CN1170924C

CN1170924C - Yeast gene engineering bacterium and endoinulase preparation and its application method

Info

Publication number: CN1170924C
Application number: CNB011284404A
Authority: CN
Inventors: 马立新; 蒋思婧
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2001-09-11
Filing date: 2001-09-11
Publication date: 2004-10-13
Anticipated expiration: 2021-09-11
Also published as: CN1341709A

Abstract

The present invention provides genetic engineering yeast of Pichia pastoris GS115/HY005, which is obtained by the following steps: screening and obtaining an endo-inulinase gene from aspergillus strains by a PCR method or a DNA Library hybridization method; cloning the endo-inulinase gene; inserting the genes into an integrant expression carrier of pichia pastoris; introducing the obtained expression carrier containing the endo-inulinase genes into the pichia pastoris; selecting genetic engineering yeast which can efficiently express the endo-inulinase from the pichia pastoris. The enzyme activity of the endo-inulinase prepared by the genetic engineering yeast can reach more than 300 u/ml, and the genetic engineering yeast can be used to hydrolyze alantin with the advantages of short reaction time and high conversion efficiency. Thus, fructo-oligosaccharide can be efficiently and economically converted and prepared.

Description

A kind of yeast gene engineering bacteria and endoinulase preparation and methods for using them

Technical field

What the present invention relates to is genetic engineering bacterium, and particularly a kind of yeast gene engineering bacteria that efficiently expresses endoinulase and inulinase preparation and endoinulase hydrolytic inulin prepare the method for oligofructose.

Background technology

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.

At present, industrial production oligofructose has two kinds of methods.A kind of is sucrose inversion, and another kind is to obtain by acid hydrolyzation or enzymolysis process for raw material with inulin (or claiming synanthrin, a kind of product of producing from jerusalem artichoke).Enzymolysis process has many good qualities, and is pollution-free, simple to operate, good in economic efficiency, is the method that praise highly countries in the world.Key in how obtaining highly active inulinase in the enzymolysis process production and be to produce.At present extract inulinase (see the 195th page of microbiology circular 1998 25 (4) phase, be called the report of " separation and purification of aspergillus niger inulinase and determination of activity thereof ", used bacterial classification is aspergillus P319) by cultivation separation and purification from aspergillus niger.Also have from yeast cultivation separation and purification extract inulinase (see the 13rd page of Tianjin microorganism magazine 94 years---26 pages, the report of " research of inulin lytic enzyme and utilisation technology thereof " by name of Tianjin industrial microorganism institute, used bacterial classification is crisp wall gram dimension yeast KF9031), above-mentioned bacterial classification is just with conventional screening, do not carry out genetic engineering modified to it, so the inulinase activity that produces generally has only 50u/ml less than 100u/ml.Effect is not very good.

Summary of the invention

The objective of the invention is to make up the yeast gene engineering bacteria that can efficiently express endoinulase, and produce endoinulase, thereby can transform the preparation oligofructose efficiently, economically from cultivation separation and purification wherein by engineered means.

The microorganism of using: the yeast gene engineering bacteria that the present invention relates to (Pichia yeast Pichia pastorisGS115/HY005), it has the characteristic that efficiently expresses endoinulase, this bacterial strain in August 31 calendar year 2001 at China typical culture collection center, preserving number is CCTCC NO:M201032 (hereinafter to be referred as HY005).

The bacteria characteristic of HY005 bacterial strain:

A, morphological character: the monogony of Pichia yeast is budding, and pseudohypha is arranged sometimes.Syngenesis produces ascus and contains 1---4 pieces of smooth circles, carnival hat shape or Saturn thecaspores.

B, physio-biochemical characteristics: Pichia yeast can utilize special material to grow for raw material, as oil, methyl alcohol, ammonia-state nitrogen, organic acid etc.The HY005 bacterial strain has the physio-biochemical characteristics of Pichia yeast, simultaneously because of having inserted the endoinulase gene, had the characteristic that efficiently expresses the endoinulase gene.

