CN1740323A - Technological process for synthesizing mycose by enzyme process - Google Patents
Technological process for synthesizing mycose by enzyme process Download PDFInfo
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- CN1740323A CN1740323A CN 200410051199 CN200410051199A CN1740323A CN 1740323 A CN1740323 A CN 1740323A CN 200410051199 CN200410051199 CN 200410051199 CN 200410051199 A CN200410051199 A CN 200410051199A CN 1740323 A CN1740323 A CN 1740323A
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- trehalose
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- sucrose phosphorylase
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Abstract
This invention discloses method synthesizing trehalose by enzymes. It includes steps as follows: a. clone huishuhua trehalose synthesizing enzyme gene and E. coli sucrose phosphorylase gene separately; b. construct expression vector of huishuhua trehalose synthesizing enzyme gene and E. coli sucrose phosphorylase gene separately; c. transform and express the expression vector of huishuhua trehalose synthesizing enzyme gene and E. coli sucrose phosphorylase gene separately in yeast, construct and purify the recombinant protein; d. add the said purified recombinant protein to synthesize trehalose in the reaction solution which has sucrose and glucose as main component.
Description
Technical field
The present invention relates to a kind of Preparation methods of trehalose, relate in particular to the processing method of a kind of using gene engineering, enzyme engineering and fermentation engineering trehalose synthesis.
Background technology
Trehalose (trehalose) is a kind of stable irreducibility disaccharide, and it is by two pyranoid ring glucose molecules and 1,1 glycosidic link link (China fir capital and interest row, 1994).Trehalose can be several solid forms exist, modal is two hydrates, fusing point reaches 97 ℃, is heated to 130 ℃, makes it lose crystal water and when becoming the anhydrous crystal body, fusing point can reach 214～216 ℃.Its properties of Aqueous Solution is stable, and colourless nothing is smelt, and mouthfeel is sweet taste slightly, can caramelize.The physico-chemical property of trehalose is very stable, can not make fehling reagent reduction, can not be by the alpha-glycosidase hydrolysis, but under strong acid condition, can be hydrolyzed to two glucose molecules.
The processing method of existing production trehalose has following several:
One, extracting trehalose from yeast
This is the method for producing trehalose the earliest, mainly is extracting from bread yeast, yeast saccharomyces cerevisiae.At present, the technology of extracting trehalose from yeast is quite ripe, but because the manufacturing cost height, its price is still higher.
Two, microbial fermentation is produced trehalose
Carry out the fermentative production trehalose and also obtained certain progress by cultivating the microorganism to produce trehalose.Fungi has 33 kinds of 20 several genus according to the literature, all is microorganism resource of exploitation trehalose.It should be noted that some mushroom bacteria especially, contained trehalose accounts for 10%～15% of its dry weight.Sight has been placed on the exploitation of Grifola frondosa in recent years both at home and abroad, its value is that not only Grifola frondosa contains polysaccharide, disaccharide, multivitamin, trace element and other effective active compositions, and the content of trehalose is all higher than mushroom, needle mushroom.Adopt selection by mutation, cytogamy or gene engineering method seed selection to produce the high bacterial strain of trehalose, adopt the substratum of high density and height to ooze fermentation then, and before fermentation ends, allow yeast " hunger " 2～3h, can obtain containing the higher culture of trehalose.(Luo Mingdian etc., 1996)
Three, utilize gene engineering method to produce trehalose
The one, utilize the gene constructed transgenic plant of trehalose with resistance, the 2nd, utilize engineered microbes and enzyme engineering to improve trehalose production.The content that the Mogen of Holland and Vanderhave company began to manage to improve trehalose in the farm crop such as beet and horse clock potato in 1992 has now obtained production patent (Yang Lin etc., 1999).The researchist of U.S. Calgene company is that the gene of trehalose imports plant by a kind of bacterium with conversion of glucose, the recombinant plant that makes up has the trehalose of production ability, and think the control (Luo Mingdian etc., 1996) that is subjected to relevant enzyme gene when conversion of glucose is trehalose fully.
The processing method cost of producing trehalose at present is all comparatively expensive, finds cheap method of producing trehalose will obtain very great economic benefit.
Summary of the invention
The object of the present invention is to provide a kind of cheapness, the processing method of using gene engineering, enzyme engineering and fermentation engineering enzyme process trehalose synthesis to be to obtain great economic benefit.
