CN108251389A - The glucose oxidase mutant that a kind of heat resistance improves - Google Patents
The glucose oxidase mutant that a kind of heat resistance improves Download PDFInfo
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- CN108251389A CN108251389A CN201710711531.5A CN201710711531A CN108251389A CN 108251389 A CN108251389 A CN 108251389A CN 201710711531 A CN201710711531 A CN 201710711531A CN 108251389 A CN108251389 A CN 108251389A
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108700042769 prolyl-leucyl-glycine Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
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- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
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Abstract
The present invention relates to genetic modification technical fields, specifically provide the glucose oxidase mutant that a kind of heat resistance improves.Compared with wild type, the heat resistance of the glucose oxidase mutant is largely increased, remnant enzyme activity improves 11.3 66.9% after 60 DEG C of 10 min of processing, remnant enzyme activity improves 23.7 105.5% after 65 DEG C of 5 min of processing, be conducive to extensive use of the glucose oxidase in feed, thus wide market.
Description
Technical field
The invention belongs to genetic modification technical fields, and in particular to the glucose oxidase mutation that a kind of heat resistance improves
Body.
Background technology
Glucose oxidase is a kind of aerobic dehydrogenase, exclusively can generate gluconic acid and peroxide by oxidation of beta-D-Glucose
Change hydrogen.Glucose oxidase usually forms an oxidation-reduction system, the oxidation of beta-D- in the presence of molecular oxygen with catalase
Glucose generates D-Glucose acid lactone, while consumes oxygen generation hydrogen peroxide.Hydrogen peroxide is decomposed and generated by catalase
Water and 1/2 oxygen, then water is combined again with glucolactone generates gluconic acid.Glucose oxidase is to β-D- glucopyranoses
Show strong specificity, the hydroxyl on glucose molecule C1 is most important to the catalytic activity of enzyme, and when hydroxyl is in β
It is 160 times higher than at α.Glucose oxidase is to L- glucose and 2-O- methyl-D-glucoses completely without activity.
Deoxygenation and antioxidation due to glucose oxidase make it in food, medicine, feed etc. using very
Extensively.In the food industry, glucose oxidase is preventing beer aging, is keeping product original as a kind of food preservative
Flavor, the aspect that extends the shelf life have remarkable result, are alternatively arranged as flour improver and Bread improver improves food quality.
In field of medicaments, hospital of China is generally using glucose oxidase electrode method, glucose oxidase-peroxidase coupling method etc.
Detect blood, glucose in serum content.As a kind of novel feed addictive, glucose oxidase can improve animal intestine
Road environment adjusts diet digestion, promotes growth of animal.
Glucose oxidase is distributed widely in animal, plant and microbial body, it is industrial mainly using black-koji mould or
Penicillium notatum is produced, but often occurs that enzyme activity is not high, stability is poor, foreign protein pollution and isolates and purifies cumbersome etc. ask
Topic.Because there are one 80~90 DEG C of short duration of hot stages during particle manufacture at present.And the grape of Aspergillus niger origin
Carbohydrate oxidase thermal stability is poor, and aqueous solution keeps the temperature 5 minutes remaining enzymatic activitys less than 40% at 65 DEG C, makes the enzyme in particle
The application of feed is restricted.At present using after feed granulating by the method in glucose oxidase liquid spray to feed not only
Increase equipment investment, and can not ensure the distribution uniformity of the stability and enzyme preparation of enzyme preparation in feed.Therefore, it carries
High glucose oxidation enzyme heat stability has important practical significance to current feed with glucose oxidase.
Invention content
The object of the present invention is to provide a kind of glucose oxidase mutant, heat resistance is significantly improved, is conducive to
Its extensive use in field of fodder.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of glucose oxidase mutant, have the amino acid sequence shown in (I), (II) or (III)
Any one in row：
(I) with the amino acid sequence SEQ ID NO of glucose oxidase:1 has the sequence of at least 95% homology；
(II) there is at least one immune epitope of the glucose oxidase described in (I), and the grape glycosyloxy
Change the amino acid sequence that the amino acid sequence of enzyme is obtained through modifying, replacing, lacking or adding one or several amino acid；
(III) by such as SEQ ID NO:When nucleotide sequence or its complementary series shown in 2 or the degeneracy because genetic code
With such as SEQ ID NO:The amino acid of the different sequential coding of the nucleotide sequence of nucleotide sequence or its complementary series shown in 2
Sequence；
It is described to be substituted by 1 amino acid of substitution in other embodiments of the present invention.
