CN106636152A - Gene of paper-making cholesterase, expression vector, strain and applications thereof - Google Patents
Gene of paper-making cholesterase, expression vector, strain and applications thereof Download PDFInfo
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- 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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- 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
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Abstract
The invention discloses a gene of paper-making cholesterase, an expression vector, a strain and applications thereof, and belongs to the field of bioengineering. A nucleotide coding sequence of the gene is shown as SEQ ID NO:1. The cholesterase gene provided by the invention, on the basis of such technologies as codon optimization, escherichia coli expression, pichia pastoris expression and the like, achieves high expression in pichia pastoris; and the enzymatic activity of the obtained cholesterase is about 0.4U/mL. The expressed cholesterase has the properties of resisting high temperature and resisting weak base; the cholesterase provided by the invention can be applied to waste paper de-inking, namely ink particles can be removed by hydrolyzing adhesives on the surface of waste paper and between paper pieces; and the stable quality of recycled paper and the operating efficiency of a paper-making machine can be guaranteed.
Description
Technical field
The invention belongs to bioengineering field, and in particular to a kind of gene for being capable of high efficient expression paper grade (stock) sterol lipase,
Expression vector, bacterial strain and its application.
Background technology
Paper industry is one of important industry of support Chinese national economy, but scarcity of resources, energy shortage and pollution
Serious the problems such as, is just restricting the development of paper industry, expands the effective way that waste paper utilization exactly solves these problems.But it is useless
Because of its complicated component, containing substantial amounts of tiny component and anionic impurity, especially gluing thing is difficult to remove paper in these impurity
Go, the presence of one side gluing thing can increase hole, dust point on paper, reduce paper quality；On the other hand, it is in shaping
Net, press section and drying section deposition easily causes disconnected paper, causes water filtering performance to be deteriorated, and the speed of paper machine lowers, thus gluing thing
Process be also key subjects in waste paper pulp-making.
In waste paper pulp-making, the source of gluing thing 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 thing.A part is derived from for encapsulating carton, paper bag and books impurity
Adhesive, another part has the Water-proof and oil-proof agent in specific use paper product, such as wax and wax compound from some；
(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.
At present, remove and control the predominantly organic tool method of method and chemical method of gluing thing in slurry.Screen, purify, washing,
The physicomechanical processes such as heat partition and flotation can remove most of primary gluing thing.And chemical method mainly passes through chemisorbed, changes
Property, dispersion, fixation, the effect such as surface passivation to be reaching the purpose of control gluing thing.With the development of biotechnology, with efficient
The biology enzyme of the advantages of property, selectivity, environmentally safe has obtained increasingly being widely applied in pulping and paper-making field.
Gluing thing also becomes a new research direction in biology enzyme control paper pulp.Most of gluing things all contain in a large number can be by gluing thing
The ester bond that links together of basic structure component, esters enzyme can be catalyzed ester linkage breaking, gluing thing be resolved into less basic
Component.And ester bond, once rupturing, the solvent of gluing thing is difficult to regroup in systems.
The graceful laboratory chemical industry (Shanghai) Co., Ltd. of Bark create for solving the problems, such as secondary stock in gluing thing most
New technology-biology enzyme Optimyze.The esters enzyme preparation can make the ester linkage breaking in gluing thing, not only decompose big gluing thing
For small gluing thing, and make the solvent of gluing thing lose or reduce stickiness, make small gluing thing be difficult to weigh in systems
New polymerization, fundamentally solves the series of problems caused due to gluing thing using the paper-making process of secondary stock.
It is the focus for processing waste paper-pulp gummy substance research in recent years using biological enzyme.Typically contain natural in ink or close
Into resinous principle, combine pigment and page.Therefore can be using esters enzyme come resin contained in partial hydrolysis ink
Or binding agent, size degradation ink so as to fiber separation.Mainly with decomposition ink and the binder of fiber surface in processing procedure
Mode discharge ink particle, the impact to fibre strength and paper pulp yield is less, at the same to remove secondary stock in other stick
Property material also has certain effect.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, it is an object of the invention 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 vector.
