CN1478887A - Yeast gene engineering fungus and beta mannosan enzyme preparation and production method of manna oligose - Google Patents
Yeast gene engineering fungus and beta mannosan enzyme preparation and production method of manna oligose Download PDFInfo
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- CN1478887A CN1478887A CNA031189849A CN03118984A CN1478887A CN 1478887 A CN1478887 A CN 1478887A CN A031189849 A CNA031189849 A CN A031189849A CN 03118984 A CN03118984 A CN 03118984A CN 1478887 A CN1478887 A CN 1478887A
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Abstract
A genetically engineered yeast Pichia pastoris (GS115/HBMO47) able to effectively express beta-mannase is prepared through screening beta-mannase gene from the environmental microbes, cloning it to the expression carrier of Pichia yeast, introducing the carrier to Pichia yeast, and screening. The process for preparing the high-activity beta-mannase and the high-purity oligomannosan from said Pichia pastoris is also disclosed.
Description
Technical field
What the present invention relates to is genetic engineering bacterium, and particularly a kind of yeast gene engineering bacteria that efficiently expresses 'beta '-mannase and beta-mannase zymin and beta-mannase enzymic hydrolysis prepare the production method of manna oligosaccharide.
Background technology
Plant is the main renewable organic resource of nature, and its main component is a Mierocrystalline cellulose, hemicellulose and xylogen.Wherein 35% of cellulose comprises plant dry weight, different mannosans extensively is distributed among the plant as second component of hemicellulose.The endosperm of seeds of leguminous plant, some vegetable jelly (as carob bean gum, melon glue, sesbania angle glue etc.), coconut meat powder, coffee berry, lettuce, Leucaena leucocephala (L.), Guan Huangmu, chickweed etc. contain abundant polygalactomannan, konjak corm, Australian Chinese fan palm, only tail grass roots etc. contain abundant glucomannan (Marga F., Ghki C.et aI.AppI.Environ.MicroboI.1996,62 (12): 4656-4658.).Exactly because abundant mannosans resource is arranged, just uses and bring wide prospect for the research of 'beta '-mannase.Mannosans can change into the oligose that people need in a large number through the enzymolysis of mannase.Oligose is meant that two to ten monosaccharide units couple together a class sugar that forms straight chain or branched chain by glycosidic link, and it has good physiological properties such as low-heat, stable, safety non-toxic, has also that probiotics increases in the intestines of making, the physiological function that harmful bacterium reduces.In foodstuff production and pharmaceutical production, broad application prospect is arranged.
In recent years, get more and more about the production of oligose and the research of application facet both at home and abroad, existing multiple functional oligose product has dropped into industrial production and has been applied in the protective foods.But most functional oligose products of putting on market at present all belong to the oligose of regular grade, be the effective constituent that only contains 30%----50% in the product butt, therefore, develop highly purified functional oligose, become the problem that the scientific worker is badly in need of solving.In Japan, the price of highly purified oligose is 5----10 a times of regular grade.Up to the present, the method for preparing the high purity oligose mainly contains: gel filtration chromatography, nanofiltration membrane method, fermentation method, the popularize law of ion-exchange look, glucose isomerase transformation approach, multi-enzyme method (glucose oxidase, catalase coupling) and glycanase enzymolysis konjaku powder method.These method costs are too high, and perhaps product is difficult to purify, and perhaps complex process scarcely is fit to large-scale industrialization production.
At present, domestic relevant R﹠D institution, extensively carried out the research of mannase and associated viscera thereof as institute of microbiology of the Chinese Academy of Sciences, Nankai University etc., but it is low that the fermenting enzyme of being reported is lived, enzymolysis product purity is low, production cost is high, especially alkaline bacterial classification also easily causes environmental pollution etc. in process of production, thereby is difficult to carry out industrialized developing.
Summary of the invention
The objective of the invention is to make up the yeast gene engineering bacteria that can efficiently express 'beta '-mannase, and produce 'beta '-mannase, thereby can transform the preparation manna oligosaccharide efficiently, economically from cultivation separation and purification wherein by engineered means.
