CN103589705B - A kind of M-Zyme and encoding gene thereof - Google Patents
A kind of M-Zyme and encoding gene thereof Download PDFInfo
- Publication number
- CN103589705B CN103589705B CN201310522569.XA CN201310522569A CN103589705B CN 103589705 B CN103589705 B CN 103589705B CN 201310522569 A CN201310522569 A CN 201310522569A CN 103589705 B CN103589705 B CN 103589705B
- Authority
- CN
- China
- Prior art keywords
- zyme
- gene
- seqidno
- nucleotide sequence
- 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
Links
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a kind of M-Zyme, is its aminoacid sequence as SEQ? ID? shown in NO.2 or at SEQ? ID? by the aminoacid sequence with keratinase activity of disappearance, sudden change, restructuring acquisition on NO.2 basis.Present invention also offers a kind of gene of M-Zyme of encoding, is its nucleotide sequence as SEQ? ID? shown in NO.1 or according to M-Zyme aminoacid deletion, sudden change, restructuring situation, the nucleotide sequence of engineer.M-Zyme provided by the invention effectively can be hydrolyzed feather, and the water-insoluble keratin substrate such as wool, can be used for leather textile industry and fodder industry.
Description
Technical field
The present invention relates to a kind of M-Zyme and encoding gene thereof, belong to enzyme genetically engineered and enzyme engineering field.
Technical background
Whole world poultry farming year produces millions of tons feather, and Keratin sulfate accounts for very large proportion in feather, up to more than 90%.Feather Analysis of Nutritional shows, aminoacids content is more than 70%.Wherein first, second limiting amino acid of animal: Methionin and methionine content are respectively up to 0.77% and 0.48%.The another kind of indispensable amino acid of animal, Glycine Levels accounts for 7.59%.And in animal diets, Methionin adding proportion is generally 0.1%-0.2%, the adding proportion of methionine(Met) is 0.02%-0.1%.Current Chinese feedstuff protein source wretched insufficiency, and China is feather output first big country, annual production more than 700,000 tons.With feather be raw material as feedstuff protein source, there is good prospect.In addition, feather keratin can also be widely used in medicine, food, composite industry.So the exploitation of feather keratin have important practical significance.
But due to keratin polypeptide interchain disulfide bond, sat linkage, the extensive existence of hydrogen bond, makes keratic structure highly stable, especially due to the existence of disulfide linkage.Conventional proteolytic enzyme, as trypsinase, papoid, stomach en-, is all difficult to Keratin sulfate of degrading.The palliating degradation degree of thermal treatment keratin is very limited, can destroy the amino acid that some have nutritive value in addition, and as Methionin, methionine(Met) and tryptophane, produce the unwanted amino acid of some animals, as lanthionine.And chemical process, mainly comprise acid-hydrolysis method and alkali hydrolysis method, also can destroy the amino acid that some have nutriture value.In addition the waste water after hydrolysis also can to environment.Based on the advantage that this and enzyme reaction have, M-Zyme degradation of feather Keratin sulfate is utilized to become a kind of method with fine prospect.M-Zyme (Keratinase) for one can the keratic enzyme of selective degradation, produced by multiple-microorganisms such as fungi, actinomycetes and bacteriums.M-Zyme at food, medicine, feed, refine and widespread use in the industry such as process hides, there is tenderization meat, production senior nutrition, immunological reagent and fodder additives and the effect such as beauty treatment, softening leather, and the degraded of Ruan's albumen (Prion) of mad cow disease and mankind's Keyashi's syndrome can be caused.More become study hotspot, application and research Huge value.
The M-Zyme gene reported at present is mainly from the kerA gene of an external strain bacillus licheniformis.This patent introduces a kind of stenotrophomonas maltophilia (StenotrophomonasmaltophiliaBBE11-1) with efficient degradation feather ability independently being screened acquisition by this laboratory; and by M-Zyme gene single expression; and carry out the research of gene and zymologic property aspect, the large-scale production and marketing for M-Zyme has far-reaching technical director's meaning.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of M-Zyme being hydrolyzed water-insoluble keratin substrate, and its aminoacid sequence passes through the aminoacid sequence with keratinase activity of disappearance, sudden change, restructuring acquisition as shown in SEQIDNO.2 or on SEQIDNO.2 basis.
