CN108048440A

CN108048440A - A kind of high-temperature resistance glucose isomerase mutant and its application

Info

Publication number: CN108048440A
Application number: CN201810007649.4A
Authority: CN
Inventors: 柳志强; 贾东旭; 郑裕国; 刘子健; 王腾; 金利群; 王远山
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-01-04
Filing date: 2018-01-04
Publication date: 2018-05-18
Anticipated expiration: 2038-01-04
Also published as: CN108048440B

Abstract

A kind of application the invention discloses high-temperature resistance glucose isomerase mutant and its in catalysis D glucose isomerizations prepare D fructose, mutant are to obtain the 89th of amino acid sequence shown in SEQ ID NO.1, the 136th, one or more of the 352nd progress single mutation or multimutation.The present invention realizes the high efficient expression of high conversion colt Glucose Isomerase Gene, enzyme activity 7.23U/mg.The optimal reactive temperature of heretofore described ToGI mutant is 110 DEG C, which is catalyzed under 100 DEG C of high temperature in the high fructose syrup of 400g/L D glucose preparation, and D fructose concentrations are up to 61.3%.

Description

A kind of high-temperature resistance glucose isomerase mutant and its application

(1) technical field

The present invention relates to a kind of glucose isomerase mutants, relate in particular to the tool prepared using mutation technique The isomerization that the method for the glucose isomerase mutant of standby superelevation reaction temperature and the mutant can be stablized under superhigh temperature The application of glucose production superelevation D-Fructose concentration high fructose syrup.

(2) background technology

Glucose isomerase (glucose isomerase, abbreviation GI, EC 5.3.1.5), is mainly used for being catalyzed in vitro D-Glucose isomerization generates D-Fructose, is the industrial key enzyme that high fructose syrup is prepared using biotransformation method.According to GI's Primary structure can be classified as two classes, i.e. I classes and II fermentoids.Compared with I classes GI, it is extra that the peptide chain N-terminal of II classes GI contains 40-50 amino acid residue (Deng H.etal., Bioprocess and Biosystems Engineering, 37：1211- 1219,2014).

High fructose syrup (high fructose corn syrup, abbreviation HFCS) is the mixture of glucose and fructose, is A kind of important sweetener.High fructose syrup has that solubility is high, chemically and thermally stability is good, osmotic pressure is big, moisture absorption and moisturizing Property it is strong, mixed with other additives and do not influence the advantages such as flavour of food products.According to the difference of its fructose content, high fructose syrup mainly has 3 kinds of products：HFCS-42, HFCS-55 and HFCS-90.Wherein, the sugariness of HFCS-55 is better than sucrose, is mainstream production in the market Product.But at present widely used glucose isomerase bio-conversion process can not a step F55 types high fructose syrup is made, it is necessary to HFCS-42 concentrations are blent again with HFCS-90 could be made HFCS-55 (MoellerS.M.et al., Journal of the American College of Nutrition,28:619-26,2009).

The D-Glucose isomerization process of GI interventions is the reaction of thermodynamical equilibrium, with the rise of isomerisation temperature, Isomerization reaction can be promoted to be carried out to fructose direction.At present, the GI for being commercially used for production high fructose syrup is mainly derived from The wild mushrooms such as Bacillus coagulans, Streptomyces murinus and Streptomyces rubiginosis (DicosimoR.et al., Chemical Society Reviews, 42：6437-6474,2013).Due to these above-mentioned GI Heat resistance it is general, can only steadily carry out catalytic reaction, fructose converting rate only 42-45% in 60-65 DEG C of isomerisation temperature. Therefore, if can be catalyzed under such as 100 DEG C of high temperature or higher isomerisation temperature, having for being generated is dense higher than 55%D- fructose The high fructose syrup of degree will be helpful to reduce the follow-up cost for being enriched with and blending, and have to the production technology change for promoting high fructose syrup Significance.

At present, have the report of some heat-resisting GI, such as Thermotoga maritima and Thermus Thermophiles etc., optimum temperature respectively reach 105 DEG C and 95 DEG C, and still, enzyme preparation success is not made for these enzymes It is invested in market.Given this background, the present invention proposes existing GI carrying out rite-directed mutagenesis, and builds height by technique for gene engineering For the genetic engineering bacterium of expression for producing high fructose syrup at high temperature, the market vacancy that high temperature resistant enzyme is lacked for filling up has weight Big meaning.