The microorganism of using: the yeast gene engineering bacteria Pichia pastorisGS115/HBM047 that the present invention relates to, it has the characteristic that efficiently expresses 'beta '-mannase, this bacterial strain on April 15th, 2003 in China's typical culture collection center preservation, preserving number is CCTCC No:M203026 (hereinafter to be referred as HBM047).
The bacteria characteristic of HBM047 bacterial strain:
A, morphological specificity: pichia spp monogony be budding, pseudohypha is arranged sometimes.Syngenesis produces ascus and contains 1----4 piece of smooth circle, carnival hat shape or Saturn thecaspore.
B, physio-biochemical characteristics: pichia spp can utilize special material to grow for raw material, as oil, methyl alcohol, ammonia-state nitrogen, organic acid etc.The HBM047 bacterial strain has the physio-biochemical characteristics of pichia spp, simultaneously because of having inserted beta-mannase gene, had the characteristic that efficiently expresses beta-mannase gene.
The present invention realizes like this.Yeast gene engineering bacteria HBM047 (Pichia pastoris) is method and the round pcr by the constructing environment genomic library, from environmental microorganism, filter out beta-mannase gene, above-mentioned beta-mannase gene is cloned in the pichia spp integrating expression vector, the beta-mannase gene expression vector that contains that obtains then imports in the pichia spp, therefrom filters out a kind of yeast gene engineering bacteria HBM047 bacterial strain that efficiently expresses 'beta '-mannase again.
Specific practice is that total DNA of extraction environmental microorganism carries out partially digested with restriction enzyme to it, reclaim the fragment of 4----10kb, be cloned in the cloning vector transformed into escherichia coli, by making substrate, filter out goal gene with the plate screening method, and check order with Rhizoma amorphophalli powder.Obtain cloning the beta-mannase gene sequence according to order-checking, use PCR method again, plasmid DNA with the positive colony that screens in the environment genomic library is a template, amplify the dna fragmentation of 1.7KB, be inserted in pichia spp (Pichia pastoris) integrating expression vector, import among the pichia spp host GS115 by chemical conversion again, therefrom filter out again and efficiently express 'beta '-mannase yeast gene engineering bacteria HBM047 bacterial strain.
With the 'beta '-mannase yeast gene engineering bacteria HBM047 that efficiently expresses, be in the substratum of carbon source with methyl alcohol, 28 ℃ of----30 ℃ of temperature, (surveying enzyme at regular intervals lived through 24----168 hour inducing culture, stopped inducing culture in 168 hours), the yeast gene engineering bacteria of finishing inducing culture is removed thalline through high speed centrifugation, collect supernatant liquor, be crude enzyme liquid, concentrate after filtration and promptly get the beta-mannase enzyme product.
Use the method that above-mentioned 'beta '-mannase crude enzyme liquid hydrolysis prepares manna oligosaccharide, the steps include:
1), the one-level Rhizoma amorphophalli powder is mixed with the konjaku aqueous solution that concentration is 10%----20% with distilled water, swelling one hour:
2), enzyme-added in the crude enzyme liquid ratio of the corresponding 1ml of every 10----20g Rhizoma amorphophalli powder;
3), above-mentioned mixed solution is in the reaction that hour is hydrolyzed of 45 ℃----55 ℃, time 1----4;
4) hydrolyzate is carried out the centrifugal residue that goes, get supernatant liquor, be the manna oligosaccharide finished product through micro-filtration, ultrafiltration, vacuum-drying again.
Hydrolyzate is carried out the centrifugal residue that goes, get supernatant liquor, weigh after micro-filtration, ultrafiltration, vacuum-drying, degradation product purity reaches 85%.
