CN104263744B - A kind of engineered glucose oxidase gene and its Expression and Application - Google Patents

Abstract

A kind of engineered glucose oxidase gene and its Expression and Application, belong to albumen or enzyme preparation improvement project and glucoseoxidase production field.Problem to be solved is that the activity of its enzyme is further improved on the basis of existing glucoseoxidase.The present invention by albumen or enzyme preparation improvement project in irrational means DNA shuffling technological transformations come from the glucose oxidase gene of aspergillus niger Z 25, selecting the genetic fragment after DNaseI is cut at random becomes complete mutant gene by fragment restructuring using DNA shuffling technologies, is integrated in the genome of yeast after being connected with shuttle expression plasmid.The screening from mutation library obtains the bacterial strain that enzyme activity is significantly improved, and its series of variation is as shown in SEQ ID NO.2.The final enzyme activity of glucoseoxidase has reached higher level in the middle of the 3L scale-ups of yeast.Finally in enzyme activity and gene aspect, verify that the high enzyme activity expression strain for obtaining has good heritability.

Description

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

Technical field

The present invention relates to irrational albumen transformation means, more particularly to using DNA shuffling technologies to aspergillus niger come The glucose oxidase gene in source is transformed, the invention further relates to the glucose oxidase gene after making a variation is in secretion production Application in glucoseoxidase, belongs to albumen or enzyme preparation improvement project and glucoseoxidase production field.

Background technology

Glucoseoxidase (Glucose Oxidase, E.C.1.1.3.4, abbreviation GOD) can be under conditions of having oxygen Specificity catalysis β-D-Glucose generates gluconic acid and hydrogen peroxide, is widely distributed in animal, plant and microbial body (Pluschkell S,Hellmuth K,Rinas U.Kinetics of glucose oxidase excretion by recombinant Aspergillus niger.Biotechnol Bioeng 1996；51:215-20.).

But as microorganism has growth and breeding speed fast, the features such as originate wide, thus microorganism to become Fructus Vitis viniferae glycoxidative The main source of enzyme, the main production bacterial strain in microorganism are aspergillus niger and penicillium sp.

Glucoseoxidase has a wide range of applications in fields such as food, medicine and biologies.

In the food industry, for removing the glucose in food, prevent brown stain；Food deoxidation is can be additionally used in, improves food The quality of product and beverage；It is additionally operable to the aspects such as the improved process of flour (Rasiah IA, Sutton KH, Low FL, Lin simultaneously 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 medical industry, for the measure and glucose in urine of blood glucose, the detection of urine ketone bodies, can be made into glycosuria test paper or test kit is used In clinical diagnosises；Oral disease and odontopathy (Wilson R, Turner APF.Glucose oxidase can also be prevented and treated: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, glucoseoxidase can reduce wine by removing glucose The concentration of essence, produces low wine (Malherbe DF, du Toit M, Cordero RR, van so as to reduce the alcoholic strength of wine 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.).

Glucoseoxidase is also used on commercial production gluconic acid, produces Fructus Vitis viniferae by reducing gluconic acid lactone Saccharic acid.Gluconic acid can be additive as food acid number is adjusted, and may be made as supplementing the various trace element of needed by human body Gluconate 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.)。

Glucoseoxidase is also applicable in textile industry, because glucoseoxidase can be produced in mechanism Hydrogen oxide, can play discoloration.The hydrogen peroxide that this mode is produced passes through checking, shows standard bleaching process Same effect.More it is noted that need not additionally add stabilizer in this bleaching process because during produce Raw gluconic acid can serve as role (Tzanov T, Silgia A, Gubitz GM, the Cavaco-Paul μ lo of stabilizer A.Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching.J Biotechnol 2002；93:87–94.).

Modal glucoseoxidase source is got by the fermentation of aspergillus niger, penicillium sp and Saccharomycodeses at present. Most business-like glucoseoxidases are isolated from the mycelium of aspergillus niger.There is the researcher of China by organic Solvent deposition, Polyethylene Glycol secondary precipitation, hydroxyapatite pillar layer separation, obtain product of the specific activity more than 190U/ml albumen Product (Chinese Journal of Pharmaceutical 1997,28 (7)).

But as the fermentation of aspergillus niger is primarily used to produce gluconic acid or gluconate, such as glucose Sour sodium, calcium gluconate, therefore glucoseoxidase occurs primarily as a kind of by-product, and this causes glucoseoxidase Production enzyme activity it is not high, be all much enzyme activity (the S.B.Bankar et al./Biotechnology of only unitss Advances27(2009)489-501)。

With the development of Protocols in Molecular Biology, researcher is attempted by Microbial Expression Systems come expression alien gene, The purpose to a certain albumen high efficient expression is reached with this.The heterologous gene expression system having been developed at present includes large intestine Bacillus, bacillus cereuss, streptomycete, aspergillosis, yeast, insecticide, cells of mamma animals etc..Under normal circumstances, made using prokaryotic cell Expression system for exogenous gene is easy to operate, and various technologies have tended to ripe, but are limited by prokaryotic system itself, very The albumen in core source can not correctly fold or lack the necessary modification after transcription and translation when expression, so as to cause Give expression to albumen, but albumen no biological activity, no further value, and it is poisonous in prokaryotic system Protein or the albumen for having antigenic action are likely to be mingled in end-product, make result be interfered.

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

And for eucaryon mammalian cell is used as host, the modification after protein expression can be carried out with ideal, but It is that, due to operating difficultiess, and later stage cultured cells has the possibility of virus infection, therefore this method does not become extensive The method for using.

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

Yeast system has become a kind of pattern operating platform at present by the development of decades, and operations have tended to It is perfect.In terms of protein expression, there is the intracellular environment for being suitable to that eukaryotic gene product is correctly folded, especially can be to egg Some white crucial glycosylation sites are modified, and enable some to give expression to activity to the albumen that glycosylation is required.

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

Yeast system has the ability of secreting, expressing first, that is to say, that can be by the protein excretion for giving expression to culture In the middle of base.This suffers from great advantage in experimentation and commercial production.Eliminate during genetic modification broken The program of cell, directly further can be studied by obtaining destination protein in culture medium fermentation liquid.In the industrial production, Albumen is very ripe with the isolation technics of thalline, so if protein product is secreted in fermentation liquid, it will save it is very big into This.

What secondly yeast expression system was taken is methanol induction mechanism, that is, is lured using alcohol oxidase promoter Lead, in shaking flask level, carry out inducing using methanol the probability that can be effectively reduced microbiological contamination.

3rd, exogenous origin gene integrator can be entered in the genome of yeast, can so be protected by the carrier operated using yeast The exogenous gene that card is introduced into stably is present in yeast system, without the existence outside offer is needed as episomal vector Pressure is ensureing presence of the carrier in yeast.The problem of spawn degeneration is avoided to a certain extent thus.

4th, the accumulation of yeast-leavened product can't yeast itself produce toxic action, and the expression bacterium of yeast Strain is easy to expand high density fermentation to from shaking flask, while not affecting the expression of exogenous gene.This 2 points are doomed to make yeast Potential quality with high density fermentation and then high level expression foreign protein (letter in biotechnology, Vol.16No.2Mar.,2005)。

After it have selected expression system, it is desirable to further improve the oxidasic enzyme activity of Exogenous Glucose only in gene water It is flat that glucose oxidase gene sequence is transformed.

Generally improve the activity of enzyme method have codon optimization, mRNA structure of modification, G/C content adjustment, random mutation, The strategies such as albumen orthogenesiss.Some methods have been achieved for extraordinary effect.Gao Zhaowei of such as Southwestern University et al. exists After being optimized to the codon of glucose oxidase gene, after 3L fermentation 120h, enzyme activity has reached 365U/ml.

But the result of codon optimization still has limitation.Because this method by all codons in gene all Transformed according to the codon preference of yeast, although can so utilize the Preference of yeast to improve the table of pheron Reach, but virtually increased the burden of yeast, it is likely that best expression effect can not be reached.Therefore consider by other Means are improving enzyme activity.

In the nature evolution of more than one hundred million years, protein defines diversified form, in respective natural ring Respective effect is played in border.Enzyme as a kind of albumen with biological activity, in productive life of the nature with the mankind Play huge effect.This just promotes people to want further to improve the utilization to enzyme.Namely want to change enzyme Make.For the artificial reconstructed means of enzyme have many kinds, optimal method is to some crucial amino acid sites in enzyme molecule Being adjusted makes its preferable macroscopic property of generation.But the bottleneck of this method is at present for enzyme molecular structure and catalysis The research of mechanism is comprehensive not enough, thus want it is purposive, rationally engineered protein also have certain difficulty.

This contradiction is directed to, enzyme molecule lactam enzyme by directional anagenesis in vitro technology is generated, according to Darwinian theory of evolution, in vitro Simulating Evolution mechanism, simulates the condition of natural evolution in the lab, and artificial screening goes out the egg that new or character is improved In vain.

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

The method of the gene random mutation for occurring earliest is fallibility PCR, and the convenient method of this principle simple operationss is rapid Find broad application.Only need on the method for PCR, the appropriate reaction condition changed in PCR system makes the guarantor of polymerase True property declines, and by producing mispairing (Chen K, Arnold FH.Proc Natl Acad Sci USA, 1993,90:5618～ 22.)。

Proposed DNA reordering techniques, i.e. DNA shuffling technologies afterwards in 1994.Theoretically, this technology can To cause gene internal to change on a large scale, so as to introduce mutation, build mutated library (Stemmer WP.Nature, 1994, 370:389～91.).On the basis of DNA shuffling technologies, the new techniques such as StEP, RPR, but principle were occurred in that again later It is similar, all it is the restructuring of genetic fragment.

Efficient sudden change means must be collocated with each other with efficient screening means.In the screening means in library, the most often See for high flux screening.High flux screening is generally carried out on 96 orifice plates, is allocated using the technology convection cell of automatization With measure.It is many that there is high flux screening the sample needed for the advantage of microanalysiss, i.e. location parameter not need, therefore can be right In laboratory scope, still the less sample of sample capacity is detected multisample.

The content of the invention

Problem to be solved by this invention is on the basis of the existing enzyme activity of glucoseoxidase further to improve enzyme activity.It is logical The glucose oxidase gene that irrational DNA shuffling technological transformations come from aspergillus niger is crossed, it is prominent from what is built afterwards Screening in becoming storehouse obtains the bacterial strain that enzyme activity is significantly improved.

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

A kind of new engineered glucose oxidase gene obtained by irrational sudden change, it is characterised in that：Tool Body nucleotide sequence is as shown in SEQ ID NO.2.

A kind of new engineered glucoseoxidase obtained by irrational sudden change, it is characterised in that：Concrete ammonia Base acid sequence is as shown in SEQ ID NO.3.

Glucose oxidase gene in the aspergillus niger Z-25 that clone is obtained by the present invention is cut at random by DNaseI Fragment restructuring is become complete piece using DNA shuffling technologies by genetic fragment, the fragment for selecting wherein 50-60bp sizes Section, 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 contain the shuttle The host cell of expression plasmid is also in the row of protection category of the invention；Wherein, described shuttle expression plasmid is escherichia coli Shuttle expression plasmid between yeast, preferably escherichia coli BMTOP10 and Pichia pastoris GS115.

A kind of high-throughput screening method of glucose oxidase gene mutation library, including：By Fructus Vitis viniferae glycosyloxy obtained above The mutation library for changing enzyme gene is connected with described shuttle expression plasmid, direct transformed yeast cell；Choose from plating medium Bacterial strain induces expression of recombinant proteins in 48 orifice plates, and enzyme activity determination is carried out in 96 orifice plates, directly with final host yeast doing High flux screening and eliminate colibacillary screening step.

The present invention is entered using the glucose oxidase gene that aspergillus niger Z-25 (Aspergillus.niger Z-25) originates Row DNA shuffling are transformed, and obtain glucose oxidase mutant gene storehouse, then mutant gene bank is connected with shuttle expression plasmid Express in switching through such as Pichia pastoris GS115, therefrom screen the bacterial strain that enzyme activity is significantly improved, afterwards its sequence is being sequenced Analysis.Test result indicate that, screen the bacterial strain for obtaining higher level has been reached in the level of expression glucoseoxidase.Separately Outward, due to using shuttle expression plasmid the gene integration for obtaining can be entered in saccharomycetic genome chromosome, therefore To live high-enzyme strain there is stable heritability.