Another technical problem that the present invention will solve is to provide a kind of gene of above-mentioned M-Zyme, its nucleotide sequence as shown in SEQIDNO.1 or according to M-Zyme aminoacid deletion, sudden change, restructuring situation, the nucleotide sequence of engineer.
In addition, present invention also offers a kind of method of M-Zyme of production, specifically comprise the steps: first to adopt the complete synthesis or PCR method of chemistry to obtain nucleotide sequence shown in SEQIDNO.1; The nucleotide sequence that clone obtains is to pET22b (+) expression vector; Then the expression vector Transformed E .coliBL21 obtained is obtained the genetic engineering bacterium of the M-Zyme of expressing; Finally by optimization for fermentation technology, the genetic engineering bacterium of acquisition 37 DEG C of liquid culture in the LB substratum of the penbritin containing 100 μ g/l are spent the night, the LB fermentation broth 37 DEG C that rear access contains the penbritin of 100 μ g/l is cultured to OD600nm=0.6, be cooled to 20 DEG C of cultivations, the inductor IPTG adding ultimate density 0.1mM induces, during 72h, clear enzyme solution in centrifugal acquisition, is crude enzyme liquid.The expression vector comprising M-Zyme gene of the present invention belongs to the scope of protection of present invention.
The clone or the genetic engineering bacterium that comprise expression vector provided by the invention are also the scope of protection of present invention.
Above-mentioned M-Zyme and encoding gene thereof are also the scope of protection of present invention in the application in process feather.
M-Zyme enzyme activity determination method, measuring principle and step are.
Principle: M-Zyme hydrolysis of keratin substrate, discharges tyrosine, is developed the color by tyrosine and forint phenol, under 660nm, measures absorbance.The size of light absorption value is directly directly proportional to the height of enzyme activity.
Determination step: 0.1mL is through the enzyme liquid of suitably dilution, add the 1%(w/v of 0.1mL) soluble keratin substrate (with the Gly-NaOH damping fluid mixing of the pH9.0 of 0.1mol/L before using), at 50 DEG C of reaction 20min, 0.2mLTCA reaction terminating protein precipitant is added in each response sample, shake up rear 10, the centrifugal 5min of 000r/min, gets the 4%(w/v that 0.2mL supernatant liquor adds 1mL) Na
2cO
3, then the forint phenol reagent (beforehand dilution 3 times) adding the purchase of 0.2mL Shanghai Sheng Gong company reacts 15 minutes at 50 DEG C.The quartz cuvette of 0.5cm is used to measure the light absorption value of clear liquid at 660nm place.Blank is that the TCA termination enzyme having added equal volume before adding substrate is lived, and other steps are identical.Enzyme work is defined as every milliliter of enzyme liquid per minute and discharges how many microgram tyrosine.
The optimum temperuture of restructuring M-Zyme provided by the invention 60 DEG C, optimal pH 9.0.50 DEG C, react with insoluble keratin substrate under pH9.0, can partial hydrolysis.At 50 DEG C, the enzymic activity transformation period is 90 minutes, 60 DEG C of half-life 62 minutes.Experimental result shows this restructuring M-Zyme and can be good at hydrolyzed feather meal, wool, has good application prospect.
