CN109337821B - Cladosporium for producing sterol esterase and enzyme production method - Google Patents

Cladosporium for producing sterol esterase and enzyme production method Download PDF

Info

Publication number
CN109337821B
CN109337821B CN201811150218.XA CN201811150218A CN109337821B CN 109337821 B CN109337821 B CN 109337821B CN 201811150218 A CN201811150218 A CN 201811150218A CN 109337821 B CN109337821 B CN 109337821B
Authority
CN
China
Prior art keywords
cladosporium
strain
sterol esterase
fermentation
enzyme
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
CN201811150218.XA
Other languages
Chinese (zh)
Other versions
CN109337821A (en
Inventor
王鹏
江晓路
乔乐克
杜春影
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.)
Ocean University of China
Original Assignee
Ocean University of China
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 Ocean University of China filed Critical Ocean University of China
Priority to CN201811150218.XA priority Critical patent/CN109337821B/en
Publication of CN109337821A publication Critical patent/CN109337821A/en
Application granted granted Critical
Publication of CN109337821B publication Critical patent/CN109337821B/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
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01013—Sterol esterase (3.1.1.13)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a screened cladosporium (a) for producing sterol esteraseCladosporiumsp.) DZ16 with a preservation number of CCTCC M2016685. The bacterial colony of the invention is velvet-shaped, and is activated by the first step of bacterial preservation and activation and the second step of bacterial plate coating. The biomass of the strain is improved by adopting a first-step low-salt liquid activated culture in the early stage of fermentation, and the strain is induced to secrete enzyme protein and improve the enzyme activity by means of a second-step multi-factor composite action in the later stage. The sterol esterase produced by the fermentation of the strain has good application potential in the fields of medical detection, food, pulping and papermaking industry, environmental protection and the like.

