid shown in SEQ ID NO. 2, and amino acid 107 is mutated from glycine to aspartic acid.
  • the present invention also provides a genetically engineered bacteria expressing the L-asparaginase mutant.
  • the method for preparing the genetically engineered bacteria is to mutate the codon encoding the 107th glycine into a codon encoding aspartic acid based on the nucleotide sequence shown in SEQ ID NO.
  • the recombinant gene is ligated to the expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a Bacillus subtilis host strain to obtain a Bacillus subtilis genetically engineered strain.
  • the expression vector is pMA5.
  • the preparation method is specifically:
  • the present invention modifies the molecular structure of L-asparaginase by site-directed mutagenesis, and the pure enzyme solution of the mutant enzyme is 83% higher than that before the mutation.
  • the substrate affinity K m of the mutant enzyme G107D was reduced by 50% compared to before the mutation, and the catalytic efficiency was increased (the ratio of k cat to K m ) was 84%.
  • the invention shows that the 107 amino acid residue has a great influence on the catalytic action of the enzyme, provides a certain basis for the research on the catalytic mechanism of the enzyme, and improves the industrial application potential of the enzyme.
  • the invention can be used for preparing medicines for treating acute lymphocytic leukemia and Hawkinson's disease, and can also be used for reducing the formation of acrylamide in fried foods.
  • the recombinant gene and pMA5 were digested with BamHI and MluI, respectively, and purified, and then ligated with T4 DNA ligase at 16 ° C overnight.
  • the ligation product chemically transforms JM109 competent cells.
  • the transformant was coated with kanamycin (50 mg/L) LB plate, the plasmid was extracted, and the constructed recombinant plasmid was verified by double digestion, and named pMA5-G107D.
  • the sequencing work was completed by Shanghai Biotech.
  • the recombinant plasmid pMA5-G107D obtained in Example 1 was chemically transformed into B. subtilis 168 competent cells by the following methods:
  • Sp-A (NH 4 ) 2 SO 4 4, K 2 HPO 4 28, sodium citrate 12Sp-B: MgSO 4 ⁇ 7H 2 O 0.4
  • yeast powder 100Sp I medium: Sp-A 49%, Sp-B 49%, 50% glucose 2%, 100 ⁇ CAYE 2%
  • Sp II medium Sp I medium 98%, 50 mmol/LCaCl 2 1%, 250 mmol/L MgCl 2 1%. Damp heat sterilization at 115 °C.
  • Example 3 High-efficiency expression and enzyme activity assay of recombinant strain pMA5-G107D/B.subtilis 168L-aspartate.
  • the recombinant strain pMA5-G107D/B.subtilis 168 constructed in Example 2 and the control strain pMA5-ansz/B.subtilis 168 expressing the unmutated enzyme were inoculated into 10 mL of LB medium containing kanamycin, respectively.
  • the culture was shaken overnight at 37 ° C, and transferred to the fermentation medium of Bacillus subtilis by 4% inoculation on the next day, and cultured at 37 ° C for 24 h.
  • the fermentation broth was centrifuged at 4 ° C, 10000 r / min for 10 min, and the supernatant was extracellular.
  • the crude enzyme solution and the cell disrupted supernatant are intracellular crude enzyme solutions for the determination of enzyme activity.
  • Fermentation medium of Bacillus subtilis soybean protein ⁇ 10g / L, K 2 HPO 4 2.3g / L, KH 2 PO 4 1.7g / L, corn syrup 15g / L, urea 3g / L, glucose 40g / L , MgSO 4 0.75 g / L, NaCl 5 g / L. Adjust pH 6.8-7.0.
  • Enzyme activity definition The amount of enzyme required to catalyze the conversion of L-aspartamide to 1 ⁇ mol of NH 3 per minute under a reaction condition of 40 ° C is an enzyme unit.
  • L-asparaginase enzyme activity assay method The enzyme activity was determined by measuring the amount of NH 3 released in the catalytic reaction using L-asparagine as a substrate.
  • the reaction mixture (1 mL) was composed of: 400 ⁇ L of 25 mM L-asparagine (dissolved in 50 mM pH 7.5 Tris-HCl); 400 ⁇ L of 50 mM pH 7.5 Tris-HCl; 100 ⁇ L of an appropriate concentration of the enzyme solution.
  • the reaction mixture was reacted at 40 ° C, pH 7.5 for 15 min, and then the reaction was terminated by adding 100 ⁇ L of a 15% (W/V%) trichloroacetic acid solution.
  • the reaction solution in which the reaction was terminated by adding trichloroacetic acid before the enzyme reaction was used as a blank control.
  • the reaction mixture was centrifuged at 20,000 g for 10 min, and 200 uL of the supernatant was added to 4.8 mL of deionized water.
  • 200 ⁇ L of the Nessler reagent was added to the above system, and the absorbance was measured at a wavelength of 450 nm, and the amount of NH 3 released by the enzyme reaction was measured by a color reaction.
  • the extracellular crude enzyme solution obtained in the step (1) is purified to obtain L-asparaginase G107D ansz , and the purified L-asparaginase G107D ansz enzymatic properties are analyzed, as shown in Table 1, substrate
  • the affinity K m was reduced by 50% before the mutation, the catalytic efficiency k cat /K m was increased by 84%, and the enzyme activity was increased by 83%. Due to the increase in catalytic efficiency, the specific enzyme activity of G107D ansz is increased.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un mutante de la L-asparaginase doté d'une activité enzymatique améliorée et son procédé de construction, le mutant étant construit sur la base de la séquence d'acides aminés telle qu'indiquée dans la SEQ ID NO.2, dans laquelle la glycine en position 107 est mutée en acide aspartique. L'invention concerne également des bactéries obtenues par génie génétique exprimant le mutant de la L-asparaginase.
PCT/CN2015/094554 2015-08-25 2015-11-13 Mutant de la l-asparaginase dote d'une activité enzymatique améliorée et son procédé de construction WO2017031839A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510526470.6A CN105062997A (zh) 2015-08-25 2015-08-25 一种酶活提高的l-天冬酰胺酶突变体及其构建方法
CN201510526470.6 2015-08-25