(3) content of the invention

The technical problems to be solved by the invention are to provide a kind of glucose isomerase enzyme mutant for possessing superelevation reaction temperature The application of body and the mutant energy desmotropism glucose production superelevation D-Fructose concentration high fructose syrup under superhigh temperature.

The technical solution adopted by the present invention is：

The present invention provides a kind of high-temperature resistance glucose isomerase mutant, and the mutant is will be shown in SEQ ID NO.1 The 89th of amino acid sequence, the 136th, one or more of the 352nd carries out single mutation or multimutation obtains.

Further, mutant of the present invention is by one, two or three in the 89th, the 136th, the 352nd Amino acids sport alanine, serine, glutamine, methionine or leucine.

Further, the mutant is one of following：(1) by the 89th of amino acid sequence shown in SEQ ID NO.1 Threonine (T) sports glutamine (Q)；(2) amino acid sequence the 89th shown in SEQ IDNO.1 threonine (T) is sported Glutamine (Q), the 136th tryptophan (W) sport serine (S)；(3) by amino acid sequence shown in SEQ ID NO.1 89 threonines (T) sport glutamine (Q), the 136th tryptophan (W) sports serine (S), while the 352nd Soviet Union Propylhomoserin (T) sports leucine (L).

The gene order of coding mutant of the present invention falls within the scope of protection of present invention.

The present invention also provides a kind of methods for producing high-temperature resistance glucose isomerase mutant：According to ToGI genes, design The mutant primer of rite-directed mutagenesis carries out rite-directed mutagenesis as template using the cloning vector for carrying GI and builds mutant, with plasmid PET28b can express the carrier of the enzyme as expression vector, and recombinant plasmid transformed Escherichia coliBL21 (DE3) is thin Born of the same parents or the host cell that can express the enzyme select the positive monoclonal after verification and carry out fermented and cultured.

The invention further relates to a kind of recombinant vector gene constructed by the high-temperature resistance glucose isomerase mutant code, And the recombination engineering bacteria obtained by recombinant vector conversion.

The invention further relates to a kind of high-temperature resistance glucose isomerase mutant D- fruits are prepared in catalysis D-Glucose isomerization Application in sugar, the application are：It is obtained with the fermented culture of the engineering bacteria of the mutant gene containing high-temperature resistance glucose isomerase Wet thallus for enzyme source, using D-Glucose as substrate, using manganese salt as auxiliary agent, with the Na of 50mM₂HPO₄/NaH₂PO₄Buffer solution (pH8.0) reaction system is formed for reaction medium, in 50-110 DEG C (preferably 100-110 DEG C, most preferably 100 DEG C), 150r/min Under the conditions of react, when reaction to the end of, reaction solution is isolated and purified, obtain D-Fructose；In the reaction system, initial substrate concentration For 50-500g/L (preferably 400g/L), the dosage of wet thallus is 10-50g/L (preferably 25g/L), the final concentration of 5- of manganese salt 25mM (preferably 20mM).

Further, the wet thallus is prepared as follows：Structure contains the high-temperature resistance glucose isomerase mutant The recombinant vector of gene converts the recombinant vector into E.coli, and the recombination engineering bacteria of acquisition carries out induced expression, Take the isolated wet thallus cell of culture solution.Specially：The engineering bacteria of the mutant gene containing high-temperature resistance glucose isomerase is connect Kind, in 37 DEG C, 150r/min culture 10h, obtains seed liquor to the LB fluid nutrient mediums for containing 50 μ g/mL kanamycins；By seed Liquid is seeded to 2% (v/v) inoculum concentration in the fresh LB culture mediums containing 50 μ g/mL kanamycins of final concentration, in 37 DEG C, 150r/min cultivates OD₆₀₀To 0.6-0.8, then the IPTG of final concentration of 1mM is added in into culture solution, 10h is induced in 28 DEG C, 8000r/min centrifuges 10min, and abandoning supernatant collects wet thallus；The LB culture mediums composition：Tryptone 10g/L, yeast Powder 5g/L, NaCl 10g/L, solvent are water, and pH value is natural.