The present invention uses HBM047 Yeast engineering bacteria fermentative production 'beta '-mannase, occupies the absolute predominance status owing to this enzyme activity height (enzymic activity reaches 1080u/ml) and in enzyme system, and therefore, the purity of its enzymolysis product can be up to more than 85%.And use the manna oligosaccharide that this enzyme produces (Mannooligosaccharibes is called for short MOS) purity height, production cost is low, is the superfine product in the functional oligose.
'beta '-mannase Yeast engineering bacteria of the present invention is compared existing high product enzyme level and lower production cost, the contingent condition of the multiple Polysaccharides that can suit again with the bacterial strain that traditional breeding technique obtains.
Embodiment
Extract total DNA of environmental microorganism, with restriction enzyme it is carried out partially digestedly, reclaim the fragment of 4----10kb, be cloned in the cloning vector, transformed into escherichia coli is by making substrate with Rhizoma amorphophalli powder, filter out goal gene with the plate screening method, and check order.Obtain cloning the beta-mannase gene sequence according to order-checking, use methods such as PCR again, said gene is inserted on pichia spp (Pichia pastoris) integrating expression vector, import among the pichia spp host GS115 by chemical conversion again, and the some transformants that obtain are carried out PCR identify.
Embodiment 1: identify that conclusive evidence has imported the GS115 transformant some (as 8----10) of 'beta '-mannase yeast genes, through shake-flask culture, makes the OD600 of cell density correspondence reach 2.0----6.0; Be transferred to methyl alcohol as in the inducing culture of sole carbon source in 28 ℃----30 ℃ of inducing culture.Inducing culture 24 hours, every sampling in 8 hours, to take a sample and carry out the SDS-PAGE electrophoretic analysis.
Select, induce, cultivate the bacterial strain HBM047 (GS115+ 'beta '-mannase) that efficiently expresses 'beta '-mannase, repeat the inducing culture that said process carries out fairly large (the bottling amount of 100----1000ml), sampling is therebetween carried out SDS-PAGE and is analyzed, reach 300u/ml when above when producing enzyme level, stop to induce the centrifugation thalline, collection contains the supernatant liquor of 'beta '-mannase, be crude enzyme liquid, crude enzyme liquid carries out enzyme activity determination, and enzymic activity is 300u/ml.
Embodiment 2: identify that conclusive evidence has imported beta-mannase gene GS115 transformant some (as 8----10), through shake-flask culture, makes the OD600 of cell density correspondence reach 2.0----6.0; Be transferred to methyl alcohol as in the inducing culture of sole carbon source in 28 ℃----30 ℃ of inducing culture.Inducing culture 72 hours, preceding therebetween 48 hours, every sampling in 8 hours, after 48 hours, every interval 12 hours sampling, to take a sample and carry out the SDS-PAGE electrophoretic analysis.
Select, induce, cultivate the bacterial strain HBM047 (GS115+ 'beta '-mannase) that efficiently expresses 'beta '-mannase, repeat the inducing culture that said process carries out fairly large (the bottling amount of 100----1000ml), sampling is therebetween carried out SDS-PAGE and is analyzed, reach 960u/ml when above when producing enzyme level, stop to induce the centrifugation thalline, collection contains the supernatant liquor of 'beta '-mannase, be crude enzyme liquid, crude enzyme liquid carries out enzyme activity determination, and enzymic activity is 960u/ml.
Embodiment 3: identify that conclusive evidence has imported the GS115 transformant some (as 8----10) of beta-mannase gene, through shake-flask culture, makes the OD600 of cell density correspondence reach 2.0----6.0; Be transferred to methyl alcohol as in the inducing culture of sole carbon source in 28 ℃----30 ℃ of inducing culture.Inducing culture 168 hours, preceding therebetween 48 hours, every sampling in 8 hours, after 48 hours, every interval 24 hours sampling, to take a sample and carry out the SDS-PAGE electrophoretic analysis.
