CN105886484A - Thermophilic cellulase, encoding gene thereof and application of thermophilic cellulase - Google Patents
Thermophilic cellulase, encoding gene thereof and application of thermophilic cellulase Download PDFInfo
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- CN105886484A CN105886484A CN201610262105.3A CN201610262105A CN105886484A CN 105886484 A CN105886484 A CN 105886484A CN 201610262105 A CN201610262105 A CN 201610262105A CN 105886484 A CN105886484 A CN 105886484A
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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Abstract
The invention relates to the field of genetic engineering, in particular to a thermophilic cellulase, an encoding gene thereof and application of the thermophilic cellulase. Amino acid sequences of the thermophilic cellulase are shown as SEQ ID NO.1 or SEQ ID NO.2. The thermophilic cellulase is a novel cellulase and can be applied to the industries of feed, foods, medicines and the like. According to the technical scheme, the thermophilic cellulase, the encoding gene and the application have the advantage that thermophilic cellulase which is excellent in property and suitable for industrial application can be produced by means of genetic engineering.
Description
Technical field
The present invention relates to genetic engineering field, be specifically related to a kind of thermophilic cellulase and encoding gene thereof and application.
Background technology
Plant cell wall is mainly made up of materials such as cellulose, hemicellulose and lignins.Cellulose is a kind of important
Polysaccharide, it is the material of plant cell support substance, is the abundantest biomass resource of nature, cellulose
Structure is defined as the linear polymers that β-D-Glucose unit is formed by connecting through β-(1 → 4) glycosidic bond, does not has in structure
Having branch, it can be glucose by cellulose degraded.
Cellulase refers to hydrolyzation of glucose glycosidic bond, and cellulose decomposition becomes a group of cellobiose and glucose
The general name of enzyme.It mainly includes three steps to the hydrolytic process of cellulose: the first step is that endo-type cellulase acts on
Amorphous region within cellulose, hydrolyzes β-(1 → 4) glycosidic bond immediately by cellulosic molecule truncate, the most circumscribed
Fiber type element enzyme, acts on cellulose linear molecule end, hydrolyzes β-(1 → 4) glycosidic bond, cuts the next one every time
Cellobiose molecule, finally, cellobiose is hydrolyzed into glucose molecule by glucosidase.
Cellulase has been widely used in food, medicine, feedstuff, papermaking, textile printing and dyeing, oil exploitation, essence
The refinement numerous areas such as work and biotechnology, is a kind of novel industrial enzyme, has the biggest potential using value.
Cellulase is widely present in the biologies such as antibacterial, actinomycetes, fungus, plant, animal.Different microorganisms is produced
Raw cellulase, its 26S Proteasome Structure and Function differs greatly.Thermophilic cellulase has all compared with room temperature cellulase
Many advantages, as catalytic efficiency is high, Substratspezifitaet is strong, and during heat treatment lignocellulose, enzyme stability is good, thus right
Reaction system has good compatibility etc..Up to the present, thermophilic cellulase multi-source, in antibacterial, mainly comes
Dwell hot spore Pseudomonas, hot ascomycetes Thermoascus aurantiacus in sea, source) etc..But, thermophilic bacteria source addicted to
Thermal fiber element expression of enzymes amount is low, it is difficult to carry out commercial production.
Current business-like cellulase is mainly produced by fungus, such as Trichoderma spp., penicillium sp, aspergillosis etc..Originated from fungus
Cellulase optimum temperature is many between 45 to 65 DEG C, the easy loss of activity when high temperature.The present invention is from thermophilic blue shape
Bacterium Talaromyces leycettanus JCM 12802 bacterial strain obtains a new cellulose enzyme gene, its coding
Cellulase have several advantages that thermophilic, acid, substrate specificity, easy fermenting and producing widely.
All these advantages can mean that neoteric cellulase is in the industries such as feedstuff, food, medicine, it will than with
The cellulase of front report more has using value.
Summary of the invention
It is an object of the invention to provide a kind of thermophilic, acid, substrate specificity and compare cellulase widely.
Another object of the present invention is to provide the gene of above-mentioned cellulase.
Another object of the present invention is to provide the recombinant vector comprising above-mentioned cellulase.
Another object of the present invention is to provide the recombinant bacterial strain comprising above-mentioned cellulose enzyme gene.
Another object of the present invention is to provide a kind of method preparing cellulase.