Compared with prior art, advantageous effect of the present invention is mainly reflected in：The present invention provides a kind of new high temperature resistant Portugals Grape sugar isomerase mutant, the mutant have super 110 DEG C of optimal reactive temperature, improve 15 DEG C than protoenzyme ToGI, belong to In the highest level of document report.High fructose syrup is produced using this mutant, in 50-110 DEG C, the conversion condition of 150r/min Under, the high conversion rate for the high fructose syrup that ToGI mutant enzymes are produced is up to 61.3%, higher than protoenzyme and turn of other mutant enzymes Change effect, while be also the highest level of document report.The ToGI mutant solves the existing enzyme of long-standing problem can not be in high temperature The technical barrier of high fructose syrup is produced, there is significant technological progress.

(4) illustrate

Fig. 1 is the optimum temperature schematic diagram of ToGI mutant；

Fig. 2 is that the E.coli of the ToGI mutant containing restructuring produces the application schematic diagram of high fructose syrup.

(5) specific embodiment

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This：

Embodiment 1：The structure of ToGI single-site mutant bodies and screening

1st, mutation construction

According to ToGI parental arrays, (amino acid sequence is shown in SEQ ID NO.1, nucleotides sequence is classified as SEQ ID NO.2 It is shown) mutant primer of design rite-directed mutagenesis, using fast PCR technology, using recombinant vector pET28b/ToGI as template, to the 89 introducing single mutation, primer are：

Forward primer CCGATGGTTNNNGCTAACCTGTTC (underscore is mutating alkali yl)

Reverse primer CAGGTTAGCNNNAACCATCGGAACTTTC (underscore is mutating alkali yl)

PCR reaction systems：2 × Phanta Max Buffer (contain Mg²⁺) 25 μ L, dNTPs 10mM, 2 μ L of forward primer, instead To primer 2 μ L, 1 μ L, Phanta Max Super-Fidelity DNA Polymerase 50U of template DNA, ddH is added in₂O To 50 μ L.

PCR amplification condition is 95 DEG C of 3min；(95 DEG C of 15s, 50 DEG C of 15s, 61 DEG C of 6.5min) 30 are cycled；72℃ 5min。

2nd, mutant conversion expression

PCR product Transformed E .coliBL21 (DE3) competent cell chooses monoclonal in the LB containing 50 μ g/mL kanamycins In fluid nutrient medium, 37 DEG C of overnight incubations.Primary dcreening operation is carried out to mutant using cysteine carbazole method development process, it is specific as follows： The positive strain containing mutation after bacterium colony PCR identifications is inoculated in LB fluid nutrient medium test tubes of the 5mL containing 50 μ g/mL kanamycins In, in 37 DEG C, 150r/min shaking table cultures to OD₆₀₀About 0.6-0.8, then IPTG to final concentration 1mM is added in into culture, 28 DEG C of Fiber differentiations stay overnight 12h, and thalline were collected by centrifugation in 1.5mL centrifuge tubes.

3rd, mutant screens

Preparing reaction mixture final concentration composition is：50mM Tris-HCl buffer solutions (pH 7.0), 1mM Co²⁺、10mM Mg²⁺, 200mM glucose.Reaction mixture 85 DEG C of heat preservations 3min, the quick 1mL that draws add in the 1.5mL centrifuge tubes containing thalline In, stop reaction in 85 DEG C, 500r/min reactions 10min, ice bath 3min after oscillator vibration mixing.With cystein-carbazol Development process screens mutant, is carried out in 1.5mL centrifuge tubes, and reactant includes 167 μ L of reaction solution, 33 μ of cysteine hydrochloride L, 70% concentrated sulfuric acid, 1000 μ L, 33 μ L of carbazole alcohol.Color change is observed after keeping the temperature 10min at 60 DEG C, with wild type glucose (gene shown in SEQ ID NO.2 is transferred to E.coli BL21 to isomerase producing bacterial strain E.coli BL21 (DE3)/pET28b/ToGI (DE3) it is made) as control, color carries out enzyme activity determination than the mutant strain of wild moldeed depth.

