CN108546660B - Chitin deacetylase high-yield strain and application thereof - Google Patents

Chitin deacetylase high-yield strain and application thereof Download PDF

Info

Publication number
CN108546660B
CN108546660B CN201810328527.5A CN201810328527A CN108546660B CN 108546660 B CN108546660 B CN 108546660B CN 201810328527 A CN201810328527 A CN 201810328527A CN 108546660 B CN108546660 B CN 108546660B
Authority
CN
China
Prior art keywords
chitin deacetylase
fermentation
rhodococcus equi
culture
chitin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201810328527.5A
Other languages
Chinese (zh)
Other versions
CN108546660A (en
Inventor
马钦元
王敏
申雁冰
夏梦雷
毕心宇
张子君
杨阳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin University of Science and Technology
Original Assignee
Tianjin University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin University of Science and Technology filed Critical Tianjin University of Science and Technology
Priority to CN201810328527.5A priority Critical patent/CN108546660B/en
Publication of CN108546660A publication Critical patent/CN108546660A/en
Application granted granted Critical
Publication of CN108546660B publication Critical patent/CN108546660B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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)
    • 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)
    • C12Y305/01041—Chitin deacetylase (3.5.1.41)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention belongs to the technical field of biology, and particularly relates to a chitin deacetylase high-yield strain and application thereof. The chitin deacetylase high-yield strain is Rhodococcus equi F6 with a preservation number of CGMCC No. 14861. The strain has extremely high speed of producing the chitin deacetylase, is the Rhodococcus equi which is reported to produce the chitin deacetylase for the first time at present, is the fastest strain for producing the chitin deacetylase by fermentation in the prior art, and is easy to culture on a large scale.

