CN104745563A - Glucose isomerase and gene, mutant, engineering bacteria and application thereof - Google Patents
Glucose isomerase and gene, mutant, engineering bacteria and application thereof Download PDFInfo
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Abstract
The invention discloses a glucose isomerase, an encoding gene, a recombinant vector, a genetically engineered bacteria and a mutant thereof and an application of the glucose isomerase and the mutant thereof in preparation of D-fructose for catalyzing D-glucose isomerization. A recombinant escherichia coli capable of efficiently expressing high-temperature resistant glucose isomerase, provided by the invention, solves the problem of low optimal reaction temperature of a common glucose isomerase, is applied to production of the glucose isomerase and has the advantages of high yield, simple process and convenient industrial application; the strain can be directly used for the high fructose syrup production without cell disruption; after finishing fermentation, the total enzyme activity for D-fructose at the temperature of 90 DEG C is 414.3U/g, the conversion rate of D-fructose is 55.4%, the remnant enzyme activity at the temperature of 90 DEG C after storage for 24h is 68% and the equilibrium of reaction is shortened to 1.5h; a good technical support for the large-scale production of the high fructose syrup and for the use of high fructose syrup as an important food additive can be provided.
Description
(1) technical field
The present invention relates to colt Glucose Isomerase Gene, mutant, and malaga sugar isomerase gene engineering bacteria and construction process and application, belong to genetically engineered field.
(2) technical background
Glucose isomerase (Glucose isomerase, be called for short GI, EC 5.3.1.5), also known as xylose isomerase (Xyloseisomerase, XI), can the isomerization reaction of catalysis D-Glucose and D-wood sugar, be separately converted to D-Fructose and D-xylulose, be one of important biological catalyst of field of food industry, especially prepare in bio-transformation in the technique of high fructose syrup and there is crucial effect.
High fructose syrup (HFCS) is glucose and fructose mixture, also claims high fructose syrup, is a kind of important sweeting agent.According to the content of fructose, high fructose syrup can be divided into three types, HFCS-42 (fructose 42%, glucose 53%, saccharan 5%), HFCS-55 (fructose 55%, glucose 42%, saccharan 3%) and HFCS-90 (fructose 90%, glucose 9%, saccharan 1%).HFCS-90 is mainly separated by HFCS-42, concentrated, purifying is obtained, directly uses less as additive, is generally used for preparation HFCS-55.HFCS-55 is mixed by HFCS-90 and HFCS-42.Be applied to field of food since the sixties in last century as a kind of sweeting agent, the market requirement of high fructose syrup increases year by year, and the method exploring its suitability for industrialized production also emerges in an endless stream.The method preparing high fructose syrup at present mainly contains chemical method, fermentation method and enzyme process.The HFCS-42 that wherein using glucose isomerase biotransformation method is produced is the main source of high fructose syrup in the market.
The isomerization of glucose isomerase enzyme catalysis D-Glucose is a thermodynamic(al)equilibrium reaction.Glucose isomerase (first kind GI) is generally reducing higher than catalytic activity when about 60 DEG C, and transformation efficiency is up to 42 ~ 45%, therefore, can only be used for obtained HFCS-42.If resistant to elevated temperatures glucose isomerase (Equations of The Second Kind GI) can be screened, temperature of reaction can be increased to more than 85 DEG C, promote that D-Glucose is to the conversion of D-Fructose, thus improve more than transformation efficiency to 55%, therefore can direct production HFCS-55.
There is the report of some high-temperature resistance glucose isomerases at present, such as come from Apollo to dwell the glucose isomerase of thermobacillus Thermotoga neapolitana DSM 5068 and Thermotoga maritima Thermotoga maritime DSM 3109, its optimal reactive temperature reaches 97 DEG C and 105 DEG C respectively.But some resistant to elevated temperatures glucose isomerase acid-fast ability is more weak, catalysis coloring matter psicose can be generated in the basic conditions.The glucose isomerase commercially produced at present is difficult to the demand meeting HFCS-55 production.Therefore filter out the resistant to elevated temperatures colt Glucose Isomerase Gene of novel expression, and had great importance in the preparation of HFCS-55 by the genetic engineering bacterium of genetic engineering technique structure high expression level.