4th, mutant enzyme activity determination

Ultrasonication is carried out to wet thallus using ultrasonication method, the wet thallus of 1g steps 2 is taken, with 20mL Tris- HCl buffer solutions (pH 7.0) suspend, the ultrasonication 20min under the conditions of 39W, and preparing cell-free extract, (i.e. ultrasound is broken Suspension after broken), centrifugation collects supernatant, and takes 1mL supernatants for reacting.Reaction system final concentration forms：Tris-HCl Buffer solution (pH 7.0), 1mM Co²⁺With 10mM Mg²⁺, 200mM D-Glucoses, common 5mL systems.Reaction condition：In 85 DEG C, 20min is reacted under the conditions of 150r/min, ice bath 10min terminates reaction, and 8000r/min centrifugation 10min take 20 μ L supernatants；It adopts The concentration of D-Glucose and D-Fructose is detected with HPLC.Analytical column is Hypersil NH₂Column (250 × 4.6mm, 5 μm) is (according to profit Special Analytical Instrument Co., Ltd, Dalian, China).2414 differential refraction detectors of Waters, Waters 1525 are pumped, Waters 717 injectors.Enzyme activity defines：It is per minute to generate D-Glucose isomerization needed for 1 μm of ol D-Fructose under 100 DEG C and pH 7.0 Enzyme amount is defined as an enzyme-activity unit (U).

The result of the embodiment is：To 115 plant weight group transformed bacteria primary dcreening operations, the mutant strain that 5 plants of enzyme activity improve is filtered out, then it is right It carries out enzyme activity determination, and concrete outcome is shown in Table 1.It is determined through analyzing, the original that remaining 110 plants of recombinant bacterium enzyme activity is remained unchanged or declined Because being that the 89th threonine (T) sports other amino acid outside A, S, Q, M and L.

The enzyme activity determination of 1 simple point mutation recombinant bacterium of table

Enzyme activity is improved into most mutant ToGI-T89Q and is denoted as ToGI-1, obtain recombinant bacterium E.coli BL21 (DE3)/ pET28b/ToGI-1。

Embodiment 2：The structure of two site mutant of glucose isomerase and screening

According to the mutant primer for the single mutant ToGI-1 sequence design rite-directed mutagenesises that embodiment 1 is built, fast PCR is utilized Technology, using recombinant vector pET28b/ToGI-1 as template, to the 136th introducing single mutation, primer is：

Forward primer CGTTGTTNNNCCGGGTCGTG (underscore is mutating alkali yl)

Reverse primer GACCCGGNNNAACAACGTAGATTTC (underscore is mutating alkali yl)

PCR amplification condition is 95 DEG C of 3min；(95 DEG C of 15s, 50 DEG C of 15s, 62 DEG C of 6.5min) 30 are cycled；72℃ 5min。

PCR product Transformed E .coliBL21 (DE3) competent cell chooses monoclonal in the LB containing 50 μ g/mL kanamycins In fluid nutrient medium, 37 DEG C of overnight incubations.Primary dcreening operation (same embodiment is carried out to mutant using cysteine carbazole method development process 1)。

Ultrasonication is carried out to wet thallus using ultrasonication method, and carries out enzyme activity determination (with embodiment 1).

The result of the embodiment is：To 97 plant weight group transformed bacteria primary dcreening operations, the mutant strain that 5 plants of enzyme activity improve is filtered out, then it is right It carries out enzyme activity determination, and concrete outcome is shown in Table 2.It is determined through analyzing, the original that remaining 92 plants of recombinant bacterium enzyme activity is remained unchanged or declined Because being that the 136th threonine (T) sports other amino acid outside A, S, Q, M and L.