Description

Chitin deacetylase high-yield strain and application thereof
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to a chitin deacetylase high-yield strain and application thereof.
Background art:
chitin, also known as chitin, (1, 4) -2-acetamido-2-deoxy- β -D-glucan, is an aminopolysaccharide that is second to cellulose in nature and is found primarily in invertebrates such as shrimps, insects, algae, fungi and yeasts. However, chitin is not soluble in water, acid, alkali and organic solvents, so that it has no great commercial value, and chitosan, which is a product obtained after deacetylation, is widely applied to industries such as medicine, food, chemical industry and cosmetics. The chemical method mainly used for producing chitosan at present has a plurality of problems, such as long reaction time, high energy consumption, unstable product quality, and particularly, huge environmental pollution caused by emissions.
The chitosan product with stable deacetylation degree and narrow molecular mass distribution range can be produced by removing acetyl on the chitin by the chitin deacetylase, and a new way is provided for solving the problems of chitosan production by a chemical method. At present, only a few documents report about the research on chitin deacetylase producing bacteria at home and abroad, and the chitin deacetylase derived from microorganisms mainly takes fungi as main materials and has few bacteria.
At present, relatively few reports on the production of chitin deacetylase by using a screened natural microbial fermentation method are reported at home and abroad, and the relevance of three domestic patents, namely CN102676485A, CN104450832A and CN104498365A, is relatively high. CN102676485A discloses that the chitin deacetylase is obtained by fermenting and culturing the nereisria bretschneideri for 90-100 hours; CN104450832A discloses that thermophilic bacillus is utilized to ferment and culture for 60-72h, and chitin deacetylase is obtained; (ii) a CN104498365A discloses that chitin deacetylase crude enzyme liquid is obtained by fermenting for 72-100h by using a strain Aspergillus versicolor X.
However, the prior microbial fermentation production of chitin deacetylase has a series of problems of long fermentation time, low enzyme activity and the like, so that the enzyme can not realize industrialization at present. Therefore, the screening of new enzyme-producing strains with excellent performance from natural environment is still one of the important problems for solving the industrial application of CDA, and has important practical value and academic research value.
The invention content is as follows:
aiming at the defects of the prior art, the invention aims to provide rhodococcus equi capable of highly producing chitin deacetylase, a fermentation method and application thereof.
The first purpose of the invention is to provide a Rhodococcus equi (Rhodococcus equi) F6 with high yield of chitin deacetylase, which is deposited in China general microbiological culture Collection center at 11/6.2017, with the address: no. 3 of Xilu No.1 of Beijing, Chaoyang, Chao code 100101 of institute of microbiology, Chinese academy of sciences, with a collection number of CGMCC No. 14861.
The physicochemical properties of said Rhodococcus equi (Rhodococcus equi) F6 are as follows: gram-positive bacteria, wherein colonies on an LB culture medium are spherical and moist, and the color of the colonies is soft pink.
The second purpose of the invention is to provide the application of the rhodococcus equi CGMCC No.14861 in the production of chitin deacetylase.
The third purpose of the invention is to provide a production method for producing chitin deacetylase by fermenting rhodococcus with CGMCC No.14861, which comprises the following steps:
(1) seed culture
The culture conditions are as follows: the stirring speed is 160-200rpm, the temperature is 30-37 ℃, and the fermentation is carried out for 12-24 h;
seed culture medium: 5-10g/L of peptone, 3-8g/L of yeast extract powder, 5-10g/L of sodium chloride and the balance of water, wherein the pH value is 6.0-7.0;
(2) fermentation culture
Fermentation conditions are as follows: the inoculation amount is 2-10%, the stirring speed is 160-;
fermentation medium: 5-10g/L of yeast extract powder, 0.5-2.0g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 0.5-2.0g/L of sodium chloride and the balance of water, wherein the pH value is 6.0-7.0;
after fermentation for 6-10h, the content of chitin deacetylase in the fermentation liquor reaches 170-180U/mL.
Has the advantages that:
the Rhodococcus equi CGMCC No.14861 provided by the invention can utilize common carbon sources and nitrogen sources to quickly carry out cell culture and accumulation of chitin deacetylase. Through condition optimization, the enzyme production can reach the maximum after fermentation for 6-10h, and the enzyme activity of each mL of fermentation liquor is 173.6U. Compared with the prior art, the Rhodococcus equi CGMCC No.14861 provided by the invention can realize the rapid accumulation of the chitosan deacetylase, achieves the effect of high yield, and has wide industrial application prospect.
Description of the drawings:
FIG. 1 is a colony picture and a photomicrograph of F6;
wherein A is a colony map; b is a photomicrograph;
FIG. 2F 6 strain phylogenetic tree;
FIG. 3 fermentation process curves.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
Example 1 screening of F6 Strain
Soil samples collected from Shandong, Shenyang, Chengdu and the like are made into suspension, then the suspension is coated on an enrichment culture medium (fine powder chitin is 2.5g/L, dipotassium hydrogen phosphate is 0.7g/L, magnesium sulfate is 0.5g/L, sodium chloride is 0.1g/L), then bacterial liquid in the enrichment culture medium is diluted by sterile water and then coated on a screening culture medium (colloidal chitin is 2.5g/L, dipotassium hydrogen phosphate is 0.7g/L, potassium dihydrogen phosphate is 0.3g/L, magnesium sulfate is 0.1g/L, p-nitroacetanilide is 2.0g/L, agar is 20g/L) for culturing for 3-5 days at 30 ℃. The resulting culture was transferred to a new solid plate and the plate streaked until a single colony appeared. And (3) performing fermentation enzyme production detection and re-screening in a triangular flask to finally obtain a pure culture, wherein the number of the pure culture is F6.
The F6 strain was subjected to 16S rDNA identification, and F6 was determined to be Rhodococcus equi (Rhodococcus equi) by constructing a phylogenetic tree (shown in FIG. 2) using a partial sequence obtained from NCBI blast and constructing the phylogenetic tree by MP method. Rhodococcus F6 was deposited in the China general microbiological culture Collection center on 6.11.2017 with the collection number of CGMCC No. 14861.
Example 2 fermentation of Rhodococcus equi CGMCC No.14861 to produce enzyme
The rhodococcus CGMCC No.14861 obtained in example 1 is subjected to fermentation culture in multiple batches.
(1) Seed culture
The culture conditions are as follows: stirring at 200rpm at 37 deg.C, and fermenting for 12 hr;
the seed culture medium is as follows: 10g/L of peptone, 5g/L of yeast extract powder, 10g/L of sodium chloride and the balance of water, wherein the pH value is 6.0-7.0;
(2) fermentation culture
Fermentation conditions are as follows: the inoculation amount is 8 percent, the stirring speed is 200rpm, and the temperature is 37 ℃;
fermentation medium: 5g/L of yeast extract powder, 0.5g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 0.5g/L of sodium chloride and the balance of water, wherein the pH value is 6.0-7.0;
fermenting for 30h, and regularly taking the fermentation liquor to carry out chitin deacetylase enzyme activity detection. The fermentation enzyme production curve is shown in figure 3, the maximum enzyme activity is reached when the fermentation lasts for 8 hours, and the enzyme production curve is determined to be 173.6U/mL.
The enzyme activity detection method comprises the following steps:
the fermentation liquor is centrifuged at 12000r/min for 5min, and then the bacteria are washed by phosphate buffer solution with pH 7.0 and crushed by an ultrasonic crusher under the conditions that: the power is 30%, the time is 55min after the power is started for 3s and stopped for 5 s. Then, the crude enzyme solution is obtained by centrifugation for 5min at 12000 r/min. Adding 0.3mL of crude enzyme solution into 0.3mL of 200mg/L paranitroacetanilide solution and 0.9mL of phosphate buffer solution with pH value of 7.0, reacting at 45 ℃ for 15min, detecting the absorbance at 400nm, and calculating the enzyme activity through a standard curve.
Example 3 enzyme production by fermentation of Rhodococcus equi CGMCC No.14861
The rhodococcus CGMCC No.14861 obtained in example 1 is subjected to fermentation culture in multiple batches.
(1) Seed culture
The culture conditions are as follows: stirring at 160rpm at 30 deg.C, and fermenting for 24 hr;
the seed culture medium is as follows: 5g/L of peptone, 8g/L of yeast extract powder, 5g/L of sodium chloride and the balance of water, wherein the pH value is 6.0-7.0;
(2) fermentation culture
Fermentation conditions are as follows: the inoculation amount is 10 percent, the stirring speed is 160rpm, and the temperature is 40 ℃;
fermentation medium: 10g/L of yeast extract powder, 2.0g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 2.0g/L of sodium chloride and the balance of water, wherein the pH value is 6.0-7.0;
when the fermentation time is up to 6 hours, the enzyme activity of the fermentation liquor is determined to reach 180.5U/mL.
Example 4 substrate catalysis assay of enzymes
The crude enzyme solution obtained in the embodiment 2 is respectively added with excessive different substrates (the substrates are shown in the table below), the substrates are dissolved by 0.2M phosphate-citric acid buffer solution with pH 4.0, the catalytic activity of the enzyme on different substrates is verified by detecting the content of acetic acid released by deacetylation through high performance liquid chromatography, the content of acetic acid generated by nitroacetanilide is taken as a standard, and the detection proves that the crude enzyme solution produced by the fermentation of the strain has certain deacetylation on colloidal chitin, chitosan with different deacetylation degrees and chitin monomer N-acetylglucosamine, and has good guiding significance for industrial application of the enzyme, and the detection results are as follows:
Figure BDA0001627269230000041
Figure BDA0001627269230000051
the above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.