Trehalose is a kind of disaccharide that broad prospect of application is arranged, and the method cost of producing trehalose at present is all comparatively expensive, finds cheap method of producing trehalose will obtain very great economic benefit.Bibliographical information is arranged, and in load seedling Grifola frondosa, the dry weight of trehalose is up to about 15～17%, and this illustrates the very capable of Grifola frondosa trehalose synthesis.Starting point of the present invention is clone and the expression of intending by the Grifola frondosa trehalose synthesize enzyme gene, with genetically engineered and enzyme engineering combination, is forming new technology and technology aspect the Production by Enzymes trehalose.
The route of synthesis of Grifola frondosa trehalose is as follows:
By above-mentioned route of synthesis as seen, TreP needs D-glucose and alpha-D-glucose-1-phosphoric acid as reaction substrate.And in intestinal bacteria, sucrose phosphorylase can be fructose and alpha-D-glucose-1-phosphoric acid (KOKI SAITO et al, 1998) with sucrose decomposition.
Thinking of the present invention is exactly the gene clone with Grifola frondosa TreP and intestinal bacteria sucrose phosphorylase, and the method by genetically engineered and fermentation engineering obtains this two kinds of enzymes.Utilize this two kinds of enzymes, can take industrially scalable production trehalose with the raw material of sucrose and these two kinds of cheapnesss of glucose.
Purpose of the present invention is achieved by the following technical programs:
The processing method of a kind of enzyme process trehalose synthesis provided by the invention comprises the following steps:
A. clone Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase gene respectively;
B. make up Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase expression carrier respectively;
C. in yeast, transform, express Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase expression carrier respectively, constitute recombinant protein, then recombinant protein is carried out purifying;
D. be in the reaction solution of main component with sucrose and glucose, adding the above-mentioned recombinant protein trehalose synthesis of purifying respectively.
The present invention can take following further measure:
The method of clone's Grifola frondosa trehalose synthesize enzyme gene is in described step a: utilize primer 1:5 ' CCGAA TTC ATG GCT CCT CCC CAC CAG-3 ', primer 2: 5 ' GC TCT AGA TCC CTGCAC ATG CAG TTC-3 ', adopt the RT-PCR method from the total RNA of Grifola frondosa, to amplify a fragment about about 2.2kb, this fragment is connected on the pMD-T carrier; The method of clone intestinal bacteria sucrose phosphorylase gene is: utilize primer 1:5 ' CC GAA TTC ATG AAA CAG AAA ATT ACG-3 ', primer 2: 5 ' GC TCT AGA TTT AAT CCA CAT AACCTG-3 ', employing RT-PCR method obtains the fragment about about 1.7kb from colibacillary total DNA, this fragment is connected on the PMD-T carrier.
In described step b with the Grifola frondosa trehalose synthesize enzyme gene with EcoRI and BamHI double digestion, be connected with pPICZ α plasmid behind the double digestion then; Intestinal bacteria sucrose phosphorylase gene with EcoRI and XbaI double digestion, is connected with pPICZ α plasmid behind the double digestion then.Obtaining mostly as need is plasmid vectors, and the expression vector that makes up among the described step b also can adopt following method to duplicate: with reference to the Sambrook method, press CaCl
2Method transforms plasmid in E.Coli.DH5 α bacterial strain, with the LB substratum transform bacteria that contains penbritin (100 μ g/ml), alkaline process extracts plasmid.
In described step c, the Grifola frondosa trehalose synthesize enzyme gene and the intestinal bacteria sucrose phosphorylase expression carrier that build are utilized electrization, it is changed in the cultured pichia spp cell, evenly be applied to after the conversion in the YPDS flat board that contains Zeocin, can see that approximately white colony occurs in 3-4 days.
Picking yeast transformant list colony inoculation places the 50ml Erlenmeyer flask in 10mlBMGY in described step c, and 30 ℃, the 250rpm shake-flask culture is to OD
600About=2-6, centrifugal collection thalline, thalline is resuspended among the 50mlBMMY to OD
600=1; Shaking culture added 100% methyl alcohol every 24 hours and carries out abduction delivering to final concentration 1%, and Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase expression of gene amount are respectively 180 mg/litre and 120 mg/litre.
Utilize the Histidine affinity column that recombinant protein is carried out purifying in described step c, the purity of protein behind the purifying reaches more than 90%.
In the described steps d at sucrose: in the reaction solution of glucose: Pi=30: 30: 1mM, add and to be dissolved in that concentration is the TreP and the intestinal bacteria sucrose phosphorylase of 1 microgram/microlitre in MES (pH6.5) damping fluid, the concentration of two kinds of enzymes in reaction solution respectively was 1 mcg/ml, in 37 ℃ of reactions 24 hours.