Another object of the present invention is to provide a kind of production bacterial strain.
Another object of the present invention is to provide a kind of restructuring sterol lipase.
It is still another object of the present invention to provide the application of above-mentioned restructuring sterol lipase.
The purpose of the present invention is achieved through the following technical solutions：
A kind of gene for being capable of high efficient expression paper grade (stock) sterol lipase, the nucleotide coding sequence of described sterol lipase CHE
Row such as SEQ ID NO：Shown in 1.
M.Nishimura etc. has cloned first streptomyces lavendulae (Streptomyces lavendulae in 1994
H646-SY2 by it, successful expression was simultaneously in Escherichia coli in 2006 for sterol lipase (CHE) gene), Hongyu Xiang etc.
Its main zymologic property is have studied, it is found that the sterol lipase has heat resistance, resistance to alkalescent, may be had and be applied to paper maker
The ideal characterisitics of industry.But hereafter see the report that production is applied to regard to the enzyme.Its reason may is that, first,
The expression of original strain is low；Although second, Gashaw Mamo etc. by its gene in expression in escherichia coli, the enzyme of expression
Work is relatively low, and less than 0.05U/mL, the enzyme can not be secreted completely the enzyme activity of secretion, has part to be distributed in empty in intracellular and pericentral siphon
Between and expression is not high, thus limit it as the further exploitation of paper industry enzyme preparation.
The present invention is realized to CHE (the sterol lipase of streptomyces lavendulae) using methods such as codon optimization, total gene synthesis
The molecular modification of gene and artificial establishment, obtain a kind of gene for being capable of high efficient expression sterol lipase.Inventor is according to state of the U.S.
Vertical Biotechnology Information center (NCBI, http://www.ncbi.nlm.nih.gov/) on announce CHE gene orders, to sequence
Row are optimized, and are substituted for the codon of Pichia pastoris preference, then by the DNAWORKS softwares of the gene order after optimization
(http://mcl1.ncifcrf.gov/lukowski.html), the primer of full genome synthesis is designed, devise 2 primers
Synthesis CHE gene orders, the nucleotide coding sequence such as SEQ ID NO of the sterol lipase gene for obtaining：Shown in 1.
The present invention provides a kind of expression vector for carrying above-mentioned CHE genes；
Described expression vector applies to the carrier expressed in Pichia pastoris.
Described expression vector is that above-mentioned CHE genes are inserted in pPICZ α A.
Described expression vector is that above-mentioned CHE genes are inserted in pPICZ α A between EcoRI and NotI restriction enzyme sites；
It is named as pPICZ α A-CHE.
By being connected into CHE genes 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 methods described full genome, in upstream and downstream limit is introduced respectively
Property restriction endonuclease EcoRI and NotI restriction enzyme site processed.Therefore by full genome PCR primer and Pichia pastoris secretion expression carrier pPICZ α
A plasmids (invitrogen) all use EcoRI and NotI double digestions, Ligation in vitro construction recombination plasmid pPICZ α A-CHE to obtain weight
Group expression vector pPICZ α A-CHE.
The present invention provides a kind of Pichi strain containing above-mentioned CHE genes again；
Described Pichi strain contains the expression vector described in one of above-mentioned expression vector.
Pichia pastoris is the yeast-like fungi in methanotrophic yeast, it is possible to use methyl alcohol is used as sole carbon source and energy
Source.As eukaryotic expression system, have the advantages that not available for many other protein expression systems：With strong ethanol 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 on pichia pastoris phaff genome, foreign gene will not occur with
Growth and breeding and lose；Expression is high, and many albumen can reach more than per liter gram of level；Meanwhile, the egg of Pichia pastoris itself secretion
In vain (background proteins) are considerably less, are conducive to purifying.