The present invention realizes like this.Yeast gene engineering bacteria (Pichia yeast Pichia pastorisGS115/HY005), preserving number is CCTCC NO:M201032, be by PCR method or DNA library hybridizing method, obtain the endoinulase gene from the Aspergillus bacterial strain screening, it is characterized in that according to aspergillus niger endoinulase gene order, utilize conventional polymerization process to synthesize a pair of primer, using conventional PCR method is template with the DNA of aspergillus NRRL3135, under the pcr amplification condition of routine, amplify 0.5---the dna fragmentation of 2Kb, be cloned into T---on the carrier pMD18-T, after order-checking confirms to be cloned into the exactness of the dna fragmentation on the pMD18-T carrier, the endoinulase gene of the NRRL3135 that the clone is obtained with BamHI and NotI scales off, be inserted among the BamHI and NotI restriction enzyme site of pichia spp (Pichia pastoris) expression vector pPIC3.5k, transform among the importing pichia spp host GS115 by electricity again, therefrom filter out the yeast gene engineering bacteria that efficiently expresses endoinulase again.

The yeast gene engineering bacteria HY005 of endoinulase will be efficiently expressed, be in the substratum of carbon source with methyl alcohol, 28 ℃ of temperature---30 ℃, through 24---(survey enzyme at regular intervals lives 148 hours inducing culture, enzymic activity reaches 250---and stop inducing culture during 450u/ml), the yeast gene engineering bacteria of finishing cultivation is removed thalline through high speed centrifugation, collect supernatant liquor, be crude enzyme liquid, concentrate after filtration and promptly get the endoinulase product.

Use the method that above-mentioned endoinulase crude enzyme liquid hydrolysis prepares oligose, the steps include:

1), the sodium-acetate buffer of---50mM, pH5.0---7.0 is mixed with the inulin solution that concentration is 10--20% with 10 with inulin;

2), in per 50---the crude enzyme liquid ratio of the corresponding 1ml of 100g inulin is enzyme-added;

3) reaction that is hydrolyzed that, above-mentioned mixed solution is at 45 ℃---60 ℃, time 4---12 hours;

4), hydrolyzate is carried out the centrifugal residue that goes, get supernatant liquor, again through micro-filtration, ultrafiltration, vacuum-drying, be the oligofructose finished product.

With the endoinulase of the yeast gene engineering bacteria preparation that efficiently expresses endoinulase, its enzymic activity can reach more than the 300u/ml, and (general method is that 100u/ml is following), thus the hydrolysis time weak point (general method is 24---48 hours) and, the transformation efficiency height.Hydrolyzate is carried out the centrifugal residue that goes, get supernatant liquor, weigh after micro-filtration, ultrafiltration, vacuum-drying, calculated yield is 65---85%, and carry out thin-layer chromatography or mass spectrum and identify---the oligofructose content of 10 sugar accounts for 85%---95% that show 2.

Embodiment

Endoinulase gene order according to the aspergillus niger of having expressed, use PCR method, DNA with Fructus Fici aspergillus NRRL3135 is a template, amplify the dna fragmentation of about 1.5kb, be cloned on the T-carrier pMD18-T, confirm that through dna sequence analysis clone's fragment includes the endoinulase gene of NRRL3135, with the aspergillus niger endoinulase gene InuB that has delivered 98% homology of having an appointment.

Be inserted in the expression box structure of pichia spp (Pichiapastoris) expression vector PIC3.5k by the endoinulase gene of subclone the NRRL3135 of being cloned into, transform among the importing pichia spp host GS115 by electricity again, and the some transformants that obtain are carried out PCR identify.

Embodiment 1: identify that conclusive evidence has imported endoinulase gene GS115 transformant some (as 8---10), through shake-flask culture, make 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 10 hours, to take a sample and carry out the SDS-PAGE electrophoretic analysis.

Select, induce, cultivate the bacterial strain HY005 (GS115+ endoinulase gene) that efficiently expresses the endoinulase effect, repeat the inducing culture that said process carries out fairly large (100---the bottling amount of 1000ml), sampling is therebetween carried out SDS-PAGE and is analyzed, reach 250u/ml when above when producing enzyme level, stop to induce, the centrifugation thalline is collected the supernatant liquor that contains endoinulase, is crude enzyme liquid.Crude enzyme liquid is carried out enzyme activity determination, and enzymic activity is 250u/ml.