Description

Cladosporium for producing sterol esterase and enzyme production method
Technical Field
The invention relates to a cladosporium strain for producing sterol esterase obtained by screening and an enzyme production method, belonging to the technical field of biological engineering.
Background
The sterol compound is a derivative of alkylene oxide polyhydrophenanthrene, is widely distributed in the biological world, plays various physiological functions in animals and plants, and plays a very important role in the metabolic process of organisms. Cholesterol is a sterol compound which is most abundant in animals, is mostly present in internal organs such as liver, kidney, intestine and the like, skin and adipose tissue, and is a precursor of five steroid hormones, vitamin D and cholic acid of organisms. Ensuring the supply of cholesterol and determining the specific content of the cholesterol are necessary for maintaining normal life activities of organisms, so that the determination of the content of the sterol in the bodies has important guiding significance in the aspect of medical clinic.
The detection methods for blood cholesterol in medical clinic mainly include enzyme chemistry methods, immunochemical analysis methods and other clinical biochemical detection methods based on instrument analysis principles. The method can be mainly classified into two categories, the first category is that large and medium-sized professional equipment is utilized, and the blood sample is detected by adopting the optical absorption principle, and the detection method has high requirements on the equipment, long measurement time and high cost; the second type is to use a special medical handheld detector for detection, mainly by means of the principles of an immunological method, an electrochemical method, a partial optical method and the like, and has the disadvantages of long operation time, high manufacturing cost and the like. Compared with the two existing detection methods, the detection method based on the enzyme method has the advantages of simple operation, good specificity, stable result and the like, and is gradually popularized and applied in more and more clinical applications.
Esterase is an enzyme that catalyzes ester bonds, and has the ability to hydrolyze or synthesize. When the hydrolysis effect is exerted, the ester bond can be specifically catalyzed to generate corresponding acid and alcohol; when the synthesis function is played, the carboxyl of acid and the hydroxyl of alcohol can be condensed and dehydrated, and the product is esters and other fragrant substances, and has good application in the aspects of numerous biochemical detection and drug synthesis. Sterol esterase is an esterase with hydrolysis action, which can hydrolyze ester bonds in cholesterol esters to produce cholesterol and the corresponding fatty acids. The related research shows that the cholesterol can generate the erythroquinone imine chromogen substance through the action of cholesterol oxidase and peroxidase, the erythroquinone imine chromogen substance can be detected at 500nm by a colorimetric method, the obtained light absorption value has a corresponding linear relation with the total cholesterol content in the sample, and the total cholesterol content in the sample can be calculated through a pre-prepared standard curve. The method does not need expensive large-scale equipment, has short detection time, less interference factors and high sensitivity, is a good sterol content detection method and has huge application potential.
The invention discloses a cladosporium strain for producing sterol esterase, which is obtained by screening, and an enzyme production method. The strain is preserved in Wuhan university in China with the preservation number of CCTCC M2016685. The strain is taken as a fermentation object, is subjected to first-step strain preservation and activation, second-step strain plate coating and subsequent low-salt liquid activation culture medium and multi-factor compound induction culture medium are combined, so that the growth biomass and the enzyme production activity of the strain are improved, and related sterol esterase enzyme preparation products are prepared, and the application value of the sterol esterase enzyme preparation products in the fields of medical detection, food, chemical industry and the like is increased.
Disclosure of Invention
The invention provides a cladosporium strain for producing sterol esterase obtained by screening and an enzyme production method. The bacterial species is namedCladosporiumsp, DZ16, the said strain is activated in two steps and through two-step induced fermentation process, and sterol esterase may be produced in high efficiency.
The specific technical scheme of the invention is as follows: separating a strain producing sterol esterase from animal liver with higher sterol content, and identifying the strain as cladosporiumCladosporiumsp. named DZ16, which was deposited in the China center for type culture Collection at 28/11/2016, and located in Wuhan university, Wuhan, China, with the deposition number: CCTCC M2016685.
The DZ16 strain is cultured on PDA plate culture medium, and after inverted culture at 28 deg.C for 2-3d, the colony is velvet, the edge is irregular, the colony and hypha are dark green, and conidia can be generated. The sterol esterase can be prepared by taking the strain as a main fermentation object through a reasonable fermentation process. The specific enzyme production process comprises the following steps.
(1) And (5) activating the strains.
The first step of strain preservation and activation, transferring the strain from a strain preservation tube frozen at the temperature of 80 ℃ below zero to a PDA culture medium for growth, and culturing for 24 to 48 hours at the temperature of 28 to 32 ℃.
And (2) coating a strain plate, selecting the liquid with vigorous growth, diluting and separating, coating the liquid on a PDA solid culture medium by using a coating rod, culturing for 48-72h at 28-32 ℃, selecting the colony with good growth, and keeping the colony for later use.
(2) And (5) culturing fermentation liquor.
The first step of low-salt liquid activated culture, inoculating the colony growing well in the plate coating to a low-salt liquid activated culture medium for culture, wherein the culture medium contains 0.1-0.3% of yeast extract, 0.2-0.5% of peptone, 0.2-0.5% of monopotassium phosphate and 0.1-0.3% of magnesium sulfate pentahydrate, and the culture conditions are 28-32 ℃, 150-180rpm and 24-48 h.