Pichia pastoris gene engineering bacterial strain Pichia Pastoris X33/pPICZ α A-CHE can be arranged by following technology
Apply realization：The linearizing recombinant plasmid pPICZ alpha A-CHE conversions of Sac I are finished by red saccharomyces pastorianus Host Strains with LiCl methods
Pichia Pastoris X33, conversion product coats MD flat boards, cultivates 2～3 days.Transformant on MD flat boards is inoculated with respectively
On Zeocin (bleomycin) the resistance YPD flat board containing variable concentrations, cultivate 3～5 days.By high concentration Zeocin-YPD flat board
The transformant picking of upper appearance its corresponding monoclonal, pastoris genomic dna is extracted as mould by Invitrogen operating guidances
Plate, carries out Yeast genome PCR identifications, obtains recombinant conversion.
Picking Jing recombinant yeast pichia pastoris bacterium colony PCR is accredited as positive transformant and is seeded to 25mL in superclean bench
In BMGY fluid nutrient mediums (250mL conical flasks liquid amount is 25mL), 30 DEG C, 250rpm shaken cultivations to OD600=2～6, together
When inoculation recombinant yeast pichia pastoris X33/pPICZ α A as negative control；Collect part nutrient solution to be centrifuged in room temperature 8000rpm
2min；Transfer thalline (250mL conical flasks liquid amount is 25mL) into 25mL BMMY culture mediums, control starting OD600=1,30
DEG C, 250rpm concussion and cultivates, per 24h sample analysis thalli growth situations and producing enzyme situation, and add 1% methyl alcohol to culture medium with
Successive induction thalline producing enzyme, terminates after 120～140h of fermentation, and sterol lipase enzyme activity is 0.4U/mL or so in zymotic fluid.
One kind restructuring sterol lipase, is that above-mentioned Pichi strain is carried out into fermentation to obtain.Described restructuring sterol lipase
Enzyme activity be 0.4U/mL.
Application of the described gene, expression vector, Pichi strain, restructuring sterol lipase in deinking.
The present invention has the following advantages and effect relative to prior art：
The sterol lipase gene that the present invention is provided is using skills such as codon optimization, Bacillus coli expression, Pichia anomala expressions
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 applicable to deinking, be made a return journey except ink particle by hydrolyzing the gluing thing between waste paper surface and paper, can be with
Ensure the operational efficiency of the stable and paper machine of recycled paper quality.
Description of the drawings
Fig. 1 is to build pPICZ α A-CHE carrier schematic diagrames.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
The specific experiment method of the present invention is referred to Molecular Cloning:A Laboratory guide (third edition, Cold Spring Harbor Publications are published)
And Pichia Fermentation Process Guidelines (Invitrogen).
In following examples unless otherwise indicated, it is normal experiment method and operating procedure in the art.
Synthesis sterol lipase gene of embodiment 1 (CHE)
Sterol lipase (CHE) gene order of streptomyces lavendulae (S.lavendulae H646-SY2) and protein structure are believed
Breath derives from US National Biotechnology Information center (NCBI, http://www.ncbi.nlm.nih.gov/) and protein data
Storehouse (PDB, http://www.rcsb.org/pdb/search/advSearch.do).
According to the CHE gene orders 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 softwares (http://mcl1.ncifcrf.gov/ lukowski.html), the primer of full genome synthesis is designed, 2 primers are devised, wherein 2 primers introduce respectively EcoRI
With NotI restriction enzyme sites and digestion protection base, primer sequence is：
CHE-F：5′-CCGGAATTCATGTCCTCCCAAGTT-3′；(horizontal line part is EcoRI restriction enzyme sites)
CHE-R：5′-ATAAGAATGCGGCCGCTTAATGG-3′；(horizontal line part is NotI restriction enzyme sites)
Enter performing PCR amplification in answering system at 25 μ L, reaction condition is：94 DEG C of denaturations 5min；94 DEG C of denaturation 30s, 55
DEG C annealing 30s, 72 DEG C extension 10min, totally 30 circulation；30th circulation, 72 DEG C of extension 10min, PCR primer gel electrophoresis
Be stored in after identification -20 DEG C it is standby.
Embodiment 2 prepares the plasmid containing CHE genes
The PCR primer electrophoresis that embodiment 1 is obtained, cuts the purpose band at glue reclaim about 700bp, recovery product Jing
The process of EcoRI and NotI double digestions, uses with same Jing EcoRI and NotI double digestions, the expression vector pPICZ α A of glue reclaim
T4DNA ligases connect.10 μ L volumetric reaction systems are as follows：The μ L (50ng) of pPICZ α A 1, add the fully synthetic μ L of gene outcome 6,
The μ L of 10 × Buffer 1 containing ATP, the μ L of T4DNA ligases 1, use ddH2O complements to 10 μ L.Slightly it is centrifuged, 16 DEG C of water-bath connections
Overnight, and in Transformed E .coli Top10 (invitrogen), in the LB flat boards containing Zeocin (bleomycin, 25 μ g/mL)
Upper screening positive transformant.Random a certain amount of transformant of picking, prepares in a small amount plasmid, Jing restriction enzymes EcoRI and
After the process of NotI double digestions, electrophoretic analysis is carried out.The plasmid of genes of interest is successfully connected Jing after digestion, it is contemplated that 700bp is obtained,
With two fragments of 3.5kb, plasmid enzyme restriction identification is correctly.Recombinant plasmid is sequenced, it was demonstrated that sterol lipase CHE gene code frames are complete
Correctly.Recombinant plasmid is named as into pPICZ α A-CHE recombinant plasmids.Structure schematic diagram such as Fig. 1 institutes of pPICZ α A-CHE carriers
Show.
High efficient expressions of the sterol lipase CHE of embodiment 3 in Pichia pastoris
The pPICZ α A-CHE that the enzymes of Jing Sac I linearize completely embodiment 2 are converted by Pichia by LiCl methods
pastoris X33(invitrogen).Conversion product is coated with MD flat boards, and 30 DEG C are cultivated 2 days.Transformant on MD flat boards is distinguished
It is inoculated in containing the μ g/mL of Zeocin 100,200 μ g/mL, 300 μ g/mL, 400 μ g/mL, on the YPD flat boards of 500 μ g/mL, 30 DEG C of trainings
Support 3 days.The transformant picking monoclonal that will occur on higher concentration Zeocin-YPD flat boards.Carry by Invitrogen operating guidances
Pastoris genomic dna is taken as template, using the PCR primer of objective gene sequence Yeast genome PCR identifications, overall reaction are carried out
Volume is 20 μ L, and Taq enzyme amount is 2U, and taking 2 μ L PCR primers carries out 0.8% agarose gel electrophoresis identification.
The agarose gel electrophoresis detection discovery of pcr amplification product Jing 1%, amplifies the purpose that expected size is 700bp
Band.
Picking Jing recombinant yeast pichia pastoris bacterium colony PCR is accredited as positive transformant and is seeded to 25mL in superclean bench
In BMGY fluid nutrient mediums (250mL conical flasks liquid amount is 25mL), 30 DEG C, 250rpm shaken cultivations to OD600=2～6, together
When inoculation recombinant yeast pichia pastoris X33/pPICZ α A as negative control；Collect part nutrient solution to be centrifuged in room temperature 8000rpm
2min；Transfer thalline (250mL conical flasks liquid amount is 25mL) into 25mL BMMY culture mediums, control starting OD600=1,30
DEG C, 250rpm concussion and cultivates, per 24h sample analysis thalli growth situations and producing enzyme situation, and add 1% methyl alcohol to culture medium with
Successive induction thalline producing enzyme, terminates after 120～140h of fermentation, and sterol lipase enzyme activity is 0.4U/mL or so in zymotic fluid.
The measuring principle of enzyme activity：Sterol lipase hydrolyzes p-nitrophenol caprylate substrate under the conditions of uniform temperature and pH,
Then high speed centrifugation, terminates enzyme reaction, and settles unhydrolysed p-nitrophenol butyrate, and reactant liquor has suction to ultraviolet light
Receive, the light absorption value under 405nm wavelength can be determined with ELIASA, further according to calibration curve enzyme activity is calculated.
The assay method of enzyme activity：
(1) p-nitrophenol butyrate substrate reactions liquid is prepared：0.523g p-nitrophenol butyrates, 0.25mL Trion
X-100,100mL ultra-pure water.High speed homogenate emulsifies 5min.It is dispensed in 2mL EP pipes with the amount of every pipe 1mL.- 20 DEG C of lucifuges are protected
Deposit.
(2) phosphate buffer of 900 μ L is taken in 2mL EP pipes, add 50 μ L fermented supernatant fluids.50 DEG C, 800rpm keeps
5min。
(3) 50 μ L substrate reactions liquids are added, 5 DEG C, 800rpm reacts 15min.To inactivate supernatant as control.
(4) reaction carries out immediately 10000rpm, 1min centrifugation terminating reactions after terminating.
(5) 200 μ L reactant liquors are taken in ELISA Plate, each sample arranges 3 Duplicate Samples, with ELIASA OD600 is surveyed.
The definition of enzyme activity is：Under conditions of 50 DEG C, pH8.0, bottom exploded thing per minute produces 1 μm of ol p-nitrophenol
For an enzyme-activity unit.
The zymologic property of embodiment 4CHE sterol lipase
The CHE sterols lipase that the present embodiment is adopted for the fermentation of embodiment 3 after supernatant.
1st, the optimum pH and optimum temperature of CHE sterols lipase
The buffer solution for taking 900 μ L pH 8.0 respectively is added in 1.5mL centrifuge tubes, adds the μ of fermented supernatant fluid 50 after purification
30 DEG C after L, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C preheating 5min, add 50 μ L substrates p-NPC (p-nitrophenol is pungent
Acid esters) reaction 5min, using the relative enzyme activity of multi-function microplate reader measurement.At ph 8.0, optimum temperature is 50 DEG C.45
DEG C～55 DEG C in the range of, enzyme activity can maintain more than 75%, even if pH9.0 conditions, temperature be 60 DEG C when, with respect to enzyme activity still
It is maintained at more than 60%.
The buffer solution for taking 900 6.0～pH of μ L pH 11.0 respectively is added in 1.5mL centrifuge tubes, adds fermentation after purification
50 DEG C of preheating 5min after the μ L of supernatant 50, add 50 μ L substrate p-NPC (p-nitrophenol caprylate) reaction 5min, using many work(
Can the relative enzyme activity of ELIASA measurement.CHE sterol lipase Optimun pH is relative enzyme activity dimension in the range of 8.0, pH7.0～9.0
Hold more than 70%；After pH ＜ 5.0 or ＞ 10.0, the enzyme activity of CHE sterol lipase drops to less than 30%.
2nd, the heat resistance and alkali resistance of CHE sterols lipase
In order to investigate the temperature stability of the recombinase, by lipase after purification respectively 30 DEG C, 35 DEG C, 40 DEG C, 45
DEG C, 50 DEG C, 55 DEG C, 60 DEG C, in 65 DEG C of water-baths after insulation 1 hour, in pH 9.0, remaining enzyme activity is measured under the conditions of temperature 50 C
Power.After CHE sterols lipase is incubated 1h at 45 DEG C, 50 DEG C and 55 DEG C, sterol lipase activity can maintain more than 80%；50℃
And when 45 DEG C, the CHE half-life be more than 6h (during 6h, 60%) vigor still retains, under the conditions of 55 DEG C, CHE long half times reach 2.5h
(during 3h, 40%) enzyme activity is maintained at.
CHE sterols lipase respectively in the buffer solution of pH5.0～pH11.0 50 DEG C process 12h after, then in optimum temperature
Relative enzyme activity change is determined under the conditions of 50 DEG C and optimal pH 8.0.Jing after above-mentioned process, more than 50% can be maintained with respect to enzyme activity,
And be maximum enzyme activity more than 80% with respect to enzyme activity during pH7.0～pH9.0, CHE sterol lipase is adapted to make under weak basic condition
With.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>South China Science ＆ Engineering University
<120>A kind of gene of paper grade (stock) sterol lipase, expression vector, bacterial strain and its application
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 684
<212> DNA
<213> Artificial Sequence
<220>
<223>The 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 (10)
1. a kind of gene for being capable of high efficient expression paper grade (stock) sterol lipase, it is characterised in that：
The nucleotide coding sequence of the gene of described sterol lipase such as SEQ ID NO：Shown in 1.
2. a kind of expression vector of the gene for being capable of high efficient expression paper grade (stock) sterol lipase containing described in claim 1.
3. expression vector according to claim 2, it is characterised in that：
Described expression vector applies to the carrier expressed in Pichia pastoris.
4. the expression vector according to Claims 2 or 3, it is characterised in that：
Described expression vector is to be inserted into the gene for being capable of high efficient expression paper grade (stock) sterol lipase described in claim 1
In pPICZ α A.
5. expression vector according to claim 4, it is characterised in that：
Described expression vector is to be inserted into the gene for being capable of high efficient expression paper grade (stock) sterol lipase described in claim 1
In pPICZ α A between EcoRI and NotI restriction enzyme sites.
6. a kind of Pichi strain of the gene for being capable of high efficient expression paper grade (stock) sterol lipase containing described in claim 1.
7. Pichi strain according to claim 6, it is characterised in that：
Described Pichi strain contains the expression vector described in any one of claim 2～5.
8. a kind of restructuring sterol lipase, it is characterised in that be to carry out the Pichi strain described in claim 6 or 7 to ferment
Arrive.
9. it is according to claim 8 restructuring sterol lipase, it is characterised in that：
The enzyme activity of described restructuring sterol lipase is 0.4U/mL.
10. described in the expression vector, claim 6 or 7 described in gene described in claim 1, any one of claim 2～5
The application in deinking of Pichi strain, the restructuring sterol lipase described in claim 8 or 9.
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| CN201611104306.7A CN106636152B (en) | 2016-12-05 | 2016-12-05 | A kind of gene, expression vector, bacterial strain and its application of paper grade (stock) sterol lipase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111718946A (en) * | 2019-03-04 | 2020-09-29 | 中国科学院天津工业生物技术研究所 | Codon-optimized lipase gene, engineering bacterium and textile application thereof |
| WO2024055376A1 (en) * | 2022-09-15 | 2024-03-21 | 华南理工大学 | Method for treating adhesive in recycled pulp by means of pectin lyase and use |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1575363A (en) * | 2001-10-23 | 2005-02-02 | 诺维信公司 | Oxidizing enzymes in the manufacture of paper materials |
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2016
- 2016-12-05 CN CN201611104306.7A patent/CN106636152B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1575363A (en) * | 2001-10-23 | 2005-02-02 | 诺维信公司 | Oxidizing enzymes in the manufacture of paper materials |
Non-Patent Citations (2)
| Title |
|---|
| NISHIMURA,M.等: "cholesterol esterase [Streptomyces lavendulae]", 《GENBANK: AAC60485.1》 * |
| 牟洪燕等: "废纸脱墨浆DCS的酶法控制研究", 《造纸科学与技术》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111718946A (en) * | 2019-03-04 | 2020-09-29 | 中国科学院天津工业生物技术研究所 | Codon-optimized lipase gene, engineering bacterium and textile application thereof |
| CN111718946B (en) * | 2019-03-04 | 2022-04-15 | 中国科学院天津工业生物技术研究所 | Codon-optimized lipase gene, engineering bacterium and textile application thereof |
| WO2024055376A1 (en) * | 2022-09-15 | 2024-03-21 | 华南理工大学 | Method for treating adhesive in recycled pulp by means of pectin lyase and use |
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| Publication number | Publication date |
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| CN106636152B (en) | 2019-01-18 |
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