In other embodiments of the present invention, the substitution includes amino acid sequence for SEQ ID NO:1 glucose
The 194th of oxidizing ferment, 200,202,219,251,254,255,258,275,276,281,290,294,303,304,305,
Any one in 313,338,339,349,358,360,362,372,385 amino acids is replaced.
In other embodiments of the present invention, the substitution includes the 194th amino acids and becomes L from R.
In other embodiments of the present invention, the substitution includes the 200th amino acids and becomes A or D or E from K.
In other embodiments of the present invention, the substitution includes the 202nd amino acids and becomes P from F.
In other embodiments of the present invention, the substitution includes the 219th amino acids and becomes P from E.
In other embodiments of the present invention, the substitution includes the 251st amino acids and becomes I from V.
In other embodiments of the present invention, the substitution includes the 254th amino acids and becomes D or K or Y from S.
In other embodiments of the present invention, the substitution includes the 255th amino acids and becomes F or K from Q.
In other embodiments of the present invention, the substitution includes the 258th amino acids and becomes D or E or K from T.
In other embodiments of the present invention, the substitution includes the 275th amino acids and becomes F or Y from H.
The present invention other embodiments in, it is described substitution include the 276th amino acids from N become E or K or T or
Y。
In other embodiments of the present invention, the substitution includes the 281st amino acids and becomes K or R from H.
In other embodiments of the present invention, the substitution includes the 290th amino acids and becomes L from A.
In other embodiments of the present invention, the substitution includes the 294th amino acids and becomes L from T.
In other embodiments of the present invention, the substitution includes the 303rd amino acids and becomes D from M.
In other embodiments of the present invention, the substitution includes the 304th amino acids and becomes P from K.
In other embodiments of the present invention, the substitution includes the 305th amino acids and becomes D or P or R from S.
In other embodiments of the present invention, the substitution includes the 313rd amino acids and becomes P from D.
In other embodiments of the present invention, the substitution includes the 338th amino acids and becomes P from S.
In other embodiments of the present invention, the substitution includes the 339th amino acids and becomes D from A.
In other embodiments of the present invention, the substitution includes the 349th amino acids and becomes Y from F.
In other embodiments of the present invention, the substitution includes the 358th amino acids and becomes P from D.
In other embodiments of the present invention, the substitution includes the 360th amino acids and becomes A from S.
In other embodiments of the present invention, the substitution includes the 362nd amino acids and becomes F or L from K.
In other embodiments of the present invention, the substitution includes the 372nd amino acids and becomes P from E.
In other embodiments of the present invention, the substitution includes the 385th amino acids and becomes D or L from H.
The present invention also provides application of the above-mentioned glucose oxidase mutant in feed.
The present invention also provides the recombinant expression carriers with above-mentioned DNA molecular.
The present invention also provides a kind of host cells, include above-mentioned recombinant expression carrier.
In some embodiments of the invention, host cell is Pichia pastoris.
The heat resistance of glucose oxidase single-point mutants provided by the invention is generally higher than wild type, 60 DEG C of processing
Remnant enzyme activity improves 11.3-66.9% after 10min, and remnant enzyme activity improves 23.7-105.5% after 65 DEG C of processing 5min.From
And illustrate that above-mentioned simple point mutation causes the heat resistance of glucose oxidase to be increased dramatically, it is more suitable as feed than wild type
Additive is conducive to extensive use of the glucose oxidase in feed, thus wide market.
Specific embodiment：
The routine techniques and method that the present invention has used genetic engineering and biology field uses, such as
MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT
Recorded method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general bibliography
Provide definition well known by persons skilled in the art and method.But those skilled in the art can be recorded in the present invention
Technical solution on the basis of, using the other conventional methods in this field, experimental program and reagent, and be not limited to of the invention specific
The restriction of embodiment.