The present invention has following beneficial effect: using gene engineering, enzyme engineering and fermentation engineering trehalose synthesis; forming new technology and technology aspect the Production by Enzymes trehalose; simultaneously realize industrially scalable production trehalose with the raw material of sucrose and these two kinds of cheapnesss of glucose; reduce cost, had great economic benefit.
Description of drawings
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but do not limit the present invention in any form.
Fig. 1 is pPICZ α-TR vector construction strategy
Fig. 2 is that the enzyme of pPICZ α-TR is cut evaluation
Among the figure: 1, pPICZ α-TR is the amplification of template
2、pPICZα-TR/EcoRI+XbaI
3、pPICZα-TR/EcoRI
4、pPICZα-TR/EcoRI
5、trehalose?synthase?gene
Fig. 3 is pPICZ α-SUC vector construction strategy
Fig. 4 is that the enzyme of pPICZ α-SUC is cut evaluation
Among the figure: 1, pPICZ α-SUC is the amplification of template
2、pPICZ?α-SUC/EcoRI+XbaI
3、pPICZα-SUC/EcoRI
4、pPICZα-TR/EcoRI
5、sucrose?phosphorylase
Fig. 5 is that the PCR that pichia spp transforms identifies
Among the figure: 1-4:pPICZ α-SUC transformant genomic dna amplification
5-7:pPICA α-TR transformant genomic dna amplification.
Fig. 6 is the purifying of sucrose phosphorylase
Among the figure: 1, low molecular weight protein standard specimen
2, Pichia/pPICZ α-fermented liquid of 4 days of SUC transformant abduction delivering
3, the sucrose phosphatization enzyme of purifying
Fig. 7 is the purifying of TreP
Among the figure: 1, low molecular weight protein standard specimen
2, Pichia/pPICZ α-fermented liquid of 4 days of TR transformant abduction delivering
3, the TreP of purifying
Fig. 8 is a Paper Chromatography enzyme analysis reaction result
Among the figure: 1, trehalose standard model
2, the synthetic result's (enzyme is by yeast expression) of trehalose
Embodiment
The clone and the sequential analysis of embodiment 1 Grifola frondosa trehalose synthesize enzyme gene
According to the Grifola frondosa trehalose synthesize enzyme gene sequences Design of gene bank website record a pair of primer: primer 1:5 ' CC GAA TTC ATG GCT CCT CCC CAC CAG-3 ', primer 2: 5 ' GC TCT AGA TCCCTG CAC ATG CAG TTC-3 ', utilize the RT-PCR method from the total RNA of Grifola frondosa, to amplify the fragment of an about 2.2kb, this fragment is connected to (called after PMD-T-TR) on the pMD-T carrier, checks order.The fragment total length 2199bp that from Grifola frondosa RNA, increases, 732 amino acid of encoding according to the DNASIS software analysis, are compared with the sequence of report, have 98.5% base identical, and aminoacid sequence then has 99.2% similarity.
The clone and the sequential analysis of embodiment 2 intestinal bacteria sucrose phosphorylase genes
Extract colibacillary total DNA, utilize primer 1:5 ' CC GAA TTC ATG AAA CAG AAAATT ACG-3 ', primer 2: 5 ' GC TCT AGA TTT AAT CCA CAT AACCTG-3 ' carries out pcr amplification, obtain the fragment of about 1.7kb, be connected to (called after PMD-T-SUC) on the PMD-T carrier, check order.This full length gene 1680bp, 559 amino acid of encoding, according to the DNASIS software analysis, sequencing result is compared with the sequence of intestinal bacteria sucrose phosphorylase among the gene BANK, and is identical.
The structure of embodiment 3 Grifola frondosa trehalose synthesize enzyme gene Yeast expression carriers
As shown in Figure 1, the trehalose synthesize enzyme gene on the PMD-T-TR carrier with EcoRI and BamHI double digestion, is connected the plasmid called after pPICZ α-TR after the connection with pPICZ α plasmid behind the double digestion.As shown in Figure 2, single endonuclease digestion result and double digestion result show that trehalose synthesize enzyme gene links to each other with carrier.With the carrier is that template is carried out the PCR reaction, has also amplified the purpose fragment, shows with sequencing result was identical in the past through sequencing result.
