CN101955953A - Glucose oxidase mutant gene, expression and application thereof - Google Patents
Glucose oxidase mutant gene, expression and application thereof Download PDFInfo
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Abstract
The invention discloses a glucose oxidase mutant gene, expression and application thereof. In the invention, 272 basic groups are changed through codon optimization and GC content change and the content of GC is reduced to 48.44% from 55.54% so as to obtain the glucose oxidase mutant gene, wherein the basic group is represented by SEQ ID NO.2. The glucose oxidase mutant gene is transferred into pichia yeast to express; the experimental result shows that the secretory expression level of the glucose oxidase mutant gene in the pichia yeast is significantly improved by comparing with the same before mutation; compared with partial research at home and abroad, the final secretory expression of the glucose oxidase mutant gene achieves high expression level, thereby building the foundation for further expansion of industrial production. The determination of enzymatic properties of the glucose oxidase mutant gene shows that the recombinant glucose oxidase protein expressed by the glucose oxidase mutant gene has good thermal stability and high enzyme activity.
Description
Technical field
The present invention relates to glucose oxidase gene, relate in particular to the glucose oxidase enzyme mutant gene after codon optimized, the invention still further relates to the application of this glucose oxidase enzyme mutant gene in the preparation glucose oxidase, belong to the preparation field of glucose oxidase.
Background technology
Glucose oxidase (Glucose Oxidase, abbreviation GOD) formal name used at school is β-D-glucose oxidoreductase (EC1.1.3.4), it can become β-D-glucose oxidase gluconic acid and hydrogen peroxide (Pluschkell S single-mindedly, Hellmuth K and Rinas U, Kinetics of glucose oxidase excretion by recombinant Aspergillus niger.Biotechnology and bioengineering, 1996,51:215-220.).GOD is widely used in many association areas (Bankar SB, Bule MV, Singhal RS, et al., Glucose oxidase-An overview.Biotechnol Adv, 2009) such as food, feed, medicine.
GOD main effect in foodstuffs industry is to remove glucose, deoxygenation, formation hydrogen peroxide, form aspect four of the gluconic acids.At present, many countries are widely used in GOD in various food and the food processing technology as the safe oxidation inhibitor of generally acknowledging, for example: (the Malherbe D such as fresh-keeping that is used for drinks deoxygenation, albumen desugar, food product pack, fruits and vegetables and goods, Du Toit M, Cordero Otero R, et al., Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production.).
GOD also can be used as green flour modifying agent and additive gradually the instead of chemical additive in the improvement industry of flour and goods, play a significant role.Potassium bromate has the applicating history of eight more than ten years in baking industry, it successfully gives necessary gluten strength of baked goods and elasticity as oxygenant, but potassium bromate has very big damaging effect to human body, so be necessary to seek a kind of additive safe, that can substitute the potassium bromate function.The sulfydryl of the hydrogen peroxide that glucose oxidase produces in oxidising process in can the oxidation mucedin forms disulfide linkage, and then improves the network result of dough, strengthened dough elasticity.Can play the identical effect of chemical additive (potassium bromate etc.), the infringement of having avoided chemical additive that human body is caused simultaneously.In wheat-flour and Semen Maydis powder, improved the biceps of flour at present, improved the feel and the shape of food such as bread, improved quality product (Chinese patent application publication number: CN 1748515A by the interpolation glucose oxidase; Denomination of invention: a kind of anti-freezing fermented dough and production method thereof that contains glucose oxidase and peroxidase).
Aspect medical, GOD is used for the external quantitative analysis of serum (slurry), urine and cerebrospinal fluid glucose as test kit, enzyme electrodes etc.; The zymin that GOD makes also can be used for removing or the formation of alleviating dental plaque, tartar and carious tooth prevents the generation of oral disease and odontopathy.In addition, owing to can catalysis generate H
2O
2, also can be used for H
2O
2Treatment (the Vodopivec M of responsive lymphadenomatous target goal, Berovi M, Jan ar J, et al., Application of Convective Interaction Media disks with immobilised glucose oxidase for on-line glucose measurements.Analytica Chimica Acta, 2000,407:105-110.).
In recent years, GOD was as a kind of novel enzyme feed additive, classified as 12 kinds by the Ministry of Agriculture and allow using at the beginning of the feed enzyme preparation additive that uses on herding, feedstuff industry.In feed, add the glucose oxidase endonuclease capable and remove a large amount of free radicals of livestock intestinal epithelial cell, protect the complete of intestinal epithelial cell.Glucose oxidase generates gluconic acid by continuous catalysis glucose, reduces GI pH value, and the slant acidity environment helps the growing multiplication of probiotic bacterium, thereby has suppressed the survival of pathogenic micro-organism, has improved the autoimmunity of livestock.In addition, because glucose oxidase can produce hydrogen peroxide in the oxidizing glucose process, when hydrogen peroxide runs up to finite concentration, can suppress growth of pathogenic bacteria breedings such as intestinal bacteria, Salmonellas, pasteurellosis bacillus, staphylococcus, vibrios, and this bacteriostatic action can not produce the thalline resistance.The continuous development for preparing purifying technique along with glucose oxidase is perfect, the continuous reduction of use cost, glucose oxidase will in feedstuff industry, play an increasingly important role (Li Yan, the glucose oxidase effect test that raises table hens. Longyan teachers training school journal, 2004,22:77-78.).