A in the present invention, R, D, C, Q, E, H, I, G, N, L, K, M, F, P, S, T, W, Y, V are alanine Ala respectively, arginine
Arg, aspartic acid Asp, cysteine Cys, glutamine Gln, glutamic acid Glu, histidine, isoleucine Ile, sweet ammonia
Sour Gly, asparagine Asn, leucine Leu, lysine Lys, methionine Met, phenylalanine Phe, proline Pro, silk ammonia
Sour Ser, threonine Thr, tryptophan Trp, tyrosine Tyr, the abbreviation of valine Val.
Experiment material used in the specific embodiment of the invention and reagent are as follows：
Bacterial strain and carrier：Bacillus coli DH 5 alpha, Pichia pastoris GS115, carrier pPIC9K, Amp, G418 are purchased from
Invitrogen companies.
Enzyme and kit：PCR enzymes and ligase purchase are from Takara companies, and restriction enzyme is purchased from Fermentas public affairs
Department, plasmid extraction kit and glue purification QIAquick Gel Extraction Kit are purchased from Omega companies, the purchase of GeneMorph II Random Mutagenesis Kits
From Beijing Bo Maisi bio tech ltd.
Culture medium prescription：
Escherichia coli culture medium (LB culture mediums)：0.5% yeast extract, 1% peptone, 1%NaCl, pH7.0)；
LB-AMP culture mediums：LB culture mediums add 100 μ g/mL ampicillins；
Yeast culture medium (YPD culture mediums)：1% yeast extract, 2% peptone, 2% glucose；
Yeast screening assay culture medium (MD culture mediums)：2% glucose, 2% agarose, 1.34%YNB, 4 × 10-5Biotin；
BMGY culture mediums：2% peptone, 1% yeast extract, 100mM kaliumphosphate buffers (pH6.0), 1.34%
YNB, 4 × 10-5Biotin, 1% glycerine；
BMMY culture mediums：2% peptone, 1% yeast extract, 100mM kaliumphosphate buffers (pH6.0), 1.34%
YNB, 4 × 10-5Biotin, 0.5% methanol.
The present invention is described in detail with reference to embodiment.
The acquisition of the heat-resisting single-point mutants of 1 glucose oxidase of embodiment
The amplification of 1.1 glucose oxidase genes
PCR amplification, PCR primer GOD-F1, GOD- are carried out by template of aspergillus niger (Aspergillus niger) genome
R1 is as follows：
GOD-F1：GGTATTGAGGCATCTTTGTTGAC
GOD-R1：TTATTGCATAGAAGCGTAATC
Glue recycle PCR product, connect pEASY-T carriers, convert into bacillus coli DH 5 alpha, the correct transformant of picking into
Row sequencing.Sequencing result shows that the nucleotides sequence of the genetic fragment expanded is classified as SEQ ID NO：2, the amino of coding
Acid sequence is SEQ ID NO：1.It is compared and found by NCBI BLAST, SEQ ID NO：1 glucose with deriving from aspergillus niger
Oxidase sequence similitude is up to 100%, so that it is determined that being glucose oxidase gene by the gene that PCR is obtained, is named as
GOD。
The amplification and synthesis of 1.2 glucose oxidase mutant genes
In order to improve the heat resistance of above-mentioned glucose oxidase GOD, applicant carries out the enzyme by directed evolution technologies
The screening of mass mutation, design PCR primer GOD-F2, GOD-R2 are as follows：
GOD-F2：GGCGAATTC(underscore identifies GGTATTGAGGCATCTTTGTTGAC for restriction enzyme EcoRI
Site)
GOD-R2：ATAGCGGCCGC(underscore identifies TTATTGCATAGAAGCGTAATC for restriction enzyme Not I
Site)
Using GOD genes as template, with above-mentioned primer GeneMorph II random mutations PCR kits (Stratagene)
Carry out PCR amplification, glue recycling PCR product, EcoRI, Not I carry out digestion processing after with the pET21a carriers after similary digestion
Connection, converts into e. coli bl21 (DE3), is coated on LB+Amp tablets, and 37 DEG C are inverted culture, after sub- appearance to be transformed,
It is chosen one by one with toothpick to 96 orifice plates, the LB+Amp culture mediums that 150ul contains 0.1mM IPTG, 37 DEG C of 220rpm is added in each hole
6h or so is cultivated, supernatant is abandoned in centrifugation, and thalline is resuspended with buffer solution, and multigelation broken wall is obtained containing the big of glucose oxidase
Coli cell lysate.