The second step of multi-factor induction enzyme production fermentation, when every 1mL of bacterial liquid in the culture medium in the first step contains 105-106When the strain spores are cultured, 5.0-10.0% (v/v) of the strain spores are inoculated into a multi-factor induction enzyme production fermentation culture medium for fermentation, and the culture medium contains 0.1-0.2% of yeast extract, 0.5-2.0% of oleic acid phytosterol ester, 0.1-0.5% of oleic acid, 0.05-0.1% of Triton X-100, 0.001-0.01% of boric acid and 0.7 multiplied by 10 of boric acid-4-2.5×10-4% sodium molybdate, 0.5X 10-4-2.0×10-4% coenzyme R, the culture conditions are 28-32 ℃, 150-180rpm and 48-96 h.
The volume of the triangular flask is 100-500mL in the fermentation process, the liquid filling amount is 20-25%, and the original pH value is obtained.
The method for measuring the enzymatic activity of sterol esterase comprises the steps of taking 0.25mL of 0.48mg/mL stigmasterol acetate solution as a substrate, adding 0.5mL of 4-AA phenol working solution, 0.25mL of 2U/mL cholesterol oxidase and 0.25mL of 12U/mL horseradish peroxidase, preheating for 5min at 40 ℃, adding 25 mu L of enzyme solution to be measured, reacting for 30min, stopping the reaction by using 0.5mL of 0.1mol/L HCl, measuring an absorbance value at OD500, and inactivating the enzyme solution to serve as a blank control group.
The crude enzyme has molecular weight of 59KDa, suitable reaction temperature of 30-35 deg.C, optimum action pH of 6.5-7.5, and trivalent metal ion (Fe)3+、Al3+) Has obvious inhibiting effect on enzyme activity. The enzyme can effectively degrade stigmasterol acetate into free stigmasterol and acetic acid, has a certain degradation effect on other types of phytosterol esters, and is widely applied to the fields of food, chemical industry and the like.
The present invention has the following advantages.
(1) The fermentation cost is low: in the whole fermentation process, the traditional fungus PDA culture medium is adopted to achieve high-density activation of strains, and in the later fermentation process, trace nutritional factors can achieve a better enzyme production effect without complex and expensive medicines or instruments.
(2) The application potential is huge: the sterol esterase produced by the self-screened strain has good capability of hydrolyzing sterol esters, and has higher value in the aspects of determination of serum total cholesterol content in clinical medicine, high-quality utilization of resin in paper industry and the like.
Description of the drawings.
FIG. 1 is a drawing ofCladosporiumsp. DZ16 colony pattern.
The detailed description is given below.
Example 1 sterol esteraseCladosporiumsp. screening and identification of strains.
(1) Primary screening: selecting a sample with high sterol ester content as a screening raw material, and dissolving part of the sample in sterilized physiological saline to ensure that the tissue distribution of the sample is uniform. The sample is inoculated and coated on a primary sieve plate culture medium taking sterol ester as a main carbon source through aseptic operation. Inverted culturing at 28-30 deg.C for 2-3 days, and observing colony morphology and culture medium transparent ring condition.
(2) Re-screening: classifying the strains with transparent circles in the primary screening culture medium, carrying out liquid shaking and re-screening, culturing for 1-2 days under the fermentation conditions of 28-30 ℃ and 150-200rpm, then collecting the fermentation liquor, and determining the enzyme activity.
(3) And (3) strain identification: the strain identification is carried out by strain genome extraction, amplification primers and subsequent PCR amplification. The similarity of the sequence of the strain and cladosporium is proved to be 99% through ITS sequence amplification comparison, and the final name isCladosporium sp. DZ16。
Example 2CladosporiumActivation of sp, DZ16 strain and enzyme production culture.
(1) And (5) activating the strains.
The first step of strain preservation and activation is to preserve and activateCladosporiumThe sp, DZ16 strains are transferred to a PDA culture medium in a 100mL triangular flask from a strain storage tube frozen at-80 ℃ for growth, the liquid loading amount is 20mL, and the culture is carried out for 24-48h at 28-32 ℃.
And (2) coating a strain plate, selecting the liquid with vigorous growth, diluting and separating, coating the liquid on a PDA (PDA) solid culture medium by using a coating rod, culturing for 48 hours at the temperature of 28-32 ℃, selecting the large colony with good growth, and keeping the large colony for later use.
(2) And (5) culturing fermentation liquor.
The first step of low-salt liquid activated culture, large colonies growing well in plate coating are inoculated into a low-salt liquid activated culture medium for culture, and the culture medium contains 0.2 percent of yeast extract, 0.3 percent of peptone, 0.2 percent of monopotassium phosphate and 0.1 percent of magnesium sulfate pentahydrate, and the culture conditions are 28 ℃, 160rpm and 48 hours.
The second step of multi-factor induction enzyme production fermentation, when the culture medium of the first step contains 10 per 1mL of bacteria solution5Inoculating 5.0% (v/v) of spore in fermentation medium for inducing multi-factor enzyme productionThe medium contains 0.2 percent of yeast extract, 2.0 percent of oleic acid soybean sterol ester, 0.2 percent of oleic acid, 0.1 percent of Triton X-100, 0.005 percent of boric acid and 1.0 multiplied by 10-4% sodium molybdate, 1.0X 10-4% coenzyme R, culture conditions are 28 ℃, 160rpm and 96 hours; the volume of the triangular flask is 250mL, the liquid loading amount is 50mL, and the original pH value is obtained in the fermentation process.
Example 3 crude enzyme enzymatic activity assay.
0.25mL of 0.48mg/mL stigmasterol acetate solution is used as a substrate, 0.5mL of 4-AA phenol working solution, 0.25mL of 2U/mL cholesterol oxidase and 0.25mL of 12U/mL horseradish peroxidase are added, 25 mu L of enzyme solution to be detected is added after preheating for 5min at 40 ℃, reaction is carried out for 30min, 0.5mL of 0.1mol/L HCl is used for stopping the reaction, the light absorption value is measured at OD500, the inactivated enzyme solution is used as a blank control group, and the enzyme activity is measured to be 0.5-0.8U/mL.