(3) summary of the invention
The object of the invention is to provide a kind of glucose isomerase, encoding gene, recombinant vectors, genetic engineering bacterium and mutant thereof, and colt Glucose Isomerase Gene engineering bacteria prepares the application in D-Fructose in the isomerization of catalysis D-Glucose, high-temperature resistance glucose isomerase by recombination bacillus coli high expression provided by the invention, solve the problem that general glucose isomerase optimal reactive temperature is low, the transformation efficiency of D-Glucose can be made to reach 55.4% through the improved glucose isomerase mutant of sudden change simultaneously, for the scale operation of high fructose syrup (HFCS-55) provides good technical support.
The technical solution used in the present invention is:
The present invention relates to a kind of glucose isomerase, the aminoacid sequence of described enzyme is for shown in SEQ ID NO.2.
The present invention relates to a kind of glucose isomerase enzyme coding gene, the nucleotide sequence of described encoding gene is as shown in SEQ IDNO.1.
The invention still further relates to a kind of recombinant vectors containing described colt Glucose Isomerase Gene, by the recombination engineering bacteria of described construction of recombinant vector.
The present invention also provides a kind of described glucose isomerase enzyme coding gene preparing the application in glucose isomerase, described is applied as: build the recombinant vectors containing described colt Glucose Isomerase Gene, described recombinant vectors is converted in intestinal bacteria, the recombination engineering bacteria obtained carries out inducing culture, gets nutrient solution and is separated the somatic cells obtained containing glucose isomerase.
The present invention relates to a kind of described glucose isomerase and prepare application in D-Fructose in the isomerization of catalysis D-Glucose, described is applied as: the wet thallus obtained through fermentation culture with the recombinant bacterial strain containing colt Glucose Isomerase Gene is for enzyme source, take D-Glucose as substrate, with magnesium salts and cobalt salt for auxiliary agent, take deionized water as reaction medium, at 55 ~ 100 DEG C (preferably 80 ~ 95 DEG C, most preferably 90 DEG C), react under 150r/min condition, after reacting completely, by reaction solution separation and purification, obtain D-Fructose; Described initial substrate concentration is 50 ~ 500g/L deionized water, and the quality consumption of wet thallus is 20g/L deionized water, and described magnesium salts final concentration is 5 ~ 25mmol/L deionized water, and cobalt salt final concentration is 0.1 ~ 5mmol/L deionized water.Further, preferred described initial substrate concentration is 100g/L deionized water, and the quality consumption of wet thallus is 20g/L deionized water, and described magnesium salts and cobalt salt amount of substance final concentration in reaction system is respectively 15mmol/L deionized water and 1mmol/L deionized water.
The preparation method that glucose isomerase of the present invention prepares enzyme source used in D-Fructose in the isomerization of catalysis D-Glucose is: will be seeded to the LB liquid nutrient medium containing final concentration 40 μ g/mL kantlex containing the recombination engineering bacteria of glucose isomerase gene, 37 DEG C, 150r/min is cultured to OD
600=0.8 ~ 1.0, obtain seed liquor; Seed liquor is proceeded in the LB liquid nutrient medium containing final concentration 40 μ g/mL kantlex with the inoculum size of volumetric concentration 2%, in 37 DEG C, cultivate OD under 150r/min condition
600=0.4 ~ 0.8, add final concentration 0.5mM IPTG, 28 DEG C, 150r/min inducing culture 10h, obtain induction broth, induction broth is centrifugal, collect wet thallus, be enzyme source.
In addition, the present invention also provides a kind of glucose isomerase mutant, described mutant 139 of aminoacid sequence shown in SEQ ID NO.2 and/or 186 amino acids is carried out single mutation or two sudden change to obtain, concrete preferred described mutant is for one of following: the tryptophane of aminoacid sequence shown in SEQ ID NO.2 the 139th is sported phenylalanine by (1), aminoacid sequence is for shown in SEQ ID NO.7, and nucleotides sequence is classified as shown in SEQ ID NO.3; (2) be Serine by the valine mutation of aminoacid sequence shown in SEQ ID NO.2 the 186th, aminoacid sequence is for shown in SEQ ID NO.8, and nucleotides sequence is classified as shown in SEQ ID NO.4; (3) be Threonine by the valine mutation of aminoacid sequence shown in SEQ ID NO.2 the 186th, aminoacid sequence is for shown in SEQ ID NO.9, and nucleotides sequence is classified as shown in SEQ ID NO.5; (4) tryptophane of aminoacid sequence shown in SEQ ID NO.2 the 139th is sported phenylalanine, be Threonine by the valine mutation of aminoacid sequence shown in SEQ ID NO.2 the 186th simultaneously, aminoacid sequence is for shown in SEQ ID NO.10, and nucleotides sequence is classified as shown in SEQ ID NO.6.