The enzyme activity determination of 2 double mutant recombinant bacterium of table

Enzyme activity is improved into most mutant ToGI-T89Q-W136S and is denoted as ToGI-2, obtains recombinant bacterium E.coli BL21 (DE3)/pET28b/ToGI-2。

Embodiment 3：The structure of three site mutant of glucose isomerase and screening

According to the mutant primer for the mutant ToGI-2 sequence design rite-directed mutagenesises that embodiment 2 is built, fast PCR skill is utilized Art, using recombinant vector pET28b/ToGI-2 as template, to the 352nd introducing single mutation, primer is：

Forward primer CGTGCTNNNGCTCTGAAAG (underscore is mutating alkali yl)

Reverse primer CAGAGCNNNAGCACGTTCAC (underscore is mutating alkali yl)

PCR amplification condition is 95 DEG C of 3min；(95 DEG C of 15s, 50 DEG C of 15s, 60 DEG C of 6.5min) 30 are cycled；72℃ 5min。

The result of the embodiment is：To 104 plant weight group transformed bacteria primary dcreening operations, the mutant strain that 5 plants of enzyme activity improve is filtered out, then it is right It carries out enzyme activity determination, and concrete outcome is shown in Table 3.It is determined through analyzing, the original that remaining 99 plants of recombinant bacterium enzyme activity is remained unchanged or declined Because being that the 352nd threonine (T) sports other amino acid outside A, S, Q, M and L.

The enzyme activity determination of 3 double mutant recombinant bacterium of table

Enzyme activity is improved into most mutant ToGI-T89Q-W136S-T352L and is denoted as ToGI-3, obtains recombinant bacterium E.coli BL21(DE3)/pET28b/ToGI-3。

Embodiment 4：Recombination bacillus coli enzymatic production

Respectively by recombinant bacterium E.coliBL21 (DE3)/pET28b/ToGI-1, E.coli BL21 of embodiment 1,2 and 3 (DE3)/pET28b/ToGI-2, E.coliBL21 (DE3)/pET28b/ToGI-3 is seeded to that is mould containing 50 μ g/mL cards of final concentration The LB fluid nutrient mediums of element, in 37 DEG C, 150r/min cultures OD₆₀₀About 0.6-0.8 obtains seed liquor；By seed liquor with 2% (v/ V) inoculum concentration is seeded in the fresh LB fluid nutrient mediums containing 50 μ g/mL kanamycins of final concentration, in 37 DEG C, 150r/min Cultivate OD₆₀₀To 0.6-0.8, then add in into culture solution the IPTG of final concentration of 0.1mM, at 28 DEG C after induced expression 10h, 4 DEG C, 8000r/min centrifugation 10min, abandoning supernatant cleans twice of wet thallus with 0.85% physiological saline, and collects wet bacterium Body, it is spare.

Embodiment 5：It is mutated the purifying of recombinant glucose isomerase

The wet thallus of each recombinant bacterium prepared by embodiment 4 is collected after ultrasonication 20min under the conditions of 39W, it will be broken mixed Liquid centrifugation is closed, takes supernatant, 15min is heat-treated at 75 DEG C, then in 4 DEG C, 8000r/min centrifugation 10min, precipitation is discarded, receives Collection supernatant purified using nickel-NTA agarose Gel columns, with level pad (20mM phosphate buffers, 300mM NaCl, 20mM imidazoles, pH 8.0) balance chromatographic column, reuse eluent (50mM phosphate buffers, 300mM NaCl, 500mM imidazoles, pH 8.0) it is eluted, it is responded according to the signal of UV detector, collects corresponding eluent, be Respective pure enzyme solution.

Embodiment 6：Purify the optimum temperature of enzyme

Using the pure enzyme solution in embodiment 5 as conversion enzyme, the optimal reactive temperature of enzyme is measured.Concrete operations are as follows： In 50mM Tris-HCl buffer solutions (pH 7.0), 200mM D-Glucoses, 1mM Co are added in²⁺、10mM Mg²⁺With the pure enzymes of 1mL Liquid, the common 5mL of system.Respectively at different conversion temperatures：60th, the work of 70,80,90,100,110,120 DEG C of measure glucose isomerases Power (with embodiment 1, the determination condition higher than 100 DEG C needs to use oil bath method).As shown in Figure 1, the optimal reaction of ToGI-3 Temperature is 110 DEG C, than protoenzyme ToGI improve 15 DEG C (Jiaet al, Enzyme Microbial Technology, 99:1- 8), while it is also the peak of document report.

Embodiment 7：The single, double and three mutation full cells of recombinant bacterium prepare high fructose syrup.