10 μ L lysates are taken out respectively to two pieces of 96 new orifice plates, one of after 70 DEG C handle 5min, two piece of 96 hole
Plate all adds in 40 μ L substrates, and after 30 DEG C are reacted 30min, DNS methods measure the reduced sugar of generation, calculate the enzyme solution phase of high-temperature process
Than in the opposite enzyme activity of untreated enzyme solution.The experimental results showed that some mutation do not have shadow to the heat resistance of glucose oxidase GOD
It rings, some mutation even make its heat resistance or enzyme activity become worse；In addition also some mutation, although it is glycoxidative to improve grape
Enzyme is to the tolerance of temperature, but significant change has occurred in its zymologic property after being mutated, these are undesirable.Finally, Shen
It asks someone to screen the heat resistance that can significantly improve glucose oxidase GOD and its enzyme activity and original zymologic property
Mutational site：R194L, K200A/D/E, F202P, E219P, V251I, S254D/K/Y, Q255F/K, T258D/E/K,
H275F/Y, N276E/K/T/Y, H281K/R, A290L, T294L, M303D, K304P, S305D/P/R, D313P, S338P,
A339D, F349Y, D358P, S360A, K362F/L, E372P, H385D/L.
PCR amplification is carried out respectively to above-mentioned mutant with primer GOD-F2, GOD-R2, and primer both ends introduce EcoRI, Not
I site.PCR reaction conditions are：94 DEG C of denaturation 5min；Then 94 DEG C of denaturation 30s, 56 DEG C of renaturation 30s, 72 DEG C of extension 1min, 30
After a cycle, 72 DEG C of heat preservation 10min.Agarose gel electrophoresis is the results show that the mutant gene that amplification obtains is size
The segment of 1800bp or so.
It expands to obtain the genetic fragment of wild type glucose oxidase GOD by above-mentioned same PCR method.
The structure of 1.3 pichia pastoris engineered strains
The glucose oxidase mutant gene that above-mentioned clone obtains is passed through into EcoRI and NotI sites and expression vector
PPIC9K is connected, construction of expression vector.
Expression vector is linearized with Sal I, expression vector linearized fragment is converted by electroporation finishes red ferment
Female GS115, screening obtains Pichia pastoris recombinant bacterial strain respectively on MD tablets, then respectively in the Geneticin containing various concentration
The transformant of multicopy is screened on YPD tablets.
Obtained transformant containing above-mentioned single-point mutants will be screened to transfer in BMGY culture mediums, 30 DEG C, 250rpm shakes
Swing culture 1d；It is transferred to again in BMMY culture mediums, 30 DEG C, 250rpm shaken cultivations；The methanol of addition 0.5% daily, induced expression
4d；Centrifugation removal thalline, obtains the fermented supernatant fluid of the mutant containing glucose oxidase；Carried out SDS-PAGE electrophoresis detections
Analysis.The results show that the molecular size range of glucose oxidase mutant is about 64kDa in fermented supernatant fluid, with Theoretical molecular
It is identical to measure size.
It builds to obtain the Pichia yeast engineering of recombinant expression wild type glucose oxidase by above-mentioned same method.
Shaking flask level is fermented, 30 DEG C, 250rpm shaken cultivations；The methanol of addition 0.5% daily, induced expression 4d；Centrifugation removal thalline,
Obtain the fermented supernatant fluid of the GOD of glucose oxidase containing wild type.
(1) definition of glucose oxidase enzyme-activity unit
It is per minute β-D-Glucose of 1 μm of ol to be oxidized to maltonic acid and peroxide under the conditions of pH6.0,30 DEG C
Change the required enzyme amount of hydrogen, be defined as 1 enzyme activity unit (IU).
(2) enzyme activity determination method
Crude enzyme liquid is directly diluted to about 10U/mL with buffer solution.Take the test tube of 4 150*15, add in 2ml buffer solutions,
0.3ml glucose, 0.4ml phenol, 0.1ml 4- amino antipyrine, 0.1ml horseradish peroxidases, 30 DEG C of preheating 5min.
A pipe adds in 0.1ml distilled water thereto, returns to zero as blank.Water-bath is placed on to be conveniently operated by spectrophotometer, to sample
0.1ml sample solutions are added in quality control, start timing at this time, after vortex mixing immediately at 500nm wavelength with 1cm cuvettes ratio
Color.Absorbance value is A0 when reading 0.5min, then after reacting 1min, reads absorbance value A1, obtains Δ A500=A1-A0.