Due to the singularity of nucleotide sequence, any SEQ ID NO.1, SEQ ID NO.3; SEQ ID NO.4, SEQ ID NO.5, the variant of polynucleotide shown in SEQ ID NO.6; as long as itself and this polynucleotide have more than 90% homology, all belong to the row of scope.The variant of described polynucleotide refers to a kind of polynucleotide sequence having one or more Nucleotide and change.The variant of these polynucleotide can make raw allelic variant or the varient of non-life, comprises and replaces varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of a multiple Nucleotide, disappearance or insertion, but can not from the amino acid whose function changing in fact its coding.
Due to the singularity of aminoacid sequence; any containing SEQ ID NO.2; SEQ ID NO.7; SEQ ID NO.8; the fragment of the polypeptide of aminoacid sequence shown in SEQ ID NO.9, SEQ ID NO.10 or its variant, as its examples of conservative variations, bioactive fragment or derivative; as long as the fragment of this polypeptide or polypeptide variants and aforementioned amino acid sequences homology, more than 95%, all belong to the row of scope.Concrete, described change can comprise amino acid whose disappearance, insertion or replacement in aminoacid sequence; Wherein, the conservative property for variant changes, and the amino acid replaced has the structure similar to original acid or chemical property, and as replaced Isoleucine with leucine, variant also can have non-conservation and change, as replaced glycine with tryptophane.
The present invention relates to described glucose isomerase mutant encoding gene, the recombinant vectors that encoding gene builds and recombinant vectors transform the recombination engineering bacteria of preparation.
The present invention relates to a kind of described glucose isomerase mutant and prepare application in D-Fructose in the isomerization of catalysis D-Glucose, described is applied as: the wet thallus obtained through fermentation culture with the recombination engineering bacteria containing glucose isomerase mutant encoding gene is for enzyme source, take D-Glucose as substrate, with magnesium salts and cobalt salt for auxiliary agent, take deionized water as reaction medium, react under 50-100 DEG C (preferably 90 DEG C), 150r/min condition, after reacting completely, by reaction solution separation and purification, obtain D-Fructose; Described magnesium salts final concentration is 5 ~ 25mmol/L deionized water (preferred 15mmol/L), and cobalt salt final concentration is 0.1 ~ 5mmol/L deionized water (preferred 2mmol/L).Further, under optimum condition, reaction 5h, transformation efficiency, higher than 55%, reacts and tends to balance at 1.5h.
Further, described glucose isomerase mutant is prepared enzyme source used in D-Fructose in the isomerization of catalysis D-Glucose and is prepared as follows: will be seeded to the LB liquid nutrient medium containing final concentration 40 μ g/mL kantlex containing the recombination engineering bacteria of glucose isomerase mutant encoding gene, 37 DEG C, 150r/min is cultured to OD
600=0.8 ~ 1.0, obtain seed liquor; Seed liquor is proceeded in the LB liquid nutrient medium containing final concentration 40 μ g/mL kantlex with the inoculum size of volumetric concentration 2%, in 37 DEG C, cultivate OD under 150r/min condition
600=0.4 ~ 0.8, add final concentration 0.5mMIPTG, 28 DEG C, 150r/min inducing culture 10h, obtain induction broth, induction broth is centrifugal, collect wet thallus, be enzyme source; Described LB liquid nutrient medium composition: Tryptones 10g/L, yeast powder 5g/L, NaCl10g/L, solvent is water, with 1M NaOH, pH is adjusted to 7.0.