The structure of embodiment 4 intestinal bacteria sucrose phosphorylase expression vectors
As shown in Figure 3, the intestinal bacteria sucrose phosphorylase gene on the PMD-T-SUC with EcoRI and XbaI double digestion, is connected the plasmid called after pPICZ α-SUC after the connection with pPICZ α plasmid behind the double digestion.As shown in Figure 4, single endonuclease digestion result and double digestion result show that intestinal bacteria sucrose phosphorylase gene links to each other with carrier.With the carrier is that template is carried out the PCR reaction, has also amplified the purpose fragment, shows with sequencing result was identical in the past through sequencing result.
The conversion of embodiment 5 pichia spp and the evaluation of transformant
The conversion plasmid that builds is utilized electrization, it is changed in the cultured yeast cell, evenly be applied to after the conversion in the YPDS flat board that contains Zeocin, can see that approximately white colony occurs in 3-4 days.The bacterium colony of picking proper number, overnight incubation is got an amount of bacterium liquid, adopts boiling method to extract genomic dna as pcr template (Liu Qiuyun, 2001).By PCR testing goal gene.As shown in Figure 5, from electrophoretogram as can be seen, be that template is carried out the PCR reaction and amplified the purpose fragment with the genomic dna, illustrate that sucrose phosphorylase gene and trehalose synthesize enzyme gene have been incorporated in the yeast genes group, the purpose fragment that amplifies is connected on the T carrier and checks order, and it is identical comparing with former sequencing result.
Embodiment 6 trehalose synthesize enzyme genes and the expression of sucrose phosphorylase gene in yeast
Picking yeast transformant list colony inoculation places the 50ml Erlenmeyer flask in 10mlBMGY, 30 ℃, the 250rpm shake-flask culture is to OD
600About=2-6, centrifugal collection thalline, thalline are resuspended among the 50mlBMMY and (are loaded in 500 milliliters of triangular flasks) to OD
600=1; Shaking culture added 100% methyl alcohol every 24 hours and carries out abduction delivering to final concentration 1%, and Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase expression of gene amount are respectively 180 mg/litre and 120 mg/litre.Do the bacteria concentration analysis every sampling in 12 hours, Protein Detection is done in sampling in per 24 hours.
The purifying of embodiment 7 TrePs and sucrose phosphorylase
PROTEIN C end with Pichia anomala expression has 6 Histidines (being with on the pPICZ α carrier), uses the Histidine affinity column, can be with the albumen fast purifying.Recombinant protein purity behind the purifying reaches more than 90% (sees Fig. 6 and Fig. 7).
The synthetic test of embodiment 8 trehaloses
At 1 milliliter reaction solution (300mM sucrose, 300mM glucose, 10mMPi), add TreP and each 1 microlitre (concentration is about 1 microgram/microlitre) of sucrose phosphorylase of being dissolved in MES (pH6.5) damping fluid, in 37 ℃ of reactions 24 hours, get 30 microlitres and do ply of paper and analyse analysis, analyse qualitative analysis by ply of paper, as shown in Figure 8, in reaction solution, all detected the existence of trehalose.TreP that proof is expressed in yeast and bacterium and sucrose phosphorylase all have enzymic activity.And illustrate that it is feasible utilizing the method for recombinant expressed TreP and sucrose phosphorylase trehalose synthesis.
In addition, obtaining mostly as need is plasmid vectors, and the expression vector that makes up among the described step b also can adopt following method to duplicate: with reference to Sambrook (Sambrook, et al.1989, Molecular cloing.Cold Spring HarborLabroratory Press.USA) method is pressed CaCl
2Method transforms plasmid in E.Coli.DH5 α bacterial strain, with the LB substratum transform bacteria that contains penbritin (100 μ g/ml), alkaline process extracts plasmid.
The scale of the foregoing description 6, embodiment 7 is amplified the technology that just can realize by application patent of the present invention reach the purpose that the cheap raw material of application takes the suitability for industrialized production trehalose.
Claims (8)
1, a kind of processing method of enzyme process trehalose synthesis comprises the following steps:
A. clone Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase gene respectively;
B. make up Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase expression carrier respectively;
C. in yeast, transform, express Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase expression carrier respectively, constitute recombinant protein, then recombinant protein is carried out purifying;
D. be in the reaction solution of main component with sucrose and glucose, adding the above-mentioned recombinant protein trehalose synthesis of purifying respectively.