As described in Example 4, recombinant bacterium E.coliBL21 (DE3)/pET28b/ToGI-1, E.coliBL21 is obtained (DE3)/pET28b/ToGI-2 and E.coliBL21 (DE3)/pET28b/ToGI-3 wet thallus are as biocatalyst, with D- Portugals Grape sugar is substrate, and bioconversion prepares high fructose syrup.100mL catalyst system and catalyzings include：With 50mM Na₂HPO₄/NaH₂PO₄Buffer solution (pH 8.0) be reaction medium, final concentration 400g/L D-Glucoses, final concentration 20mM Mn²⁺, final concentration 25g/L wet thallus.In 100 DEG C, 150r/min react 8h.1mL reaction solutions are taken every 1h, are centrifuged, with HPLC detection D-Fructoses after 0.22 μm of membrane filtration Concentration.As shown in Figure 2, E.coliBL21 (DE3)/pET28b/ToGI-3 in 2h substrate conversion efficiencies up to 61.3%, higher than protoenzyme With the conversion ratio of other mutant enzymes, while also be document report highest level.

Sequence table

<110>Zhejiang Polytechnical University

<120>A kind of high-temperature resistance glucose isomerase mutant and its application

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Met Tyr Glu Pro Lys Pro Glu His Lys Phe Thr Phe Gly Leu Trp Thr

1 5 10 15

Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg Glu Lys

20 25 30

Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu Gly Val Tyr

35 40 45

Gly Ile Asn Leu His Asp Glu Asp Leu Ile Pro Arg Gly Thr Pro Pro

50 55 60

Ala Glu Arg Asp Arg Ile Val Arg Arg Phe Arg Lys Ala Leu Glu Glu

65 70 75 80

Thr Gly Leu Lys Val Pro Met Val Thr Ala Asn Leu Phe Ser Asp Pro

85 90 95

Ala Phe Lys Asp Gly Ala Phe Thr Ser Pro Asp Pro Trp Val Arg Ala

100 105 110

Tyr Ala Leu Arg Lys Ser Leu Glu Thr Met Asp Leu Gly Ala Glu Leu

115 120 125

Gly Ala Glu Ile Tyr Val Val Trp Pro Gly Arg Glu Gly Ala Glu Val

130 135 140

Glu Ala Thr Gly Lys Ser Arg Arg Val Trp Gly Trp Val Arg Glu Ala

145 150 155 160

Leu Asn Phe Met Ala Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg

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Phe Ala Leu Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe

180 185 190

Ala Thr Val Gly Ser Phe Leu Ala Phe Ile Tyr Thr Leu Asp Gln Pro

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Glu Arg Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly

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Leu Asn Phe Val His Ala Val Ala Gln Val Leu Asp Ala Gly Lys Leu

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Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu Val Asp

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Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe Asp Ala His

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Cys Met Arg Thr Tyr Leu Ile Phe Lys Glu Lys Ala Gln Ala Phe Arg

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Glu Asp Pro Glu Val Arg Ser Leu Leu Glu Glu Tyr Tyr Gly Glu Asp

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Pro Gln Ala Leu Gly Leu Leu Gly Pro Tyr Ser Arg Glu Arg Ala Thr

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Ala Leu Lys Glu Val Ala Leu Pro Leu Glu Ala Lys Arg Arg Arg Gly

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Tyr Ala Leu Glu Arg Leu Asp Gln Leu Val Val Glu His Leu Leu Gly

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ggtcgtgacc cgttcggtga cgctgttcgt gaaaaactgg acccggttta cgttgttcac 120