The screening of glucose isomerase mutant of the present invention, expression and enzyme activity determination method are:
(1) screening of recombinant bacterium: with kalamycin resistance gene on described recombinant expression plasmid pET-28b-tegi, if recombinant plasmid has been transformed in intestinal bacteria, recombinant bacterial strain has kalamycin resistance, can at the grow on plates containing 40 μ g/mL kantlex; Picking positive transformant, is glucose isomerase recombinant bacterium BL21 (DE3)/pET-28b-tegi.
(2) structure of glucose isomerase mutant expression strain
With the plasmid of recombinant bacterium BL21 (DE3)/pET-28b-tegi for template, carry out two-wheeled sudden change PCR continuously, 139th tryptophane is sported phenylalanine, be Threonine by the 186th valine mutation, mutant plasmid proceeds in intestinal bacteria after Dpn I enzyme cuts template, recombinant bacterial strain has kalamycin resistance, can at the grow on plates containing 40 μ g/mL kantlex, picking positive transformant checks order, sequence shows successfully suddenly change 139 sites and 186 sites, is glucose isomerase mutant bacterium BL21 (DE3)/pET-28b-mut-tegi.
(3) recombinant mutant bacterium is through culture expression glucose isomerase
LB liquid nutrient medium (g/L) forms: Tryptones 10, yeast powder 5, NaCl 10, and solvent is water, with 1MNaOH, pH is adjusted to 7.0; LB solid medium adds 20g/L agar; Autoclaving; Final concentration 40 μ g/mL kantlex is added before using.
Glucose isomerase mutant bacterium is seeded to the LB liquid nutrient medium of final concentration 40 μ g/mL kantlex, 250mL triangular flask, liquid amount is 50mL, and culture temperature 37 DEG C, shaking speed 150r/min, be cultured to OD
600=0.8 ~ 1.0, obtain seed liquor; Seed liquor is proceeded to the inoculum size of volumetric concentration 2% in the 500mL triangular flask of the fermention medium (LB liquid nutrient medium) that 100mL final concentration 40 μ g/mL kantlex is housed, in 37 DEG C, cultivate 2 ~ 3h (OD under 150r/min condition
600=0.4 ~ 0.8), add final concentration 0.5mM IPTG, 28 DEG C, 150r/min inducing culture 10h, obtain induction broth.
(4) glucose isomerase enzyme is lived and is measured
Get induction broth, 4 DEG C, the centrifugal 10min of 8000r/min, be dissolved in by 0.4g bacterial sediment in 20mL deionized water (pH 6.8), fully mixing suspension thalline, adds final concentration 1mM Co
2+, final concentration 10mM Mg
2+with the D-Glucose of final concentration 100g/L, 90 DEG C, react 1h, ice bath 5min termination reaction under 150rpm condition, the centrifugal 10min of 8000r/min, gets supernatant; HPLC is adopted to detect the concentration of D-Glucose and D-Fructose.Analytical column is Hi-Plex Ca ion exchange column (300 × 7.7mm, the U.S., Agilent), Waters 2414 differential refraction detector, Waters 1525 pump, Waters 717 sampler.Enzyme (U) alive is defined as per minute under this condition and generates enzyme amount needed for 1 μm of ol D-Fructose; Be U/g wet thallus than Mei Huo unit.
Glucose isomerase of the present invention and mutant can both withstand high temperatures 80 ~ 95 DEG C, and when concentration of substrate is 100g/L, temperature of reaction 90 DEG C, the enzyme of glucose isomerase is lived and reached 207.4U/g, and transformation efficiency reaches 52.9%; The enzyme of glucose isomerase mutant is lived and is reached 414.3U/g, and transformation efficiency reaches 55.4%.
The invention has the advantages that the recombination bacillus coli constructing a plant height efficient expression high temperature resistant (80 ~ 95 DEG C) glucose isomerase, solve the problem that general glucose isomerase optimal reactive temperature is low; Have that output is high further across the improved glucose isomerase mutant of sudden change, technique is simple, be convenient to the advantages such as industrial applications, this bacterial strain can be directly used in the production of high fructose syrup, and does not need cytoclasis.Live to the total enzyme of D-Glucose under 90 DEG C of conditions after fermentation ends and reach 414.3U/g, transformation efficiency reaches 55.4%, this be the scale operation of high fructose syrup and its provide good technical support as important foodstuff additive.