Claims (2)

1. Cladosporium (A) for producing sterol esteraseCladosporiumsp.) DZ16 characterized by: the culture is preserved in China center for type culture Collection in 2016, 11 months and 28 days, and is located in the university of Wuhan, China, with the preservation number: CCTCC M2016685.
2. A sterol esterase producing Cladosporium species (A), (B) according to claim 1Cladosporiumsp.) DZ16, characterized in that: the sterol esterase crude enzyme is prepared by activation, aerobic fermentation and centrifugal separation.
CN201811150218.XA 2018-09-29 2018-09-29 Cladosporium for producing sterol esterase and enzyme production method Active CN109337821B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811150218.XA CN109337821B (en) 2018-09-29 2018-09-29 Cladosporium for producing sterol esterase and enzyme production method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811150218.XA CN109337821B (en) 2018-09-29 2018-09-29 Cladosporium for producing sterol esterase and enzyme production method

Publications (2)

Publication Number Publication Date
CN109337821A CN109337821A (en) 2019-02-15
CN109337821B true CN109337821B (en) 2022-06-24

Family

ID=65307927

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811150218.XA Active CN109337821B (en) 2018-09-29 2018-09-29 Cladosporium for producing sterol esterase and enzyme production method

Country Status (1)

Country Link
CN (1) CN109337821B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113684195B (en) * 2020-05-19 2022-07-22 中国海洋大学 Sterol esterase and coding gene and mutant thereof
CN116376708B (en) * 2022-12-05 2024-06-07 青岛农业大学 Cladosporium fungus and application thereof

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4491631A (en) * 1981-06-25 1985-01-01 Toyo Jozo Kabushiki Kaisha Assay method for lipid component, assay composition, and process for production of enzyme used therefor
US5919807A (en) * 1995-05-12 1999-07-06 Dr. Karl Thomae Gmbh Benzothiazoles and benzoxazoles, drugs containing them, their use and methods of preparing them
CN1701115A (en) * 2002-09-26 2005-11-23 诺维信北美公司 Fermentation processes and compositions
CN104222098A (en) * 2013-06-08 2014-12-24 江西珀尔农作物工程有限公司 Preparation method for formamide phenazine biological fungicide
CN105238703A (en) * 2015-10-27 2016-01-13 江南大学 Cladosporiumc cladosporioides for high yield of feruloyl esterase and application of Cladosporiumc cladosporioides in edible vinegar brewing
CN106754430A (en) * 2017-02-13 2017-05-31 浙江师范大学 Dendritic bacterial strains of cladosporium Cc 106 of high yield monascus purpureus and application thereof
CN108753637A (en) * 2018-04-25 2018-11-06 大连大学 A kind of bacterial strain and its fermentation process producing low temperature sterol esterase
CN109456953A (en) * 2018-11-13 2019-03-12 青岛海洋生物医药研究院股份有限公司 A kind of cholesterol esterase and its application

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6740498B2 (en) * 2001-12-06 2004-05-25 Paradigm Genetics, Inc. Methods for the identification of inhibitors of histidinol-phosphate as antibiotics