The application of GOD has expanded to the textiles industry.Textiles needs to use a large amount of hydrogen peroxide in process of production, i.e. hydrogen peroxide, the hydrogen peroxide that glucose oxidase produces in mechanism can be used for the bleaching of textile industry to be handled.Employing has the immobilized glucose oxidase of high-recovery and produces hydrogen peroxide, and concentration of hydrogen peroxide can reach 3.5g/L, need not add hydrogen peroxide stabilizer during processing, handles back fabrics feel soft, plentiful.And handle the back immobilized glucose oxidase and can reclaim, use down again in the processing of batch product, thereby can effectively reduce the production cost of textile industry, and improve the quality of yarn fabric.China is as textile production and consumption big country, the application of glucose oxidase in the industry has good prospect (Chinese patent application publication number: CN 1884682A, Chen Jian, Yan Hejing, stifled state becomes, Hua Zhaozhe, denomination of invention: a kind of method of boiling off cotton fabric of using glucose oxidase preparation).
GOD is widely distributed at organic sphere, found in aspergillus niger and Penicillum glaucum prior to 1904, the glucose oxidase of research is mainly derived from mould fungi microorganism and the insect body at present, in bacterium such as weak oxidized acetic acid bacteria, also there is (Murray FR, Llewellyn DJ, Peacock WJ, et al., Isolation of the glucose oxidase gene from Talaromyces flavus and characterisation of its role in the biocontrol of Verticillium dahliae.Curr Genet, 1997,32:367-375.).But its industrial general employing aspergillus niger and Penicillium bacterial strain do to produce bacterium.But the natural bacterial strain of aspergillus niger and Penicillium notatum can produce catalase during the fermentation, cellulase, other multiple by products such as amylase (Frederick K, Tung J, Emerick R, et al., Glucose oxidase from Aspergillus niger.Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme.Journal of Biological Chemistry, 1990,265:3793.), a large amount of foreign proteins causes very big difficulty to the separation and purification work in later stage, has also increased cost.In addition, the biosynthesizing of zymoprotein is checked by inducing with the catastatic of high concentration glucose of glucose in the natural mould production process, and the glucose in the GOD that produces in the fermenting process energy oxidation substratum generates gluconic acid, and gluconic acid is synthetic also inhibited to enzyme.In order to address this problem, utilize the recombinant expressed glucose oxidase of genetic engineering bacterium to be subjected to researchist's favor in recent years.Whittington H has expressed the GOD gene that derives from aspergillus niger in yeast saccharomyces cerevisiae, obtained to have glucose oxidase (the Whittington H of biologic activity, Kerry-Williams S, Bidgood K, et al., Expression of the Aspergillus niger glucose oxidase gene in A.niger, A.nidulans and Saccharomyces cerevisiae.Curr Genet, 1990,18:531-536.); Mother respects the mould glucose oxidase gene of having expressed the aspergillus niger source of employing Rui Shi wood such as strongly fragrant, output be 25U/ml (it is strongly fragrant that mother respects, Wang Qiao, Yang Chunzhong, the mould expression aspergillus niger glucose oxidase of Rui Shi wood., the biotechnology journal, 2006,22:82-86.); People such as Zhou Yafeng and Zhang Xianen has carried out the glucose oxidase gene in aspergillus niger source recombinant expressed in pichia spp, induce secondary fermentation liquid enzyme to live and be 30-40U/ml (Zhou YF, Zhang XE, Liu H, et al., Cloning and expression of Aspergillus niger glucose oxidase gene in methylotrophic yeast.Sheng Wu Gong Cheng Xue Bao, 2001,17:400-405.); Silvia Crognale is the glucose oxidase in successful expression mould source in pichia spp also, output is 50U/ml (Silvia Crognale, Valentina Pulci and Brozzoli V, Expression of Penicillium variabile P16 glucose oxidase gene in Pichia pastoris and characterization of the recombinant enzyme.Enzyme and Microbial Technology 392006,1230-1235).From above-mentioned document as can be known, though adopt engineered means to realize the heterogenous expression of glucose oxidase at present, expression amount is improved on the original strain basis, utilize particularly that pichia spp allos secreting, expressing GOD has high cell density fermentation, albumen can be secreted into born of the same parents and express outward, and foreign protein is few; Have translation post-treatment modification system, the immunogenicity of glycoprotein is lower; Advantage (Macauley-Patrick S such as heredity and expression stability are good, Fazenda M, McNeil B, et al., Heterologous protein production using the Pichia pastoris expression system.Yeast, 2005,22:249-270.), but the secreting, expressing amount is all lower, usually at 50-100U/ml, this just causes zymin to cost an arm and a leg, and has limited it and has been extensive use of.