(4) accompanying drawing explanation
Fig. 1 is the PCR primer agarose gel electrophoresis figure of tegi.M:DL2000DNA Marker; 1: the tegi gene fragment increased for primer PCR with tegi-NMK-F and tegi-NMK-R; 2: the tegi gene fragment increased for primer PCR with tegi-MA-F and tegi-MA-R.
Fig. 2 is the restriction enzyme digestion and electrophoresis figure of plasmid pP43NMK-tegi (a) and pMA0911-tegi (b).(a): M1:FastDL10000DNA Marker (500 ~ 10000); 1: plasmid pP43NMK-tegi by the fragment after Pst I and Hind III double digestion; 2: plasmid pP43NMK by the fragment after Pst I and Hind III double digestion; M2:DL2000DNAMarker; (b): M1:DL2000DNA Marker; 1: plasmid pMA0911-tegi by the fragment after EcoR I and BamH I double digestion; 2: plasmid pMA0911-tegi by the fragment after EcoR I single endonuclease digestion; M2:FastDL10000DNA Marker (500 ~ 10000).
Fig. 3 is the physical map of recombinant expression plasmid pP43NMK-tegi (a) and pMA0911-tegi (b).
Fig. 4 is the PCR primer agarose gel electrophoresis figure of tegi.M:DL2000DNA Marker; 1: the tegi gene fragment increased for primer PCR with tegi-F and tegi-R.
Fig. 5 is the restriction enzyme digestion and electrophoresis figure of positive recombinant expression plasmid pET-28b-tegi.M1:DL2000DNA Marker; The Nco I of 2:pET-28b-tegi and Xho I double digestion band; M2:Fast DL10000DNA Marker (500 ~ 10000).
Fig. 6 is the physical map of pET-28b-tegi recombinant expression plasmid.
Fig. 7 is rite-directed mutagenesis pcr amplification product agarose gel electrophoresis figure.M：DL10000DNA Marker；1：pET-28b-tegi；2：pET-28b-tegi-W139F；3：pET-28b-tegi-V186T；4：pET-28b-tegi-V186S；5：pET-28b-tegi-W139F/V186T。
Fig. 8 is glucose isomerase mutant colony PCR amplification product agarose gel electrophoresis figure.M：DL2000DNA Marker；1：tegi；2，3，4：tegi-W139F；5，6，7：tegi-V186S 8，9，10：tegi-W139F/V186T。
Fig. 9 is that recombinant glucose isomerase expresses SDS-PAGE figure.M: molecular weight of albumen Marker; 1:E.coliBL21 (DE3); 2:E.coli BL21 (DE3)/pET-28b (+); 3: E.coliBL21 (the DE3)/pET-28b-tegi do not induced; 4:0.5mM BL21 (the DE3)/pET-28b-tegi of IPTG induction; BL21 (the DE3)/pET-28b-tegi cytoclasis precipitation of 5:0.5mMIPTG induction; BL21 (DE3)/pET-28b-tegi cytoclasis supernatant that 6:0.5mM IPTG induces; 7: the TEGI after ni-sepharose purification.
Figure 10 is the thermostability of glucose isomerase and glucose isomerase mutant.Remnant enzyme activity after glucose isomerase and mutant thereof are incubated 24h at 90 DEG C is 68%, 1.21 times of wild-type glucose isomerase 56%.
Figure 11 is glucose isomerase and glucose isomerase mutant catalysis D-Fructose reaction process.Glucose isomerase mutant makes catalyzed reaction reach balance in 1.5h, and transformation efficiency reaches 55.4%, and wild-type glucose isomerase need react more than 3h.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the gene chemical synthesis of glucose isomerase
The ctg00047 sequence fragment that the gene source of glucose isomerase checks order in the full-length genome air gun of thermophilic ethanol bacillus Thermoanaerobacter ethanolicusCCSD1, wherein from 7706bp to 9022bp, base is the sequence (GenBank No.EEU61835.1, GI 256748793) of encodes glucose isomerase gene.The albumen with His-tag can be expressed after being connected to carrier pET-28b to make this gene, excise its terminator codon, and with the codon preference of B.subtilis 168 for reference carries out sequence optimisation to its sequence and conventional restriction enzyme enzyme recognition site BamHI, Xho I, Pst I, Hind III and Nco I, the sequence of newly-designed colt Glucose Isomerase Gene (tegi) is as shown in SEQ ID NO.1, gene chemical synthesis working delegation Shanghai Xu Guan biotechnology Development Co., Ltd completes, and is connected in cloning vector pES after gene chemical synthesis.
