CN101157900B - Alkaline pectic enzyme producing engineering strain and its construction and method for producing alkaline pectic enzyme with the same - Google Patents
Alkaline pectic enzyme producing engineering strain and its construction and method for producing alkaline pectic enzyme with the same Download PDFInfo
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Abstract
An engineering bacterium of producing alkaline pectinase, a construction thereof and a method of producing alkaline pectinase by using the alkaline pertain to the technical field of bioengineering. The invention uses nonpathogenic bacillus sp WSHB04-02 genes group as a model and adopts amplification technology of PCR. And then the sequence pel containing 1.2kb coding alkaline pectinase genes section is obtained. At the same time from the point of the demand for industrial production, the invention makes use of a shuttle expression carrier to restructure yeast Pichiapastoris(pel), which means to produce engineering bacterium CGMCC No.2143 of alkaline pectinase (E.C.4.2.2.2). The invention also provides CGMCC No.2143 as the method of producing alkaline pectinase for fermented yeast strain. The genes engineering bacterium aims at exocytosis of protein and the species of extracellular protein is few, which simplifies the purification technique of producing alkaline pectinase by using microorganism and reduces the cost and shoetens the time for ferment. Besides, the invention improves the production efficiency and lays a foundation for industrialization of the ferment method of producing alkaline pectinase by using microorganism.
Description
Technical field
One strain product alkaline pectin enzyme engineering bacteria and structure thereof and the method for producing alkaline pectase with this bacterium belong to technical field of bioengineering.
Background technology
Polygalacturonase is meant the class of enzymes of energy decompose pectin matter, and alkaline pectase (E.C.4.2.2.2) is meant a class polygalacturonase that has greater activity in alkaline range.Alkaline pectase has become vital biological enzyme formulation in the cotton fabric preprocessing process except being applied at aspects such as the purifying of plant virus, association with pulp bleaching.The use of alkaline pectase will change traditional alkali refining process, not only reduce environmental pollution, save a large amount of process waters, and can improve the quality of cotton-spinning fabric.
Over past ten years, the foreign study personnel attempt to utilize genetic engineering means to make up pectin ester lyase superior strain always, derive from Erwinia, alkaline pectase such as Pseudomonas and Bacillus gene has obtained expression by genetically engineered, these expression mainly are expressive host with E.coli., its phraseology nearly all with IPTG as inductor.Though IPTG applies to laboratory study widely,, therefore limited it in industrial use because it costs an arm and a leg.
The constructed alkaline pectase gene overwhelming majority of foreign study expresses in the born of the same parents, and this has brought inconvenience to this enzyme of industrialized extraction.Therefore, construct high yield, effectively to be secreted into born of the same parents outer and to induce the genetic engineering bacterium of mode cheapness be research topic anxious to be solved.
China was also studied alkaline pectase since the beginning of the nineties in last century, generally all rested on bacterial screening and reached the enzyme characteristic research stage, and domestic almost to report about the engineering research of alkaline pectin ester lyase gene.
Summary of the invention
The method that the purpose of this invention is to provide strain product alkaline pectin enzyme engineering bacteria and structure thereof and produce alkaline pectase with this bacterium, the industrialization of producing alkaline pectase for biological process lays the first stone.
Technical scheme of the present invention:
Alkaline pectase (E.C.4.2.2.2) engineering bacteria is produced in one strain, and its called after recombinant yeast pichia pastoris Pichiapastoris (pel) that classifies has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.2143.
Produce the preparation method of alkaline pectase (E.C.4.2.2.2) engineering bacteria CGMCC No.2143, be to utilize round pcr from genus bacillus (Bacillus sp) WSHB04-02, to amplify the coding alkaline pectase gene pel of 1.2kb, it is linked among the expression vector pPIC9K, obtain recombinant plasmid pPIC9K-pel, recombinant plasmid is incorporated on pichia spp (Pichia pastoris) karyomit(e) and obtains recombinant yeast pichia pastoris bacterium Pichiapastoris (pel), is to produce alkaline pectase (E.C.4.2.2.2) engineering bacteria CGMCC No.2143.