Claims (3)

1. The Rhodococcus equi for high yield of the chitin deacetylase is characterized in that the Rhodococcus equi is Rhodococcus equi (Rhodococcus equi) F6 with the preservation number of CGMCC No. 14861.
2. Use of the Rhodococcus equi for producing chitin deacetylase in high yield according to claim 1 for producing chitin deacetylase.
3. The use of Rhodococcus equi for the production of chitin deacetylase according to claim 2, wherein the method for the fermentative production of chitin deacetylase comprises the following steps:
(1) seed culture
The culture conditions are as follows: the stirring speed is 160-200rpm, the temperature is 30-37 ℃, and the fermentation is carried out for 12-24 h;
seed culture medium: 5-10g/L of peptone, 3-8g/L of yeast extract powder, 5-10g/L of sodium chloride and the balance of water, wherein the pH value is 6.0-7.0;
(2) fermentation culture
Fermentation conditions are as follows: the inoculation amount is 2-10%, the stirring speed is 160-;
fermentation medium: 5-10g/L of yeast extract powder, 0.5-2.0g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 0.5-2.0g/L of sodium chloride and the balance of water, and the pH value is 6.0-7.0.
CN201810328527.5A 2018-04-13 2018-04-13 Chitin deacetylase high-yield strain and application thereof Active CN108546660B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810328527.5A CN108546660B (en) 2018-04-13 2018-04-13 Chitin deacetylase high-yield strain and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810328527.5A CN108546660B (en) 2018-04-13 2018-04-13 Chitin deacetylase high-yield strain and application thereof

Publications (2)

Publication Number Publication Date
CN108546660A CN108546660A (en) 2018-09-18
CN108546660B true CN108546660B (en) 2021-05-25