2, the processing method of enzyme process trehalose synthesis according to claim 1, the method of clone's Grifola frondosa trehalose synthesize enzyme gene is in described step a: utilize primer 1:5 ' CC GAA TTC ATG GCT CCTCCC CAC CAG-3 ', primer 2: 5 ' GC TCTAGA TCC CTG CAC ATG CAG TTC-3 ', adopt the RT-PCR method from the total RNA of Grifola frondosa, to amplify a fragment about about 2.2kb, this fragment is connected on the pMD-T carrier; The method of clone intestinal bacteria sucrose phosphorylase gene is: utilize primer 1:5 ' CC GAA TTC ATG AAA CAG AAA ATT ACG-3 ', primer 2: 5 ' GC TCT AGA TTT AATCCA CAT AACCTG-3 ', employing RT-PCR method obtains the fragment about about 1.7kb from colibacillary total DNA, this fragment is connected on the PMD-T carrier.
3, the processing method of enzyme process trehalose synthesis according to claim 1, in described step b with the Grifola frondosa trehalose synthesize enzyme gene with EcoRI and BamHI double digestion, be connected with pPICZ α plasmid behind the double digestion then; Intestinal bacteria sucrose phosphorylase gene with EcoRI and XbaI double digestion, is connected with pPICZ α plasmid behind the double digestion then.
4, the processing method of enzyme process trehalose synthesis according to claim 1, the expression vector that makes up among the described step b also can adopt following method to duplicate: with reference to the Sambrook method, press CaCl
2Method transforms plasmid in E.Coli.DH5 α bacterial strain, with the LB substratum transform bacteria that contains penbritin (100 μ g/ml), alkaline process extracts plasmid.
5, the processing method of enzyme process trehalose synthesis according to claim 1, in described step c, the Grifola frondosa trehalose synthesize enzyme gene and the intestinal bacteria sucrose phosphorylase expression carrier that build are utilized electrization, it is changed in the cultured pichia spp cell, evenly be applied to after the conversion in the YPDS flat board that contains Zeocin, can see that approximately white colony occurs in 3-4 days.
6, the processing method of enzyme process trehalose synthesis according to claim 1, picking yeast transformant list colony inoculation places the 50ml Erlenmeyer flask in 10mlBMGY in described step c, and 30 ℃, the 250rpm shake-flask culture is to OD
600About=2-6, centrifugal collection thalline, thalline is resuspended in OD among the 50mlBMMY
600=1; Shaking culture added 100% methyl alcohol every 24 hours and carries out abduction delivering to final concentration 1%, and Grifola frondosa trehalose synthesize enzyme gene and intestinal bacteria sucrose phosphorylase expression of gene amount are respectively 180 mg/litre and 120 mg/litre.
7, the processing method of enzyme process trehalose synthesis according to claim 1 utilizes the Histidine affinity column that recombinant protein is carried out purifying in described step c, and the purity of protein behind the purifying reaches more than 90%.
8, the processing method of enzyme process trehalose synthesis according to claim 1, in the described steps d at sucrose: in the reaction solution of glucose: Pi=30: 30: 1mM, add and to be dissolved in that concentration is the TreP and the intestinal bacteria sucrose phosphorylase of 1 microgram/microlitre in MES (pH6.5) damping fluid, the concentration of two kinds of enzymes in reaction solution respectively was 1 mcg/ml, in 37 ℃ of reactions 24 hours.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952786A (en) * | 2012-12-11 | 2013-03-06 | 中国热带农业科学院橡胶研究所 | Plant stress tolerance associated protein and application of coding gene thereof |
| CN107475271A (en) * | 2017-09-20 | 2017-12-15 | 国家***第三海洋研究所 | 6 phosphotrehalose UDP-transglucosylase synzyme caused by the microbacterium of deep-sea and 6 phosphotrehalose UDP-transglucosylase phosphates |
| CN108841899A (en) * | 2018-07-13 | 2018-11-20 | 安徽民祯生物工程有限公司 | A kind of method of enzymatic conversion production trehalose |
-
2004
- 2004-08-24 CN CN 200410051199 patent/CN1740323A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952786A (en) * | 2012-12-11 | 2013-03-06 | 中国热带农业科学院橡胶研究所 | Plant stress tolerance associated protein and application of coding gene thereof |
| CN107475271A (en) * | 2017-09-20 | 2017-12-15 | 国家***第三海洋研究所 | 6 phosphotrehalose UDP-transglucosylase synzyme caused by the microbacterium of deep-sea and 6 phosphotrehalose UDP-transglucosylase phosphates |
| CN108841899A (en) * | 2018-07-13 | 2018-11-20 | 安徽民祯生物工程有限公司 | A kind of method of enzymatic conversion production trehalose |
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