Embodiment 2: identify that conclusive evidence has imported endoinulase gene GS115 transformant some (as 8---10), through shake-flask culture, make 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 10 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 HY005 (GS115+ endoinulase gene) that efficiently expresses the endoinulase effect, repeat the inducing culture that said process carries out fairly large (100---the bottling amount of 1000ml), sampling is therebetween carried out SDS-PAGE and is analyzed, reach 350u/ml when above when producing enzyme level, stop to induce the centrifugation thalline, collection contains the supernatant liquor of endoinulase, be crude enzyme liquid, crude enzyme liquid is carried out enzyme activity determination, enzymic activity is 350u/ml.

Embodiment 3: identify that conclusive evidence has imported endoinulase gene GS115 transformant some (as 8---10), through shake-flask culture, make 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 10 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 HY005 (GS115+ endoinulase gene) that efficiently expresses the endoinulase effect, repeat the inducing culture that said process carries out fairly large (100---the bottling amount of 1000ml), sampling is therebetween carried out SDS-PAGE and is analyzed, reach 450u/ml when above when producing enzyme level, stop to induce the centrifugation thalline, collection contains the supernatant liquor of endoinulase, be crude enzyme liquid, crude enzyme liquid is carried out enzyme activity determination, enzymic activity is 450u/ml.

Crude enzyme liquid hydrolysis time: with 100---the 1000g extracting in the inulin of jerusalem artichoke with 10---the inulin solution of the pH5.0 of 50mM---7.0 sodium-acetate buffer is mixed with 10---20%, again in 50---the ratio of the thick liquid of the corresponding 1ml of 100g inulin is enzyme-added,---60 ℃ are carried out 4---hydrolysis reaction of 12 hours in 45 ℃.

The said hydrolyzed thing is carried out the centrifugal residue that goes, getting supernatant liquor weighs after micro-filtration, ultrafiltration, the vacuum-drying successively, calculated yield is 65---85%, get the dry thing of part and be dissolved in by a certain percentage again in the aquae destillata, carry out thin-layer chromatography or mass spectrum and identify---the oligofructose content of 10 sugar accounts for 85%---95% that show 2.

Claims

1, a kind of yeast gene engineering bacteria (Pichia yeast Pichia pastoris GS115/HY005), preserving number is CCTCC NO:M201032, be by PCR method or DNA library hybridizing method, obtain the endoinulase gene from the Aspergillus bacterial strain screening, it is characterized in that according to aspergillus niger endoinulase gene order, utilize conventional polymerization process to synthesize a pair of primer, using conventional PCR method is template with the DNA of aspergillus NRRL3135, under the pcr amplification condition of routine, amplify the dna fragmentation of 0.5----2Kb, be cloned on the T----carrier pMD18--T, after order-checking confirms to be cloned into the exactness of the dna fragmentation on the pMD18--T carrier, the endoinulase gene of the NRRL3135 that the clone is obtained with BamHI and NotI scales off, be inserted among the BamHI and NotI restriction enzyme site of pichia spp (Pichia pastoris) expression vector pPIC3.5k, transform among the importing pichia spp host GS115 by electricity again, therefrom filter out the yeast gene engineering bacteria that efficiently expresses endoinulase again.

2, a kind of endoinulase preparation by the described yeast gene engineering bacteria of claim 1, it is characterized in that to efficiently express the yeast gene engineering bacteria of endoinulase, be in the substratum of carbon source with methyl alcohol, 28 ℃-30 ℃ of temperature, through 24-148 hour inducing culture, surveying enzyme at regular intervals lives, when reaching 250-450u/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 endoinulase product.

3, by the method for the described endoinulase hydrolysis of claim 2 oligose, it is characterized in that:

1), inulin is mixed with the inulin solution that concentration is 10--20% with the sodium-acetate buffer of 10----50mM, pH5.0----7.0;

2), enzyme-added in the crude enzyme liquid ratio of the corresponding 1ml of every 50----100g inulin;

3), above-mentioned mixed solution is in the reaction that hour is hydrolyzed of 45 ℃----60 ℃, time 4----12;

4) hydrolyzate is carried out the centrifugal residue that goes, gets supernatant liquor, again through micro-filtration, ultrafiltration, vacuum-drying, be the oligofructose finished product.