Family

ID=63514930

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810328527.5A Active CN108546660B (en) 2018-04-13 2018-04-13 Chitin deacetylase high-yield strain and application thereof

Country Status (1)

Country Link
CN (1) CN108546660B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110628679B (en) * 2019-10-09 2022-05-24 江苏海洋大学 Raoultella ornithinolytica G10, enzyme production method, product and application
CN110699344B (en) * 2019-10-24 2022-03-18 山东理工大学 Comprehensive utilization process of citric acid fermentation tailings
CN111154747B (en) * 2020-01-19 2022-04-08 天津科技大学 Method for improving chitin deacetylase yield through mixed fermentation
CN111172141A (en) * 2020-01-19 2020-05-19 天津科技大学 Chitin deacetylase

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2171047A1 (en) * 2007-07-23 2010-04-07 Henkel AG & Co. KGaA Removal of by-products from crosslinkable preparations
CN104109636A (en) * 2014-06-30 2014-10-22 浙江树人大学 Aspergillus versicolor SD-3 and its application in preparation of chitin deacetylase
CN104498365A (en) * 2014-11-17 2015-04-08 华南理工大学 Bacterial strain capable of producing chitin deacetylase and application of bacterial strain in production of chitin deacetylase through fermentation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2171047A1 (en) * 2007-07-23 2010-04-07 Henkel AG & Co. KGaA Removal of by-products from crosslinkable preparations
CN104109636A (en) * 2014-06-30 2014-10-22 浙江树人大学 Aspergillus versicolor SD-3 and its application in preparation of chitin deacetylase
CN104498365A (en) * 2014-11-17 2015-04-08 华南理工大学 Bacterial strain capable of producing chitin deacetylase and application of bacterial strain in production of chitin deacetylase through fermentation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
高活性甲壳素脱乙酰基酶产生菌种筛选;活泼;《浙江农业科学》;20141231(第3期);第390-391页 *

Also Published As

Publication number Publication date
CN108546660A (en) 2018-09-18

Similar Documents

Publication Publication Date Title
CN108546660B (en) Chitin deacetylase high-yield strain and application thereof
Hu et al. Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing
Lee et al. Bioethanol production from sweet potato by co-immobilization of saccharolytic molds and Saccharomyces cerevisiae
CN103468624B (en) Genetic engineering bacteria used for high efficient production of mycose
CN110484471B (en) Acid-resistant strain for high yield of bacterial cellulose and method for producing bacterial cellulose by using acid-resistant strain
CN101555461A (en) Bacterial strain LT3 producing alkalescence cellulase and breeding method and initial optimization of cellulase production conditions thereof
CN111100827B (en) Bacillus capable of producing high-activity alginate lyase and application thereof
CN106811438B (en) Straw degradation acidification microbial inoculum and preparation method thereof
CN104498365A (en) Bacterial strain capable of producing chitin deacetylase and application of bacterial strain in production of chitin deacetylase through fermentation
CN103571772A (en) Novel strain for producing butanol and method for producing butanol
CN108823116B (en) Rhodococcus equi mutant strain for producing chitin deacetylase and application thereof
CN110093298B (en) Microbacterium estericum MCDA02 and method for producing chitin deacetylase by using same
CN111154747B (en) Method for improving chitin deacetylase yield through mixed fermentation
CN113652379B (en) Bacillus cell wall depolymerization GIEC and application thereof
CN110684691A (en) Preparation process of microbial agent based on directional screening of microorganisms
CN107118980B (en) Microbacterium keratanolyticum MCDA02 from sea, and its enzyme production method and product
CN102690773B (en) Enterobacteria strain FY-07 and method thereof for producing bacterial cellulose by static liquid submerged fermentation
CN112574920A (en) Fibroblastic microbacterium PX02 and method and application thereof for producing dextranase
CN108913629B (en) Bacterium for producing cellulase, preparation method and application thereof
CN108728370B (en) Salmonidae nephrobacter strain QD-01 capable of efficiently producing chitosanase and fermentation method and application thereof
CN112458022B (en) Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof
CN102051385B (en) Method for producing lactic acid by fermentation of acorn powder
CN107164246A (en) A kind of thermotolerant yeast bacterium and its application
CN110093297B (en) Nitrate reducing bacteria MCDA3-3 and method for producing chitin deacetylase by using same
CN110358687B (en) Gibberellin for producing D-pantolactone hydrolase and application and fermentation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant