CN102943050A - Method for producing xylitol by recombined candida tropicalis - Google Patents
Method for producing xylitol by recombined candida tropicalis Download PDFInfo
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- CN102943050A CN102943050A CN2012104523678A CN201210452367A CN102943050A CN 102943050 A CN102943050 A CN 102943050A CN 2012104523678 A CN2012104523678 A CN 2012104523678A CN 201210452367 A CN201210452367 A CN 201210452367A CN 102943050 A CN102943050 A CN 102943050A
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Abstract
The invention provides a method for producing xylitol by recombined candida tropicalis in a whole cell method and belongs to the technical field of bioengineering technology. A genetically engineered bacterium used for producing the xylitol provided by the invention is the candida tropicalis formed by recombining the encoding genes of exogenous xylanase and xylosidase and can be used for producing the xylitol in the whole cell method. According to the recombined candida tropicalis bacterial strain protected by the invention, on the basis of retaining the original conversion characteristics of the xylitol, an xylan degradation way is reconstructed in the original bacterial strain through introducing the encoding genes of the exogenous xylanase and the xylosidase, the coupling of xylan degradation and xylitol production is realized, and the characteristic of producing the xylitol through fermentation in the whole cell method directly by taking natural hemicellulose raw material as a substrate is obtained. The method for producing the xylitol by the recombined candida tropicalis in the whole cell method is simple in process and low in cost, and has a mild condition, less energy consumption and less pollution discharge, and an efficient clean pollution-free industrial application technology is created for producing the xylitol.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of structure and the application in full cell method production Xylitol thereof of the candida tropicalis engineering bacteria of recombinating.
Background technology
Xylitol (English name: xylitol, chemical name: 1,2,3,4,5-pentahydroxypetane), claim again pentitol.Xylitol is a kind of naturally occurring five-carbon sugar alcohol as one of intermediate product of xylose metabolism.Xylitol is a kind of high sugariness natural plant sweetening agent low in calories, is a kind of polyvalent alcohol of high added value.Xylitol has non-cariogenicity, its in vivo metabolism do not rely on Regular Insulin, and have the effect of liver protecting, be widely used in the industrial circles such as oral hygiene, pharmaceutical prod, foodstuff additive.
At present commercial Xylitol production all is to adopt chemical transformation.The hemicellulose that will derive from first different biomass materials is processed through acid or basic hydrolysis, make pentosan wherein be hydrolyzed to the D-wood sugar, then remove detrimental impurity through operations such as complicated detoxification, decolourings, under the nickel catalyzator effect, the wood sugar in the hydrolyzed solution is carried out hydrogenating reduction again and obtain Xylitol.Chemical method is produced Xylitol and is yielded poorly, and needs in process of production High Temperature High Pressure, and complicated component in the last catalytic liquid, the extraction purification ratio of Xylitol be difficulty.Whole Xylitol complex manufacturing, energy consumption is high, quantity of wastewater effluent is high, and production cost is high, has seriously limited production and the application of Xylitol.
The Xylitol mild condition is produced in microbial transformation, and has the advantages such as specificity is good, transformation efficiency is high, cost is lower, is the desirable approach that the instead of chemical method is produced.In general, yeast is considered to best Xylitol microbial.At present for the production of the yeast strain of Xylitol comprise can fermenting xylose the unartificial yeast bacterium, and the recombinant Saccharomyces cerevisiae bacterium that utilizes the Xylose reductase gene (XYL1) of trunk pichia (Pichiastipitis) to make up.These yeast strains transform when producing Xylitol, and its substrate wood sugar still will obtain by sulfuric acid or basic hydrolysis hemicellulose raw material, and technological process still need be discharged a large amount of waste water, is subject to the strict restriction of national environmental protection index.Simultaneously, in the hydrolyzed solution that sulfuric acid or basic hydrolysis hemicellulose raw material obtain, often contain toxicant and other antinutritional factor such as phenol, furfural, the growth of yeast cell is had great toxic action, and then affect the ability of strain fermentation production Xylitol.
Therefore, directly take the hemicellulose raw material as substrate, adopt full cell method cleaner production Xylitol to become the inexorable trend of following Xylitol industrial development, can solve the high energy consumption of half biological synthesis method, the industry issue of high blowdown, with the energy-saving and emission-reduction of implementation procedure, promote the progress of biomass resource utilization overall technology.
Summary of the invention
The objective of the invention is the above-mentioned deficiency for prior art, a kind of method of utilizing the full cell method of restructuring candida tropicalis to produce Xylitol is provided.
For achieving the above object, technical scheme of the present invention provides a kind of restructuring candida tropicalis engineering strain, clone respectively zytase (XYN) and xylosidase (XYL) encoding gene, utilize Protocols in Molecular Biology that the expressed intact box of these two gene fragments is imported among the Yeast expression carrier pAUR123, change over to behind the construction recombination plasmid pAUR123-xyn-xyl in the xylose utilization type candida tropicalis (Candida tropicalis) and obtain engineering strain.Restructuring Candida tropicalis strain C.tropicalis PNL energy coexpression zytase of the present invention and xylosidase, these two kinds of enzymes are the exocytosis type, and are active high.This recombinant bacterium can directly utilize the corn cob hemicellulose to be substrate, realizes full cell method production Xylitol, has reduced environmental pollution.
The application of the Candida tropicalis strain C.tropicalis PNL of described restructuring in full cell method production Xylitol.
(1) preparation of thalline seed liquor: the liquid amount of pressing 10-50% in shaking flask adds seed culture medium, and used seed culture based formulas is: wood sugar (5-50gL), peptone (1-10gL), yeast extract paste (1-10g/L), K
2HPO
4(1-5g/L), MgSO
47H
2O (0.1-0.5g/L), CaCl
2(0.1-0.5g/L), pH4.5-6.5, behind 100-130 ℃ of lower sterilization 10-30min, the single bacterium colony of picking C.tropicalis PNL accesses the above-mentioned shaking flask on activating flat board under aseptic condition, culture temperature is 30-40 ℃, shaking speed is 150-300rpm, and incubation time is 12-30h, obtains the thalline seed liquor for subsequent use.
Preferably, the seed culture medium Xylose Content is 5-25gL, and peptone content is 3-8gL, and yeast extract paste content is 2-6gL, K
2HPO
4Content is 1-3g/L, MgSO
47H
2O content is 0.1-0.3g/L, CaCl
2Content is 0.1-0.3g/L, pH4.5-5.5.
Preferably, the seed culture medium liquid amount is 15-35%.
Preferably, culture temperature is 32-38 ℃.
Preferably, shaking speed is 160-200rpm.
(2) the full cell method fermentative production of Xylitol: the thalline seed liquor that under aseptic condition, above-mentioned steps (1) is prepared, fill the shaking flask of fermention medium by the access of the inoculum size of 1-15%, liquid amount is 20-80%.Used fermentative medium formula is: glucose 5-40g/L, peptone 5-40g/L, yeast extract paste 5-30gL, corn cob meal 10-50g/L, pH4.5-6.5.The controlled fermentation temperature is 30-40 ℃, and shaking speed is 150-300rpm, and fermentation time is 24-120h.
Preferably, the fermention medium glucose content is 10-25g/L, and peptone content is 1-25g/L, and yeast extract paste content is 5-15g/L, and corn cob meal content is 10-30g/L, pH4.5-5.5.
Preferably, the fermention medium liquid amount is 30-60%.
Preferably, inoculum size is 3-10%.
Preferably, culture temperature is 32-38 ℃.
Preferably, shaking speed is 160-200rpm.
Preferably, fermentation time is 60-100h.
The present invention adopts the thought of metabolic engineering, obtains candida tropicalis engineering strain---the C.tropicalis PNL of restructuring by genetic engineering means, and its starting strain C.tropicalis CICC1779 is Bioconversion of D-xylose To Produce Xylitol efficiently.Recombinant bacterium is on the basis that keeps original Xylitol conversion characteristic, by importing Exogenous-xylanase Additive and xylosidase encoding gene, in original strain reconstruct the xylan degrading approach, realize the coupling that xylan degrading and Xylitol are produced, obtained take the natural hemicellulose raw material as substrate directly to carry out the characteristic of the full cell method of Xylitol fermentative production.
The advantage of restructuring Candida tropicalis strain is among the present invention: xylanase gene and xylosidase gene all place under the control of yeast complex type strongly expressed promotor PADH1, do not need to add inductor, needn't change carbon source, zymotechnique is simple, mild condition; Recombinase energy expression-secretion is to fermented liquid, and active high, the Xylitol transformation efficiency is higher; Recombinant bacterial strain can be take natural hemicellulose raw materials such as corn cobs as substrate, and full cell fermentation is produced Xylitol, and whole technological process cost is low, less energy consumption, blowdown are few, has the commercial application prospect.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples are used for explanation the present invention, but are not used for limiting the scope that the present invention protects.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
Embodiment 1:
The structure of restructuring candida tropicalis engineering strain
(1) from terreus, clones xylanase gene (atn) fragment, compare as BLAST with the xylanase sequence of having announced on the Genbank, carry out homology analysis, the result shows, clone's atn gene is a new gene (Genbank registration sequence number be JQ087496).
(2) from terreus, clone xylosidase gene (atl) fragment, make the BLAST compare of analysis with the xylosidase gene order of having announced on the Genbank, the result shows, the xylobiase sequence is in full accord among the A.terreus NIH2624 that has reported among clone's atl gene order and the Genebank.
(3) utilize genetic engineering means that xylanase gene (atn) and xylosidase gene (atl) are cascaded, import in the pAUR123 carrier, make up the pAUR-atn-atl plasmid, this plasmid is imported in the candida tropicalis, make up new bacterial strain, make zytase and xylosidase coexpression, detailed process is as follows:
The atn gene is connected on the multiple clone site of yeast carrier for expression of eukaryon pAUR123, obtains recombinant plasmid pAUR-atn;
According to the base sequence design primer at PADH1 and TADH1 two ends, and at primer two ends interpolation Dra III restriction enzyme site, take the pAUR-atn plasmid as template, by the expression cassette PADH1-atn-TADH1 of PCR method amplification atn gene;
The atl gene is connected on the multiple clone site of yeast carrier for expression of eukaryon pAUR123, obtains recombinant plasmid pAUR-atl;
PADH1-atn-TADH1 expression casette fragment and pAUR-atl plasmid are carried out enzyme with Dra III respectively cut, with the T4DNA ligase enzyme the two is connected, thereby make up its co-expression carrier pAUR-atn-atl.
(4) Construction and identification of restructuring candida tropicalis engineering strain
The above-mentioned expression vector pAUR-atn-atl that successfully constructs is transformed among the candida tropicalis C.tropicalisCICC1779 by electric shocking method, by golden load element (AureobasidinA, AbA) resistance screening and bacterium colony PCR screening obtains positive transformant.
(5) xylan culture medium flat plate Screening and Identification
The C.tropicalis PNL-At transformant of the picking colony PCR positive is inoculated on the screening culture medium flat board take xylan as sole carbon source, and inoculating simultaneously one does not have the original bacterium of candida tropicalis that transforms in contrast.If the contrast bacterium can not grow, and the engineering strain that transforms can be grown, and illustrates that recombinant bacterium can utilize xylan preferably.Screening is dull and stereotyped behind congo red staining, can produce transparent circle around the recombinant bacterium, can according to the size of transparent circle, determine the ability of its metabolism xylan.Select can produce larger transparent circle recombinant bacterium as the seed bacterial strain, carry out further fermenting experiment.
Embodiment 2:
Xylanase gene (atn) and expression identification and the activation analysis of xylosidase gene (atl) in candida tropicalis
Select the recombinant bacterium that embodiment 1 filters out and carry out shake-flask culture, the culture supernatants after centrifugal is measured recombinant expressed zytase and xylosidase vigor as crude enzyme liquid, selects the highest bacterial strain of recombinase vigor.
Embodiment 3:
The application of Candida tropicalis strain C.tropicalis PNL-At in Xylitol is produced of recombinating as described in Example 1
Seed liquor is cultivated: the single bacterium colony of picking C.tropicalis PNL-At from the flat board is inoculated in 50mL seed culture medium (wood sugar 20g/L, peptone 5g/L, yeast extract paste 3g/L, K is housed
2HPO
42.5g/L, MgSO
47H
2O0.25g/L, CaCl
20.25gL, in 250mL shaking flask pH5.0), 35 ℃, 170rpm concussion cultivation 24h.
The shake flask fermentation experiment: the inoculum size switching seed liquor by 5% is to the 250mL shaking flask that fills 100mL fermention medium (glucose 20gL, peptone 20gL, yeast extract paste 10gL, corn cob meal 15g/L, pH5.0).The controlled fermentation temperature is 35 ℃, and shaking speed is 170rpm, and fermentation time is 96h.Every 12h sampling once, measure the content of Xylitol in the fermented liquid, the result shows, restructuring candida tropicalis engineering strain C.tropicalis PNL-At can effectively utilize the xylan in the corn cob hemicellulose to produce Xylitol, Determination of Xylitol reaches maximum value 3.17g/L in the fermented liquid when fermentation 84h, because xylan content is 30% in the used corn cob, then calculating the transformation efficiency of knowing wood sugar conversion Xylitol is 70.4%.
Embodiment 4:
The application of Candida tropicalis strain C.tropicalis PNL-At in Xylitol is produced of recombinating as described in Example 3, difference is:
The fermention medium that uses in the step (2) is: glucose 10gL, peptone 15g/L, yeast extract paste 7gL, corn cob meal 10g/L, pH5.5; Liquid amount is 30% in the step (2); Fermentation is in the step (2): 30 ℃ of leavening temperatures, shaking speed 180rpm.
Analyze through HPLC, Determination of Xylitol reaches maximum value 1.99g/L in the fermented liquid when fermentation 72h, because xylan content is 30% in the used corn cob, then calculating the transformation efficiency of knowing wood sugar conversion Xylitol is 66.3%.
Claims (9)
1. restructuring candida tropicalis engineering strain C.tropicalis PNL, it is characterized in that, zytase and xylosidase expression casette are cascaded, import among the Yeast expression carrier pAUR123, construction recombination plasmid pAUR123-xyn-xyl imports this plasmid in the xylose utilization type candida tropicalis.
2. restructuring candida tropicalis engineering strain C.tropicalis PNL as claimed in claim 1 is characterized in that, makes up the xylan degrading approach in xylose utilization type candida tropicalis, and described recombinant bacterial strain can coexpression zytase and xylosidase.
3. restructuring candida tropicalis engineering strain C.tropicalis PNL as claimed in claim 1, it is characterized in that, realize the coupling that xylan degrading and Xylitol are produced, can carry out take the natural hemicellulose raw material as substrate full cell method direct production Xylitol.
4. application in utilizing the full cell method of corn cob xylitol zymolysis production such as claim 1 or 2 or 3 described candida tropicalis engineering strains.