Select, induce, cultivate the bacterial strain HBM047 (GS115+ beta-mannase gene) that efficiently expresses the 'beta '-mannase effect, repeat the inducing culture that said process carries out fairly large (the bottling amount of 100----1000ml), sampling is therebetween carried out SDS-PAGE and is analyzed, reach 1080u/ml when above when producing enzyme level, stop to induce the centrifugation thalline, collection contains the supernatant liquor of 'beta '-mannase, be crude enzyme liquid, crude enzyme liquid carries out enzyme activity determination, and enzymic activity is 1080u/ml.
Embodiment 4
Use the method that above-mentioned 'beta '-mannase crude enzyme liquid hydrolysis prepares manna oligosaccharide, the steps include:
1), one-level Rhizoma amorphophalli powder 100g is mixed with concentration with distilled water is 10% the konjaku aqueous solution, swelling one hour;
2), enzyme-added in the crude enzyme liquid ratio of the corresponding 1ml of every 10g Rhizoma amorphophalli powder;
3), above-mentioned mixed solution is in the reaction that is hydrolyzed of 45 ℃, 1 hour time;
4) hydrolyzate is carried out the centrifugal residue that goes, get supernatant liquor, be H-manna oligosaccharide finished product through micro-filtration, ultrafiltration, vacuum-drying again.
Embodiment 5
1), one-level Rhizoma amorphophalli powder 100g is mixed with concentration with distilled water is 15% the konjaku aqueous solution, swelling one hour;
2), enzyme-added in the crude enzyme liquid ratio of the corresponding 1ml of every 15g Rhizoma amorphophalli powder;
3), above-mentioned mixed solution is in the reaction that is hydrolyzed of 52 ℃, 3 hours time;
4) hydrolyzate is carried out the centrifugal residue that goes, get supernatant liquor, be H-manna oligosaccharide finished product through micro-filtration, ultrafiltration, vacuum-drying again.
Embodiment 6
1), one-level Rhizoma amorphophalli powder 100g is mixed with concentration with distilled water is 20% the konjaku aqueous solution, swelling one hour;
2), enzyme-added in the crude enzyme liquid ratio of the corresponding 1ml of every 20g Rhizoma amorphophalli powder;
3), above-mentioned mixed solution is in the reaction that is hydrolyzed of 55 ℃, 4 hours time;
4) hydrolyzate is carried out the centrifugal residue that goes, get supernatant liquor, be H-manna oligosaccharide finished product through micro-filtration, ultrafiltration, vacuum-drying again.
The said hydrolyzed thing is carried out the centrifugal residue that goes, to weigh after supernatant liquor micro-filtration successively, ultrafiltration, the vacuum-drying, calculated yield is 85%, get the dry thing of part and be dissolved in by a certain percentage again in the distilled water, carry out the evaluation of thin-layer chromatography or mass spectrum and show that the manna oligosaccharide content of 5----10 sugar is greater than 60%.
Claims (4)
1, a kind of 'beta '-mannase yeast gene engineering bacteria (Pichia pastoris GS115/HBM047) is method and the round pcr by the constructing environment genomic library, it is characterized in that from environmental microorganism, filtering out beta-mannase gene, above-mentioned beta-mannase gene is cloned in the pichia spp integrating expression vector, the beta-mannase gene expression vector that contains that obtains then imports in the pichia spp, therefrom filters out a kind of yeast gene engineering bacteria HBM047 bacterial strain that efficiently expresses 'beta '-mannase again.
2, the yeast gene engineering bacteria of 'beta '-mannase according to claim 1, it is characterized in that extracting total DNA of environmental microorganism, carry out partially digested with restriction enzyme to it, reclaim the fragment of 4----10kb, be cloned in the cloning vector, transformed into escherichia coli, by making substrate with Rhizoma amorphophalli powder, filter out goal gene with the plate screening method, and check order, obtaining cloning the beta-mannase gene sequence according to order-checking, use PCR method again, is template with the plasmid DNA of the positive colony that screens in the environment genomic library, amplify the dna fragmentation of 1.7KB, be inserted in pichia spp (Pichia pastoris) integrating expression vector, import among the pichia spp host GS115 by chemical conversion again, therefrom filter out again and efficiently express 'beta '-mannase yeast gene engineering bacteria HBM047 bacterial strain.