Accompanying drawing explanation
Fig. 1 recombinant bacterium builds agarose gel electrophoresis figure
M:DNAmarker, 1: M-Zyme total length reading frame gene fragment (1923bp), 2: M-Zyme gene PCR product, 3: amplification plasmid pMD19T+KerSMD, 4: plasmid pMD19T+KerSMD double digestion (Nco I/Xho I) 5: expression plasmid pET22b+KerSMD, 6: expression plasmid pET22b+KerSMD double digestion (Nco I/Xho I)
M-Zyme SDS-PAGE figure is produced in the fermentation of Fig. 2 recombinant bacterium
M:proteinmarker, 1: induction 12h fermentation supernatant, 2: induction 24h fermented liquid supernatant, 3: induction 48h fermented liquid supernatant, 4: the M-Zyme of purifying, in figure, total arrow is depicted as target protein.
The enzymic activity of Fig. 3 M-Zyme KerSMD under different pH.PH4-6 is sodium-acetate buffer, and pH6-7 is phosphoric acid buffer, and pH7-9 is Tris-HCl damping fluid, and pH9-12 is Gly-NaOH damping fluid.
Fig. 4 M-Zyme KerSMD enzymic activity at different temperatures.Use pH7.0 damping fluid.
Fig. 5 M-Zyme KerSMD enzyme stability at different temperatures.Use pH7.0 damping fluid, preservation M-Zyme measures enzyme and lives under differing temps after different time.
Embodiment
The separating clone program of embodiment 1 M-Zyme gene.
Preservation mechanism is submitted in the patent application of StenotrophomonasmaltophiliaBBE11-1(before this seminar, on April 3rd, 2011 is preserved in China typical culture collection center (Wuhan University), deposit number is CCTCCNo.2011193) at LB liquid medium (peptone 10g/L, sodium-chlor 10g/L, yeast extract 10g/L) middle cultivation 12h, the centrifugal 5min of 10000rpm collects thalline, sterilized water washs, and uses genome to extract test kit (purchase of Shanghai Sheng Gong company) and extracts STb gene.
Utilize primer NdeI-kerD(GGAATTC
cATATGand XhoI-kerD(CCG ATGGCCGCAGTGTTG)
cTCGAGcTGCGTGGCGAGGATG), with StenotrophomonasmaltophiliaBBE11-1 genome for masterplate, pcr amplification M-Zyme gene.Reaction conditions is: enter after 95 DEG C of denaturation 5min and circulate: 95 DEG C of sex change 50s, 60 DEG C of annealing 30s, and 72 DEG C extend 110s, 30 circulations; 72 DEG C extend 10min.Amplification obtains the PCR fragment of 1923bp, cuts glue and reclaims.Reclaim segment to be connected with pMD19-Tsimple carrier, connect product conversion e. coli jm109, converted product coating is dull and stereotyped containing the LB of 100mg/L penbritin.37 DEG C of overnight incubation, picking list bacterium colony, access LB liquid nutrient medium, extracts plasmid after 8h.This plasmid is carried out sequencing, and result shows this gene open reading frame total length 1902 Nucleotide, 634 amino acid of encoding, and the kerD gene of StenotrophomonasmaltophiliaYHYJ-1 has 7 amino acid whose differences.
The construction procedures of embodiment 2 M-Zyme gene on coli expression carrier.
PET22b (+) for building the plasmid of coli expression carrier.Utilize primer NocI-p-kerD(CATG
cCATGGand XhoI-kerD(CCG CCGGGTTGCCGAC)
cTCGAGcTGCGTGGCGAGGATG) gene of amplification M-Zyme KerSMD is carried out.PET22b (+) plasmid and M-Zyme gene are carried out Nco I and Xho I double digestion, after digestion products cuts glue recovery, connect with T4 ligase enzyme 16 DEG C again and spend the night, connect product conversion E.coliJM109 competent cell, through 37 DEG C of overnight incubation, select transformant 100mg/L penbritin LB and carry out liquid culture, then extracting plasmid, obtain KerSMD-pET22b (+) plasmid of enrichment, building process electrophorogram is shown in Fig. 1.
Embodiment 3 escherichia coli host transforms and screens the program of recombinant bacterium.