Enzyme activity calculation formula：
Enzyme activity X1 (U/mL or U/g) is calculated according to equation below in sample：
X1=Δs A500 × f × B × 1000/ (887 × t × A × d)=33.82 × Δ A500 × f
In formula：
F--------------------- enzyme solution extension rates
B-------------------- reaction solutions volume (3ml)
1000---------------- extinction coefficient unit conversion factors
887----------------- extinction coefficients (Lmol-1cm-1)
T--------------------- reaction time (min), i.e., the time difference 1min between reading A1 and A0.
A-------------------- adds in sample volume (0.1ml)
The thickness (cm) of d-------------------- cuvettes
(3) enzyme activity determination result
The enzyme activity determination of fermented supernatant fluid is carried out according to the method described above, is as a result shown：Recombinantly express wild type grape glycosyloxy
The enzyme activity for changing the Pichia pastoris fermented supernatant fluid of enzyme is 105U/ml, and recombinantly expresses the complete red ferment of glucose oxidase mutant
The enzyme activity of female fermented supernatant fluid is about 102-154U/mL.
1.4 fermentation verifications
Carry out the fermentation for the Pichia yeast engineering that above-mentioned structure obtains, the training used of fermenting respectively on 10 liters of fermentation tanks
Foster based formulas is：Calcium sulfate 1.1g/L, potassium dihydrogen phosphate 5.5g/L, ammonium dihydrogen phosphate 55g/L, potassium sulfate 20.3g/L, magnesium sulfate
16.4g/L, potassium hydroxide 1.65g/L, antifoaming agent 0.05%.
Zymotechnique：PH value 5.0,30 DEG C of temperature, stir speed (S.S.) 300rpm, ventilation quantity 1.0-1.5 (v/v), dissolved oxygen control
More than 20%.
Entire fermentation process is divided into three phases：First stage be thalline cultivation stage, in 7% ratio access seed, 30
DEG C culture 24-26h, using mended glucose for indicate；Second stage is the hungry stage, after glucose has been mended, does not flow plus appoints
What carbon source, terminates, by a definite date about 30-60min when dissolved oxygen rose to for 80% stage indicated above；Phase III is induced expression rank
Section, stream plus methanol induction, and keep dissolved oxygen incubation time is between 150-180h more than 20%.After fermentation, it ferments
Liquid obtains crude enzyme liquid after being handled by flame filter press.
Enzyme activity assay is carried out to crude enzyme liquid using in the embodiment 1 1.3 glucose oxidase enzyme activity determination methods, as a result
It has been shown that, the fermentation enzyme activity that the Pichia pastoris of recombinant expression wild type glucose oxidase is final is 3050U/ml, and is recombinantly expressed
The fermentation enzyme activity that the Pichia pastoris of glucose oxidase mutant is final reaches 3030-3590U/ml.
1.5 glucose oxidase zymologic properties measure
1st, most suitable action pH
It is respectively 2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0 phosphorus using pH value
Sour disodium hydrogen-citrate buffer solution, it is glycoxidative to carry out grape for fermentation crude enzyme liquid described in embodiment 1 1.4 under the conditions of 30 DEG C
Enzyme activity determination using highest enzyme activity as 100%, calculates opposite enzyme activity, as a result shows wild type glucose oxidase GOD and mutation
The Optimun pH of body is all 6.0, and the horizontal difference of opposite enzyme activity under condition of different pH is little.
2nd, thermal stability analysis
After the acetic acid-sodium acetate buffer solution dilution of pH 6.0 of above-mentioned crude enzyme liquid, at 60 DEG C of processing 10min, 65 DEG C
After managing 5min, enzyme activity is measured respectively, using the enzyme activity of untreated samples as 100%, calculates remnant enzyme activity.The results are shown in table below.
It can be seen that compared with wild type from the data in table, the heat resistance of 41 single-point mutants provided by the invention
It is significantly increased, remnant enzyme activity improves 11.3-66.9% after 60 DEG C of processing 10min, and 65 DEG C handle remnant enzyme activity after 5min
Improve 23.7-105.5%.So as to illustrate that above-mentioned simple point mutation causes the heat resistance of glucose oxidase to be increased dramatically,
Feed addictive is more suitable as than wild type, is conducive to extensive use of the glucose oxidase in feed, market prospects are wide
It is wealthy.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Qingdao Weilan Biology Group Co., Ltd.