Usually improve the secreting, expressing of foreign gene in pichia spp strategies such as codon optimized, mRNA structure of modification, the adjustment of GC content are arranged.Wherein, the codon of optimizing foreign gene be main means (Nie Dongsong, Liang Songping. the strategy that foreign protein efficiently expresses in pichia pastoris., the journal .2001 of University Of Jishou, 22, (3), 40-44).
What is called is codon optimized promptly to be: because the degeneracy of codon, the codon of each amino acid correspondence can have a plurality of.Discover that species have codon-bias, promptly different plant species is different to the frequency of utilization of codon, some codon even never use, and those often do not use even are called rare codon or non-preference type codon from obsolete codon.The reason that non-preference type codon forms mainly is the tRNA that lacks these codons of identification in the cell.For different expression systems such as pichia spp and intestinal bacteria, its codon preference is different.If foreign gene contains the more non-preference type of host bacterium codon, occur especially continuously, will have a strong impact on its expression amount in the host bacterium.In order to eliminate the influence of rare codon to expressing quantity, can adopt codon to heterologous gene to be optimized realizes, promptly under the situation that does not change the foreign gene aminoacid sequence, the rare codon in the foreign gene is replaced with the codon that the host uses always.This method has simple, the characteristics of good reproducibility, obtained using widely (Sharp P, Tuohy T and Mosurski K, Codon usage in yeast:cluster analysis clearly differentiates highly and lowly expressed genes.Nucleic Acids Research, 1986,14:5125.).
It is very big that the zymoprotein foreign gene of different sources its expression amount in pichia spp after codon optimized improves degree difference, for example: Teng etc. are by optimizing β-1,3-1, the codon of 4 dextranases is formed, make its expression amount in pichia spp improve 10 times of (Teng D, Fan Y, Yang YL, et al., Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase gene and its expression in Pichia pastoris.Appl Microbiol Biotechnol, 2007,74:1074-1083); After the lipase codon that Chang will derive from candiyeast is optimized, expression amount in pichia spp improves 4.6 times of (Chang SW, Lee GC and Shaw JF, Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase.J Agric Food Chem, 2006,54:815-822.).
Summary of the invention
Technical problem to be solved by this invention is the lower defective of existing secreting, expressing amount when overcoming glucose oxidase gene and expressing in host cell, the codon of glucose oxidase gene is optimized and reduces its GC content, obtain the glucose oxidase enzyme mutant gene that a kind of secreting, expressing amount is significantly increased.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of glucose oxidase enzyme mutant gene, its base sequence are shown in the SEQ ID NO.2.
The present invention will clone the some notatin gene of acquisition under the prerequisite that does not change protein amino acid sequence, take all factors into consideration codon usage frequency, the adjustment of GC content, the influence factors such as deletion of unstable sequence, preference codon according to pichia pastoris phaff is transformed the glucose oxidase gene sequence, 272 bases have been changed altogether, relate to 96 amino acid, GC content reduces to 48.44% by 55.54%, and the secreting, expressing amount of the glucose oxidase enzyme mutant gene after the optimization in pichia spp is significantly increased.
Contain the recombinant expression vector of mutator gene of glucose oxidase and the row that the host cell that contains this recombinant expression vector also belongs to protection category of the present invention certainly; Preferably, described recombinant expression vector is a recombinant eukaryon expression vector, more preferably the restructured Pichia pastoris in expression carrier; Described host cell is preferably yeast cell, more preferably pichia spp (Pichia pastoris) cell.
The present invention also provides a kind of method for preparing glucose oxidase, comprising: being connected with expression vector of the glucose oxidase enzyme mutant gene operability shown in the SEQ ID NO.2 obtained recombinant expression vector; With described recombinant expression vector transformed host cell, get recombinant bacterial strain; Cultivate recombinant bacterial strain, induce the expression of reorganization glucose oxidase, reclaim and the expressed glucose oxidase of purifying, promptly.
In the aforesaid method, described recombinant expression vector is preferably recombinant eukaryon expression vector, more preferably the restructured Pichia pastoris in expression carrier; Described host cell is preferably yeast cell, more preferably pichia spp (Pichia pastoris) cell.
The glucose oxidase gene (SEQ ID NO.1) that the present invention will put mould (Penicillium notatum) source obtains glucose oxidase enzyme mutant gene (SEQ ID NO.2) through comprehensive reformations such as codon optimized, GC content changes.The present invention is transferred to the glucose oxidase enzyme mutant gene in the pichia spp and expresses, test-results shows, before sudden change, the secreting, expressing amount of glucose oxidase enzyme mutant gene in pichia spp is significantly increased, compare with domestic and international part Study, the final expression amount of glucose oxidase enzyme mutant gene of the present invention reaches higher expression level, lays a good foundation for further industrialization enlarges to produce.The glucose oxidase zymologic property is measured and is shown that the expressed reorganization glucose oxidase zymoprotein of glucose oxidase enzyme mutant gene of the present invention has good thermostability and higher enzyme activity.
Description of drawings
Fig. 1 GOD protogene codon usage frequency.
The improved GOD-M gene codon of Fig. 2 frequency of utilization.