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4491631A (en) * 1981-06-25 1985-01-01 Toyo Jozo Kabushiki Kaisha Assay method for lipid component, assay composition, and process for production of enzyme used therefor
US5919807A (en) * 1995-05-12 1999-07-06 Dr. Karl Thomae Gmbh Benzothiazoles and benzoxazoles, drugs containing them, their use and methods of preparing them
CN1701115A (en) * 2002-09-26 2005-11-23 诺维信北美公司 Fermentation processes and compositions
CN104222098A (en) * 2013-06-08 2014-12-24 江西珀尔农作物工程有限公司 Preparation method for formamide phenazine biological fungicide
CN105238703A (en) * 2015-10-27 2016-01-13 江南大学 Cladosporiumc cladosporioides for high yield of feruloyl esterase and application of Cladosporiumc cladosporioides in edible vinegar brewing
CN106754430A (en) * 2017-02-13 2017-05-31 浙江师范大学 Dendritic bacterial strains of cladosporium Cc 106 of high yield monascus purpureus and application thereof
CN108753637A (en) * 2018-04-25 2018-11-06 大连大学 A kind of bacterial strain and its fermentation process producing low temperature sterol esterase
CN109456953A (en) * 2018-11-13 2019-03-12 青岛海洋生物医药研究院股份有限公司 A kind of cholesterol esterase and its application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
甾醇酯酶菌株Chryseobacterium sp.的筛选及酶学性质研究;曾诚等;《林产化学与工业》;20130628(第03期);全文 *

Also Published As

Publication number Publication date
CN109337821A (en) 2019-02-15

Similar Documents

Publication Publication Date Title
CN1935977B (en) Levo lactone hydrolase producing fungus, and its method for preparing chiral hydroxy acid
CN109337821B (en) Cladosporium for producing sterol esterase and enzyme production method
CN114250157B (en) Saccharomycopsis fibuligera cx-3 strain for high yield of aroma substances and application thereof
CN111763636B (en) Method for producing coenzyme Q10 by industrial fermentation
CN103937691B (en) One plant production β fructosidases aspergillus oryzae strain and its cultural method and application
Cunha et al. Indirect method for quantification of cellular biomass in a solidscontaining medium used as pre-culture for cellulase production
CN108929865A (en) A method of induction Produced from Pleurotus ostreatus accelerates to produce laccase
CN101250492A (en) Agrobacterium strain and method for preparing left-lateral lactone compounds thereby
CN105018410B (en) A method of the mould aging bacterial strain rapid, high volume of three spore cloth Laplaces of induction produces spore
CN113122460A (en) Rice straw degrading bacterium and screening method and application thereof
CN108587923B (en) Method for improving malic acid fermentation performance
CN1821380A (en) High efficiency compound microbe prepn for treating peltate yam
JP2710834B2 (en) FO-608A substance and method for producing the same
CN109097435A (en) A method of screening malaga carbohydrate oxidase strain from air
Aghera et al. Biosynthesis of citric acid using distillery spent wash as a novel substrate
RU2712703C1 (en) Method of producing lactic acid
Zhang et al. Screening and identification of a cutinase-producing Rhodotorula mucilaginosa and properties of the cutinase
CN116083307B (en) Lactococcus garvieae for producing esterase and ester compounds and application thereof
CN102417884B (en) Aspergillus niger strain and application thereof in enrichment and extraction of 1-deoxynojirimycin (1-DNJ) from white mulberry root-bark
CN114958654B (en) Propionic acid-producing proteophilic bacteria derived from white spirit brewing pit mud and application thereof
CN117004495B (en) Trichoderma atroviride T280, screening method and application thereof
Dong Separation and screening of Acetobacter producing alcohol dehydrogenase
JP5461026B2 (en) β-glucosidase-producing bacterium and method for producing β-glucosidase
CN101215531A (en) Method for separating extracellular cholesterol oxidase producing strain
AL-Temime et al. Isolation, identification of some fugal isolates and testing their ability for lipoxygenase production

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