Another object of the present invention is to provide the application of above-mentioned cellulase.
The most to be solved technical problem is that of the present invention overcomes the deficiencies in the prior art, it is provided that a kind of good properties,
It is suitable in the industries such as feedstuff, food, medicine the new cellulase of application, its aminoacid sequence such as SEQ ID
NO.1:
Wherein, 409 aminoacid of this enzyme total length, 18 aminoacid of N end are signal peptide sequence " MKFSNVILAA
SASSLVLA”。
Therefore, the theoretical molecular of ripe thermophilic cellulase is 42.5kDa, its aminoacid sequence such as SEQ ID
NO.2:
The optimum pH of this cellulase is 3.5, in the range of pH2.5-pH5.0, this enzyme be able to maintain that its 60% with
On enzyme activity；Optimum temperature 80 DEG C, still has the enzyme activity of 30% when 85 DEG C, processes 60min at 70 DEG C,
Enzyme is lived and is not lost, and processes 5min, it is possible to keep the enzyme activity of more than 70%, locate at 80 DEG C at 75 DEG C
Reason 2min, it is possible to keep the enzyme activity of more than 60%, there is good heat stability.
Present invention also offers the gene encoding above-mentioned cellulase.The complete genome sequence of this enzyme such as SEQ ID NO.3
Shown in:
The present invention passes through this cellulose enzyme gene of the method separating clone of PCR, DNA complete sequence analysis result table
Bright, cellulase structural gene total length 1464bp, containing 4 introns ,+96～162 ,+374～429 ,+495～551,
+ 650～7.3 is its intron sequences, the long 1230bp of cDNA, and its cDNA sequence is as shown in SEQ ID NO.4:
Wherein, the base sequence of signal peptide is:
“ATGAAGTTTT CCAACGTGAT TCTTGCGGCC AGCGCGTCGA GCCTGGTGCT CGCT”
Therefore, the coded sequence of ripe gene is
Shown in SEQ ID NO.5:
This enzyme belongs to glycosyl hydrolase the 5th family.By cellulose enzyme gene cDNA sequence and the aminoacid sequence derived
It is listed in GenBank and carries out BLAST comparison and determine that this cellulase is a kind of new cellulase.
Present invention also offers the recombinant vector comprising above-mentioned cellulose enzyme gene, preferably yeast expression vector.
The cellulose enzyme gene of the present invention is inserted between the suitable restriction enzyme site of expression vector so that it is nucleotide
Sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, preferably
For between SnaBI and NotI restriction enzyme site that cellulose enzyme gene cDNA is inserted on plasmid pPIC9,
Make this nucleotide sequence be positioned at the downstream of AOXl promoter and be regulated and controled by it, obtain expression of recombinant yeast plasmid.
Present invention also offers the recombinant bacterial strain comprising above-mentioned cellulose enzyme gene, preferably recombinant pichia yeast strain.
Present invention also offers a kind of method preparing cellulase, comprise the following steps:
1) with above-mentioned recombinant vector transformed host cell, recombinant bacterial strain is obtained；
2) recombinant bacterial strain is cultivated, the expression of induction recombinant fiber element enzyme；And
3) cellulase also expressed by purification is reclaimed.
Wherein, the most described host cell is Pichia sp. (Pichia pastoris) cell, beer yeast
(Saccharomyces cerevisiae) cell or Hansenula polymorpha (Hansenula polymorpha) cell, preferably will
Expression of recombinant yeast Plastid transformation Pichia pastoris (Pichic pastoris) GS115, obtains recombinant bacterial strain.
Present invention also offers the application of above-mentioned cellulase.Use genetic engineering means to carry out industrialization and produce cellulose
Enzyme.The invention provides a new cellulase, the industry such as feedstuff, food, medicine can be applied to.According to
Technical scheme just can realize utilizing the fiber of the excellent applicable commercial Application of genetic engineering means nature of production
Element enzyme.
Accompanying drawing explanation
The SDS-PAGE that Fig. 1 cellulase is expressed in Pichia sp. analyzes.
The optimum pH of Fig. 2 recombinant fiber of the present invention element enzyme.
The pH stability of Fig. 3 cellulase of the present invention.
Fig. 4 cellulase of the present invention optimal reactive temperature.
Fig. 5 cellulase of the present invention heat stability.