Embodiment 2: the structure of glucose isomerase recombined bacillus subtilis
On the basis of embodiment 1, respectively for different subtilis expression vectors design primer, utilize round pcr, for template, colt Glucose Isomerase Gene (tegi) is increased with the nucleotide sequence synthesized (shown in SEQ ID NO.1).For expression vector pP43NMK, design primer tegi-NMK-F:5 '-AAAA
cTGCAGaTGGAATACTTCAAAAACG-3 ' and tegi-NMK-R:5 '-CCC
aAGCTTtTATTCAGAGAAAAGGTATTGG-3 ', and introduce Pst I and Hind III restriction enzyme site respectively; For expression vector pMA0911, design primer tegi-MA-F:5 '-CCG
gAATTCaTGGAATACTTCAAAAACG-3 ' and tegi-MA-R:5 '-CGC
gGATCCtTATTCAGAGAAAAGGTATTGG-3 ', and introduce EcoR I and BamH I restriction enzyme site respectively.
PCR reaction system (50 μ L) is: 10 × Pfu PCR buffer 5 μ L, dNTP Mixture 8 μ L; Template DNA 1 μ L; The each 2 μ L of upstream and downstream primer; Pfu archaeal dna polymerase 0.5 μ L; Sterilized water 31.5 μ L.The program of PCR reaction is 94 DEG C of denaturation 5min; 30 circulations (72 DEG C extend 120s for 94 DEG C of sex change 30s, 50 DEG C of annealing 30s); 72 DEG C extend 10min.Reclaim pcr amplification product with 0.8% agarose gel electrophoresis with 1% agarose gel electrophoresis checking, result increases the tegi gene fragment (Fig. 1 swimming lane 1 and swimming lane 2) conformed to SEQ ID No.1 sequence size.
Tegi gene after purifying with restriction enzyme Pst I and Hind III difference double digestion and carrier pP43NMK; With restriction enzyme EcoR I and BamH I respectively double digestion purify after tegi gene and carrier pMA0911, respectively with T4DNA ligase enzyme in 16 DEG C of connections of spending the night.Ligation system is (20 μ L): goal gene fragment 12 μ L, carrier DNA 5 μ L, 10 × T4DNA ligase enzyme buffer 2 μ L, T4DNA ligase enzyme 1 μ L.
Connect product conversion escherichia coli jm109 competent cell, coat on the LB flat board containing 40 μ g/mL kantlex, cultivate 12 ~ 18h for 37 DEG C.Picking positive colony, 37 DEG C, 150r/min shaking culture spends the night, and extracts plasmid, cuts qualification (qualification result is shown in Fig. 2) through enzyme, obtain prokaryotic expression plasmid pP43NMK-tegi and pMA0911-tegi of correct structure, its physical map is shown in Fig. 3.By plasmid pP43NMK-tegi and pMA0911-tegi respectively electricity forward in subtilis WB800 competent cell, method for transformation is: get and connect product 5 ~ 10 μ L, join in 100 μ L subtilis WB800 competent cells, after abundant mixing, proceed in the 0.2cm electricity revolving cup of precooling, precooling 10min in ice bath; Take out electric revolving cup, suck the moisture on surface with paper handkerchief, put into sample cell; Be 2.5kv by parameter setting, 25 μ F, 200 Ω, the electric shock time is 4 ~ 5ms; Electric shock, adds 1mL LB substratum immediately, 30 DEG C, 150r/min cultivates 2h; Be coated on the LB flat board containing 40 μ g/mL kantlex, cultivate 12 ~ 24h for 37 DEG C, picking transformant, carries out plasmid extraction and digestion verification, obtains correct recombined bacillus subtilis (B.subtilis) WB800/pP43NMK-tegi and WB800/pMA0911-tegi built.LB substratum consists of: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L, and solvent is water, with 1M NaOH, pH is adjusted to 7.0.