Fig. 3 glucose oxidase optimized gene salvage and restriction enzyme site synoptic diagram.
The design of graphics of Fig. 4 yeast recombinant expression plasmid pPIC9-GOD and pPIC9-GOD-M.
The PCR of Fig. 5 yeast transformant identifies electrophorogram (1: negative control, 2-11: the PCR result who changes GOD; 12-21: the PCR result who changes GOD-M; M:marker).
The screening of Fig. 6 flat band method has the yeast transformant of glucose oxidase activity.
Fig. 7 changes glucose oxidase enzyme slip-knot fruit in GOD and 3 liters of fermentor tanks of GOD-M yeast strain.
Expression amount (the M: molecular weight of albumen marker of glucose oxidase in the methanol induction different time in Fig. 8 fermentor tank; 1-9: methanol induction 24,36,48,60,72,84,96,108,120h fermented liquid).
Fig. 9 glucose oxidase enzyme typical curve alive.
Figure 10 glucose oxidase optimal pH curve.
Figure 11 glucose oxidase pH beta stability line.
Figure 12 glucose oxidase optimum temperuture curve.
Figure 13 glucose oxidase enzyme heat stability curve.
Figure 14 metal ion and chemical reagent are to the influence of enzymic activity.
Figure 15 glucose oxidase oxidizing glucose double reciprocal curve.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Illustrate: the genetic recombination of using learns a skill and is routine techniques in this area in the following specific embodiment.The technology that does not describe in detail in following examples is all carried out according to related Sections in following laboratory manual or the document or part, comprising: people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With Current Protocols in Molecular Biology (people such as Ausubel compiles, 1994).
The optimization design of embodiment 1 glucose oxidase gene and synthetic
1.1 bacterial strain and plasmid
Point mould (Peniciliium notatum) is preserved by this laboratory of contriver;
Intestinal bacteria (Escherichia coli) bacterial strain TOP10 and cloning vector pSP72 are available from the Beijing Quanshijin Biotechnology Co., Ltd; The gene small segment is synthetic by Beijing AudioCodes biotech company.
1.2 the optimization design of glucose oxidase gene
At first the sequence of the glucose oxidase original gene that clone from a mould (Penicillium notatum) is obtained analyzes that (clone of gene please refer to: Li Zhuofu (Master's thesis), the clone of glucose oxidase gene and efficiently expressing. Changchun University of Science and Technology, 2008), under the prerequisite that does not change protein amino acid sequence, take all factors into consideration codon usage frequency, the adjustment of GC content, the influence factors such as deletion of unstable sequence are transformed the glucose oxidase gene sequence according to the preference codon of pichia pastoris phaff.
The glucose oxidase prochymosin gene total length 1815bp in some mould source, encode altogether 604 amino acid and a terminator codon.18 amino acid of wherein preceding 54 alkali yl codings are signal peptide sequence, need remove the signal peptide sequence that carries when expressing in pichia spp, are signal peptide with the alpha factor on the expression vector pPIC9.Therefore the glucose oxidase gene (GOD) of removing self signal peptide is by 1761 based compositions, encode 586 amino acid and a terminator codon (SEQ ID NO.1).Glucose oxidase gene (GOD-M) sequence (SEQ ID NO.2) after under the prerequisite that does not change aminoacid sequence the GOD gene being optimized has changed 272 bases altogether, relates to 96 amino acid, and GC content reduces to 48.44% by 55.54%.(table 1).Codon service condition before and after the GOD gene optimization as shown in Figure 1, 2.
The variation of GC content before and after table 1 glucose oxidase gene is optimized
1.3 the glucose oxidase gene after codon optimized (GOD-M) is synthetic
1.3.1 the design of oligonucleotide small segment and synthetic
The GOD-M gene two strands that designs is divided into A, B, C, D, E, F, 7 big fragments of G, each big fragment is about about 300bp, then with the positive-sense strand of every section greatly and some segments that antisense strand is divided into about 50-60bp again, the overlapped paired of 20-30bp zone is wherein arranged between each segment of two chains approximately, and between each big section, select proper restriction site to be beneficial to synthetic (table 2) of gene.The oligonucleotide small segment is by Beijing AudioCodes biotech company synthetic (Fig. 3).
1.3.2 the big segmental splicing of oligo DNA
1) the A fragment comprises 16 oligonucleotide segments, and with each segment deionized water dissolving, making its final concentration is 20 μ mol/L.
2) each oligonucleotide small segment being carried out phosphorylation handles.The phosphorylation system is as follows:
T
4Oligonucleotide phosphokinase 2U
(T
4PNK)
10 * damping fluid, 1 μ L
ATP(1μmol/ml) 1μL
Totally 10 μ L, 37 ℃ of water bath heat preservation 1h
3) 16 sections are merged in the EP pipe through the oligonucleotide small segment after the phosphorylation processing, totally 160 μ l, place 95 ℃ of water-baths to carry out denaturing treatment in mixture, behind the sex change 5min, the sex change sample is taken out, and naturally cooling is annealed, treat that temperature is reduced to room temperature after, annealing steps finishes, and the product of will annealing places-20 ℃ of preservations standby.