By plasmid KerSMD-pET22b (+) at 42 DEG C of thermal shock transformed competence colibacillus cell E.coliBL21 (DE3) Host Strains, through 37 DEG C of overnight incubation on penbritin (100mg/L)-LB flat board, select transformant (recombinant bacterium KerSMD-pET22b (+)/E.coliBL21 (DE3)) at LB substratum (peptone 10g/L, yeast extract 5g/L, NaCl10g/L) in, 37 DEG C of liquid culture are spent the night, and rear access LB fermentation broth 37 DEG C is cultured to OD
600be cooled to 20 DEG C of cultivations after=0.6, add IPTG(isopropylthio β D galactoside, final concentration is 0.1mM) induction, collected by centrifugation supernatant during 72h, the work of supernatant enzyme is for reaching as high as 720U/ml.Electrophorogram is shown in Fig. 2, apparent molecular weight 46kDa.
Claims (7)
1. a M-Zyme, is characterized in that its aminoacid sequence is as shown in SEQIDNO.2.
2. the gene of M-Zyme described in coding claim 1, is characterized in that its nucleotide sequence is as shown in SEQIDNO.1.
3. express the genetic engineering bacterium of M-Zyme described in claim 1.
4. the expression vector containing M-Zyme gene described in claim 2.
5. the method for M-Zyme described in production claim 1, is characterized in that comprising the steps:
1) the complete synthesis or PCR method of chemistry is adopted to obtain nucleotide sequence shown in SEQIDNO.1;
2) nucleotide sequence shown in SEQIDNO.1 is cloned to pET22b (+) expression vector;
3) by step 2) the expression vector Transformed E .coliBL21 (DE3) that obtains obtains the genetic engineering bacterium of express keratin enzyme;
4) by step 3) genetic engineering bacterium that obtains 37 DEG C of liquid culture in the LB substratum of the penbritin containing 100 μ g/l spend the night, and the LB fermentation broth 37 DEG C that rear access contains the penbritin of 100 μ g/l is cultured to OD
600nm=0.6, be cooled to 20 DEG C of cultivations, the inductor IPTG adding ultimate density 0.1mM induces, and during 72h, clear enzyme solution in centrifugal acquisition, is crude enzyme liquid.
6. the application of M-Zyme described in claim 1 in process feather.
7. the application of proteinase gene described in claim 2 in Keratin sulfate process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310522569.XA CN103589705B (en) | 2013-10-29 | 2013-10-29 | A kind of M-Zyme and encoding gene thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310522569.XA CN103589705B (en) | 2013-10-29 | 2013-10-29 | A kind of M-Zyme and encoding gene thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103589705A CN103589705A (en) | 2014-02-19 |
| CN103589705B true CN103589705B (en) | 2016-04-13 |
Family
ID=50080019
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310522569.XA Active CN103589705B (en) | 2013-10-29 | 2013-10-29 | A kind of M-Zyme and encoding gene thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103589705B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475229B (en) * | 2015-03-18 | 2019-12-24 | 江南大学 | Keratinase mutant with improved substrate specificity and preparation method thereof |
| CN105316306B (en) * | 2015-11-26 | 2018-10-16 | 江南大学 | A kind of fermentation process efficiently producing keratinase using recombination bacillus coli |
| CN105754974A (en) * | 2016-04-28 | 2016-07-13 | 中国水产科学研究院黄海水产研究所 | Protease and preparation and application thereof |
| CN105884871B (en) * | 2016-04-28 | 2019-10-01 | 中国水产科学研究院黄海水产研究所 | A kind of bacillus marinus albumen with anti-tumor activity and its preparation and application |
| CN106636042B (en) * | 2016-10-18 | 2019-08-06 | 江南大学 | A kind of keratinase mutant of thermal stability and catalysis activity raising |
| CN115820612B (en) * | 2022-07-05 | 2024-07-19 | 山东龙昌动物保健品股份有限公司 | Keratinase, bile acid enzyme preparation containing keratinase and application of bile acid enzyme preparation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864115A (en) * | 2012-06-15 | 2013-01-09 | 江南大学 | Gene engineering bacterium for producing keratinase escherichia coli and application thereof |