aaactggctg aactgggtgt ttacggtatc aacctgcacg acgaagacct gatcccgcgt 180

ggtaccccgc cggctgaacg tgaccgtata gttcgtaggt tccgtaaagc tctcgaagaa 240

accggtctga aagttccgat ggttaccgct aacctgttct ctgacccggc gttcaaagac 300

ggtgcgttca cctctccgga cccgtgggtt cgtgcttacg ctctgcgtaa atctctggaa 360

accatggacc tgggtgctga actgggtgct gaaatctacg ttgtttggcc gggtcgtgaa 420

ggtgctgaag ttgaagctac cggtaaatct cgtcgtgttt ggggttgggt tcgtgaagct 480

ctgaacttca tggctgctta cgctgaagac cagggttacg gttaccgttt cgctctggaa 540

ccgaaaccga acgaaccgcg tggtgacatc tacttcgcta ccgttggttc tttcctggct 600

ttcatctaca ccctcgacca gccagaaagg ttcggtctga acccagaatt cgctcacgaa 660

accatggctg gtctgaactt cgttcacgct gttgctcagg ttctggacgc tggtaaactg 720

ttccacatcg acctgaacga ccagcgtatg tctcgtttcg accaggacct gcgtttcggt 780

tctgaaaacc tgaaagctgc tttcttcctg gttgacctgc tggaatcttc tggttaccag 840

ggtccgcgtc acttcgacgc tcacgctctg cgtaccgaag acgaagaagg tgtttgggct 900

ttcgctcgtg gttgcatgcg tacctacctg atcttcaaag aaaaggcgca ggcgttccgt 960

gaagacccag aagttcgttc tctgctggaa gaatactacg gtgaagaccc gcaggctctg 1020

ggtctgctgg gtccgtactc tcgtgaacgt gctaccgctc tgaaagaagt tgctctgccg 1080

ctggaagcta aacgtcgtcg tggttacgct ctggaacgtc tggaccagct ggttgttgaa 1140

cacctgctgg gtgttcgtgg tcaccaccac caccaccac 1179

Claims

1. a kind of high-temperature resistance glucose isomerase mutant, it is characterised in that the mutant is by ammonia shown in SEQ ID NO.1 The 89th of base acid sequence, the 136th, one or more of the 352nd carries out single mutation or multimutation obtains.

2. high-temperature resistance glucose isomerase mutant as described in claim 1, it is characterised in that the mutant be by the 89th, 136th, the amino acid mutation of the 352nd be one kind in alanine, serine, glutamine, methionine or leucine.

3. high-temperature resistance glucose isomerase mutant as described in claim 1, it is characterised in that the mutant is one of following： (1) the 89th threonine of amino acid sequence shown in SEQ ID NO.1 is sported into glutamine；(2) by SEQ ID NO.1 institutes The threonine for showing amino acid sequence the 89th sports glutamine, the 136th tryptophan sports serine, while the 352nd Position threonine sports leucine.

4. high-temperature resistance glucose isomerase mutant described in a kind of claim 1 prepares D-Fructose in catalysis D-Glucose isomerization In application.

5. application as claimed in claim 4, it is characterised in that the method for the application is：To contain high-temperature resistance glucose isomerase The wet thallus that the fermented culture of recombinant bacterium of mutant gene obtains is enzyme source, using D-Glucose as substrate, using manganese salt as auxiliary agent, With pH 8.0, the Na of 50mM₂HPO₄/NaH₂PO₄Buffer solution forms reaction system for reaction medium, in 50-110 DEG C, 150r/min Under the conditions of react, when reaction to the end of, reaction solution is isolated and purified, obtain D-Fructose.

6. application as claimed in claim 5, it is characterised in that in the reaction system, initial substrate concentration 50-500g/L, The dosage of wet thallus is 10-50g/L, the final concentration of 5-25mM of manganese salt.

7. application as claimed in claim 5, it is characterised in that the reaction temperature is 100-110 DEG C.

8. application as claimed in claim 5, it is characterised in that the wet thallus is prepared as follows：High temperature resistant grape will be contained The engineering bacteria of sugared isomerase mutant gene is seeded to the LB fluid nutrient mediums containing 50 μ g/mL kanamycins, in 37 DEG C, 150r/ Min cultivates 10h, obtains seed liquor；Seed liquor with 2% inoculum concentration of volumetric concentration is seeded to and fresh contains 50 μ g/ of final concentration In the LB culture mediums of mL kanamycins, in 37 DEG C, 150r/min cultures OD₆₀₀To 0.6-0.8, then addition is dense eventually into culture solution The IPTG for 1mM is spent, 10h, 8000r/min centrifugation 10min are induced in 28 DEG C, abandoning supernatant collects wet thallus；The LB trainings Support base composition：Tryptone 10g/L, dusty yeast 5g/L, NaCl 10g/L, solvent are water, and pH value is natural.