4) other 6 big sections are synthesized respectively according to above-mentioned steps.
Restriction enzyme site (as shown in Figure 3) according to A (XhoI, PvuII double digestion), B (PvuII, SphI double digestion), C (SphI, PstI double digestion), D (PstI, XbaI double digestion), E (XbaI, BamHI double digestion), F (BamHI, KpnI double digestion), 7 fragment designs of G (KpnI, ClaI double digestion), used cloning vector pSP 72 is carried out enzyme respectively cut processing, after enzyme is cut digestion fully, glue reclaims enzyme and cuts the back carrier segments, is used for being connected with each big fragment.
1.3.3 the oligo DNA fragment is connected and conversion with carrier
Each big fragment of oligonucleotide annealing generation is cut the back carrier segments with corresponding enzyme respectively be connected, the mol ratio of nucleotide fragments and carrier is 5: 1, and linked system is as follows:
Nucleotide fragments 0.5 μ mol
Carrier segments 0.1 μ mol
T
4DNALigase 1μL
10 * damping fluid, 1 μ L
Moisturizing to 10 μ L
Take out behind 16 ℃ of connection 8h and connect product
System joins in the 50 μ l Top10 competent escherichia coli cells with the enzyme disjunctor, and 42 ℃ of thermal shocks transform, and coating contains the LB flat board of penbritin (100 μ g/ml), is inverted in 37 ℃ of incubator overnight incubation.After treating that transformant grows in the LB flat board, the picking transformant is cultivated and is extracted plasmid and identifies positive colony by the PCR reaction.The PCR system is as follows:
dNTP(2.5μmol/mL?each) 1.5μL
Upstream universal primer T7 (10 μ mol/L) 0.5 μ L
Downstream universal primer SP6 (10 μ mol/L) 0.5 μ L
10 * damping fluid, 2 μ L
Taq archaeal dna polymerase 0.5 μ L
Moisturizing to 20 μ L
The pcr amplification program is as follows:
95 ℃ of pre-sex change 10min
95 ℃ of sex change 45s
55 ℃ of annealing 30s
72 ℃ are extended 90s
After 30 PCR circulations, continue 72 ℃ and extend 5min, take out the PCR sample, electrophoresis detection, positive colony is sent to Beijing three rich polygala root biotech companies and carries out sequencing.
1.3.4GOD-M full length gene splicing
With A, B, C, D, E, F, 7 big fragments of G are connected on the cloning vector pSP72, and transformed into escherichia coli TOP10, identify that through order-checking the subclone that sequence is correct is called after pSP72-A, pSP72-B, pSP72-C, pSP72-D, pSP72-E, pSP72-F, pSP72-G. respectively.The plasmid that extracts 7 subclones respectively (A-B-C-D-E-F-G) in order reclaims DNA fragment successively and all is connected on the cloning vector pSP72, identifies the correct clone's called after pSP72-GOD-M that contains the GOD-M full length gene through order-checking.
The structure and the screening of embodiment 2 glucose oxidase recombinant pichia yeast strains
2.1 bacterial strain and plasmid
The Top10 competent escherichia coli cell is available from the Beijing Quanshijin Biotechnology Co., Ltd;
Expression vector pPIC9 and pichia spp F-strain GS115 are Invitrogen company product.
Have the plasmid pBluescriptSK-GOD of raw glucose enzyme gene and have the plasmid pSP72-GOD-M that transforms back glucase gene and make up for inventor laboratory.
2.2 substratum and other solution
YPD substratum: peptone 20g/L, yeast extract 10g/L, glucose 20g/L (solid medium contains 1.5% agar powder), 108 ℃ of sterilization 15min.
10 * YNB (yeast does not have the amino acid nitrogenous source): 134g YNB solid is dissolved in the 1L deionized water, filtration sterilization, 4 ℃ of preservations;
500 * vitamin H: vitamin H 20mg/10omL water, filtration sterilization, 4 ℃ of preservations.
10 * glucose: 200gD-glucose is dissolved in 1000mL water, filtration sterilization, 4 ℃ of preservations.
MD substratum: YNB13.4g/L, glucose 20g/L, vitamin H 4 * 10
-4G/L, agarose 20g/L.
MM substratum: YNB13.4g/L, methyl alcohol 0.5%, vitamin H 4 * 10
-4G/L, agarose 20g/L.
BMGY substratum: peptone 20g/L, yeast extract 10g/L, YNB 13.4g/L, vitamin H 4 * 10
-4G/L, glycerine 10ml/L is with the preparation of pH6.0 phosphate buffered saline buffer.
BMMY substratum: peptone 20g/L, yeast extract 10g/L, YNB 13.4g/L, vitamin H 4 * 10
-4G/L, methyl alcohol 5mL/L is with the preparation of pH6.0 phosphate buffered saline buffer.
1mol/L sorbyl alcohol: 182.1g D-sorbyl alcohol is dissolved in the 1L water filtration sterilization, 4 ℃ of preservations.
