CN106636152B - A kind of gene, expression vector, bacterial strain and its application of paper grade (stock) sterol lipase - Google Patents
A kind of gene, expression vector, bacterial strain and its application of paper grade (stock) sterol lipase Download PDFInfo
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- CN106636152B CN106636152B CN201611104306.7A CN201611104306A CN106636152B CN 106636152 B CN106636152 B CN 106636152B CN 201611104306 A CN201611104306 A CN 201611104306A CN 106636152 B CN106636152 B CN 106636152B
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- 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)
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- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- 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)
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- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
- D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
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- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
- D21C5/02—Working-up waste paper
- D21C5/025—De-inking
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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/102—Plasmid DNA for yeast
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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/64—Paper recycling
Abstract
The present invention discloses gene, expression vector, bacterial strain and its application of a kind of paper grade (stock) sterol lipase, belongs to bioengineering field.The nucleotide coding sequence of the gene is as shown in SEQ ID NO:1.Sterol lipase gene provided by the invention realizes the high expression in Pichia pastoris using technologies such as codon optimization, Bacillus coli expression, Pichia anomala expressions；Obtained sterol lipase enzyme activity is 0.4U/mL or so.The sterol lipase of expression has high temperature resistant, resistance to weak base property；Sterol lipase of the present invention can be applied to deinking, remove ink particle by the gluing object between hydrolysis waste paper surface and paper, it is ensured that the stabilization of recycled paper quality and the operational efficiency of paper machine.
Description
Technical field
The invention belongs to bioengineering fields, and in particular to one kind be capable of high efficient expression paper grade (stock) sterol lipase gene,
Expression vector, bacterial strain and its application.
Background technique
Paper industry is one of the important industry of support Chinese national economy, but scarcity of resources, energy shortage and pollution
The problems such as serious, is just restricting the development of paper industry, and expanding waste paper utilization is exactly the effective way solved these problems.But it is useless
Paper is because of its complicated component, and containing a large amount of tiny component and anionic impurity, especially gluing object is difficult to remove in these impurity
It goes, the presence of one side gluing object will increase hole, dust point on paper, reduce paper quality；On the other hand, it is shaping
Net, press section and drying section deposition easily cause disconnected paper, and water filtering performance is caused to be deteriorated, and the speed of paper machine lowers, thus gluing object
Processing be also waste paper pulp-making in key subjects.
In waste paper pulp-making, the source of gluing object is more complicated, mainly there is following source: (1) natural resin.Useless
It is mainly some remaining deinking agents in paper pulp；(2) heat melts object.A part is from for encapsulating carton, paper bag and books impurity
Adhesive, another part is from certain Water-proof and oil-proof agent in specific use paper product, such as wax and wax compound；
(3) pressure-sensitive adhesive agent.Label and valve bag are packed for from various；(4) coating adhesive.From processing paper production process
Used in various coating adhesives；(5) sizing agent.From various sized papers；(6) ink connection material and residual ink；
(7) inorganic filler and fiber fines.
Currently, the method for gluing object mainly has Mechanical Method and chemical method in removal and control slurry.Screen, purification, wash,
The physicomechanical processes such as heat partition and flotation can remove most of primary gluing object.And chemical method mainly passes through chemisorption, changes
Property, dispersion, fixation, surface passivation the effects of come achieve the purpose that control gluing object.With the development of biotechnology, have efficient
The biological enzyme of the advantages that property, specificity, no pollution to the environment is more and more widely used in pulping and paper-making field.
Gluing object also becomes a new research direction in biological enzyme control paper pulp.Most of gluing objects all contain largely can be by gluing object
The ester bond that links together of basic structure component, esters enzyme can be catalyzed ester linkage breaking, gluing object be resolved into lesser basic
Component.And ester bond, once being broken, the basic component of gluing object is difficult to regroup in systems.
Bark graceful laboratory chemical industry (Shanghai) Co., Ltd. creates for solving the problems, such as that gluing object is most in secondary stock
New technology-biological enzyme Optimyze.The esters enzyme preparation can make the ester linkage breaking in gluing object, not only decompose big gluing object
For small gluing object, and the basic component of gluing object is made to lose or reduce stickiness, small gluing object is made to be difficult to weigh in systems
New polymerization, fundamentally solves paper-making process a series of problems due to caused by gluing object using secondary stock.
It is the hot spot of processing waste paper-pulp gummy substance research in recent years using biological enzyme.It typically contains natural in ink or closes
At resin component, combine pigment and page.Therefore can use esters enzyme carrys out resin contained in partial hydrolysis ink
Or binder, size degradation ink make itself and fiber separation.Binder in treatment process mainly to decompose ink and fiber surface
Mode release ink particle, the influence to fibre strength and paper pulp yield is smaller, while glutinous to other in removal secondary stock
Property substance also has certain effect.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide one kind being capable of high efficient expression papermaking
With the gene of sterol lipase.The gene can encode high temperature resistant, alkaline-resisting, efficient stable sterol lipase.
Another object of the present invention is to provide a kind of expression vectors.
Another object of the present invention is to provide a kind of production bacterial strains.
Another object of the present invention is to provide a kind of recombination sterol lipase.
A further object of the present invention is to provide the applications of above-mentioned recombination sterol lipase.
The purpose of the invention is achieved by the following technical solution:
A kind of gene for capableing of high efficient expression paper grade (stock) sterol lipase, the nucleotide coding sequence of the sterol lipase CHE
Column are as shown in SEQ ID NO:1.
M.Nishimura etc. has cloned streptomyces lavendulae (Streptomyces lavendulae in 1994 for the first time
H646-SY2 sterol lipase (CHE) gene), by it, successful expression was simultaneously in Escherichia coli in 2006 by Hongyu Xiang etc.
Its main zymologic property is had studied, it is found that the sterol lipase has heat resistance, resistance to alkalescent, may have applied to paper maker
The ideal characterisitics of industry.But the report for being applied to production about the enzyme is not hereafter seen.Its reason may is that, first,
The expression quantity of original strain is low；Second, although Gashaw Mamo etc. is by its gene in expression in escherichia coli, the enzyme of expression
Work is relatively low, and less than 0.05U/mL, which cannot secrete the enzyme activity of secretion completely, has part to be distributed in empty in intracellular and pericentral siphon
Between and expression quantity it is not high, thus limit its further exploitation as paper industry enzyme preparation.
The present invention uses the methods of codon optimization, total gene synthesis, realizes to CHE (the sterol lipase of streptomyces lavendulae)
The molecular modification of gene and artificial establishment, obtain a kind of gene for capableing of high efficient expression sterol lipase.Inventor is according to state of the U.S.
The CHE gene order announced on vertical Biotechnology Information center (NCBI, http://www.ncbi.nlm.nih.gov/), to sequence
Column optimize, and are substituted for the codon of Pichia pastoris preference, then by the DNAWORKS software of the gene order after optimization
(http://mcl1.ncifcrf.gov/lukowski.html), the primer of design full genome synthesis, devises 2 primers
CHE gene order is synthesized, the nucleotide coding sequence of obtained sterol lipase gene is as shown in SEQ ID NO:1.
The present invention provides a kind of expression vector for carrying above-mentioned CHE gene；
The expression vector is the carrier expressed suitable for Pichia pastoris.
The expression vector is that above-mentioned CHE gene is inserted into pPICZ α A.
The expression vector is inserted into above-mentioned CHE gene in pPICZ α A between EcoRI and NotI restriction enzyme site；
It is named as pPICZ α A-CHE.
By being connected into CHE gene in commercial Pichia pastoris secretion expression carrier pPICZ α A, recombinant plasmid pPICZ alpha is constituted
A-CHE。
Expression vector pPICZ α A-CHE can be realized by following technical measures:
When synthesizing CHE using first goal of the invention the method full genome, limit is introduced respectively in upstream and downstream
Property restriction endonuclease EcoRI and NotI restriction enzyme site processed.Therefore by full genome PCR product and Pichia pastoris secretion expression carrier pPICZ α
A plasmid (invitrogen) all uses EcoRI and NotI double digestion, and Ligation in vitro construction recombination plasmid pPICZ α A-CHE obtains weight
Group expression vector pPICZ α A-CHE.
The present invention provides a kind of Pichi strain containing above-mentioned CHE gene again；
The Pichi strain contains expression vector described in one of above-mentioned expression vector.
Pichia pastoris is the yeast-like fungi in methanotrophic yeast, can use methanol as sole carbon source and energy
Source.As eukaryotic expression system, have the advantages that not available for many other protein expression systems: there is strong ethyl alcohol oxygen
Change enzyme (AOX1) gene promoter, can strictly regulate and control the expression of foreign protein；Yeast growth reproduction speed is fast, nutritional requirement is low,
Culture medium is cheap；Foreign gene can by plasmid integration to pichia pastoris yeast genome, foreign gene will not occur with
Growth and breeding and lose；Expression quantity is high, and many albumen can reach every liter of gram-grade or more；Meanwhile the egg of Pichia pastoris itself secretion
White (background proteins) are considerably less, are conducive to purify.
Pichia pastoris gene engineering bacterial strain Pichia Pastoris X33/pPICZ α A-CHE can be arranged by following technology
It applies realization: the recombinant plasmid pPICZ alpha A-CHE conversion that Sac I is linearized is finished by red saccharomyces pastorianus host strain with LiCl method
Pichia Pastoris X33, conversion product are coated on MD plate, cultivate 2~3 days.Transformant on MD plate is inoculated with respectively
In on Zeocin (bleomycin) resistance YPD plate containing various concentration, cultivate 3~5 days.By high concentration Zeocin-YPD plate
Its corresponding monoclonal of the transformant picking of upper appearance extracts pastoris genomic dna as mould by Invitrogen operating guidance
Plate carries out Yeast genome PCR identification, obtains recombinant conversion.
Picking is accredited as positive transformant through recombinant yeast pichia pastoris bacterium colony PCR and is seeded to 25mL in superclean bench
In BMGY fluid nutrient medium (250mL conical flask liquid amount is 25mL), 30 DEG C, 250rpm shaken cultivation to OD600=2~6, together
When be inoculated with recombinant yeast pichia pastoris X33/pPICZ α A as negative control；Collection part culture solution is centrifuged in room temperature 8000rpm
2min；Thallus (250mL conical flask liquid amount is 25mL) into 25mL BMMY culture medium is shifted, controls and originates OD600=1,30
DEG C, 250rpm shake culture, per sampling analysis thalli growth situation and producing enzyme situation for 24 hours, and add 1% methanol to culture medium with
Successive induction thallus producing enzyme terminates after the 120~140h that ferments, and sterol lipase enzyme activity is 0.4U/mL or so in fermentation liquid.
A kind of recombination sterol lipase, is to be fermented to obtain by above-mentioned Pichi strain.The recombination sterol lipase
Enzyme activity be 0.4U/mL.
Application of the gene, expression vector, Pichi strain, recombination sterol lipase in deinking.
The present invention compared with the existing technology, have following advantages and effects
Sterol lipase gene provided by the invention utilizes the skills such as codon optimization, Bacillus coli expression, Pichia anomala expression
Art realizes the high expression in Pichia pastoris；The sterol lipase of expression has high temperature resistant, resistance to weak base property；Steroid of the present invention
Alcohol lipase can be applied to deinking, remove ink particle by the gluing object between hydrolysis waste paper surface and paper, can be with
Guarantee the stabilization of recycled paper quality and the operational efficiency of paper machine.
Detailed description of the invention
Fig. 1 is building pPICZ α A-CHE carrier schematic diagram.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Specific experiment method of the invention is referred to Molecular Cloning:A Laboratory guide (third edition, Cold Spring Harbor Publications are published)
And Pichia Fermentation Process Guidelines (Invitrogen).
It in following embodiment unless otherwise indicated, is routine experiment method and operating procedure in the art.
Embodiment 1 synthesizes sterol lipase gene (CHE)
Sterol lipase (CHE) gene order and protein structure of streptomyces lavendulae (S.lavendulae H646-SY2) are believed
Breath derives from US National Biotechnology Information center (NCBI, http://www.ncbi.nlm.nih.gov/) and protein data
Library (PDB, http://www.rcsb.org/pdb/search/advSearch.do).
According to the CHE gene order announced on NCBI, sequence is optimized, is all substituted for the close of Pichia pastoris preference
Numeral, then by the gene order after optimization with DNAWORKS software (http://mcl1.ncifcrf.gov/ lukowski.html), the primer of design full genome synthesis devises 2 primers, wherein 2 primers introduce EcoRI respectively
Base, primer sequence are protected with NotI restriction enzyme site and digestion are as follows:
CHE-F:5 '-CCGGAATTCATGTCCTCCCAAGTT-3′；(horizontal line part is EcoRI restriction enzyme site)
CHE-R:5 '-ATAAGAATGCGGCCGCTTAATGG-3′；(horizontal line part is NotI restriction enzyme site)
It is answered in 25 μ L hair and carries out PCR amplification, reaction condition in system are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55
DEG C annealing 30s, 72 DEG C of extensions 10min, totally 30 recycle；30th circulation, 72 DEG C of extension 10min, PCR product gel electrophoresis
Be stored in after identification -20 DEG C it is spare.
The plasmid of the preparation of embodiment 2 gene containing CHE
The PCR product electrophoresis that embodiment 1 is obtained, the purpose band at gel extraction about 700bp, recovery product warp
The processing of EcoRI and NotI double digestion is used with the expression vector pPICZ α A equally recycled through EcoRI and NotI double digestion, glue
The connection of T4DNA ligase.10 μ L volumetric reaction systems are as follows: fully synthetic 6 μ L of gene product is added in 1 μ L (50ng) of pPICZ α A,
10 × Buffer, 1 μ L, T4DNA ligase, 1 μ L containing ATP, uses ddH2O complements to 10 μ L.It is slightly centrifuged, 16 DEG C of water-bath connections
Overnight, and in Transformed E .coli Top10 (invitrogen), in the LB plate for containing Zeocin (bleomycin, 25 μ g/mL)
Upper screening positive transformant.Random a certain amount of transformant of picking, prepares plasmid in a small amount, through restriction enzyme EcoRI and
After the processing of NotI double digestion, electrophoretic analysis is carried out.The plasmid of target gene is successfully connected after digestion, it is contemplated that 700bp can be obtained,
With two segments of 3.5kb, plasmid enzyme restriction identification is correctly.Recombinant plasmid is sequenced, it was demonstrated that sterol lipase CHE gene encoder block is complete
Correctly.Recombinant plasmid is named as pPICZ α A-CHE recombinant plasmid.Building schematic diagram such as Fig. 1 institute of pPICZ α A-CHE carrier
Show.
High efficient expression of the 3 sterol lipase CHE of embodiment in Pichia pastoris
The pPICZ α A-CHE that complete embodiment 2 is linearized through I enzyme of Sac is converted into Pichia by LiCl method
pastoris X33(invitrogen).Conversion product is coated with MD plate, and 30 DEG C are cultivated 2 days.By the transformant difference on MD plate
It is inoculated in containing 100 μ g/mL of Zeocin, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL, on the YPD plate of 500 μ g/mL, 30 DEG C of trainings
It supports 3 days.The transformant picking monoclonal that will occur on higher concentration Zeocin-YPD plate.It is mentioned by Invitrogen operating guidance
It takes pastoris genomic dna as template, carries out Yeast genome PCR identification, overall reaction using the PCR primer of objective gene sequence
Volume is 20 μ L, and Taq enzyme amount is 2U, and 2 μ L PCR products is taken to carry out 0.8% agarose gel electrophoresis identification.
Pcr amplification product detects discovery through 1% agarose gel electrophoresis, amplifies the purpose that expected size is 700bp
Band.
Picking is accredited as positive transformant through recombinant yeast pichia pastoris bacterium colony PCR and is seeded to 25mL in superclean bench
In BMGY fluid nutrient medium (250mL conical flask liquid amount is 25mL), 30 DEG C, 250rpm shaken cultivation to OD600=2~6, together
When be inoculated with recombinant yeast pichia pastoris X33/pPICZ α A as negative control；Collection part culture solution is centrifuged in room temperature 8000rpm
2min；Thallus (250mL conical flask liquid amount is 25mL) into 25mL BMMY culture medium is shifted, controls and originates OD600=1,30
DEG C, 250rpm shake culture, per sampling analysis thalli growth situation and producing enzyme situation for 24 hours, and add 1% methanol to culture medium with
Successive induction thallus producing enzyme terminates after the 120~140h that ferments, and sterol lipase enzyme activity is 0.4U/mL or so in fermentation liquid.
The measuring principle of enzyme activity: sterol lipase hydrolyzes p-nitrophenol caprylate substrate under the conditions of certain temperature and pH,
Then high speed centrifugation terminates enzyme reaction, and settles unhydrolysed p-nitrophenol butyrate, and reaction solution has suction to ultraviolet light
It receives, the light absorption value under 405nm wavelength can be measured with microplate reader, calculates enzyme activity further according to standard curve.
The measuring method of enzyme activity:
(1) p-nitrophenol butyrate substrate reactions liquid: 0.523g p-nitrophenol butyrate, 0.25mL Trion is prepared
X-100,100mL ultrapure water.High speed homogenate emulsifies 5min.It is dispensed into 2mL EP pipe with the amount of every pipe 1mL.- 20 DEG C are protected from light guarantor
It deposits.
(2) it takes the phosphate buffer of 900 μ L in 2mL EP pipe, 50 μ L fermented supernatant fluids is added.50 DEG C, 800rpm is kept
5min。
(3) 50 μ L substrate reactions liquids are added, 5 DEG C, 800rpm reacts 15min.To inactivate supernatant as control.
(4) 10000rpm is carried out immediately after reaction, 1min centrifugation terminates reaction.
(5) take 200 μ L reaction solutions in ELISA Plate, 3 Duplicate Samples are arranged in each sample, survey OD600 with microplate reader.
Enzyme activity is defined as: under conditions of 50 DEG C, pH8.0, bottom exploded object per minute generates 1 μm of ol p-nitrophenol
For an enzyme-activity unit.
The zymologic property of embodiment 4CHE sterol lipase
The CHE sterol lipase that the present embodiment uses for the fermentation of embodiment 3 after supernatant.
1, the optimum pH and optimum temperature of CHE sterol lipase
It takes the buffer of 900 μ L pH 8.0 to be added in 1.5mL centrifuge tube respectively, 50 μ of fermented supernatant fluid after purification is added
30 DEG C after L, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C of preheating 5min, be added 50 μ L substrate p-NPC (p-nitrophenol is pungent
Acid esters) reaction 5min, opposite enzyme activity is measured using multi-function microplate reader.At ph 8.0, optimum temperature is 50 DEG C.45
DEG C~55 DEG C within the scope of, enzyme activity can maintain 75% or more, even if pH9.0 condition, when temperature is 60 DEG C, opposite enzyme activity is still
It is maintained at 60% or more.
It takes the buffer of 900 6.0~pH of μ L pH 11.0 to be added in 1.5mL centrifuge tube respectively, fermentation after purification is added
50 DEG C of preheating 5min after 50 μ L of supernatant are added 50 μ L substrate p-NPC (p-nitrophenol caprylate) and react 5min, use more function
It can the opposite enzyme activity of microplate reader measurement.CHE sterol lipase Optimun pH is that opposite enzyme activity is tieed up in the range of 8.0, pH7.0~9.0
It holds 70% or more；After pH < 5.0 or 10.0 >, the enzyme activity of CHE sterol lipase drops to 30% or less.
2, the heat resistance and alkali resistance of CHE sterol lipase
In order to investigate the temperature stability of the recombinase, by lipase after purification respectively in 30 DEG C, 35 DEG C, 40 DEG C, 45
DEG C, 50 DEG C, 55 DEG C, 60 DEG C, keep the temperature 1 hour in 65 DEG C of water-baths after, remaining enzyme activity is measured under the conditions of pH 9.0, temperature 50 C
Power.For CHE sterol lipase after 45 DEG C, 50 DEG C and 55 DEG C heat preservation 1h, sterol lipase activity can maintain 80% or more；50℃
And at 45 DEG C, CHE half-life period be 6h or more (when 6h, 60%) vigor still retains, under the conditions of 55 DEG C, CHE long half time reach 2.5h
(when 3h, 40%) enzyme activity is maintained at.
