CN104263744A - Artificially modified glucose oxidase gene and expression application thereof - Google Patents

Abstract

The invention relates to an artificially modified glucose oxidase gene and expression application thereof, belonging to the fields of protein or enzyme preparation modification engineering and glucose oxidase production. The artificially modified glucose oxidase gene aims to further enhance enzyme activity on the basis of the existing glucose oxidase. An irrational means DNA (deoxyribonucleic acid) shuffling technique in protein or enzyme preparation modification engineering is utilized to modify a glucose oxidase gene derived from Aspergillus niger Z-25; and a gene segment subjected to DNaseI random cutting is subjected to the DNA shuffling technique to recombine the segment into a complete variant gene, and the complete variant gene is connected with a shuttling expression plasmid and integrated into the yeast genome. The strain with obviously higher enzyme activity is screened from the mutant library, and the variant sequence is disclosed as SEQ ID NO.2. In a 3L scale-up experiment of yeast, the end enzyme activity of the glucose oxidase reaches the higher level. Finally, in the aspect of enzyme activity and genes, the obtained high-enzyme-activity expression strain has favorable hereditary.

Description

A kind of engineered glucose oxidase gene and Expression and Application thereof

Technical field

The present invention relates to irrational albumen transformation means, particularly relate to and utilize the glucose oxidase gene of DNA shuffling technology to Aspergillus niger origin to transform, glucose oxidase gene after the invention still further relates to variation produces the application in glucose oxidase in secretion, belong to albumen or zymin improvement project and glucose oxidase production field.

Background technology

Glucose oxidase (Glucose Oxidase, E.C.1.1.3.4, be called for short GOD) under the condition having oxygen, gluconic acid and hydrogen peroxide can be generated by specificity catalysis β-D-Glucose, be distributed in (Pluschkell S in animal, plant and microbe widely, Hellmuth K, Rinas U.Kinetics of glucose oxidase excretion by recombinant Aspergillus niger.Biotechnol Bioeng 1996; 51:215-20.).

But due to microorganism, to have growth and breeding speed fast, and the feature such as to originate wide, so microorganism becomes the main source of glucose oxidase, the main production bacterial strain in microorganism is aspergillus niger and mould.

Glucose oxidase has a wide range of applications in food, medicine and the field such as biological.

In the food industry, for removing the glucose in food, brown stain is prevented; Also can be used for food deoxidation, improve the quality of food and drink; Simultaneously also for aspect (the Rasiah IA such as improved technology of flour, Sutton KH, Low FL, Lin HM, Gerrard JA.Crosslinking of wheat dough proteins by glucose oxidase and theresulting effects on bread and croissants.Food Chem 2005; 89:325 – 32.; Crueger A, Crueger W.Glucose transforming enzymes.In:Fogarty WM, Kelly CT, editors.Microbial enzymes and biotechnology.New York:Elsevier; 1990.p.177 – 226.).In medicine industry, for the detection of the mensuration of blood sugar and glucose in urine, urine ketoboidies, can be made into glycosuria test paper or test kit for clinical diagnosis; Also can prevent and treat oral disease and odontopathy (Wilson R, Turner APF.Glucose oxidase:and ideal enzyme.Biosens Bioelectron1992; 7:165-85; Etemadzadeh H, Ainamo J, Murtomaa H.Plaque growth-inhibiting effects of an abrasive fluoride – chlorhexidine toothpaste and a fluoride toothpaste containing oxidative enzymes.J Clin Periodontol 1985; 7:607-16.).In alcoholic beverage industry, glucose oxidase can reduce the concentration of alcohol by removing glucose, thus reduce the alcoholic strength of wine and production low alcohol (Malherbe DF, du Toit M, Cordero RR, van Rensburg P, Pretorius IS.Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production.Appl Microbiol Biotechnol 2003; 5-6:502-11 [My paper].; Pickering GJ, Heatherbell DA, BarnesMF.Optimising glucose conversion in the production of reduced alcohol wine using glucose oxidase.Food Res Int 1998; 31 (10): 685-92.).

Glucose oxidase is also used on industrial production gluconic acid, produces gluconic acid by reduction Gluconolactone.Gluconic acid can be additive as adjustment food acid number, also can be made into the gluconate of the various trace element of supplementary needed by human body and production and sales (Nakao K, Kiefner A, Furumoto K, Harada T.Production of gluconic acid with immobilized glucose oxidase in airlift reactors.Chem Eng Sci 1997; 52:4127 – 33.; Klein J, Rosenberg M, Markos J, Dolgos O, KroslakM, Kristofikova L.Biotransformation of glucose to gluconic acid by Aspergillus niger-study of mass transfer in an airlift bioreactor.Biochem Eng J 2002; 3568:1 – 9.).

Glucose oxidase is also used in textile industry, because glucose oxidase can produce hydrogen peroxide in mechanism, can play bleaching action.The hydrogen peroxide that this mode produces through checking, shows the effect that standard bleaching process is same.More it is worth mentioning that, do not need additionally to add stablizer in this bleaching process, because the gluconic acid produced in process can serve as role (the Tzanov T of stablizer, Silgia A, Gubitz GM, Cavaco-Paul μ lo A.Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching.J Biotechnol 2002; 93:87 – 94.).

Current modal glucose oxidase enzyme source is got by the fermentation of aspergillus niger, mould and Saccharomycodes.Most business-like glucose oxidase is separated to obtain from the mycelium of aspergillus niger.There is the investigator of China by organic solvent deposit, polyoxyethylene glycol secondary sedimentation, hydroxyapatite pillar layer separation, obtain the product (Chinese Journal of Pharmaceutical 1997,28 (7)) of specific activity more than 190U/ml albumen.

But the fermentation due to aspergillus niger is mainly used as to produce gluconic acid or gluconate, such as Sunmorl N 60S, calglucon, therefore glucose oxidase mainly occurs as a kind of by product, and this causes the production enzyme of glucose oxidase alive not high, be all much only have the enzyme of units to live (S.B.Bankar et al./Biotechnology Advances27 (2009) 489-501).

Along with the development of Protocols in Molecular Biology, investigator attempts to carry out expression alien gene by Microbial Expression Systems, reaches the object to a certain albumen high expression with this.The heterologous gene expression system developed at present comprises intestinal bacteria, genus bacillus, streptomycete, aspergillus, yeast, insect, cells of mamma animals etc.Under normal circumstances, use prokaryotic cell prokaryocyte easy and simple to handle as the expression system of foreign gene, various technology has been tending towards ripe, but by the restriction of prokaryotic system itself, the albumen of eukaryotic source can not correctly fold when expressing or necessity modification after lacking transcription and translation, thus cause having given expression to albumen, albumen does not but have biological activity, there is no further utility value, and toxic protein in prokaryotic system or have the albumen of antigenic action to be mingled in possibly in end product, result is interfered.

But prokaryotic expression is still significant, before can expressing as eukaryotic system, verify that a kind of albumen can depart from autogenous system and the means expressed by another kind of system.

And for eucaryon mammalian cell as host, the modification after protein expression can be carried out ideally, but due to operational difficulty, and later stage culturing cell has the possibility of virus infection, and therefore this method does not become widely used method.

So the protein expression system of main flow is yeast system at present.

Yeast system has become a kind of pattern service platform at present by the development of decades, and operations has been tending towards perfect.In protein expression, there is the intracellular environment being suitable for eukaryotic gene product and correctly folding, especially can modify the crucial glycosylation site of some of albumen, enable some have the albumen of requirement to give expression to activity to glycosylation.

In addition, the Yeast system advantage that also has other system incomparable.

First Yeast system has the ability of secreting, expressing, that is, and can by the protein excretion that gives expression in the middle of substratum.This has great advantage in experimentation and industrial production.Eliminating the program of smudge cells in the process of genetic modification, can directly further studying by obtaining target protein in substratum fermented liquid.In the industrial production, the isolation technique of albumen and thalline is very ripe, if therefore protein product is secreted in fermented liquid, will save very large cost.

What secondly yeast expression system was taked is methanol induction mechanism, namely utilizes alcohol oxidase promotor to induce, and in shaking flask level, utilizes methyl alcohol to carry out inducing the probability that effectively can reduce microbiological contamination.

3rd, utilize the carrier that yeast operates, can exogenous origin gene integrator be entered in the genome of yeast, can ensure that the foreign gene introduced stably exists in yeast system like this, and not need the free carrier needs of picture to provide the survival pressure of outside to ensure the existence of carrier in yeast.So just avoid the problem of spawn degeneration to a certain extent.

4th, the accumulation of yeast-leavened product can't produce toxic action by yeast self, and the expression strain of yeast is easy to expand high density fermentation to from shaking flask, does not affect the expression level of foreign gene simultaneously.Be doomed for these 2 to make yeast have the potential quality (letter in biotechnology, Vol.16No.2Mar., 2005) of high density fermentation and then high level expression foreign protein.

After have selected expression system, want to improve the oxidasic enzyme work of Exogenous Glucose further and only at gene level, glucose oxidase gene sequence is transformed.

The method of the activity of usual raising enzyme has the strategies such as codon optimized, mRNA structure of modification, the adjustment of GC content, random mutation, albumen orthogenesis.Some method has achieved extraordinary effect.The people such as the Gao Zhaowei of such as Southwestern University are after being optimized the codon of glucose oxidase gene, and after 3L fermentation 120h, enzyme is lived and reached 365U/ml.

But codon optimized result still has limitation.Because all codons in gene are all transformed according to the codon preference of yeast by this method, although the Preference of yeast can be utilized like this to improve the expression of zymoprotein, but virtually increase the weight of the burden of yeast, probably can not reach best expression effect.Therefore consider that improving enzyme by other means lives.

In the nature evolution of more than one hundred million years, protein defines diversified form, plays respective effect in respective physical environment.Enzyme has bioactive albumen as one, in the productive life of nature and the mankind, play huge effect.This just impels people to want the utilization improved further enzyme.Namely want to transform enzyme.Artificial reconstructed means for enzyme have a variety of, and optimal method makes it produce desirable macroscopic property to the amino acid sites adjustment of some key in enzyme molecule.But the bottleneck of this method is that the research at present for enzyme molecular structure and catalytic mechanism is comprehensive not enough, therefore wants purposive, that engineered protein is certain in addition reasoningly difficulty.

Be directed to this contradiction, create enzyme Directed Molecular Evolution in Vitro technology, according to Darwinian evolution, Simulating Evolution mechanism in vitro, the condition of simulating nature evolution in the lab, the albumen that artificial screening makes new advances or proterties is improved.

The orthogenesis basic ideas of enzyme molecule are divided into two steps: the foundation of mutated library and the screening of high-quality mutant.

The method of the gene random mutation occurred the earliest is fallibility PCR, and this principle simple operations easily method is found broad application rapidly.Only need on the method for PCR, the reaction conditions in appropriate change PCR system, makes the fidelity of polysaccharase decline, and produces mispairing and gets final product (Chen K, Arnold FH.Proc Natl Acad Sci USA, 1993,90:5618 ~ 22.).

DNA reordering technique is proposed afterwards, i.e. DNA shuffling technology in 1994.Theoretically, this technology can cause gene internal to change on a large scale, thus introduces sudden change, builds mutated library (Stemmer WP.Nature, 1994,370:389 ~ 91.).On the basis of DNA shuffling technology, occurred again the new technologies such as StEP, RPR afterwards, but principle being similar, is all the restructuring of gene fragment.

Efficient sudden change means must be arranged in pairs or groups mutually with efficient screening means.In the screening means in library, the most common is high flux screening.High flux screening carries out usually on 96 orifice plates, adopts the technology of automatization carry out distributing to fluid and measure.High flux screening has the advantage of trace analysis, and the sample namely needed for location parameter does not need a lot, but therefore can detect by the sample less to multiple sample sample capacity in laboratory scope.

Summary of the invention

Problem to be solved by this invention on the existing enzyme of glucose oxidase lives basis, improves enzyme further live.Come from the glucose oxidase gene of aspergillus niger by irrational DNA shuffling technological transformation, from the mutation library built, screening obtains the enzyme bacterial strain significantly improved alive afterwards.

Problem to be solved by this invention is realized by following experimental program:

By the new engineered glucose oxidase gene that irrational sudden change obtains, it is characterized in that: concrete nucleotide sequence is as shown in SEQ ID NO.2.

By the new engineered glucose oxidase that irrational sudden change obtains, it is characterized in that: concrete aminoacid sequence is as shown in SEQ ID NO.3.

Glucose oxidase gene in the aspergillus niger Z-25 that clone obtains by the present invention is cut into gene fragment at random by DNaseI, the fragment selecting wherein 50-60bp size utilizes DNA shuffling technology that fragment restructuring is become complete fragment, obtains the mutation library of glucose oxidase gene.

The shuttle expression plasmid of the glucose oxidase gene obtained containing above-mentioned irrational sudden change and the host cell containing this shuttle expression plasmid are also at the row of protection category of the present invention; Wherein, described shuttle expression plasmid is the shuttle expression plasmid between intestinal bacteria and yeast, is preferably intestinal bacteria BMTOP10 and Pichia pastoris GS115.

A high-throughput screening method for glucose oxidase gene mutation library, comprising: be connected with described shuttle expression plasmid by the mutation library of glucose oxidase gene obtained above, direct transformed yeast cell; From plate culture medium, choose bacterial strain induce expression of recombinant proteins in 48 orifice plates, in 96 orifice plates, carry out enzyme activity determination, directly do high flux screening with final host yeast and eliminate colibacillary screening step.

The glucose oxidase gene that the present invention utilizes aspergillus niger Z-25 (Aspergillus.niger Z-25) to originate carries out DNA shuffling transformation, obtain glucose oxidase mutant gene storehouse, being connected with shuttle expression plasmid in mutator gene storehouse transforms as expressed in Pichia pastoris GS115 again, therefrom screen the enzyme bacterial strain significantly improved alive, afterwards sequencing analysis is being carried out to its sequence.Experimental result shows, screens the bacterial strain obtained and reach higher level in the level expressing glucose oxidase.In addition, because the gene integration obtained can enter in saccharomycetic genome karyomit(e) by the shuttle expression plasmid used, the live high-enzyme strain therefore obtained has stable heredity.

Accompanying drawing explanation

Fig. 1 aspergillus niger Z-25 glucose oxidase enzymatic amplification electrophorogram

Fig. 2 DNaseI enzyme cuts aspergillus niger Z-25 glucose oxidase electrophorogram

Fig. 3 glucose oxidase gene fragment reunion electrophorogram

Fig. 4 glucose oxidase gene mutation library and shuttle expression plasmid connection diagram

Figure 54 8 deep-well plates induced glucose oxydase mutator gene transformant schematic diagram

Figure 69 6 orifice plate high flux screening glucose oxidase mutant gene transformant schematic diagram

Fig. 7 height enzyme mutator gene transformant alive 3 liters of fermented grape carbohydrate oxidase enzymes graphic representation alive

Fig. 8 height enzyme mutator gene transformant alive 3 liters of fermented liquid supernatant protein content graphic representations

Fig. 9 height enzyme mutator gene transformant 3 liters alive fermentation graphic representation more alive than enzyme

Figure 10 height enzyme mutator gene transformant alive 3 liters of fermentation biomass graphic representations

Figure 11 height enzyme mutator gene transformant alive 3 liters of fermented cells dry weight graphic representations

Figure 12 height enzyme mutator gene transformant alive genetic stability electrophorogram

Embodiment

Details of the present invention is set forth further below in conjunction with specific embodiment.But embodiment is exemplary in nature, any restriction is not formed to scope of the present invention.Those skilled in that art are construed as, and can modify to the technical scheme details of invention and form or replace in the spirit and scope of the present invention, but these amendments and replacement are all within protection scope of the present invention.

Illustrate: gene manipulation techniques involved in following specific embodiment is the routine techniques of the maturation of present stage in this area.Remove the technology be not described in detail, all carry out according to the related Sections in following experiments handbook or document: the concentration that is not particularly illustrated in following examples is volumetric concentration.

EasySelect ^tMpichia Expression Kit A Manual of Methods for Expression of Recombinant Proteins Using pPICZ and pPICZ α in Pichia pastoris Catalog no.K1740-01; Molec μ lar (word is correct) Cloning:a laboratory manual on the web.

The amplification of embodiment 1 aspergillus niger Z-25 glucose oxidase gene

1.1 bacterial strains and plasmid

Aspergillus niger Z-25 (Aspergillus.niger Z-25);

Intestinal bacteria BWTOP10 (Escherichia.coli BMTOP10) bacterial strain is buied by the limited company limited of Beijing Bo Maide development in science and technology;

The synthesis of amplimer is completed by Hua Da gene;

Sequencing vector pMD19-T is purchased from Takara company;

The amplification of 1.2 glucose oxidase genes

First the genome extracting aspergillus niger in the aspergillus niger Z-25 mycelium that glucose oxidase fermenting experiment uses is carried out.

Adopt and improve Benzyl chloride method extraction aspergillus niger Z-25 genome.

(1) mycelium pellet that obtains in Cha Shi substratum of suction filtration, blots the moisture in substratum with filter paper as far as possible

(2) in liquid nitrogen, the thalline collected is ground into powder ,-20 DEG C of preservations

(3) get 0.2g thalline in 1.5ml centrifuge tube, add the extracting solution that 500 μ l have been preheated to 50 DEG C, vibration fully mixes, and leaves standstill 2min, adds 100 μ l 10%SDS, and mixing, add 300 μ l Benzyl Chlorides, thermal agitation makes Guan Zhongcheng emulsus

(4) 50 DEG C of water-bath 1h, every 10min turn upside down vibration mixing once, make to react completely

(5) take out centrifuge tube, add 300 μ lNaAc, mixing ice bath 15min, every 4-5min puts upside down mixing once

(6) 10 DEG C of centrifugal 15min of 6000rpm, collect in supernatant to new centrifuge tube

(7) chloroform that volume ratio is 23:1:1 is added: primary isoamyl alcohol: phenol (), mixing, 10 DEG C, 10000rpm, 15min, collected by centrifugation supernatant

(8) add chloroform: primary isoamyl alcohol (24:1) extracting 2-3 time, collect whole supernatant

(9) in supernatant, add isopyknic freezing primary isoamyl alcohol, put upside down a little, precipitation at room temperature 20min

(10) 10 DEG C, 10000rpm, centrifugal 5min

(11) abandon supernatant, stay precipitation, by 70% washing with alcohol 2 times, each 10 DEG C, 10000rpm, centrifugal 10min, finally wash once with dehydrated alcohol

(12) 4 DEG C, 10000rpm, centrifugal 15min, removes ethanol, and super clean bench dries up

(13) 100 μ l water dissolution are added ,-20 DEG C of preservations

The amplification of glucose oxidase gene is carried out by following primer:

Upstream	5-CTCCTAGGATGCAGACTCTCCTTGTGAGCT-3
		Downstream	5-ATGCGGCCGCTCACTGCATGGAAGCATAATCC-3

After aspergillus niger Z-25 glucose oxidase gene removing signal peptide, length is 1746bp, 582 amino acid of encoding, and this gene is registered on GeneBank, and accession number is FJ979866.1 (SEQ ID NO.1).

The structure in embodiment 2 glucose oxidase mutant gene storehouse

2.1DNaseI cuts glucose oxidase gene at random

First utilize DNaseI to cut at random the glucose oxidase obtained that increases, DNaseI can carry out Non-specific cleavage to any gene, and system is as follows:

Glucose oxidase gene	10μl
		DNaseI damping fluid	2μl
DNaseI	0.8μl
		ddH 2O	7.2μl
Condition	37 DEG C of constant temperature 1h

2.2 glucose oxidase enzyme fragments meet again (DNA shuffling)

Get a certain amount of glucose oxidase fragment as substrate, add the PCR that enzyme and dNTP carry out without primer, this process is the process of DNA shuffling.

Concrete system is as follows:

Tag?Mix	10μl
		ddH2O	3μl
Gene fragments	7μl

Shuffling PCR program is:

1	94℃	3min
			2	94℃	30s

3	56.5℃	30s
			4	72℃	30s
5	Repeat 2-4 step	55 times
			6	72℃	10min

In above shuffling product, add the variation group (GOD-Mtt) that primer obtains glucose oxidase afterwards, concrete system is as follows:

Shuffling product	20μμl
		Upstream primer	4μl
Downstream primer	4μl
		ddH 2O	12μl
Tag?Mix	40μlμl

Difficulty maximum in whole experiment flow can be run in the process of the reunion of glucose oxidase enzyme fragment, namely how can regain complete gene fragment from the gene fragments after DNaseI digestion, and affect a lot of because have of reunion effect, wherein the size of fragment occupies principal element.

Through multiple trial in this experiment, successively test the length of 10bp, 30bp, 50bp, 60bp, 100bp, 200bp, 400bp and 600bp, finally, experimental result shows, the gene fragment of 50-60bp length can be more satisfactory meet again as complete gene fragment, and can activity be demonstrated in the middle of follow-up importing yeast strain.

Therefore, under the prerequisite overcoming difficulty of meeting again, follow-up experimental implementation is proceeded.

2.3GOD-Mtt and shuttle expression plasmid pPIC9K double digestion

Successively carry out AvrII and NotI substep enzyme to GOD-Mtt and shuttle expression plasmid pPIC9K to cut, it is as follows that enzyme cuts system:

The structure of 2.4 recombinant plasmids

Be connected with gene by carrier after double digestion, 16 DEG C, connection of spending the night, linked system is:

T4 ligase enzyme	0.5μl
		Ligase enzyme damping fluid	1μl
Double digestion pPIC9K-(A-N)	2μl
		Double digestion GOD-Mtt-(A-N)	6.5μl

Composition Variation gene pool after glucose oxidase mutant gene is connected with shuttle expression plasmid pPIC9K, called after pPIC9K-GOD-Mtt.

Glucose oxidase mutant gene storehouse is imported Pichia pastoris GS115 and screening thereof by embodiment 3

3.1 bacterial strains and plasmid

Yeast expression bacterial strain Pichia pastoris GS115;

Shuttle expression carrier pPIC9K is purchased from Invitrogen company;

Sequencing vector pMD19-T is purchased from Takara company.

3.2 substratum and enzyme activity determination substrate

The conventional medium YPD, the yeast enrichment substratum BMGY that cultivate for yeast and the concrete composition of yeast inducing culture BMMY are with reference to EasySelect ^tMpichia Expression Kit A Manual of Methods for Expression of Recombinant Proteins Using pPICZ and pPICZ α in Pichia pastoris Catalog no.K1740-01.

High enzyme for filtering out is lived and is expressed saccharomycetic 3L amplification fermentative medium formula with reference to substratum composition involved in " research of pichia spp fermentative production recombination human serum albumin high-density culturing condition " of the people such as Liu Yanli.

Enzyme activity determination substrate:

First configure the first five, 30 DEG C of incubation 10min, use HCl termination reaction afterwards again.Its absorbancy is measured at 540nm.The consumption of above-mentioned listed every substrate adjusts to adapt to 96 orifice plate methods of high flux screening.

The drafting of 3.2 glucose oxidase enzymes typical curve alive

Buy glucose oxidase standard substance by Sigma company, be diluted to certain gradient drawing standard curve.

The gradient of dilution is as follows: 0.4,0.8,1.0,2.0,4.0U/ml, measures absorbancy OD540 according to after the substrate proportioning mixing insulation described in 3.2.

With the work of glucose oxidase standard substance enzyme for ordinate zou, take OD540 as X-coordinate, linear return two times is carried out to loose point and does typical curve.

According to the result of typical curve, draw the calculation formula of glucose oxidase Mei Huo unit:

Enzyme (U/ml)=(4.792x-0.637) × N alive

Wherein x be reaction terminating after absorbancy at OD540 place; N is the extension rate of sample.