Basal salts (fermention medium): KH
2PO
410g/L, NH
4H
2PO
4100g/L, K
2SO
436.4g/L, MgSO
47H
2O 29.7g/L, Ca
2SO
41.86g/L, KOH 3.0g/L.
Used trace salt solution (PTM) in the fermentation: copper sulfate 6.0g/L, sodium iodide 0.08g/L, manganous sulfate 3.0g/L, key acid sodium 0.2g/L, boric acid 0.02g/L, cobalt chloride 0.5g/L, zinc chloride 20g/L, ferrous sulfate 65g/L, vitamin H 0.25g/L, sulfuric acid 5ml/L; Filtration sterilization, 4 ℃ of preservations.
Dianisidine solution: the 0.1g dianisidine, be dissolved in 10ml methyl alcohol, be storage liquid, the 0.1ml storage liquid is got in 4 ℃ of preservations before the experiment, be dissolved in the 0.1mol/L of 12ml, in Sodium phosphate dibasic-citrate buffer solution of pH 6.2.
Plate screening colour developing liquid:
Solution I: dianisidine storage liquid (the 0.1g dianisidine is dissolved in 10ml methyl alcohol, 4 ℃ of preservations)
Solution II: 18% glucose (18g glucose is dissolved in the 100ml deionized water);
The horseradish peroxidase of solution III: 90U/ml;
1% the agarose of 10ml is dissolved the back add the 2ml solution II, 200 μ l solution I and 400 μ l solution III are colour developing liquid (matching while using).
2.3 the structure of glucose oxidase yeast recombinant expression vector
Extract pBluescriptSK-GOD, pSP72-GOD-M and pPIC9 plasmid respectively, and with EcoRI and NotI these three recombinant plasmids are carried out double digestion respectively and handle, glucose oxidase gene GOD that will not transform respectively and the GOD-M through optimize transforming and expression vector pPIC9 enzyme are cut product and are reclaimed and be connected, cut and check order positive colony is identified by enzyme, made up yeast recombinant expression vector pPIC9-GOD and pPIC9-GOD-M (Fig. 4) thus respectively.
2.4 the expression of glucose oxidase in pichia spp
Cut recombinant expression plasmid pPIC9-GOD and pPIC9-GOD-M with the BglII enzyme respectively, make plasmid linearization, respectively these two expression plasmids are transformed pichia spp host bacterium GS115 according to the Pichia anomala expression handbook.Coat on the MD flat board with every plate 200 μ L bacterium liquid measures, 28 ℃ are cultured to and grow transformant.Respectively select 10 clone's at random and extract genome, utilize the CTAB method to extract genomic dna, be template then with the genomic dna, carry out pcr amplification with the special primer (5 ' GCATGCACGGTTCCGTCAGT) of the not special primer of modifying gene GOD (5 ' TCAATCTGGGCGGCAGTCGG) and modifying gene GOD-M with universal primer 5 ' AOX (TACGTAATGAAGCTCCTCTCTGTTGCT) on the carrier respectively and detect, the purpose stripe size is about 1kb (Fig. 5).The positive rate that is tentatively drawn transformant by Fig. 5 is about more than 95% than higher.
2.5 malaga carbohydrate oxidase recombinant yeast pichia pastoris is in the screening of shaking table level
At first adopt the plate screening method that transformant is carried out primary dcreening operation.With aseptic toothpick with transformant longer on the MD flat board, copy on the MM flat board with numbering, be replicated in simultaneously on the MD flat board with identical numbering, place 28 ℃ of incubators to cultivate MM replica plate and MD replica plate, after cultivating 24h, place 4 ℃ of refrigerators to preserve the taking-up of MD replica plate, after taking out the MM flat board, the liquid that will develop the color is poured on the MM plate, room temperature 10-20min can develop the color, positive strain can show brown on flat board, determines positive rate and glucose oxidase Pichiapastoris expression strain (Fig. 6) in view of the above.Transformant to dull and stereotyped primary dcreening operation carries out multiple sieve subsequently.With aseptic toothpick with primary dcreening operation have corresponding clone that enzyme lives choose in the 10mL BMGY substratum shaking table cultivate 48h (28 ℃, 200rpm), centrifugal then (6000rpm, 8min), discard the substratum supernatant, add 5mL BMMY, shaking table is cultivated (28 ℃, 200rpm), add 0.5% methyl alcohol every 24h during this time, induce centrifugal (6000rpm, 8min behind the 60h, 4 ℃), collect supernatant liquor and measure enzyme activity.Sieve the result again and carry out revision test once more, confirm enzyme activity.
Respectively screening assay change each 800 discovery of yeast transformant of GOD and GOD-M, changeing on average has 30% transformant to have the activity of glucose oxidase approximately in 800 yeast transformants of GOD, and the yeast transformant enzyme that changes GOD is lived generally lower, wherein the highest 53# transformant is lived at the enzyme of shaking table shaker screen secreting, expressing and is also had only 10.8U/ml, there is 35% transformant to have enzyme to live in 800 yeast transformants of GOD-M approximately and change, wherein have the enzyme work of 65% transformant to be higher than the not activity of modifying gene (GOD) 53# approximately, the enzyme work of the highest DG16# is 53# 3.2 times (table 3).