Description of the drawings

Fig. 1 aspergillus niger Z-25 glucoseoxidases expand electrophoretogram

Fig. 2 DNaseI enzyme action aspergillus niger Z-25 glucoseoxidase electrophoretograms

Fig. 3 glucose oxidase gene fragment reunion electrophoretograms

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

48 deep-well plates induced glucoses of Fig. 5 aoxidize enzyme mutant gene transformant schematic diagram

96 orifice plate high flux screening glucose oxidase mutant gene transformant schematic diagrams of Fig. 6

3 liters of fermented grape carbohydrate oxidase enzyme activity curve charts of the high enzyme activity mutant gene transformants of Fig. 7

3 liters of fermented liquid supernatant protein content curve charts of the high enzyme activity mutant gene transformants of Fig. 8

3 liters of fermentation specific enzyme activity curve charts of the high enzyme activity mutant gene transformants of Fig. 9

Figure 10 3 liters of fermentation biomass curve charts of high enzyme activity mutant gene transformant

3 liters of fermented cells dry weight curve charts of the high enzyme activity mutant gene transformants of Figure 11

The high enzyme activity mutant gene transformant hereditary stability electrophoretograms of Figure 12

Specific embodiment

The details of the present invention is expanded on further below in conjunction with specific embodiment.But embodiment is exemplary in nature, not Any restriction is constituted to the scope of the present invention.Those skilled in that art are construed as, in the spirit and scope of the present invention The technical scheme details of invention and form can be modified or replaced, but these modifications and replacement are in the guarantor of the present invention Within the scope of shield.

Explanation：In specific examples below, involved gene manipulation techniqueses are the normal of maturation at this stage in the art Rule technology.The technology that removing is not described in detail, is carried out according to following experiments handbook or the related Sections in document：Following reality The concentration that is not particularly illustrated in applying example is volumetric concentration.

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

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

1.1 bacterial strains and plasmid

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

Escherichia coli BWTOP10 (Escherichia.coli BMTOP10) bacterial strain is limited by Beijing Bo Maide developments in science and technology Company limited buys；

The synthesis of amplimer is completed by Hua Da gene；

Sequencing vector pMD19-T is purchased from Takara companies；

The amplification of 1.2 glucose oxidase genes

The base of aspergillus niger is extracted in carrying out the aspergillus niger Z-25 mycelium used by glucoseoxidase fermenting experiment first Because of group.

Aspergillus niger Z-25 genomes are extracted using Benzyl chloride method is improved.

(1) moisture in culture medium is blotted by the mycelium pellet that sucking filtration is obtained in czapek's medium as far as possible with filter paper

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

(3) 0.2g thalline are taken into 1.5ml centrifuge tubes, adds 500 μ l to have been warmed up to 50 DEG C of extracting solution, vibration is abundant Mixing, stands 2min, adds 100 μ l 10%SDS, mixes, and adds 300 μ l benzyl chlorides, and acutely vibration makes Guan Zhongcheng emulsus

(4) 50 DEG C of water-bath 1h, turn upside down vibration mixing once, make reaction complete per 10min

(5) centrifuge tube is taken out, add 300 μ lNaAc, mix ice bath 15min, mixing is overturned once per 4-5min

(6) 10 DEG C of 6000rpm are centrifuged 15min, and collection supernatant is into new centrifuge tube

(7) volume ratio is added to be 23:1:1 chloroform：Isoamyl alcohol：Phenol, mixes, 10 DEG C, 10000rpm, 15min, centrifugation Collect supernatant

(8) add chloroform：Isoamyl alcohol (24:1) extract 2-3 time, collect whole supernatant

(9) isopyknic freezing isoamyl alcohol is added in supernatant, somewhat overturn, precipitation at room temperature 20min

(10) 10 DEG C, 10000rpm is centrifuged 5min

(11) supernatant is abandoned, stays precipitation, with 70% washing with alcohol 2 times, 10 DEG C every time, 10000rpm is centrifuged 10min, finally Washed once with dehydrated alcohol

(12) 4 DEG C, 10000rpm is centrifuged 15min, goes ethanol, super-clean bench to dry up

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

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 genes remove signal peptide, length is 1746bp, encodes 582 amino Acid, the gene are registered on GeneBank, and accession number is FJ979866.1 (SEQ ID NO.1).

The structure in 2 glucose oxidase mutant gene storehouse of embodiment

2.1DNaseI cuts glucose oxidase gene at random

The glucoseoxidase that amplification is obtained is cut at random first with DNaseI, DNaseI can be to any base Because carrying out Non-specific cleavage, system is as follows：

Glucose oxidase gene	10μl
		DNaseI buffer	2μl
DNaseI	0.8μl
		ddH2O	7.2μl
Condition	37 DEG C of constant temperature 1h

2.2 glucoseoxidase fragments meet again (DNA shuffling)

A certain amount of glucoseoxidase fragment is taken as substrate, adds enzyme the PCR without primer to be carried out with dNTP, this process The as process of DNA shuffling.

Specific system is as follows：

Tag Mix	10μl
		ddH2O	3μl
Gene fragments	7μl

Shuffling PCR programs are：

1	94℃	3min
			2	94℃	30s

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

In the above shuffling product primer is added to obtain the variation group (GOD-Mtt) of glucoseoxidase afterwards, Concrete system is as follows：

Shuffling products	20μμl
		Forward primer	4μl
Downstream primer	4μl
		ddH2O	12μl
Tag Mix	40μlμl

The difficulty of maximum in whole experiment flow can be run into during the reunion of glucoseoxidase fragment, that is, Complete genetic fragment how is regained in the gene fragments from after digesting through DNaseI, and affects reunion effect Factor have much, the size of wherein fragment occupies principal element.

Through various trials in this experiment, 10bp, 30bp, 50bp, 60bp, 100bp, 200bp, 400bp have successively been tested And the length of 600bp, finally, experimental result shows, the genetic fragment of 50-60bp length can be more satisfactory meet again and be Complete genetic fragment, and can show that activity in the middle of yeast strain in follow-up importing.

Therefore, proceed follow-up experimental implementation on the premise of difficulty of meeting again is overcome.

2.3GOD-Mtt and shuttle expression plasmid pPIC9K double digestions

AvrII and NotI substep enzyme action are successively carried out to GOD-Mtt and shuttle expression plasmid pPIC9K, enzyme action system is such as Under：

The structure of 2.4 recombiant plasmid

Carrier after double digestion is connected with gene, 16 DEG C, is overnight connected, linked system is：

T4 ligases	0.5μl
		Ligase buffer solution	1μl
Double digestion pPIC9K- (A-N)	2μl
		Double digestion GOD-Mtt- (A-N)	6.5μl

Glucoseoxidase mutant gene be connected with shuttle expression plasmid pPIC9K after Composition Variation gene bank, be named as pPIC9K-GOD-Mtt。

Glucoseoxidase mutant gene storehouse is imported Pichia pastoris GS115 and its screening by embodiment 3

3.1 bacterial strains and plasmid

Yeast expression bacterial strain Pichia pastoris GS115；

Shuttle expression carrier pPIC9K is purchased from Invitrogen companies；

Sequencing vector pMD19-T is purchased from Takara companies.

3.2 culture medium and enzyme activity determination substrate

For conventional medium YPD of yeast culture, yeast enrichment culture medium BMGY and yeast inducing culture The concrete compositions of base BMMY are with reference to EasySelect^TM Pichia Expression Kit A Manual of Methods for Expression of Recombinant Proteins Using pPICZ and pPICZαin Pichia pastoris Catalog no.K1740-01。

High enzyme activity for filtering out is expressed saccharomycetic 3L and amplifies fermentative medium formula with reference to Liu Yanli's et al.《Finish The research of red yeast fermenting and producing recombination human serum albumin high-density culturing condition》In involved culture medium composition.

Enzyme activity determination substrate：

First five is configured first, 30 DEG C of incubation 10min use HCl terminating reactions afterwards again.Its absorbance is determined in 540nm.On The consumption for stating listed items substrate is adjusted to adapt to 96 orifice plate methods of high flux screening.

The drafting of 3.3 glucoseoxidase enzyme activity standard curves

Glucoseoxidase standard substance are bought by Sigma companies, certain gradient is diluted to and is drawn standard curve.

The gradient of dilution is as follows：0.4,0.8,1.0,2.0,4.0U/ml, protect according to the substrate proportioning mixing described in 3.2 Mensuration absorbance OD540 after using warming therapy.

With glucoseoxidase standard substance enzyme activity as vertical coordinate, with OD540 as abscissa, quadratic linear is carried out to scatterplot and is returned Return and do standard curve.

According to the result of standard curve, the computing formula of glucoseoxidase enzyme-activity unit is drawn：

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

Wherein x is the absorbance after reaction terminating at the OD540；Extension rates of the N for sample.

Expression of the 3.4 glucoseoxidase mutant genes in Pichia pastoris GS115

Glucoseoxidase mutant gene is carried for the ease of shuttle plasmid to enter in Pichia sp., while facilitating access for ferment Gene in female bacterium can be integrated in saccharomycetic genome, first mutant gene storehouse pPIC9K-GOD-Mtt is carried out with PmeI Linearized enzyme digestion.

The transformed yeast bacterium by the way of electroporated afterwards.Transformed bacteria solution is coated on MD flat boards 30 DEG C to cultivate 4 days To growing transformant.It is deep that random choose transformant is seeded to containing BMGY fluid mediums 48 together with control yeast strain Thalline is enriched with 24 hours in orifice plate；Collects thalline and in transferring them to 48 deep-well plates containing BMMY fluid mediums using first Alcohol is induced.Induction is centrifuged thalline after 72 hours, determine the glucoseoxidase enzyme activity of supernatant.

By bacterial strain purification amplification culture after the bacterial strain that discovery enzyme activity is significantly increased compared with matched group.First from pure Say colony lift into 4mlYPD liquid tube culture medium on the flat board of change.Culture was transferred with 1% inoculum concentration after 24 hours 50mlBMGY, cultivate 24 hours afterwards collects thalline the stream of methanol carried out in being transferred to 100mlBMMY culture medium add induction.Induction Enzyme activity is determined afterwards within 72 hours.

The bacterial strain that enzyme activity is increased really is can determine after 100ml induction fermentations are amplified to, is with this bacterial strain Send out bacterium and amplify the glucose oxidase gene for wherein making a variation, carry out DNA shuffling again.

The above-mentioned correlation step of repetition, after two-wheeled DNA shuffling, screens about 1000 plants altogether containing mutation base The bacterial strain of cause.The bacterial strain that one plant of enzyme activity is significantly increased compared with matched group is obtained, ZC#4 is named as.

The amplification high density fermentation of 3.5 live high-enzyme strains

The bacterial strain ZC#4 that screening is obtained is amplified to the high density fermentation of 3L fermentation tank levels.

Liquid bacterium solution is seeded in 200mlBMGY culture medium with 1% inoculum concentration carries out seed culture.General 48 hours 20 or so seeds are reached to OD600 afterwards to form, be inoculated in 3L fermentation tanks.

Fermentation condition is controlled by fermentation tank switch board.Thalline concentration stage be pH6.0,28 DEG C of temperature, ventilation 8L/ Min, mixing speed 300rpm.Methanol is added to be induced when dissolved oxygen drops to start to flow when minimum processus aboralis gets over back high level. Now mixing speed rises to 500rpm.The concentration for controlling methanol in fermentation tank is 0.8% or so.

Induction period, sampled per 12 hours, the glucoseoxidase enzyme activity, egg after sample is centrifuged in measure supernatant The Biomass of Bai Hanliang, OD600, while drying thalline, determines dry cell weight.As induction time increases, enzyme activity constantly rises, Induction stopped induction after 132 hours, and fermentation is completed.

Can obtain from the result of 3L fermentations, enzyme activity reached afterwards in methanol induction 132 hours to screen the ZC#4 transformants that obtain 767U/ml (Fig. 7), protein secretion are 0.89mg/ml (Fig. 8), and specific enzyme activity after conversion reaches 858U/mg (Fig. 9), Biomass OD600 is up to 311 (Figure 10), and dry cell weight is finally 85g/L (Figure 11).

Through compared with known glucoseoxidase production at this stage or modification scheme, it can be seen that two-wheeled After DNAshuffling transformations, can screen and obtain enzyme activity and have the bacterial strain for significantly improving.