5. application as claimed in claim 4 is characterized in that, described candida tropicalis engineering strain C.tropicalis PNL can be applied to Xylitol production as follows:
(1) preparation of thalline seed liquor: the liquid amount of pressing 10-50% in shaking flask adds seed culture medium, and the seed culture based formulas is: wood sugar (5-50g/L), peptone (1-10g/L), yeast extract paste (1-10g/L), K
2HPO
4(1-5gL), MgSO
47H
2O (0.1-0.5g/L), CaCl
2(0.1-0.5gL), pH4.5-6.5 is behind 100-130 ℃ of lower sterilization 10-30min, picking list bacterium colony accesses the above-mentioned shaking flask on activating flat board under aseptic condition, and culture temperature is 30-40 ℃, and shaking speed is 150-300rpm, incubation time is 12-30h, obtains the thalline seed liquor for subsequent use.
(2) the full cell method fermentative production of Xylitol: the thalline seed liquor that under aseptic condition, above-mentioned steps 1 is prepared, fill the shaking flask of fermention medium by the access of the inoculum size of 1-15%, liquid amount is 20-80%.Used fermentative medium formula is glucose 5-40gL, peptone 5-40g/L, yeast extract paste 5-30g/L, corn cob meal 10-50gL, pH4.5-6.5.The controlled fermentation temperature is 30-40 ℃, and shaking speed is 150-300rpm, and fermentation time is 24-120h.
6. application as claimed in claim 5 is characterized in that, the seed culture medium Xylose Content described in the step (1) is 5-25gL, and peptone content is 3-8gL, and yeast extract paste content is 1-5gL, K
2HPO
4Content is 1-3g/L, MgSO
47H
2O content is 0.1-0.3g/L, CaCl
2Content is 0.1-0.3g/L, pH4.5-5.5.
7. application as claimed in claim 5 is characterized in that, the culture condition described in the step (1) is: the seed culture medium liquid amount is 15-35%, and culture temperature is 32-38 ℃, and shaking speed is 160-200rpm.
8. application as claimed in claim 5 is characterized in that, the fermention medium glucose content described in the step (2) is 10-25gL, and peptone content is 1-25gL, and yeast extract paste content is 5-15g/L, and corn cob meal content is 10-30gL, pH4.5-5.5.
9. application as claimed in claim 5 is characterized in that, the culture condition described in the step (2) is: inoculum size is 3-10%, the fermention medium liquid amount is 30-60%, culture temperature is 32-38 ℃, and shaking speed is 160-200rpm, and fermentation time is 60-100h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152392A (en) * | 2014-07-31 | 2014-11-19 | 上海交通大学 | Hemicellulase-based engineering bacteria and realization method thereof |
| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1400311A (en) * | 2002-08-27 | 2003-03-05 | 河北大学 | Method for preparing xylitol by using saccharomycetes to make fermentation |
| CN1982460A (en) * | 2005-09-30 | 2007-06-20 | 广西壮族自治区中国科学院广西植物研究所 | Separation of tropical candiyeast strain and production of xylitol |
| CN101899479A (en) * | 2010-07-02 | 2010-12-01 | 北京化工大学 | Clean production method for preparing xylitol by using agricultural and forestry waste |
-
2012
- 2012-11-13 CN CN2012104523678A patent/CN102943050A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1400311A (en) * | 2002-08-27 | 2003-03-05 | 河北大学 | Method for preparing xylitol by using saccharomycetes to make fermentation |
| CN1982460A (en) * | 2005-09-30 | 2007-06-20 | 广西壮族自治区中国科学院广西植物研究所 | Separation of tropical candiyeast strain and production of xylitol |
| CN101899479A (en) * | 2010-07-02 | 2010-12-01 | 北京化工大学 | Clean production method for preparing xylitol by using agricultural and forestry waste |
Non-Patent Citations (1)
| Title |
|---|
| X X GUO等: "A novel pathway construction in Candida tropicalis for direct xylitol conversion from corncob xylan", 《BIORESOURCE TECHNOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152392A (en) * | 2014-07-31 | 2014-11-19 | 上海交通大学 | Hemicellulase-based engineering bacteria and realization method thereof |
| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
| US11840500B2 (en) | 2016-02-19 | 2023-12-12 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
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Application publication date: 20130227 |