3, press the 'beta '-mannase of the yeast gene engineering bacteria preparation of claim 1 or 2, it is characterized in that to efficiently express the yeast gene engineering bacteria of 'beta '-mannase, be in the substratum of carbon source with methyl alcohol, 28 ℃ of temperature--30 ℃, (surveying enzyme at regular intervals lived through 24----168 hour inducing culture, when reaching 300-1080u/ml, enzymic activity stops inducing culture), the yeast gene engineering bacteria of finishing inducing culture is removed thalline through high speed centrifugation, collect supernatant liquor, be crude enzyme liquid, concentrate after filtration and promptly get the beta-mannase enzyme product.
4, prepare the method for manna oligosaccharide by the described beta-mannase enzymic hydrolysis of claim 3, it is characterized in that the steps include:
1), the one-level Rhizoma amorphophalli powder is mixed with the konjaku aqueous solution that concentration is 10%---20%, swelling one hour with distilled water;
2), enzyme-added in the crude enzyme liquid ratio of the corresponding 1ml of every 10----20g Rhizoma amorphophalli powder;
3), above-mentioned mixed solution is in the reaction that hour is hydrolyzed of 45 ℃----55 ℃, time 1----4;
4) hydrolyzate is carried out the centrifugal residue that goes, get supernatant liquor, be the manna oligosaccharide finished product through micro-filtration, ultrafiltration, vacuum-drying again.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008024B (en) * | 2007-01-30 | 2010-06-02 | 武汉泰运投资担保有限公司 | Process for producing high-purity kanjak mannan-oligosaccharides |
| CN102732494A (en) * | 2011-04-11 | 2012-10-17 | 中国农业大学 | Beta-mannanase and preparation method thereof |
| CN103060214B (en) * | 2007-12-10 | 2014-09-03 | 东方酵母工业株式会社 | Yeast having immunopotentiating effect and food or feed containing thereof |
| CN104404109A (en) * | 2014-12-10 | 2015-03-11 | 江南大学 | Method for preparing mannan-oligosaccharide through enzymolysis of fine konjak powder |
| CN105087717A (en) * | 2015-07-31 | 2015-11-25 | 丽江玉元食品有限公司 | Method of utilizing acidic mannase to hydrolyze refined konjac powder to prepare konjac oligosaccharide |
| CN107129958A (en) * | 2017-06-09 | 2017-09-05 | 华南理工大学 | A kind of screening technique of β mannosans enzyme engineering bacteria |
-
2003
- 2003-04-22 CN CNA031189849A patent/CN1478887A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008024B (en) * | 2007-01-30 | 2010-06-02 | 武汉泰运投资担保有限公司 | Process for producing high-purity kanjak mannan-oligosaccharides |
| CN103060214B (en) * | 2007-12-10 | 2014-09-03 | 东方酵母工业株式会社 | Yeast having immunopotentiating effect and food or feed containing thereof |
| CN102732494A (en) * | 2011-04-11 | 2012-10-17 | 中国农业大学 | Beta-mannanase and preparation method thereof |
| CN102732494B (en) * | 2011-04-11 | 2014-06-11 | 中国农业大学 | Beta-mannanase and preparation method thereof |
| CN104404109A (en) * | 2014-12-10 | 2015-03-11 | 江南大学 | Method for preparing mannan-oligosaccharide through enzymolysis of fine konjak powder |
| CN105087717A (en) * | 2015-07-31 | 2015-11-25 | 丽江玉元食品有限公司 | Method of utilizing acidic mannase to hydrolyze refined konjac powder to prepare konjac oligosaccharide |
| CN107129958A (en) * | 2017-06-09 | 2017-09-05 | 华南理工大学 | A kind of screening technique of β mannosans enzyme engineering bacteria |
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