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

4.1ZC#4's passes on

Microbiological contamination can be caused using liquid YPD medium due to simple, and bacterial strain occurs using YPD plating mediums merely The situation of degeneration.Therefore passing on for bacterial strain is passed on using the admittedly alternate method of liquid.

Bacterium colony complete picking is seeded in 4mlYPD fluid mediums first from flat board, at 30 DEG C, 180rpm's Under the conditions of cultivate 24 hours, carry out streak culture afterwards on the YPD flat boards containing G418, complete a wheel purification.So hand over Pass on for five wheels are carried out.

First generation thalline is named as ZC#4-1, after passing on five instead of for bacterial strain ZC#4-5.

4.2ZC#4-1 and the fermentation enzyme activity checking of ZC#4-5 hereditary stabilityes

The yeast strain for choosing ZC#4-1 and ZC#4-5 from G418 resistant panels respectively is seeded to 4mlYPD liquid cultures In base, it is seeded in 50mlBMGY fluid mediums with 1% inoculum concentration and is enriched with thalline 24 hours；Collects thalline is simultaneously shifted Abduction delivering is carried out using 1% methanol into 100mlBMMY fluid mediums.Induction is centrifuged thalline after 72 hours, determine supernatant Glucoseoxidase enzyme activity in liquid.

The measurement result of enzyme activity shows that the enzyme activity of ZC#4-1 and ZC#4-5 is respectively 21.66U/ml and 23.87U/ml.The Although the bacterial strain in five generations has been risen with the bacterial strain enzyme activity phase specific enzyme activity of the first generation, rise amplitude range of error it It is interior.

Therefore, can be drawn by enzyme-activity data, screen the live high-enzyme strain for obtaining and good effect is kept in enzyme activity.

4.3ZC#4-1 verify with ZC#4-5 hereditary stabilityes PCR

The yeast strain of the ZC#4-1 chosen from G418 resistant panels respectively in 4.2 and ZC#4-5 is seeded to 4mlYPD liquid In body culture medium, lift saccharomycetic genome using the thalline for obtaining, the genome for obtaining is extracted as template, therefrom expand Glucoseoxidase mutant gene.

The electrophoresis result of PCR shows (Figure 12), all expands and obtained corresponding in genomes of the ZC#4-1 with ZC#4-5 Gene.Show that the bacterial strain in the 5th generation has good stability on hereditism with the bacterial strain of the first generation, be integrated into yeast genes Gene in group can stably entail offspring.

Claims (8)

1. a kind of new engineered glucose oxidase gene obtained by irrational sudden change, it is characterised in that：Nucleic acid Sequence is as shown in SEQ ID NO.2.

2. a kind of new engineered glucoseoxidase obtained by irrational sudden change, it is characterised in that：Aminoacid sequence Row are as shown in SEQ ID NO.3.

3. it is various containing the aminoacid sequence being related in claim 2 be SEQ ID NO.3 shown in aminoacid sequence Portugal Glucoseoxidase expression strain.

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

5. the application according to gene described in claim 4 in secreting, expressing glucoseoxidase, it is characterised in that：

By the described new engineered glucose oxidase gene obtained by irrational sudden change in prokaryotic cell, with The shuttle expression plasmid carried out used by genetic manipulation for prokaryotic cell connects and composes recombinant expression plasmid；By recombinant expression plasmid Be transformed into eukaryotic cell expression albumen and recombination yeast is obtained in being integrated into gene of eucaryote cell group；Culture recombination yeast, induction The secreting, expressing of glucoseoxidase.

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

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

Eukaryotic cell expression albumen is Pichia sp. Pichia pastoris GS115 expressing proteins.

8. according to the application described in claim 5, it is characterised in that：By it is described by irrational sudden change obtain it is new artificial The glucose oxidase gene of transformation is connected with shuttle expression plasmid in escherichia coli Escherichia coli BMTOP10 Constitute recombinant expression plasmid；Recombinant expression plasmid is transformed into into Pichia sp. Pichia pastoris GS115 and which is integrated into Recombination yeast is obtained in genome；Culture recombination yeast, the oxidasic secreting, expressing of induced glucose.