Embodiment 3: the enzyme activity determination of recombined bacillus subtilis
(containing kantlex 40 μ g/mL) in 50mL LB liquid nutrient medium is seeded to respectively, 37 DEG C, 150r/min shaking culture is to OD by the recombinant bacterium B.subtilis WB800/pP43NMK-tegi after digestion verification and sequence verification and B.subtilis WB800/pMA0911-tegi
600=0.8 ~ 1.0; Nutrient solution is inoculated into fresh in the 100mL LB liquid nutrient medium of 40 μ g/mL kantlex with 2% (v/v) inoculum size, 37 DEG C, 150r/min shaking culture 10 ~ 24h.
Get bacterial culture fluid, the centrifugal 5min of 1000r/min, gets bacterial sediment and supernatant liquor respectively, add 20 μ LSDS damping fluid mixing respectively, boiling water bath heating 5min, carries out SDS-PAGE analysis, compare with carrier-free empty host, result does not find obvious glucose isomerase protein band; Accordingly, do not detect that the enzyme of glucose isomerase is lived yet.Show that glucose isomerase fails expression in subtilis, or the glucose isomerase of expressing is easily degraded by proteases.
Embodiment 4: the structure of glucose isomerase recombination bacillus coli
On the basis of embodiment 1, utilize round pcr, with the nucleotide sequence synthesized (shown in SEQ ID NO.1) for template, with tegi-F:5 '-CATG
cCATGGtAGAATACTTCAAAAACG-3 ' and tegi-R:5 '-CCG
cTCGAGtTCAGAGAAAAGGTATTGG-3 ' is primer, increases to colt Glucose Isomerase Gene (tegi), and introduces Nco I and Xho I restriction enzyme site respectively at its 5' end and 3'.PCR reaction system (50 μ L) is: 10 × Pfu PCR buffer 5 μ L, dNTP Mixture 8 μ L; Template DNA 1 μ L; The each 2 μ L of upstream and downstream primer; Pfu archaeal dna polymerase 0.5 μ L; Sterilized water 31.5 μ L.The program of PCR reaction is 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 57 DEG C of annealing 90s, 72 DEG C extend 1.5min (30 circulations); 72 DEG C extend 10min.Reclaim pcr amplification product with 0.8% agarose gel electrophoresis with 1% agarose gel electrophoresis checking, result increases the tegi gene fragment (Fig. 4 swimming lane 1) conformed to SEQ ID No.1 sequence size.
Tegi gene (pcr amplification product namely reclaimed) after purifying with restriction enzyme Nco I and Xho I double digestion and carrier pET-28b (+), to spend the night in 16 DEG C with T4DNA ligase enzyme and connect.Ligation system is (20 μ L): goal gene fragment 12 μ L, carrier DNA 5 μ L, 10 × T4DNA ligase enzyme buffer 2 μ L, T4DNA ligase enzyme 1 μ L.Connect product conversion e. coli bl21 (DE3) competent cell, method for transformation is: get and connect product 5 ~ 10 μ L, join in 100 μ L e. coli bl21 (DE3) competent cells, fully after mixing, and ice bath 30min; The Eppendorf pipe that mixture is housed is placed in 42 DEG C of water-bath heat-shocked 90s, then transfers to cooled on ice 2min immediately; Xiang Guanzhong adds 600 μ L LB liquid nutrient mediums, is placed in 37 DEG C, 150r/min constant-temperature table cultivates 2 ~ 4h, then coat on the LB flat board containing 40 μ g/mL kantlex, cultivates 12 ~ 18h for 37 DEG C.Picking positive colony, 37 DEG C, 150r/min shaking culture spends the night, and extracts plasmid, cuts qualification (qualification result is shown in Fig. 5) through enzyme, obtain the prokaryotic expression plasmid pET-28b-tegi of correct structure, its physical map is shown in Fig. 6.Positive colony containing plasmid pET-28b-tegi is correct recombination bacillus coli BL21 (the DE3)/pET-28b-tegi built, called after TEGI.