3.3 expression of glucose oxidase mutant gene in Pichia pastoris GS115

Carrying glucose oxidase mutant gene for the ease of shuttle plasmid enters in pichia spp, is convenient to the gene entered in yeast simultaneously and can be integrated in saccharomycetic genome, first carry out linearizing enzyme with PmeI to mutant gene storehouse pPIC9K-GOD-Mtt and cut.

Adopt electroporated mode transformed yeast bacterium afterwards.Transformed bacteria liquid is coated on dull and stereotyped upper 30 DEG C of MD and cultivates 4 days to growing transformant.Enrichment thalline 24 hours during random choose transformant is seeded to containing BMGY liquid nutrient medium 48 deep-well plates together with contrast yeast strain; Collecting thalline and transferring them in 48 deep-well plates containing BMMY liquid nutrient medium utilizes methyl alcohol to induce.Induce centrifugal thalline after 72 hours, the glucose oxidase enzyme measuring supernatant liquor is lived.

By bacterial strain purifying amplification culture after finding the bacterial strain that enzyme work is significantly increased compared with control group.First from the flat board colony lift of purifying in 4mlYPD liquid tube substratum.To cultivate after 24 hours with 1% inoculum size switching 50mlBMGY, cultivate collection thalline after 24 hours and be transferred to the stream carrying out methyl alcohol in 100mlBMMY substratum and add induction.Mensuration enzyme is alive afterwards to induce 72 hours.

Can determine that after being amplified to 100ml induction fermentation enzyme is lived the bacterial strain that really increases, be that the bacterium that sets out amplifies the glucose oxidase gene wherein made a variation with this bacterial strain, again carry out DNA shuffling.

Repeat above-mentioned correlation step, after two-wheeled DNA shuffling, altogether screen the bacterial strain that about 1000 strains contain mutator gene.Obtain the bacterial strain that a strain enzyme work is significantly increased compared with control group, called after ZC#4.

The amplification high density fermentation of 3.4 live high-enzyme strains

To the high density fermentation screening the bacterial strain ZC#4 that obtains and be amplified to 3L fermentation tank level.

Liquid bacteria liquid is seeded in 200mlBMGY substratum by the inoculum size with 1% carries out seed culture.Reach about 20 seeds to OD600 after general 48 hours to form, be inoculated in 3L fermentor tank.

Fermentation condition is controlled by fermentor tank housing.Be pH6.0 at thalline concentration stage, temperature 28 DEG C, air flow 8L/min, stirring velocity 300rpm.To start stream time Schwellenwert processus aboralis gets over back high level and add methyl alcohol when dissolved oxygen drops to and induce.Now stirring velocity rises to 500rpm.The concentration controlling methyl alcohol in fermentor tank is about 0.8%.

Induction period, sampling in every 12 hours, by the biomass of centrifugal for the sample glucose oxidase enzyme work measured afterwards in supernatant liquor, protein content, OD600, dries thalline simultaneously, measures dry cell weight.Along with induction time increases, enzyme is lived and is constantly risen, and induce after 132 hours and stop induction, fermentation completes.

Can obtain from the result of 3L fermentation, screen the ZC#4 transformant obtained and reach 767U/ml (Fig. 7) in methanol induction enzyme work afterwards in 132 hours, protein secretion is 0.89mg/ml (Fig. 8), live than enzyme after conversion and reach 858U/mg (Fig. 9), biomass OD600 is up to 311 (Figure 10), and dry cell weight is finally 85g/L (Figure 11).

To produce or compared with modification scheme through the glucose oxidase known with present stage, after can finding out that two-wheeled DNAshuffling transforms, can screen and obtain enzyme work and have the bacterial strain significantly improved.

The checking of embodiment 4ZC#4 bacterial strain genetic stability

4.1ZC#4's goes down to posterity

Owing to using merely liquid YPD medium to cause microbiological contamination, and YPD plate culture medium is used merely to there will be the situation of strain degeneration.Therefore going down to posterity of bacterial strain adopts the liquid-solid method replaced to go down to posterity.

First on flat board, complete for bacterium colony picking is seeded to 4mlYPD liquid nutrient medium, at 30 DEG C, cultivates 24 hours under the condition of 180rpm, carry out streak culture afterwards on the YPD flat board containing G418, complete one and take turns purifying.So hocket and five take turns and go down to posterity.