Detailed description of the invention
Test material and reagent
1, bacterial strain and carrier: Pichia sp. (Pichia pastoris GS115) is that this laboratory preserves；Pichia sp. table
Reach carrier pPIC9 and bacterial strain GS115 purchased from Invitrogen company.
2, enzyme and other biochemical reagents: restriction endonuclease is purchased from TaKaRa company, ligase is public purchased from Invitrogen
Department, other is all domestic reagent (all can be commercially available from common biochemical Reagent Company).
3, culture medium:
(I) culture medium: 30g/L Testa Tritici, 30g/L maize cob meal, 30g/L bean cake, 5g/L Fructus Hordei Vulgaris Portugal gathers
Sugar, 5g/L (NH4)SO4, 1g/L KH2PO4, 0.5g/LMgSO4·7H2O, 0.01g/L FeSO4·7H2O, 0.2
g/L CaCl2In 1L deionized water, 121 DEG C, sterilization treatment 20min under the conditions of 15 pounds
(2) Escherichia coli culture medium LB (126 peptones, 0.5% yeast extract, 126NaCI, pH7.O).
(3) BMGY culture medium；1% yeast extract, 2% peptone, 1.34%YNB, 0.000049 < Biotin,
1% glycerol (v/v).
(4) BMMY culture medium: replacing glycerol divided by 0.5% methanol, remaining composition is all identical with BMGY, pH4.0.
Illustrate: following example are not made the experimental methods of molecular biology illustrated, all with reference to " molecular cloning
Experiment guide " concrete grammar listed in (third edition) J. Pehanorm Brooker one book carries out, or according to test kit
Carry out with product description.
The clone of embodiment 1 cellulase encoding genes
Extract Talaromyces leycettanus JCM 12802 genomic DNA, design cloning primer F:
Atgaagttttccaacgtgattcttgcggc and R:ctacaggcactggtagtaataggggttcag, with Talaromyces
Leycettanus JCM 12802 STb gene is that template carries out PCR amplification.PCR response parameter is: 95 DEG C of 5min；
94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 60sec, 30 circulations, 72 DEG C of 10min.Obtain an about 1.5k bp sheet
Section, obtains full-length gene after order-checking is correct.
Extract Talaromyces leycettanus JCM 12802 total serum IgE, utilize Oligo (dT)20And reverse transcription
Obtaining a chain of cDNA, then design expands primers F and the R of open reading frame, expands this strand cDNA,
Obtaining the cDNA sequence of cellulase, amplification obtains sending order-checking after product reclaims.
By to finding after the genome sequence of cellulase and cDNA sequence comparison that this cellulase structural gene is complete
Long 1464bp, containing 4 introns, the long 1230bp of cDNA, encodes 409 aminoacid and a termination codon
Son, 18 aminoacid of N end are its signal peptide sequence, prove from Talaromyces leycettanus JCM through comparison
In 12802, the gene of the encoding cellulase that separating clone obtains is new gene.
The structure of embodiment 2 cellulase engineered strain
(1) expression vector structure and in the expression of yeast
With the cDNA of the correct cellulase of order-checking as template, design has synthesized and has limited with SnaB I and Not I
The primer cdna-sF:act of property restriction enzyme sitetacgtaGctcccaagagcaagaccaagcgcacatc and cdnaR:
atagcggccgcCtacaggcactggtagtaataggggttcag, expands the coding region of the maturation protein of cellulase
Increase.And utilize SnaB I and Not I enzyme action PCR primer, connect and enter expression vector pPIC9 (Invitrogen, San
Diego), the sequence of cellulase maturation protein is inserted into the downstream of the signal peptide sequence of above-mentioned expression vector, with signal
Peptide forms correct reading frame, is built into Yeast expression carrier, converts competent escherichia coli cell Trans1.
Positive transformant carries out DNA sequencing, and order-checking shows that transformant that sequence is correct is for preparing recombiant plasmid in a large number.With
Restricted enzyme Bgl II carries out linearisation expression plasmid carrier DNA, and electroporated yeast GS115 competence is thin
Born of the same parents, cultivate 2-3 days for 30 DEG C, and the transformant that picking grows on MD flat board further expresses experiment, tool
Gymnastics refer to Pichia anomala expression workbook.
Build the expression vector of the cDNA of cellulase signal peptide sequence in the same way, and convert.