CHE sterol lipase is respectively in the buffer of pH5.0~pH11.0 after 50 DEG C of processing 12h, then in optimum temperature
50 DEG C change with opposite enzyme activity is measured under the conditions of optimal pH 8.0.After above-mentioned processing, opposite enzyme activity can maintain 50% or more,
And when pH7.0~pH9.0, opposite enzyme activity is maximum 80% or more enzyme activity, and CHE sterol lipase is suitble to make under weak basic condition
With.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>South China Science & Engineering University
<120>a kind of gene, expression vector, bacterial strain and its application of paper grade (stock) sterol lipase
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 684
<212> DNA
<213> Artificial Sequence
<220>
<223>nucleotide coding sequence of sterol lipase CHE
<400> 1
atgtcctccc aagttcgggg aggtactaga tggaagagat ttgctttggt tatggttcct 60
tctattgctg ctactgctgc tgttggagtt ggtttggctc aaggggcttt ggctgcttcc 120
ttttccgtta gtggtcaaga ttttaaggtt tccgctgata agttggatgg agataacttg 180
attcaatacg gaggtattgc tgaaggtcat gatttgaagg gaaacgctca acatcatcca 240
gttactattt caggtttttc tcatgctgaa attactaata tgtgtcaatc cttggttact 300
ccaactccat tgggtaacat tactttgcaa ttgagaactg gtcataaggg aaagccagct 360
gttgctgata acatctactt ggatgttgct gaattggata ctgatgctga gtttaagaac 420
ttggatattg gtgttgctgt tggagaccct aaccataaga ctaagccaca agctggaact 480
gttgcttctc catacgcttt ttcacaacgt gctcatcaag ataaggctta cttgattttg 540
actaacgtta gacaaaaggc ttgggctact actagggctc attttcaaaa gggaactttt 600
aagttgccag atttgaagtt gagattgttg ggtggagatc aaagacattt tgatcaacca 660
tgttaccaag atattaagga ttaa 684
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> CHE-F
<400> 2
ccggaattca tgtcctccca agtt 24
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> CHE-R
<400> 3
ataagaatgc ggccgcttaa tgg 23
Claims (8)
1. the gene that one kind is capable of high efficient expression paper grade (stock) sterol lipase, it is characterised in that:
The nucleotide coding sequence of the gene of the sterol lipase is as shown in SEQ ID NO:1.
2. a kind of expression vector containing the gene described in claim 1 for capableing of high efficient expression paper grade (stock) sterol lipase.
3. expression vector according to claim 2, it is characterised in that:
The expression vector is the carrier expressed suitable for Pichia pastoris.
4. expression vector according to claim 2 or 3, it is characterised in that:
The expression vector is to be inserted into the gene described in claim 1 for capableing of high efficient expression paper grade (stock) sterol lipase
In pPICZ α A.
5. expression vector according to claim 4, it is characterised in that:
The expression vector is to be inserted into the gene described in claim 1 for capableing of high efficient expression paper grade (stock) sterol lipase
In pPICZ α A between EcoRI and NotI restriction enzyme site.
6. a kind of Pichi strain containing the gene described in claim 1 for capableing of high efficient expression paper grade (stock) sterol lipase.
7. Pichi strain according to claim 6, it is characterised in that:
The Pichi strain contains the described in any item expression vectors of claim 2~5.
8. described in the described in any item expression vectors of gene described in claim 1, claim 2~5, claim 6 or 7
Application of the Pichi strain in deinking.
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| cholesterol esterase [Streptomyces lavendulae];Nishimura,M.等;《GenBank: AAC60485.1》;19941108;全文 * |
| 废纸脱墨浆DCS的酶法控制研究;牟洪燕等;《造纸科学与技术》;20081231;第27卷(第5期);第11-14、22页 * |
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