Table 3 part is changeed glucose oxidase modifying gene zymic enzyme activity determination result
2.6 the fermentation of glucose oxidase recombinant yeast pichia pastoris
With the glucose oxidase recombinant yeast pichia pastoris bacterium 53# that do not carry out genetic modification is control strain, and 2 strains that comparison in the multiple sieve experiment is significantly improved according to the work of bacterial strain enzyme are changeed the pichia spp recombinant bacterial strain DG16# of GOD-M and the DG19# 3 liters of fermentor tank levels in chamber that experimentize and induced the product enzymic fermentation.
Respectively 53#, DG16# and DG19# yeast transformant are chosen in the 40ml YPD substratum with aseptic toothpick, and the shaking table cultivation (28 ℃, 200rpm) behind the 48h, be transferred in the 200ml YPD substratum, (28 ℃, 200rpm) 24h is inoculated in 3 liters of fermentor tanks as ferment-seeded bacterium liquid in the shaking table cultivation.
The fermentor tank parameter is set to pH5.5,30 ℃ of temperature, stir speed (S.S.) is 1000rpm, air flow is 200, the initial inoculation amount is a 200ml bacterium liquid, when dissolved oxygen drops to minimumly, begin again then to rise at 100% o'clock, begin to mend sugar (40% glucose of 400ml+10mlPTM salt), after mending sugared 8-10h, beginning mixed feeding (40% glucose of 100ml+4mlPTM salt+18ml methyl alcohol) behind the mixed feeding 4h, begins stream and adds methyl alcohol (500ml methyl alcohol+12.5mlPTM salt) and induce the product enzyme, between inductive phase by regulating the flow velocity of adding of methyl alcohol, dissolved oxygen level in the fermentor tank is controlled between the 25%-50%, after beginning to induce, gets fermented sample every 12h and measure thalline weight in wet base and sample enzyme and live and keep sample, enzyme work increases along with the prolongation of induction time, when enzyme work begins to descend, stop fermentation, following jar, with the centrifugal (10000rpm of fermented liquid, 10min, 4 ℃), clear enzyme solution in the collection.From 3 liters of lab scale level fermentation results as can be known, the activity of changeing yeast transformant 53# glucose oxidase behind methanol induction 132h of protogene GOD is 130U/ml, then reached 614U/ml and 582U/ml respectively after 132h is induced in the enzyme work of changeing two yeast transformant DG16# of full genetic modification and DG19#, the enzyme work of transformant DG16# behind 3 liters of horizontal methanol induction 132h of fermentor tank is 4.7 times (Fig. 7) of contrast 53#.
This shows that the glucose oxidase in some mould source has significantly improved the secreting, expressing amount of this zymoprotein in pichia spp behind comprehensive reformations such as codon optimized, GC content change.Compare with domestic and international part Study, the final expression amount of the glucose oxidase that this experiment obtains reaches higher expression level, produces lay a good foundation (table 4) for further industrialization enlarges.
The output of table 4 bibliographical information glucose oxidase
3.1 concentrating of glucose oxidase sample
The content of the glucose oxidase of Pichia anomala expression in fermented liquid supernatant accounts for more than 90% of total protein, therefore fermented liquid supernatant just can be used for later stage property testing (Fig. 8) by ultrafiltration and concentration.Earlier with the centrifugal 10min of fermented liquid supernatant 10000rpm, remove bacterial sediment, last clear enzyme solution adopts PALL company tangential flow membrane filtration system to concentrate, and at first adopts 0.1 μ m microfiltration membrane to filter, with the impurity of removing cell debris residual in the clear enzyme solution and may existing, collect filtered solution.Be that the ultra-filtration membrane of 10kDa filters through molecular weight cut-off again with filtered solution then, discard filtered solution, obtain the mensuration that spissated enzyme liquid is used for zymologic property.
3.2 glucose oxidase enzyme activity determination method
3.2.1 the making of typical curve
With reference to SIGMA Glucose (GO) Assay Kit, improve a little.Do typical curve with sigma company glucose oxidase standard substance.The glucose oxidase standard substance are diluted to 0.4,0.8,1.2,1.6,2.0 2.4U/ml adds 2.5ml dianisidine solution in test tube, the 18%D-glucose that adds 0.3ml, add 0.1ml90U/ml horseradish peroxidase solution, 35 ℃ of insulation 2min add 0.lml and dilute good glucose oxidase standard substance, the 2mol/L sulfuric acid termination reaction that adds 2ml behind the reaction 3min, assaying reaction liquid light absorption value under 540nm.See Table 5.
The drafting of table 5 glucose oxidase typical curve
According to glucose oxidase standard substance experimental result, be ordinate zou with glucose oxidase standard substance enzyme activity, with OD
540Light absorption value be X-coordinate, do the enzyme activity typical curve, as shown in Figure 9,
According to the result of typical curve, the calculation formula that draws glucose oxidase unit alive is:
Enzymic activity (U/ml)=(4.3769x-0.3034) * N
X:OD
540Light absorption value;
N: diluted sample multiple.