(2) screening of high-cellulose enzymatic activity transformant
There is picking list bacterium colony the MD plate of transformant with sterilized toothpick from long, first put down to MD according to numbering
On plate, MD flat board is placed in 30 DEG C of incubators cultivation 1～2 day, grows to bacterium colony.Put down from MD by number
During on plate, picking transformant is inoculated in the centrifuge tube equipped with 3mL BMGY culture medium, 30 DEG C, the training of 220rpm shaking table
Support 48h；Shaking table is cultivated the bacterium solution 3 of 48h, and 000 × g is centrifuged 15min, removes supernatant, adds 1mL in centrifuge tube
BMMY culture medium containing 0.5% methanol, at 30 DEG C, 220rpm inducing culture；After inducing culture 48h,
3,000 × g is centrifuged 5min, takes supernatant for Enzyme assay, therefrom filters out the transformant of high-cellulose enzymatic activity,
Concrete operations refer to Pichia anomala expression workbook.
The preparation of embodiment 3 recombinant fiber element enzyme
(1) cellulose enzyme gene great expression of shaking flask level in Pichia sp.
Filter out enzyme higher transformant alive, be inoculated in the 1L triangular flask of 300mL BMGY fluid medium,
30 DEG C, 220rpm shaking table shaken cultivation 48h；5,000rpm are centrifuged 5min, softly abandon supernatant, then to thalline
Add 100mL and contain the BMMY fluid medium of 0.5% methanol, 30 DEG C, 220rpm inducing culture 72h.
During inducing culture, interval 24h adds methanol solution to compensate the loss of methanol, makes methanol concentration keep
About 0.5%；(3) 12,000 × g are centrifuged 10min, collect supernatant fermentation liquid, and detection enzymatic activity is also carried out
SDS-PAGE protein electrophoresis analyzes (Fig. 1).
(2) purification of recombinant fiber element enzyme
Collect the recombinant fiber element enzyme supernatant that shaking flask is expressed, concentrated by 10kDa film bag, use less salt simultaneously
Buffer exchange culture medium therein, then further concentrates with 10kDa super filter tube.Concentration can be diluted to one
Determine the recombinant fiber element enzyme of multiple, be purified by ion-exchange chromatography.Specifically, recombinant fiber element enzyme is taken dense
Contracting liquid 2.0mL is cloudy with HiTrap Q Sepharose XL equilibrated for 20mM Tris-HCl (pH 7.5) through in advance
Ion column, then carries out linear gradient elution with the NaCl of 0-1mol/L, detects the eluent of Fraction collection
Enzymatic activity and the mensuration carrying out protein concentration.
Embodiment 4 recombinant fiber element enzyme some properties is analyzed
Use DNS method that the cellulase of the present invention is carried out activity analysis.Concrete grammar is as follows: at pH 3.5,80 DEG C
Under the conditions of, the reaction system of 1mL includes dilution enzyme liquid suitable for l00 μ L, 900 μ L substrates, reacts l0rnin,
Add 1.5mL DNS and terminate reaction, boiling water boiling 5min.After cooling, 540nm measures OD value.Cellulase activity
Property unit definition: under certain condition, decomposition carboxymethyl cellulose per minute generates the enzyme needed for l μm ol reducing sugar
Amount is 1 active unit (U).
(1) optimum pH of cellulase and pH stability
It is the suitableeest to measure it that the cellulase that purified embodiment 4 is expressed carries out enzymatic reaction under different pH
pH.Buffer used is pH 1.0～3.0 glycine-HCI buffer, the citric acid one phosphoric acid hydrogen of pH2.2～8.0
Disodium series of buffer, pH 8.0～9.0Tris-HC buffer, lpH9.0～12 glycine-NaoH series of buffer.
The cellulase of purification is in the buffer system of different pH.PH adaptive result (Fig. 2) measured at 80 DEG C shows:.
The optimum pH of this cellulase is 3.5, in the range of pH2.5-pH5.0, this enzyme be able to maintain that its more than 60%
Enzyme activity.
Enzyme liquid is processed in the buffer of different pH value at 37 DEG C 60min, then measures enzymatic activity with studying enzyme
PH stability.Result shows (Fig. 3), and analysis result shows that this enzyme is stable between pH2.0-pH11.0, tool
There is excellent pH stability.