<120>The glucose oxidase mutant that a kind of heat resistance improves
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 581
<212> PRT
<213> 1
<400> 1
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 2
<211> 1746
<212> DNA
<213> 2
<400> 2
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accactgccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acgcttacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttgg ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctcacaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctgct gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccgctgg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746
Claims (8)
1. a kind of glucose oxidase mutant, with any of amino acid sequence shown in (I), (II) or (III)：
(I) with the amino acid sequence SEQ ID NO of glucose oxidase:1 has the sequence of at least 95% homology；
(II) there is at least one immune epitope of glucose oxidase described in (I), and the amino of the glucose oxidase
The amino acid sequence that acid sequence is obtained through modifying, replacing, lacking or adding one or several amino acid；
(III) by such as SEQ ID NO:When nucleotide sequence or its complementary series shown in 2 or the degeneracy because genetic code with such as
SEQ ID NO:The amino acid sequence of the different sequential coding of the nucleotide sequence of nucleotide sequence or its complementary series shown in 2
Row.
2. glucose oxidase mutant as described in claim 1, which is characterized in that described is substituted by 1 amino of substitution
Acid.
3. glucose oxidase mutant as claimed in claim 2, which is characterized in that the substitution includes amino acid sequence
For SEQ ID NO:The 194th of 1 glucose oxidase, 200,202,219,251,254,255,258,275,276,281,
Any one in 290,294,303,304,305,313,338,339,349,358,360,362,372,385 amino acids occurs
Substitution.
4. glucose oxidase mutant as claimed in claim 3, which is characterized in that the substitution includes R194L,
K200A, K200D, K200E, F202P, E219P, V251I, S254D, S254K, S254Y, Q255F, Q255K, T258D,
T258E, T258K, H275F, H275Y, N276E, N276K, N276T, N276Y, H281K, H281R, A290L, T294L,
M303D, K304P, S305D, S305P, S305R, D313P, S338P, A339D, F349Y, D358P, S360A, K362F,
Any one in K362L, E372P, H385D, H385L.
5. encode the DNA molecular of claim 1-4 any one of them glucose oxidase mutant.
6. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier carries the DNA described in claim 5
Molecule.
7. a kind of host cell, which is characterized in that the recombinant expression that the host cell includes described in claim 6 carries
Body.
8. application of the claim 1-4 any one of them glucose oxidase mutant in feed preparation.
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| CN201710711531.5A CN108251389A (en) | 2017-08-18 | 2017-08-18 | The glucose oxidase mutant that a kind of heat resistance improves |
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Family
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| CN (1) | CN108251389A (en) |
Cited By (5)
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| WO2020125700A1 (en) * | 2018-12-20 | 2020-06-25 | 南京百斯杰生物工程有限公司 | Glucose oxidase mutant and use thereof in industrial production |
| CN112143717A (en) * | 2019-06-26 | 2020-12-29 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant with improved specific activity |
| CN113874498A (en) * | 2019-05-31 | 2021-12-31 | 南京百斯杰生物工程有限公司 | Thermostable glucose oxidase |
| EP4006149A4 (en) * | 2019-07-26 | 2023-05-31 | Feed Research Institute, Chinese Academy of Agricultural Sciences | Mutant glucose oxidase (god) having improved thermal stability and gene and application thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| WO2020125700A1 (en) * | 2018-12-20 | 2020-06-25 | 南京百斯杰生物工程有限公司 | Glucose oxidase mutant and use thereof in industrial production |
| CN113874498A (en) * | 2019-05-31 | 2021-12-31 | 南京百斯杰生物工程有限公司 | Thermostable glucose oxidase |
| CN112143717A (en) * | 2019-06-26 | 2020-12-29 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant with improved specific activity |
| CN112143717B (en) * | 2019-06-26 | 2023-02-03 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant with improved specific activity |
| EP4006149A4 (en) * | 2019-07-26 | 2023-05-31 | Feed Research Institute, Chinese Academy of Agricultural Sciences | Mutant glucose oxidase (god) having improved thermal stability and gene and application thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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