First-generation thalline called after ZC#4-1, after five generations of going down to posterity is bacterial strain ZC#4-5.

4.2ZC#4-1 and ZC#4-5 genetic stability fermenting enzyme is lived and is verified

The yeast strain choosing ZC#4-1 and ZC#4-5 G418 resistant panel is respectively seeded in 4mlYPD liquid nutrient medium, and the inoculum size with 1% to be seeded in 50mlBMGY liquid nutrient medium enrichment thalline 24 hours; Collecting thalline and transferring them in 100mlBMMY liquid nutrient medium utilizes 1% methyl alcohol to carry out abduction delivering.Induce centrifugal thalline after 72 hours, measure glucose oxidase enzyme in supernatant liquor and live.

The measurement result display that enzyme is lived, the enzyme of ZC#4-1 and ZC#4-5 is lived and is respectively 21.66U/ml and 23.87U/ml.Although enzyme is lived and risen to some extent compared with the bacterial strain in the 5th generation is alive with the bacterial strain enzyme of the first-generation, the amplitude risen is within limit of error.

Therefore, can be drawn by enzyme live data, screen the live high-enzyme strain obtained and keep good effect on enzyme is lived.

4.3ZC#4-1 verify with ZC#4-5 genetic stability PCR

The yeast strain of ZC#4-1 and the ZC#4-5 chosen respectively G418 resistant panel in 4.2 is seeded in 4mlYPD liquid nutrient medium, the thalline obtained is utilized to mention saccharomycetic genome, the genome obtained with extraction is for template, and therefrom increase glucose oxidase mutant gene.

Electrophoresis result display (Figure 12) of PCR, all increases and obtains corresponding gene in the genome of ZC#4-1 and ZC#4-5.Show that the bacterial strain in the 5th generation and the bacterial strain of the first-generation have good stability on genetics, be integrated into gene in Yeast genome can genetic stability to offspring.

Sequence table

Claims (9)

1. by the new engineered glucose oxidase gene that irrational sudden change obtains, it is characterized in that: nucleotide sequence is as shown in SEQ ID NO.2.

2. by the new engineered glucose oxidase that irrational sudden change obtains, it is characterized in that: aminoacid sequence is as shown in SEQ ID NO.3.

3. the various glucose oxidase expression strain that is the aminoacid sequence shown in SEQ ID NO.3 containing the aminoacid sequence related in claim 2.

4. the application of gene described in claim 1 in secreting, expressing glucose oxidase.

5., according to the application of gene described in claim 4 in secreting, expressing glucose oxidase, it is characterized in that:

By the described glucose oxidase gene obtained by irrational sudden change in prokaryotic cell prokaryocyte, connect and compose recombinant expression plasmid with the shuttle expression plasmid carrying out genetic manipulation used for prokaryotic cell prokaryocyte; Recombinant expression plasmid is transformed into eukaryotic cell expression albumen and is integrated in gene of eucaryote cell group and obtain recombination yeast; Cultivate recombination yeast, the oxidasic secreting, expressing of induced glucose.

6., according to the application described in claim 5, prokaryotic cell prokaryocyte is intestinal bacteria Escherichia coli BMTOP10.

7., according to the application described in claim 5, it is characterized in that:

Eukaryotic cell expression albumen is pichia spp Pichia pastoris GS115 expressing protein.

8. according to application according to claim 5, it is characterized in that: the described glucose oxidase gene obtained by irrational sudden change is connected and composed recombinant expression plasmid with shuttle expression plasmid in intestinal bacteria Escherichia coli BMTOP10; Recombinant expression plasmid is transformed into pichia spp Pichia pastoris GS115 and is integrated in its genome and obtain recombination yeast; Cultivate recombination yeast, the oxidasic secreting, expressing of induced glucose.

9. the preparation method of gene described in claim 1, it is characterized in that: the glucose oxidase gene in aspergillus niger Z-25 is cut into gene fragment at random by DNaseI, the fragment selecting wherein 50-60bp size utilizes DNA shuffling technology that fragment restructuring is become complete fragment, obtains glucose oxidase gene.