3.2.2 the mensuration of glucose oxidase activity
Add 2.5ml dianisidine solution in the test tube, 18% glucose of 03ml, 0.1ml the 90U/ml peroxidase, behind 35 ℃ of insulation 2min, in test tube, add the good enzyme liquid sample 0.1ml (adding) of dilution, behind the reaction 3min, add 2M sulfuric acid termination reaction (being that each 10s adds equally) every 10s, take out test tube, survey OD
540Light absorption value, do blank with the enzyme liquid of heat inactivation.According to the result of typical curve, calculate the glucose oxidase enzyme activity unit.
3.3 the optimal pH of glucose oxidase and pH stability
Measure the activity of glucose oxidase under the damping fluid of different pH, as Figure 10, as can be seen from the figure, the optimal pH of glucose oxidase is 6.2, can remain on more than 60% at enzyme activity between the pH4.0-7.0.
Under condition of different pH behind the treat enzyme liquid 1h, under optimal pH, measure the residue vigor of enzyme liquid, in enzyme the maximum data 100% alive, calculate relative vigor, as can be seen from Figure 11, glucose oxidase is basicly stable between pH4.0-7.0, and enzyme activity remains on more than 80%, and enzyme activity also can remain on more than 60% after pH 3.0 handles.The sour tolerance that this glucose oxidase is described is relatively good, and enzyme work has only 24% pH 7.5 time, and enzymic activity completely loses time the pH 8.0.
3.4 the optimum temperuture of glucose oxidase and temperature stability
As can be seen from Figure 12, the optimal reactive temperature of glucose oxidase is 35 ℃, and between 30-50 ℃ when reaction enzyme activity all more than 80%.
Behind 50 ℃ of insulation 2h, enzyme activity still keeps more than 70%, shows that glucose oxidase has thermostability (Figure 13) preferably.
3.5 metal ion and part chemical reagent are to the influence of enzymic activity
Add different metal ion or chemical reagent in enzymatic reaction system, final concentration is 1m mol/L, is contrast with the enzymatic reaction that does not add metal ion and chemical reagent, and vigor counts 100%.The result as shown in figure 14, common metal ion and chemical reagent do not have big influence to enzymic activity, enzyme activity is constant substantially, but SDS is bigger to the influence of enzyme activity, as can be seen from the figure, when the SDS that 1mmol/L is arranged in the enzymatic reaction system existed, the residual enzyme vigor had only about 40%.
3.6 enzyme kinetics
As substrate, in Sodium phosphate dibasic-citrate buffer solution of pH6.2, measure corresponding speed of response for 35 ℃, with different concns glucose according to double-reciprocal plot method (Lineweaver-Burk), as shown in figure 15.The negative inverse of transverse axis intercept is the Km value of enzyme among the figure, and the Km value of glucose oxidase is 83.21mmol/L as calculated, V
MaxBe 2170 μ mol/min/mg.
Claims (10)
1. glucose oxidase enzyme mutant gene, it is characterized in that: its base is shown in the SEQ ID NO.2.
2. the recombinant expression vector that contains the described glucose oxidase enzyme mutant gene of claim 1.
3. according to the described recombinant expression vector of claim 2, it is characterized in that: described recombinant expression vector is a recombinant eukaryon expression vector.
4. according to the described expression vector of claim 3, it is characterized in that: described recombinant eukaryon expression vector is that reorganization is than red yeast (Pichia pastoris) expression vector.
5. the host cell that contains any one described recombinant expression vector of claim 2-4.
6. according to the described host cell of claim 5, it is characterized in that: described host cell is a yeast cell.
7. according to the described host cell of claim 6, it is characterized in that: described yeast cell is pichia spp (Pichia pastoris) cell.
8. the application of the described glucose oxidase enzyme mutant gene of claim 1 in the preparation glucose oxidase.
9. according to the described application of claim 8, it is characterized in that, comprising: being connected with expression vector of the described glucose oxidase enzyme mutant gene of claim 1 operability obtained recombinant expression vector; With described recombinant expression vector transformed host cell, get recombinant bacterial strain; Cultivate recombinant bacterial strain, induce the expression of reorganization glucose oxidase, reclaim and the expressed glucose oxidase of purifying.
10. according to the described application of claim 9, it is characterized in that: described recombinant expression vector is a recombinant eukaryon expression vector, is preferably yeast expression vector; Described host cell is a yeast cell, is preferably pichia spp cell (Pichia pastoris).
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101348794A (en) * | 2007-07-19 | 2009-01-21 | 中国科学院武汉病毒研究所 | Encoding gene of high activity glucose oxidase, preparation and use thereof |
-
2010
- 2010-09-09 CN CN 201010277121 patent/CN101955953A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101348794A (en) * | 2007-07-19 | 2009-01-21 | 中国科学院武汉病毒研究所 | Encoding gene of high activity glucose oxidase, preparation and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库》 20100831 郜赵伟 葡萄糖氧化酶基因密码子优化及其在毕赤酵母中的高效表达 A006-86 1-10 第2010卷, 第08期 2 * |
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