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Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434612B (zh) * 2016-10-21 2020-01-17 江南大学 一种天冬酰胺酶突变体及其应用
CN107828768B (zh) * 2017-12-13 2020-10-09 江南大学 一种l-天冬酰胺酶突变体及其构建方法
CN108094976B (zh) * 2017-12-15 2020-03-06 江南大学 一株嗜热l-天冬酰胺酶在高温油炸食品中的应用
WO2019113965A1 (fr) * 2017-12-15 2019-06-20 江南大学 Mutant de l-asparaginase thermophile et procédé de criblage et de fermentation associé
CN107988194B (zh) * 2017-12-15 2020-08-04 江南大学 一种酶活提高的L-天冬氨酸α-脱羧酶变体及其构建方法
CN108070581B (zh) * 2017-12-15 2020-09-04 江南大学 一种酶活提高的L-天冬氨酸β-脱羧酶突变体及其应用
CN108559734B (zh) * 2018-01-15 2020-09-04 江南大学 一种催化效率提高的l-乳酸脱氢酶突变体及其应用
CN109266635B (zh) * 2018-11-20 2020-12-01 江南大学 一种酶活提高的l-天冬酰胺酶突变体及其构建方法
CN112941059B (zh) * 2021-02-23 2022-03-15 江南大学 一种l-天冬酰胺酶突变体及其在枯草芽孢杆菌中的表达

Citations (5)

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CN102864163A (zh) * 2012-09-20 2013-01-09 江南大学 一种高效表达枯草芽孢杆菌l-天冬酰胺酶的方法
CN103243063A (zh) * 2013-05-24 2013-08-14 江南大学 枯草芽孢杆菌L-天冬酰胺酶(ansZ)在大肠杆菌中的高效表达
WO2014170811A2 (fr) * 2013-04-16 2014-10-23 Sonawane Avinash Nouvelles l-asparaginases mutées
CN104371993A (zh) * 2014-10-22 2015-02-25 江南大学 一种酶活提高的天冬酰胺酶突变体
CN104611317A (zh) * 2015-03-09 2015-05-13 江南大学 一种提高l-天冬酰胺酶分泌表达的方法

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CN103060299B (zh) * 2011-10-19 2017-05-03 北京安百胜生物科技有限公司 活性提高的l‑天冬酰胺酶变体

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CN102864163A (zh) * 2012-09-20 2013-01-09 江南大学 一种高效表达枯草芽孢杆菌l-天冬酰胺酶的方法
WO2014170811A2 (fr) * 2013-04-16 2014-10-23 Sonawane Avinash Nouvelles l-asparaginases mutées
CN103243063A (zh) * 2013-05-24 2013-08-14 江南大学 枯草芽孢杆菌L-天冬酰胺酶(ansZ)在大肠杆菌中的高效表达
CN104371993A (zh) * 2014-10-22 2015-02-25 江南大学 一种酶活提高的天冬酰胺酶突变体
CN104611317A (zh) * 2015-03-09 2015-05-13 江南大学 一种提高l-天冬酰胺酶分泌表达的方法

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CHEN, JIANHUA ET AL.: "Effects of Lysl96 in antigenic epitopes of recombinant E. coli L-asparaginase on its antigenicity", JOURNAL OF CHINA PHARMACEUTICAL UNIVERSITY, vol. 37, no. 03, 25 June 2006 (2006-06-25), pages 277 - 280, ISSN: 1000-5408 *

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