CN102965384A - Novel lipase gene, lipase production strain and application - Google Patents
Novel lipase gene, lipase production strain and application Download PDFInfo
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- CN102965384A CN102965384A CN2012103880555A CN201210388055A CN102965384A CN 102965384 A CN102965384 A CN 102965384A CN 2012103880555 A CN2012103880555 A CN 2012103880555A CN 201210388055 A CN201210388055 A CN 201210388055A CN 102965384 A CN102965384 A CN 102965384A
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- pichia pastoris
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- 108090001060 Lipase Proteins 0.000 title claims abstract description 63
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- 235000019421 lipase Nutrition 0.000 title claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 title abstract description 4
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Classifications
-
- 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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- Enzymes And Modification Thereof (AREA)
Abstract
Belonging to the field of enzymatic deinking, the invention discloses a novel lipase gene, a lipase production strain and application. The novel lipase gene is obtained by codon optimization, and can realize high expression in Pichia Pastoris. By connecting the gene to a Pichia Pastoris expression plasmid pPICZ alpha, a plasmid pPICZ alpha A-ARL can be obtained. Then the plasmid pPICZ alpha A-ARL is employed to convert a Pichia Pastoris host bacterium Pichia Pastoris X33, thus obtaining the lipase production strain Pichia Pastoris X33/pPICZ alpha A-ARL. The strain can achieve high-efficiency lipase expression. The expressed lipase has an activity of 2500U/mL, and has medium and high temperature resistance as well as alkali resistance, thus being applicable to old newspaper deinking effectively.
Description
Technical field
The present invention relates to the Enzymatic Deinking field, the Pichi strain and this gene and the application of Pichi strain in the waste newsprint deinking that are specifically related to a kind of novel lipase gene and produce this enzyme.
Background technology
The pulping and paper-making industry is mainstay of the national economy industry, in recent years in order to alleviate the pressure of resource, the energy and environmental protection aspect, one after another sight is turned to the recycling of secondary stock, and along with the more participations of ultimate consumer in the paper circulation, provide more old newspaper (ONP).The key of ONP recycling is deinking.Traditional Chemical Deinking makes deinking wastewater COD value high owing to adopt a large amount of strong basicity pharmaceutical chemicalss, and pollution load is large; Chemical Deinking efficient is low in addition, and the drawbacks such as the fibre strength loss is large, Deinking Pulp poor quality are along with significantly raising and the complicated of waste paper kind of waste paper usage quantity are become clear day by day.The biological enzyme deinking is to utilize one or more zymin instead of chemical medicines of cellulase, zytase, lipase to process waste paper, but facts have proved this method Effective Raise printing ink removal efficiency, improve whiteness and the yield of Deinking Pulp, improve the physicals of Deinking Pulp, what is more important can effectively reduce the pollution load of waste water, belongs to the environmental type project that current pulping and paper-making industry is badly in need of.
At present, domestic only have a few patents to propose Enzymatic Deinking, and most of Biologic Deinking Agent main component that proposes is cellulase, zytase.These two kinds of enzymes are used for deinking, because own direct effect Mierocrystalline cellulose itself causes that paper pulp fiber decomposes, so there is the deficiency that reduces paper pulp difficulty and yield.
1998, the scholars such as Ming Chuan Hong are from radioprotective acinetobacter calcoaceticus CMC-1(Achetobacter radioresistens CMC-1 lipase) found a kind of alkaline lipase, its molecular weight 45kDa(SDS-PAGE measures), iso-electric point about 5.2, optimum reaction conditions is middle temperature alkaline environment, show 1,3 specificity during take three oleoyl glyceride as substrate.In alkaline environment, this lipase shows very high pH adaptive and stability, and is not subjected to Fe
2+, Ca
2+, Mg
2+, Ni
2+, Cu
2+, Mn
2+, Cd
2+Deng cation recognition, and the organic solvent such as EDTA, 2 mercapto ethanol, dithiothreitol (DTT) in demonstrate high stability.1998, TA-JUNG WANG etc. carried out a batch fermentative production ARL with the radioprotective acinetobacter calcoaceticus as producing bacterial strain, and output is 30U/mL.Calendar year 2001, Chen-You Li etc. carries out radioprotective acinetobacter calcoaceticus feed supplement batch fermenting experiment take tween 80 as carbon source, and ARL output also only reaches 120U/mL.But after this do not see the report that is applied to produce about more these enzymes, its reason may be that the expression amount at original strain is low thereby limit its further exploitation as industrial enzymes.
Summary of the invention
The shortcoming that one of purpose of the present invention is to overcome prior art provides a kind of novel lipase gene with not enough, and this novel lipase gene can efficiently express in pichia spp.
Another object of the present invention is to provide a kind of plasmid that contains above-mentioned novel lipase gene.