Building process is:
(1) clone of gene pel: synthesize the required primer of PCR according to pel genes involved sequences Design:
P1：5′-GCTTACGTAGCTGATTTAGGCCACCAGACG-3′
P2：5′-AACGCGGCCGCTTAATTTAATTTACCCGCACCCG-3′
Or
P1’：5′-GCTTACGTAGCTGATTTAGGCCATCAAACG-3′
P2’：5′-AACGCGGCCGCTTAATTTAATTTACCAGCACCCG-3′
SnaB I and Not I site are all introduced in the primer two ends, with genus bacillus (Bacillus sp) WSHB04-02DNA is that template is finished the PCR reaction: add following composition: Buffr 5 μ L in 200 μ L PCR reaction tubess, 2.5mmol/L DNTPs 4 μ L, template DNA 1 μ L, Taq DNAPolymerase 1 μ L, moisturizing to 50 μ L; Reaction conditions: 95 ℃ of sex change 5min, through 95 ℃ of 50s, 42 ℃ of 90s, 35 circulations of 72 ℃ of 5min, the PCR product that obtains is confirmed through electrophoretic analysis, behind PCR product purification test kit purifying, SnaB I and NotI enzyme are cut, reclaim the wherein fragment of 1.2kb, connection carrier pPIC9K obtains recombinant plasmid pPIC9K-pel;
(2) the gene bacterium makes up: will transform pichia spp after linearization of recombinant plasmid pPIC9K-pel and be integrated on the karyomit(e), coating contains kantlex G418 flat board, the picking positive transformant, obtain recombinant yeast pichia pastoris, reorganization bacterium Pichia pastoris (pel) is and produces alkaline pectin enzyme engineering bacteria CGMCC No.2143.
The technology of utilizing engineering bacteria CGMCC No.2143 to produce alkaline pectase is:
(1) starting strain: CGMCC No.2143;
(2) seed culture:
Seed culture medium is formed: in g/1000mL, and peptone 10-20, yeast extract 5-10, glucose 10-20, sodium-chlor 5-15;
Seed culture: the 250mL triangular flask, liquid amount 50mL, 28 ℃-30 ℃ of culture temperature, shaking speed 200r/min cultivates 24h (stationary phase);
(3) yeast culture
The yeast culture base is formed: in g/1000mL, and yeast nitrogen basic medium 12-15, vitamin H 0.0002-0.0005, glycerine 7-10.
Yeast culture: centrifugal collection seed, physiological saline washing 2 times changes in the 100mL substratum (adding 1mL 100 * vitamin H); 28 ℃～30 ℃ of culture temperature, shaking speed 200r/min cultivates 48h;
(4) fermentation culture:
Fermention medium is formed: in g/1000mL, and yeast nitrogen basic medium 12-15, vitamin H 0.0002-0.0005, methyl alcohol 10-50, (NH
4)
2SO
41-3, MgSO
47H
2O 0.1-0.4, FeSO
47H
2O0.003-0.007, CaCl
20.1-0.2, trace element solution 0.5-20ml, with 2mol/L KOH adjust pH 6.5-7.5,121 ℃ of sterilization 15min;
Described trace element solution: in mg/1000mL, MnSO
44H
2O 100, ZnCl
270, Na
2MoO
42H
2O 35, H
3BO
360, CoCl
26H
2O 200, CuSO
45H
2O 29, NiCl
26H
2O 25,37% hydrochloric acid 0.9ml;
Fermentation culture: 500ml triangular flask, liquid amount 30-80ml, 30 ℃ of leavening temperatures, shaking speed 150-250r/min, fermentation time: 3-4 days.
Beneficial effect of the present invention: the invention provides a kind of novel gene engineering bacteria CGMCC No.2143, with the method that this bacterium produces alkaline pectase, the output of fermented liquid neutral and alkali polygalacturonase can reach 300U/mL.The advantage of the inventive method is that this genetic engineering bacterium target protein exocytosis and extracellular protein kind are few, simplified the purifying technique of microorganisms producing alkaline pectase, reduced production cost, shortened fermentation time, improved production efficiency, laid a good foundation for producing the industrialization of alkaline pectase microbe fermentation method.
The biological material specimens preservation
Recombinant yeast pichia pastoris Pichia pastoris (pel) bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 30th, 2007, be called for short CGMCC, and deposit number is CGMCC No.2143.
Embodiment
Embodiment 1 reorganization is produced alkaline pectin enzyme engineering bacteria CGMCC No.2143 and is made up
(1) clone of gene pel: synthesize the required primer of PCR according to pel genes involved sequences Design:
P1：5′-GCTTACGTAGCTGATTTAGGCCACCAGACG-3′
P2：5′-AACGCGGCCGCTTAATTTAATTTACCCGCACCCG-3′
SnaB I and Not I site are all introduced in the primer two ends, with genus bacillus (Bacillus sp) WSHB04-02DNA is that template is finished the PCR reaction: add following composition: Buffer 5 μ L in 200 μ LPCR reaction tubess, 2.5mmol/L DNTPs 4 μ L, template DNA 1 μ L, Taq DNAPolymerase 1 μ L, moisturizing to 50 μ L; Reaction conditions: 95 ℃ of sex change 5min, through 95 ℃ of 50s, 42 ℃ of 90s, 35 circulations of 72 ℃ of 5min, the PCR product that obtains is confirmed through electrophoretic analysis, SnaB I and Not I enzyme are cut behind PCR product purification test kit purifying, reclaim the wherein fragment of 1.2kb, connection carrier pPIC9K obtains recombinant plasmid pPIC9K-pel;
(2) the gene bacterium makes up: will transform pichia spp after linearization of recombinant plasmid pPIC9K-pel and be integrated on the karyomit(e), coating contains kantlex G418 flat board, the picking positive transformant, obtain recombinant yeast pichia pastoris, reorganization bacterium Pichia pastoris (pel) is and produces alkaline pectin enzyme engineering bacteria CGMCC No.2143.
Embodiment 2 reorganization are produced alkaline pectin enzyme engineering bacteria CGMCC No.2143 and are made up
Primer is used instead:
P1’：5′-GCTTACGTAGCTGATTTAGGCCATCAAACG-3′
P2’：5′-AACGCGGCCGCTTAATTTAATTTACCAGCACCCG-3′
All the other working method are with embodiment 1.
The technology that embodiment 3 utilizes engineering bacteria CGMCC No.2143 to produce alkaline pectase is:
(1) starting strain: CGMCC No.2143;
(2) seed culture:
Seed culture medium is formed: in g/1000mL, and peptone 10-20, yeast extract 5-10, glucose 10-20, sodium-chlor 5-15;
Seed culture: the 250mL triangular flask, liquid amount 50mL, 28 ℃-30 ℃ of culture temperature, shaking speed 200r/min cultivates 24h (stationary phase);
(3) yeast culture
The yeast culture base is formed: in g/1000mL, and yeast nitrogen basic medium 12-15, vitamin H 0.0002-0.0005, glycerine 7-10.
Yeast culture: centrifugal collection seed, physiological saline washing 2 times changes in the 100mL substratum (adding 1mL 100 * vitamin H); 28 ℃-30 ℃ of culture temperature, shaking speed 200r/min cultivates 48h;
(4) fermentation culture:
Fermention medium is formed: in g/1000mL, and yeast nitrogen basic medium 12-15, vitamin H 0.0002-0.0005, methyl alcohol 10-50, (NH
4)
2SO
41-3, MgSO
47H
2O 0.1-0.4, FeSO
47H
2O0.003-0.007, CaCl
20.1-0.2, trace element solution 0.5-20mL, with 2mol/L KOH adjust pH 6.5-7.5,121 ℃ of sterilization 15min;
Described trace element solution: in mg/1000mL, MnSO
44H
2O 100, ZnCl
270, Na
2MoO
42H
2O 35, H
3BO
360, CoCl
26H
2O 200, CuSO
45H
2O 29, NiCl
26H
2O 25,37% hydrochloric acid 0.9mL;
Fermentation culture: 500mL triangular flask, liquid amount 30-80mL, 30 ℃ of leavening temperatures, shaking speed 150-250r/min, fermentation time: 3-4 days.
Claims (2)
1. the alkaline pectin enzyme engineering bacteria is produced in a strain, and its called after recombinant yeast pichia pastoris bacterium Pichia pastoris (pel) that classifies has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.2143.
2. with the method for the described product alkaline pectin of claim 1 enzyme engineering bacteria CGMCC No.2143 fermentation production of alkaline pectic enzyme, it is characterized in that:
(1) starting strain: CGMCC No.2143;
(2) seed culture:
Seed culture medium is formed: in g/L, and peptone 10-20g/L, yeast extract 5-10g/L, glucose 10-20g/L, sodium-chlor 5-15g/L;
Seed culture: the 250mL triangular flask, liquid amount 50mL, 28 ℃-30 ℃ of culture temperature, shaking speed 200r/min cultivates 24h;
(3) yeast culture
The yeast culture base is formed: in g/L, and yeast nitrogen basic medium 12-15g/L, vitamin H 0.0002-0.0005g/L, glycerine 7-10g/L;
Yeast culture: centrifugal collection seed, physiological saline washing 2 times changes in the 100mL yeast culture base that is added with 1mL100 * vitamin H; 28 ℃-30 ℃ of culture temperature, shaking speed 200r/min cultivates 48h;
(4) fermentation culture:
Fermention medium is formed: in g/L, and yeast nitrogen basic medium 12-15g/L, vitamin H 0.0002-0.0005g/L, methyl alcohol 10-50g/L, (NH
4)
2SO
41-3g/L, MgSO
47H
2O 0.1-0.4g/L, FeSO
47H
2O 0.003-0.007g/L, CaCl
20.1-0.2g/L, trace element solution 0.5-20mL, with 2mol/LKOH adjust pH 6.5-7.5,121 ℃ of sterilization 15min;
Described trace element solution: in mg/L, MnSO
44H
2O 100mg/L, ZnCl
270mg/L, Na
2MoO
42H
2O 35mg/L, H
3BO
360mg/L, CoCl
26H
2O 200mg/L, CuSO
45H
2O29mg/L, NiCl
26H
2O 25mg/L, 37% hydrochloric acid 0.9mL;
Fermentation culture: 500mL triangular flask, liquid amount 30-80mL, 30 ℃ of leavening temperatures, shaking speed 150-250r/min, fermentation time: 3-4 days.
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Families Citing this family (7)
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| CN102399764B (en) * | 2011-11-04 | 2013-04-03 | 中国农业科学院饲料研究所 | Alkaline pectinase PL-STR as well as gene and application thereof |
| CN102604977B (en) * | 2011-11-22 | 2013-08-14 | 湖北大学 | Optimized nucleotide sequence of alkaline pectinase pell68s and high-level expression method thereof |
| CN102911956B (en) * | 2012-08-07 | 2014-05-14 | 江南大学 | Optimized alkaline pectinase gene and application thereof |
| CN104711246B (en) * | 2015-04-07 | 2017-11-07 | 河南中烟工业有限责任公司 | Mould and saccharomycete mixed fungus fermentation produce pectase and its application |
| CN105754884A (en) * | 2016-03-23 | 2016-07-13 | 江南大学 | Strain capable of efficiently expressing alkaline pectinase and application of strain |
| CN105802867B (en) * | 2016-05-23 | 2019-09-17 | 江南大学 | A kind of alkaline pectase secretes enhanced bacterial strain and its application |
| CN110484457A (en) * | 2019-08-21 | 2019-11-22 | 河南省医药科学研究院 | A kind of saccharomyces cerevisiae engineered yeast, purposes and the catalyst of cell surface display pectase |
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| CN1648236A (en) * | 2004-12-15 | 2005-08-03 | 江南大学 | Method or improving alkaline pectase enzyme activity in preparing alkaline pectase process by fermenting method |
| CN1699563A (en) * | 2005-05-12 | 2005-11-23 | 江南大学 | Method for improving stability of alkaline pectase in cotton spinning and refining process |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| ES2220152A1 (en) * | 2001-01-30 | 2004-12-01 | Universidade De Santiago De Compostela | Expression of PGU-1 gene for Pichia pastoris methylotrophic yeast comprises isolation from saccharomyces cerevisiae yeast to give a polygalacturonase pectinase |
| CN1648236A (en) * | 2004-12-15 | 2005-08-03 | 江南大学 | Method or improving alkaline pectase enzyme activity in preparing alkaline pectase process by fermenting method |
| CN1699563A (en) * | 2005-05-12 | 2005-11-23 | 江南大学 | Method for improving stability of alkaline pectase in cotton spinning and refining process |
Non-Patent Citations (4)
| Title |
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| 张健红 等.一株碱性果胶酶高产细菌的分离、***发育分析和产酶条件的初步优化.应用与环境生物学报11 3.2005,11(3),354~358. |
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