(2) cellulase reaction optimum temperature and heat stability
The cellulase of purification, under the conditions of pH 3.5, measures the enzymatic activity under different temperatures (40-90 DEG C), analyzes
Test result indicate that display, optimum temperature 80 DEG C, still there is when 85 DEG C the enzyme activity (Fig. 4) of 30%,
Processing 60min at 70 DEG C, enzyme is lived and is not lost, and processes 5min, it is possible to keep the enzyme of more than 70% at 75 DEG C
Vigor, processes 2min, it is possible to keep the enzyme activity of more than 60% at 80 DEG C, has good heat stability (figure
5)。
Claims (7)
1. a thermophilic cellulase, it is characterised in that the aminoacid sequence of described thermophilic cellulase such as SEQ ID
Shown in NO.1 or SEQ ID NO.2.
2. a thermophilic cellulase gene, it is characterised in that coding thermophilic cellulase described in claim 1.
Thermophilic cellulase gene the most according to claim 2, it is characterised in that its nucleotide sequence such as SEQ
Shown in ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.
4. comprise the recombinant expression carrier of fine thermophilic cellulase gene described in claim 2.
5. comprise the recombinant bacterial strain of fine thermophilic cellulase gene described in claim 2.
6. the preparation method of the thermophilic cellulase described in a claim 1, it is characterised in that the method bag
Include following steps:
1) structure comprises the recombinant expression carrier of fine thermophilic cellulase gene described in claim 2；
2) with the recombinant expression carrier transformed host strain obtained；
3) express and separate the thermophilic cellulase described in claim 1.
7. the application of the thermophilic cellulase described in claim 1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967701A (en) * | 2017-04-13 | 2017-07-21 | 中国农业科学院饲料研究所 | Acid high temperature-resisting cellulase Cel5 and its gene and application |
| CN107022535A (en) * | 2017-04-24 | 2017-08-08 | 中国农业科学院饲料研究所 | The Multidomain acidic cellulase and its gene of originated from fungus and application |
| CN107022536A (en) * | 2017-04-24 | 2017-08-08 | 中国农业科学院饲料研究所 | The cellulase variants and its gene of high catalytic efficiency and application |
| CN107988190A (en) * | 2018-01-08 | 2018-05-04 | 中国农业科学院饲料研究所 | A kind of acid protease and its encoding gene and application |
| WO2019076021A1 (en) * | 2018-04-25 | 2019-04-25 | 邦泰生物工程(深圳)有限公司 | Preparation method for hesperetin, preparation method for hesperetin intermediate, and biological enzyme used for preparing hesperetin |
| CN116426504A (en) * | 2022-10-10 | 2023-07-14 | 大理大学 | Acidophilic, halophilic, thermophilic and ion-tolerant cellulase and application thereof |
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| CN106967701A (en) * | 2017-04-13 | 2017-07-21 | 中国农业科学院饲料研究所 | Acid high temperature-resisting cellulase Cel5 and its gene and application |
| CN107022535A (en) * | 2017-04-24 | 2017-08-08 | 中国农业科学院饲料研究所 | The Multidomain acidic cellulase and its gene of originated from fungus and application |
| CN107022536A (en) * | 2017-04-24 | 2017-08-08 | 中国农业科学院饲料研究所 | The cellulase variants and its gene of high catalytic efficiency and application |
| CN107022535B (en) * | 2017-04-24 | 2020-07-14 | 中国农业科学院饲料研究所 | Multi-domain acid cellulase derived from fungi as well as gene and application thereof |
| CN107022536B (en) * | 2017-04-24 | 2020-09-18 | 中国农业科学院北京畜牧兽医研究所 | Cellulase mutant with high catalytic efficiency, and gene and application thereof |
| CN107988190A (en) * | 2018-01-08 | 2018-05-04 | 中国农业科学院饲料研究所 | A kind of acid protease and its encoding gene and application |
| CN107988190B (en) * | 2018-01-08 | 2020-01-21 | 中国农业科学院饲料研究所 | Acid protease and coding gene and application thereof |
| WO2019076021A1 (en) * | 2018-04-25 | 2019-04-25 | 邦泰生物工程(深圳)有限公司 | Preparation method for hesperetin, preparation method for hesperetin intermediate, and biological enzyme used for preparing hesperetin |
| CN116426504A (en) * | 2022-10-10 | 2023-07-14 | 大理大学 | Acidophilic, halophilic, thermophilic and ion-tolerant cellulase and application thereof |
| CN116426504B (en) * | 2022-10-10 | 2024-07-26 | 大理大学 | Acidophilic, halophilic, thermophilic and ion-tolerant cellulase and application thereof |
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Application publication date: 20160824 |