A further object of the present invention is to provide a kind of bacterial strain that carries above-mentioned novel lipase gene.
Another purpose of the present invention is to provide a kind of lipase to produce bacterial strain.
The present invention also aims to provide the application of above-mentioned novel lipase gene.
Purpose of the present invention is achieved through the following technical solutions: a kind of novel lipase gene, its nucleotide sequence is shown in SEQ ID NO.1.
The nucleotide sequence of described novel lipase gene is with the state-run biotechnology (NCBI of information center of the U.S., http://www.ncbi.nlm.nih.gov/) the upper Achetobacter radioresistens CMC-1lipase(GenBank:AF073953.1 that announces) gene order is the basis, this sequence is optimized, replace to the codon of pichia spp preference, obtain by full gene is synthetic again.This novel lipase gene can efficiently express in pichia spp.
A kind of plasmid pPICZ α A-ARL that contains the novel lipase gene obtains by the novel lipase gene being connected among the Pichia anomala expression plasmid pPICZ α.
A kind of bacterial strain intestinal bacteria TOP10/pPICZ α A-ARL Escherichiacoli TOP10/pPICZ α A-ARL that carries the novel lipase gene, this bacterial strain on August 14th, 2012 in the center preservation of Chinese Typical Representative culture collection, preserving number is: CCTCC NO:M 2012309, the preservation address is Wuhan City, Hubei Province Wuhan University (430072).
Described bacterial strain Escherichia coli TOP10/pPICZ α A-ARL carries plasmid pPICZ α A-ARL.
A kind of lipase is produced bacterial strain Pichia Pastoris X33/pPICZ α A-ARL, obtains by plasmid pPICZ α A-ARL being transformed pichia spp Host Strains Pichia Pastoris X33.
Above-mentioned novel lipase gene, plasmid pPICZ α A-ARL, bacterial strain Escherichia coli TOP10/pPICZ α A-ARL or the application of bacterial strain Pichia Pastoris X33/pPICZ α A-ARL in the waste newsprint deinking.
Pichia spp is the yeast-like fungi in the methyl alcohol nutritional type yeast, can utilize methyl alcohol as sole carbon source and the energy.As eukaryotic expression system, has the not available advantage of many other protein expression systems: have strong alcohol oxidase (AOX1) gene promoter, can strictly regulate and control the expression of foreign protein; The yeast growth reproduction speed is fast, nutritional requirement is low, substratum is cheap; Foreign gene can be by plasmid integration to the pichia spp genome, and foreign gene can not occur to lose with growth and breeding; Expression amount is high, and many albumen can reach more than every liter of gram level; Simultaneously, the albumen (background albumen) of pichia spp self secretion is considerably less, is conducive to purifying.
The present invention has following advantage and effect with respect to prior art:
(1) novel lipase gene provided by the invention can be realized high expression level in pichia spp after codon optimized, and vigor is 2500U/mL, and the lipase of expression has anti-middle high temperature, alkaline-resisting character.
(2) lipase of bacterial strain Pichia Pastoris X33/pPICZ α A-ARL production provided by the invention is used for waste newsprint (ONP) deinking, evidence, the gained pulp brightness is 51.49%ISO, residual ink value 472.06mg/kg, tearability 14.32mN.m
2/ g, bursting strength 2.41KPa.m
2/ g, tensile strength 30.52N.m/g.Simultaneously, use this lipase-deinked, greatly reduce chemical oxygen demand (COD) (the Chemical Oxygen Demand in the waste water, the discharging of pollutent such as COD), in the deinking wastewater, suspended solid SS(suspend solid) be 187.69mg/L, COD is 269.50mg/L, total organic carbon TOC(Total organic carbon) 3.24g/L, the waste water load index is lower than chemical deinking more than 50%.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited to this.
Synthesizing of embodiment 1 novel lipase gene
With the state-run biotechnology (NCBI of information center of the U.S., http://www.ncbi.nlm.nih.gov/) the upper Achetobacter radioresistens CMC-1lipase(GenBank:AF073953.1 that announces) gene order is the basis, this sequence is optimized, the codon that replaces to the pichia spp preference obtains the novel lipase gene, its sequence obtains plasmid pUC57-ARL with the novel lipase gene clone by the full gene of biotech company's (Jin Sirui bio tech ltd) is synthetic again to the pUC57 plasmid shown in SEQ ID NO.1.
Embodiment 2 contains the plasmid pPICZ α A-ARL of novel lipase gene and carries the structure of the bacterial strain Escherichia coli TOP10/pPICZ α A-ARL of novel lipase gene
(1) according to the increase primer of this gene of the sequences Design of novel lipase gene:
Upstream primer F1:5 '-CG
GAATTCTGTAATGACGACCACGACGA-3 ' contains EcoRI restriction enzyme site (illustrating with underscore) and protection base;
Downstream primer R1:5 '-ATTAAATA
GCGGCCGCCTGAATTGGCATAAGACT-3 ' contains NotI restriction enzyme site (illustrating with underscore) and protection base.
(2) the plasmid pUC57-ARL in the embodiment 1 carries out pcr amplification as template, F1 and R1 as primer; Pcr amplification program: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 68 ℃ are extended 90s, totally 30 circulations; 68 ℃ of extensions of the 30th circulation 7min.
(3) with PCR product and plasmid pPICZ α A(available from Invitrogen) with EcoR I and Not I double digestion, 16 ℃ of lower connections with the T4 ligase enzyme spend the night.
(4) connect product and transform e. coli host bacteria TOP10, through the Zecoin(bleomycin) resistant panel screening positive transformant, transformant extracts plasmid and obtain recombinant plasmid pPICZ alpha A-ARL after double digestion (EcoR I and Not I double digestion) is identified correctly.
Identify that correct positive transformant is the bacterial strain intestinal bacteria TOP10/pPICZ α A-ARL Escherichia coli TOP10/pPICZ α A-ARL that carries the novel lipase gene, this bacterial strain on August 14th, 2012 in the center preservation of Chinese Typical Representative culture collection, preserving number is: CCTCC NO:M 2012309, the preservation address is Wuhan City, Hubei Province Wuhan University (430072).
Embodiment 3 lipase are produced the structure of bacterial strain Pichia Pastoris X33/pPICZ α A-ARL
Adopt lithium chloride (LiCl) method to use Sac I enzyme enzyme to cut the pPICZ α A-ARL(of linearizing embodiment 2) Plasmid Transformation Pichia pastoris X33 (available from Invitrogen), at the Zeocin(of 0.1～2mg/mL bleomycin) resistance YPD flat board screens.The genomic dna of the transformant that obtains with screening is as template, and upstream primer F1 and downstream primer R1 are that primer carries out PCR and identifies that PCR identifies that correct transformant is that lipase is produced bacterial strain Pichia Pastoris X33/pPICZ α A-ARL.
Embodiment 4Pichia Pastoris X33/pPICZ α A-ARL fermentative production lipase
The single colony inoculation of picking Pichia Pastoris X33/pPICZ α A-ARL is in the YPD shaking flask, and 30 ℃, 250rpm are cultivated about 20h and carried out actication of culture; With the bacterium liquid of activation in the 6%(volume percent) ratio inoculates in the YPD shaking flask 30 ℃, 250rpm and cultivates about 20h; Then adopt the flame inoculation method in the 8%(volume percent) ratio be inoculated into (initial fermentation volume 5L) in the fermentor tank that 10L is equipped with the BSM substratum, and add 4.35mL/L PTM1 and replenish salts solution, begin fermentation.In the glycerine batch fermentation stage, pH is 5.5 with the control of mass percentage concentration 25% ammoniacal liquor, about 10L/min that ventilates, and culture temperature is 30 ℃.Begin stream when cultivating about 19h and add 50%(w/v) glycerine (containing PTM1 12mL/L), the about 11.1gL of glycerine feed rate
-1h
-1, be cultured to thalline and reach finite concentration (OD
600Be about 120～200).The glycerine feed supplement is complete regulates pH to 6.0 afterwards with mass percentage concentration 25% ammoniacal liquor, and temperature is down to 25 ℃, and hungry about 30min residual glycerol to the fermented liquid exhausts (take the dissolved oxygen numerical stability as index), begins to carry out methanol induction.Use first that (10～15g/h) streams add methyl alcohol, and behind the cell adapted methyl alcohol, every approximately 2h suitably increases methanol feeding speed in fermented liquid, and finally methanol feeding is about 30 ± 2g/h than slug flow speed the initial stage of inducing.Lipase enzyme enzyme is lived and is 2500U/mL in the fermented liquid.This fermented liquid supernatant can directly be applied to follow-up ONP deinking as lipase enzyme liquid and process.
The measuring principle of lipase activity: lipase is hydrolysis substrate p-nitrophenyl phenolic ester under certain condition, generate p-NP and lipid acid, the amount of p-NP and reaction solution shade are directly proportional within the specific limits, survey its absorbancy under 405nm, thereby calculate lipase activity.
The measuring method of lipase activity: enzyme liquid adding distil water is diluted to suitable extension rate, get 50 μ L in 900 μ LTris-HCl damping fluids (pH9.0), the p-NP octanoate that adds 50 μ L50mM, 50 ℃ of lower accurate response 5min, after taking out immediately the flowing water cooling, centrifugal rear its absorbancy of measuring under 405nm is according to the absorbancy of test solution, by the concentration of the p-NP in the p-NP typical curve calculation sample, calculate lipase activity.
Lipase activity power is defined as: under 50 ℃, the condition of pH9.0, per minute is degraded from the p-NP octanoate solution of 50mM, and to discharge the needed enzyme amount of 1mol p-NP be an enzyme activity unit.
The zymologic property of embodiment 5 lipase is measured
The lipase that present embodiment adopts is the fermented liquid supernatant after embodiment 4 fermentations.
(1) optimum pH of lipase
Use different pH damping fluids (6.0,7.0,8.0,9.0,10.0,11.0) (pH≤9.0 Tris-HCl damping fluid, pH〉9.0 use the glycine sodium hydroxide solution) prepare respectively p-NP octanoate substrate solution and the dilution lipase enzyme liquid of 50mM, measure the relative enzyme work of lipase under various different pH values.Lipase has good activity at alkaline condition, and Optimun pH is 9.0, is about 85% of maximum in the work of pH8.0 enzyme.
(2) optimum temperuture of lipase
At 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, measure the relative enzyme of lipase under the condition of pH9.0 and live.Under pH9.0, optimum temperature is 55 ℃.In 45 ℃～60 ℃ scopes, enzyme is lived and all can be maintained more than 85%.
The application of embodiment 6 lipase in the waste newsprint deinking
The lipase that present embodiment adopts is the fermented liquid supernatant after embodiment 4 fermentations.
At first the secondary stock raw material is carried out pre-treatment: old newsprint (ONP) is issued a magazine more than 3 months, 60 ℃ of two weeks of baking oven ageing, be shredded into 20mm * 20mm small pieces.Raw material is in water after the soaked overnight (starching dense is mass percent 10%), then move into pulping engine and carry out the enzyme processing, starch the dense 10%(mass percent that still is controlled at), the lipase consumption is the 1.5U/g oven dry stock, control pH value is 9.0, temperature 50 C behind the treatment time 25min, changes in the horizontal flotation instrument of 12L and adopts floatation deinking.Flotation process is that the mechanism of using variable grain to have different surface propertys is carried out deinking, will add tensio-active agent (select tween 80 herein, add concentration volume percent 1%) during the enzyme deinking.During flotation, reduction paste concentration is that mass percent 1%, temperature are increased to 60 ℃ (being no more than 65 ℃), and flotation instrument air flow quantity is 4～6m
3/ h collects slurry behind the flotation 7min, discongests 10000 and turns, and then copies in flakes at paper machine, and oven dry is put in sealed bag, is detected as deinking efficiency and the paper performance of paper.
Behind above-mentioned lipase treatment, pulp brightness is 51.49%ISO, residual ink value 472.06mg/kg, tearability 14.32mN.m
2/ g, bursting strength 2.41KPa.m2/g, tensile strength 30.52N.m/g.Simultaneously, use above-mentioned lipase-deinked, greatly reduce the discharging of the pollutents such as chemical oxygen demand (COD) (COD) in the waste water, in the deinking wastewater, suspended solid SS(suspend solid) be 187.69mg/L, COD is 269.50mg/L, total organic carbon TOC(Total organic carbon) 3.24g/L, the waste water load index is lower than chemical deinking more than 50%.This is because chemical deinking has added the pharmaceutical chemicalss such as caustic soda, water glass, hydrogen peroxide, and it is larger to cause the waste water load index to raise.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. novel lipase gene, it is characterized in that: the nucleotide sequence of described novel lipase gene is shown in SEQ ID NO.1.
2. a plasmid pPICZ α A-ARL who contains novel lipase gene claimed in claim 1 is characterized in that obtaining by the novel lipase gene being connected among the plasmid pPICZ α A.
3. carry the bacterial strain Escherichia coli TOP10/pPICZ α A-ARL of novel lipase gene claimed in claim 1, preserving number is CCTCC NO:M2012309.
4. a bacterial strain Pichia Pastoris X33/pPICZ α A-ARL who produces lipase is characterized in that obtaining by plasmid pPICZ α A-ARL claimed in claim 2 being transformed pichia spp Host Strains Pichia Pastoris X33.
5. novel lipase gene claimed in claim 1, plasmid pPICZ α A-ARL claimed in claim 2, bacterial strain Escherichia coli TOP10/pPICZ α A-ARL claimed in claim 3 or the application of bacterial strain Pichia Pastoris X33/pPICZ α A-ARL claimed in claim 4 in the waste newsprint deinking.
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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104278004A (en) * | 2013-07-12 | 2015-01-14 | 丰益国际有限公司 | Escherichia F6 for expressing lipase, F6 lipase and production and application of F6 lipase |
| CN103437231A (en) * | 2013-08-30 | 2013-12-11 | 华南理工大学 | Alkaline biological enzyme deinking agent and application technology thereof in waste paper deinking |
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| 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 |
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