| CN103074318A (en) * | 2012-11-01 | 2013-05-01 | 广东溢多利生物科技股份有限公司 | Keratinase, and coding gene and application thereof |
-
2013
- 2013-10-29 CN CN201310522569.XA patent/CN103589705B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864115A (en) * | 2012-06-15 | 2013-01-09 | 江南大学 | Gene engineering bacterium for producing keratinase escherichia coli and application thereof |
| CN103074318A (en) * | 2012-11-01 | 2013-05-01 | 广东溢多利生物科技股份有限公司 | Keratinase, and coding gene and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Biodegradation of wool waste and keratinase production in scale-up fermenter with different strategies by Srenotrophomonas maltophilia BBE11-1;Zhen Fang等;《Bioresource Technology》;20130731;第286-291页 * |
| keratinase, partial [Stenotrophomonas maltophilia];Fang,Z.等;《GeneBank》;20130428 * |
| Stenotrophomonas maltophilia strain BBE11-1 keratinase gene, partial cds;Fang,Z.等;《GeneBank》;20130428 * |
| 嗜麦芽窄食单胞菌角蛋白酶基因(KerF)的克隆及其在大肠杆菌中的表达;邹晓凤等;《农业生物技术学报》;20111231;第363-368页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103589705A (en) | 2014-02-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103589705B (en) | A kind of M-Zyme and encoding gene thereof | |
| CN105614023B (en) | Fermentation enzymolysis agent for soybean meal fermentation and application thereof | |
| CN104726436B (en) | Keratinase mutant that a kind of substrate specificity improves and preparation method thereof | |
| CN104017791B (en) | Keratinase mutant with improved thermal stability and preparation method thereof | |
| CN103820352A (en) | Bacillus cereus YSQ08 and application thereof | |
| CN105494890A (en) | Enzymolysis and fermentation combined method for improving feeding nutritional value of rapeseed meal | |
| CN102757947A (en) | Xylanase xyn-CDBFV-m with modified thermal stability, gene thereof, and application thereof | |
| US20210277374A1 (en) | Xylanase-containing feed additives for cereal-based animal feed | |
| CN101285086A (en) | Feather products, processing technology and use thereof | |
| CN105379947A (en) | Animal feed and preparation method thereof | |
| Xie et al. | A thermophilic fungal GH36 α-galactosidase from Lichtheimia ramosa and its synergistic hydrolysis of locust bean gum | |
| CN102676561B (en) | Recombinant bacterium for expressing keratinase and fermentation method thereof | |
| Colombatto et al. | In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage: II. Effects on rate of acidification, fibre degradation during ensiling and rumen fermentation | |
| CN105255771A (en) | Staphylococcus succinus for generating collagenase and application thereof | |
| Zayed et al. | Evaluation of cellulolytic exogenous enzyme-containing microbial inoculants as feed additives for ruminant rations composed of low-quality roughage | |
| CN105316306A (en) | Fermentation method for efficiently producing keratinase through recombinant escherichia coli | |
| CN102224938B (en) | Application of cold-adapted protease MCP-01 for tenderizing meat | |
| CN108977401A (en) | Using the method for lignocellulosic culture microalgae | |
| CN108588053A (en) | Animal protein hydrolyzes specific enzyme and preparation method thereof | |
| CN102864115B (en) | Gene engineering bacterium for producing keratinase escherichia coli and application thereof | |
| Wang et al. | Preparation of peptone from chicken bone residue by using natural pancreas as catalyst | |
| CN102978187B (en) | High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof | |
| CN103525789A (en) | Whole cell xylanase for promoting rumen fermentation, and preparation method thereof | |
| CN103898081B (en) | A kind of M-Zyme mutant and application thereof | |
| CN1847400A (en) | Improved xylanase with high specific activity, its gene expression vector and recombinant yeast cell, and expression method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |