CN101037686B - Method for preparing recombinant heat-resistant xylose/dextrose isomerase by employing pHsh expression system - Google Patents
Method for preparing recombinant heat-resistant xylose/dextrose isomerase by employing pHsh expression system Download PDFInfo
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Abstract
A preparing method of recombination heat resistance xylose/glucose isomerase, including: first, extracting genome DNA of thermozymes, amplificating PCR to obtain isomerase gene, inserting the gene in gene to express carrier pHsh, inverting coliform bacteria to obtain isomerase expression plasmid; second, mutagisming gene, changing usage frequency of codon and two-level structure of mRNA to obtain optimized isomerase expression plasmid; third, shifting the isomerase expression plasmid or optimized isomerase expression plasmid into coliform bacteria to obtain gene strains, and expressing xylose/glucose isomerase by thermally stimulating and inducing; finally, collecting cells, rupturing and centrifugating to obtain rough enzyme liquid, further purifying to obtain 90% purness of recombination heat resistance xylose/glucose isomerase. The invention first uses expression carrier pHsh to efficent express heat resistance xylose/glucose isomerase gene of thermozymes, producibility of recombination enzyme using this technque is more than 200 mg/L, the product has good stability and the method is easy to industrialized produce.
Description
One, technical field
The present invention relates to fields such as molecular biology, zymetology, information biology and genetically engineered.Be specifically related to a kind of method for preparing recombinant heat-proof property xylose with the pHsh expression system.
Two, background technology
High fructose syrup is also claimed high fructose syrup (High-Fructose Corn Syrup; HFCS) or the isomery syrup; It is with the saccharification liquid of the enzyme process starch saccharification gained isomerization through xylose; Will be wherein a part of glucose isomerase become fructose, a kind of mixing sugar syrup of forming by glucose and fructose.The colourless nothing of high fructose syrup is smelt, and normal temperature current downflow property is good, easy to use; Wide material sources; Sugariness and sucrose are suitable than sucrose is low for price, can part in drink production and food-processing even whole substituting saccharoses, so it more and more receives people's attention and welcome.The market requirement of high fructose syrup constantly increases at present; Specification of quality to product is increasingly high, therefore, and one of key enzyme that xylose is produced as high fructose syrup; Become a big focus (Bandlish et al. of people's research; Biotechnologyand Bioengineering, 2002,80: 185-194).
A lot of mikrobes can produce xylose, and present widely used xylose is mainly from streptomyces (Streptomyces), actinoplanes (Actinoplanes), Flavobacterium (Flavobacterum) etc.These xylose thermostabilitys from the normal temperature mikrobe are relatively poor; (as more than 70 ℃) easy inactivation at high temperature, therefore, the isomerization reaction of glucose generally carries out about 60 ℃; Fructose content is generally about 42% in the syrup of being produced; The high density fructose syrup that desire production sugariness and sucrose are suitable (being generally 55%) need concentrate through chromatographic separating process, thereby production cost increases substantially.There is research to point out,, just can omits enrichment step and directly obtain 55% high fructose syrup if the isomerization reaction temperature reaches more than 90 ℃.Therefore, the focus of research is to seek a kind of thermotolerance xylose both at home and abroad.
Many research work are round utilizing rite-directed mutagenesis, random mutation that the normal temperature xylose is improved, though these methods have increased the understanding of people to the thermotolerance mechanism of these enzymes, the thermotolerance of enzyme not be improved significantly.In recent years work has turned to the research of the thermotolerance xylose of thermophile bacteria (Bandlish etal., Biotechnology and Bioengineering, 2002,80: 185-194; Sriprapundh et al., Protein Engineering, 2003,16: 683-690; Li Song etc., food research and development, 2006,27 (8): 84-86.).
Thermophile bacteria such as thermobacillus (Thermotoga spp.), thermophilic anaerobic bacillus (Thermoanaerobacter sp.) and thermophilic anaerobic genus bacillus (Thermoanaerobacterium sp.) etc. are the extreme microorganisms that is grown in 55 ℃-90 ℃ the thermophilic environment.The various enzymes that thermophile bacteria produced have good thermostability usually, wherein in the xylose that has good stability more than 90 ℃ great application potential are arranged in high fructose syrup production undoubtedly.But, because the growth conditions of thermophile bacteria is harsh, adding that the natural expression amount of xylose is very low, people are difficult to obtain a large amount of industrial enzymes from natural bacterial strain.Simultaneously, because the gene of character such as codon preference, gene structure at normal temperatures in the thermophile bacteria gene and normal temperature bacterium differs greatly, the expression level of its natural gene in genetic engineering bacterium is lower usually.
Three, summary of the invention
The purpose of this invention is to provide a kind of method for preparing recombinant heat-proof property xylose with the pHsh expression system.
We use efficient expression vector pHsh; Thermotolerance xylose gene is efficiently expressed; And change the secondary structure of codon frequency of usage and mRNA through the gene directional transformation; Realize xylose efficiently expressing in intestinal bacteria, thereby obtained that output is high, cost is low, be easy to the method for the production recombinant heat-proof property xylose of purifying.Expression vector pHsh is by the novel plasmid of identification of the intestinal bacteria sigma32 factor and regulation and control, through heat-inducible genetic expression, avoids the use of chemical inducer; Have background and express the end, the advantage that recombinant protein output is high (Shao, W.et.al.; 0verexpression Vectors Regulated bv A Sigma Factor inEscherichia coli; 2005,6,105
ThGeneral Meeting American Society forMicrobiology, Atlanta, USA).
1. method for preparing recombinant heat-proof property xylose with the pHsh expression system; It is characterized in that; Express thermotolerance xylose gene or its two mutants with expression vector pHsh; Obtain recombinant heat-proof property xylose, the method steps for preparing this enzyme is following:
(1) genomic dna of extraction thermophile bacteria from thermophile bacteria obtains thermotolerance xylose gene through pcr amplification, and this gene is inserted expression vector pHsh, is built into isomerase expression plasmid;
(2) the mRNA translation initiation district potential secondary structure and the codon frequency in the isomerase expression plasmid are carried out on-line analysis; And through gene mutagenesis break mRNA loop-stem structure, reduce free energy and reduce rare codon, the isomerase expression plasmid that is optimized;
(3) the isomerase expression plasmid transformed into escherichia coli with isomerase expression plasmid or optimization obtains genetic engineering bacterium, and in the process of growth of genetic engineering bacterium, carries out heat-inducible, makes xylose obtain expressing;
(4) collecting cell, broken wall and centrifugal acquisition crude enzyme liquid;
(5) recombinase in the above-mentioned crude enzyme liquid is carried out purifying and obtain purified recombinant thermotolerance xylose.
Thermophile bacteria described in the above-mentioned steps (1) is Thermotoga maritima (Thermotoga maritima), Naples thermobacillus (Thermotoga neapolitana), thermophilic anaerobic ethanol bacterium (Thermoanaerobacter ethanolicus) and thermophilic anaerobic genus bacillus (Thermoanaerobacterium sp.).
Gene mutafacient system described in the above-mentioned steps (2) is: design changes the mutant primer of the inner base of gene and does to the gene xylA of Thermotoga maritima or Naples thermobacillus; Primer 1:5 '-TATAT CTCCT TCTTG TCGACTTTTA ACAGG TGATT GGATC ATGG-3 '; Primer 2: 5 '-CCCAT GGCCG AATTC TTCCCCGAAATTCCAAAGAT TCAGT TTGAA GGT-3 '; With the isomerase expression plasmid is that template is carried out site-directed mutagenesis, the isomerase expression plasmid that is optimized.
Intestinal bacteria described in the above-mentioned steps (3) are coli strain K12 or its derivative strain.The derivative strain of coli strain K12 is JM109, BL21 or DH5 α etc.
Purification process described in the above-mentioned steps (4) is with behind the ultrasonic disruption cell, handles 15min, the centrifugal recombinant heat-proof property xylose that obtains purity 90% for 90 ℃.
Genetic manipulations such as the extraction of thermophile bacteria genomic dna and pcr amplification are all by " (the Sambrook and Russel l that the standard method on the molecular cloning handbook third edition is carried out in the above-mentioned steps (1); 2001; CSHL Press, ColdSpring Harbor, New York)).
Heat shock inducing method described in the above-mentioned steps (3) is referring to patent documentation: Chinese invention patent ZL200410065776.8.
Method of the present invention has the following advantages:
(1) the present invention uses expression vector pHsh that the thermotolerance xylose gene from thermophile bacteria is efficiently expressed first, and the xylose gene of 4 different sourcess being tested has all obtained efficiently expressing in pHsh.
(2) among the present invention, isomerase expression plasmid can be carried out rite-directed mutagenesis and directional transformation to gene in position, and the sequence of mRNA is optimized, thereby further improves the expression level of thermotolerance xylose in pHsh.The determination of activity of isomerase and SDS-polyacrylamide gel electrophoresis analytical results show that the expression level ratio through the isomerase expression plasmid of optimizing improves 80% (Fig. 2).
(3), can receive that output is high, cost is low, recombinase is easy to purifying, help beneficial effect such as industrial fermentation with method production thermotolerance xylose of the present invention.
Four, description of drawings
Fig. 1 is the structure iron of the isomerase expression plasmid pHsh-xylAII of optimization among the embodiment 1.
Fig. 2 efficiently expresses in intestinal bacteria for the Thermotoga maritima xylose, the whole-cell protein SDS-polyacrylamide gel electrophoresis figure that obtains.M: molecule marker; 1: the reorganization bacterium that contains the pHsh carrier; 2: the reorganization bacterium that contains isomerase expression plasmid pHsh-xylA; 3: the isomerase expression plasmid pHsh-xylAII reorganization bacterium that contains optimization.
Fig. 3 is SDS-polyacrylamide gel electrophoresis figure, shows the purification effect that the Thermotoga maritima xylose reaches through 90 ℃ of thermal treatments.M: molecule marker; The 1:90 ℃ of enzyme liquid of handling behind the 15min; 2: the cell extract after efficiently expressing.
Five, embodiment
Employed in the present invention term only if other explanation is arranged, generally has the implication of genetically engineered field those of ordinary skill common sense.Below in conjunction with concrete embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit scope of the present invention by any way.
In following embodiment; Thermotoga maritima, Naples thermobacillus, thermophilic anaerobic ethanol bacterium are purchased in American Type Culture Collecti (article No. ATCC43589, ATCC49049, ATCC31936), and the thermophilic anaerobic genus bacillus is bought (article No. DSM8685) from German DSMZ.The cultivation of Thermotoga maritima and Naples thermobacillus see reference document (Jiang, Y.et al., FEMS Microbiol Lett 2006,259: 254-259); The cultural method of thermophilic anaerobic ethanol bacterium see reference document (Wiegel, J.et al., Arch Microbiol 1981,128: 343-348); The cultural method of thermophilic anaerobic genus bacillus see reference document (Shao, w.and J.wiegel, Applied andEnvironmental Microbiology, 1995,61: 937-940).The character of used plasmid pHsh or source are referring to document (Shao, W.et al., 0verexpression vectors regulated by a sigma factorinEscherichia coli.2005.6,105
ThGeneral Meeting American Society forMicrobiology Atlanta, USA; US patent application No.11/641,626).Various processes and the method do not described in detail among the embodiment are ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moity person indicate when occurring first that all used thereafter identical reagent is like no specified otherwise, and is all identical with the content of indicating first.
Embodiment 1: the preparation method of Thermotoga maritima recombinant heat-proof property xylose, and its preparation process is following:
(1) cultivates Thermotoga maritima by ordinary method, from Thermotoga maritima, extract genomic dna; According to known thermotolerance xylose gene (xylA; GenBank Access No.:TM16667) design primer; Genomic dna with Thermotoga maritima is a template, carries out pcr amplification with the synthetic primer, obtains the original gene xylA of xylose; Wherein genetic manipulations such as the extraction of genomic dna and pcr amplification are by " (Sambrook and Russell is carried out in the standard method on the molecular cloning handbook third edition; 2001, CSHL Press, Cold SpringHarbor; New York), primer is synthetic by biosynthesizing center, Chinese Academy of Sciences Shanghai; The used toolenzyme of genetic manipulation is available from precious biotechnology (Dalian) ltd; To pcr amplification product and carrier pHsh carry out respectively enzyme cut with purifying after, connect with the T4DNA ligase enzyme, and change intestinal bacteria over to and obtain isomerase expression plasmid pHsh-xylA;
(2) secondary structure and the codon frequency of usage to the mRNA translation initiation district that derives from Thermotoga maritima xylose gene carries out on-line analysis; And through rite-directed mutagenesis and directional transformation it is optimized, be that the mutant primer of stencil design is with pHsh-xylA:
Primer 1:5 '-TATAT CTCCT TCTTG TCGAC TTTTA ACAGG TGATT GGATC ATGG-3 ', primer 2: 5 '-CCCAT GGCCG AATTC TTCCC CGAAA TCCCA AAGAT TCAGT TTGAAGGT-3 ', wherein; Italic is represented the mutational site; With pHsh-xylA is that template is carried out site-directed mutagenesis, the isomerase expression plasmid pHsh-xylAII that is optimized (Fig. 1), and gene site-directed mutagenesis method can be with reference to " (the Sambrook and Russell of the standard method on the molecular cloning handbook third edition; 2001; CSHL Press, Cold Spring Harbor, New York).
3. genetic expression
With isomerase expression plasmid pHsh-xylA or pHsh-xylAII transformed into escherichia coli bacterial strain JM109; Transformant changes the LB substratum that contains the 100mg/L penbritin over to; After 37 ℃ of overnight cultures, be transferred to 200mL by 1% and contain and continue in the L β nutrient solution of 100mg/L penbritin to cultivate 0D
600When reaching 1.0 left and right sides, the fermentor tank A that 3L contains the TB substratum of 100mg/L penbritin is equipped with in the bacterial classification access, is cultured to 0D in 30 ℃
6001.0 about, the nutrient solution among the fermentor tank A is injected 42 ℃ the fermentor tank B of being preheated to that the same substratum of lL is housed, and (the heat shock method is referring to patent documentation: Chinese invention patent ZL 200410065776.8), continue to cultivate about 9 hours;
4. the purifying of recombinase and analysis
Centrifugal collecting cell after heat-inducible is expressed and accomplished with the phosphoric acid buffer suspension cell of 40mL pH 7.0, and adds Mg
2+And Co
2+Be respectively 10mmol/L and 1mmol/L to ultimate density; Behind the ultrasonic disruption cell, handle 15min, centrifugal acquisition soluble recombining thermotolerance xylose for 90 ℃; Purity reaches 90% (Fig. 3), and the output of recombinant protein reaches 200 milligrams of every liter of fermented liquids (mg/L).
Working method and embodiment 1 are basic identical; Difference is that genomic dna derives from Naples thermobacillus; And according to the xylose gene order (xylA, GenBank Access No.L389941) of Naples thermobacillus design primer, other is the same.
Working method and embodiment 1 are basic identical; Difference is that genomic dna derives from thermophilic anaerobic ethanol bacterium; And according to the xylose gene order (xylA, GenBank Access No.AF001974) of thermophilic anaerobic ethanol bacterium design primer and mutant primer, other is the same.
Embodiment 4
Working method and embodiment 1 are basic identical; Difference is that genomic dna derives from the thermophilic anaerobic genus bacillus; And according to the xylose gene order (xylA, GenBank Access No.U21678) of thermophilic anaerobic genus bacillus design primer and mutant primer, other is the same.
Claims (4)
1. method for preparing recombinant heat-proof property xylose with the pHsh expression system; It is characterized in that; Express thermotolerance xylose gene or its two mutants with expression vector pHsh; Obtain recombinant heat-proof property xylose, the method steps for preparing this enzyme is following:
(1) from thermophile bacteria, extracts genomic dna, obtain thermotolerance xylose gene, and this gene is inserted expression vector pHsh, be built into isomerase expression plasmid through pcr amplification;
(2) the mRNA translation initiation district potential secondary structure and the codon frequency in the isomerase expression plasmid are carried out on-line analysis; And through gene mutagenesis break mRNA loop-stem structure, reduce free energy and reduce rare codon, the isomerase expression plasmid that is optimized; The method of wherein said gene mutagenesis is: design changes the mutant primer of the inner base of gene and does to the gene xylA of Thermotoga maritima or Naples thermobacillus; Primer 1:5 '-TATAT CTCCT TCTTGTCGAC TTTTA ACAGG TGATT GGATC ATGG-3 '; Primer 2: 5 '-CCCAT GGCCG AATTCTTCCC CGAAA TTCCA AAGAT TCAGT TTGAA GGT-3 ' is that template is carried out site-directed mutagenesis with the isomerase expression plasmid;
(3) the isomerase expression plasmid transformed into escherichia coli with isomerase expression plasmid or optimization obtains genetic engineering bacterium, and in the process of growth of genetic engineering bacterium, carries out heat-inducible, makes xylose obtain expressing;
(4) collecting cell, broken wall and centrifugal acquisition crude enzyme liquid;
(5) recombinase in the above-mentioned crude enzyme liquid is carried out purifying and obtain purified recombinant thermotolerance xylose.
2. preparation method as claimed in claim 1 is characterized in that in the thermophile bacteria described in the step (1) be Thermotoga maritima (Thermotoga maritima), Naples thermobacillus (Thermotoga neapolitana), thermophilic anaerobic ethanol bacterium (Thermoanaerobacter ethanolicus) or thermophilic anaerobic genus bacillus (Thermoanaerobacterium sp.).
3. preparation method as claimed in claim 1 is characterized in that the intestinal bacteria described in the step (3) be coli strain K12 or its derivative strain JM109, BL21 or DH5 α.
4. preparation method as claimed in claim 1, it is characterized in that the purification process described in the step (5) be with the ultrasonic disruption cell after, handle 15min, the centrifugal recombinant heat-proof property xylose that obtains purity 90% for 90 ℃.
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| KR101868194B1 (en) * | 2013-11-19 | 2018-06-18 | 씨제이제일제당 (주) | Composition for non-phosphate sugar epimerization comprising sugar epimerase derived from thermophilic bacteria |
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| CN1451753A (en) * | 2003-05-21 | 2003-10-29 | 江南大学 | Technical scheme for increasing maricolous thermobacillus xylanase B gene expression level by large margin |
| CN1609223A (en) * | 2004-08-09 | 2005-04-27 | 南京师范大学 | Colibacillus plasmid vector and its application method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1451753A (en) * | 2003-05-21 | 2003-10-29 | 江南大学 | Technical scheme for increasing maricolous thermobacillus xylanase B gene expression level by large margin |
| CN1609223A (en) * | 2004-08-09 | 2005-04-27 | 南京师范大学 | Colibacillus plasmid vector and its application method |
Non-Patent Citations (4)
| Title |
|---|
| 侯进等.木糖异构酶在酿酒酵母表面表达及对木糖代谢影响的初步研究.生物加工过程4 1.2006,4(1),30-34. |
| 侯进等.木糖异构酶在酿酒酵母表面表达及对木糖代谢影响的初步研究.生物加工过程4 1.2006,4(1),30-34. * |
| 刘思园等.系列木聚糖降解酶基因同向串连表达的研究.南京师大学报（自然科学版）29 1.2006,29(1),93-97. |
| 刘思园等.系列木聚糖降解酶基因同向串连表达的研究.南京师大学报(自然科学版)29 1.2006,29(1),93-97. * |
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