Embodiment 5: the structure of glucose isomerase single-site mutant body
Recombination bacillus coli BL21 (the DE3)/pET-28b-tegi built in embodiment 4 is extracted plasmid, using plasmid pET-28b-tegi as template, design mutant primer the W139F-F:5 '-CTAAAACAAAAGTTCTT of three pairs of rite-directed mutagenesises according to parental array
tTCgGCACAGCTAACC-3 ' and W139F-R:5 '-GAGAAAAGGTTAGCTGTGCC
gAAaAGAACTTTTG-3 ', V186T-F:5 '-GAACTTGGCGGCCAAAACTAC
aCTtTCTGGGGCG-3 ' and V186T-R:5 '-CCTTCACGGCCGCCCCAGAA
aGTgTAGTTTTGGC-3 ', V186S-F:5 '-GAACTTGGCGGCCAAAACTAC
tCTtTCTGGGGCG-3 ' and V186S-R:5 '-CCTTCACGGCCGCCCCAGAA
aGAgTAGTTTTGGC-3 ' (underscore is mutating alkali yl) utilizes fast PCR technology to carry out first round sudden change.PCR reaction system is:
gC Buffer 25 μ L, dNTPs (each 2.5mmol/L) 4 μ L, forward primer (10 μMs) 1 μ L, reverse primer (10 μMs) 1 μ L, template DNA 1 μ L,
hS DNA Polymerase (2.5U/ μ L) 0.5 μ L, adds distilled water to 50 μ L.Pcr amplification program is: 98 DEG C of denaturation 3min; Carry out 30 circulations (98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 7min) subsequently; 72 DEG C extend 10min; Last 4 DEG C of insulations.PCR primer is verified through 0.8% agarose gel electrophoresis, and result increases the gene fragment (Fig. 7 swimming lane 2, swimming lane 3 and swimming lane 4) conformed to object carrier size.
PCR primer is after Dpn I enzyme cuts template 3h, transformation of E. coli BL21 (DE3) competent cell (detailed process is shown in embodiment 4), competent cell is after LB solid medium (containing kantlex 40 μ g/mL) overnight incubation, choose mono-clonal and carry out bacterium colony PCR checking (Fig. 8), after the bacterium being verified as positive colony is inoculated in and cultivates 8h in LB liquid nutrient medium (containing kantlex 40 μ g/mL), extract plasmid, serve the order-checking of extra large Sani bio tech ltd, sequencing result display is correct, the single mutant glucose isomerase built is called after TEGI-W139F respectively, TEGI-V186T, TEGI-V186S.
Embodiment 6: the structure of glucose isomerase double-site mutant body
Recombination bacillus coli BL21 (the DE3)/pET-28b-tegi built in embodiment 4 is extracted plasmid, using plasmid pET-28b-tegi as template, according to mutant primer the W139F-F:5 '-CTAAAACAAAAGTTCTT of parental array design rite-directed mutagenesis
tTCgGCACAGCTAACC-3 ' W139F-R:5 '-GAGAAAAGGTTAGCTGTGCC
gAAaAGAACTTTTG-3 ' (underscore is mutating alkali yl) utilizes fast PCR technology to carry out first round sudden change.Carry out second for template take turns sudden change to introduce W139F catastrophe point again, primer is V186T-F:5 '-GAACTTGGCGGCCAAAACTAC
aCTtTCTGGGGCG-3 ' V186T-R:5 '-CCTTCACGGCCGCCCCAGAA
aGTgTAGTTTTGGC-3 '.PCR reaction system is:
gC Buffer 25 μ L, dNTPs (each 2.5mmol/L) 4 μ L, forward primer (10 μMs) 1 μ L, reverse primer (10 μMs) 1 μ L, template DNA 1 μ L,
hS DNA Polymerase (2.5U/ μ L) 0.5 μ L, adds distilled water to 50 μ L.Pcr amplification condition is: 98 DEG C of denaturation 3min; Carry out 30 circulations (98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 7min) subsequently; 72 DEG C extend 10min; Last 4 DEG C of insulations.PCR primer is verified through 0.8% agarose gel electrophoresis, and result increases the gene fragment (Fig. 7 swimming lane 5) conformed to object carrier size.
PCR primer is after Dpn I enzyme cuts template 3h, transformation of E. coli BL21 (DE3) competent cell (detailed process is shown in embodiment 4), competent cell is after LB solid medium (containing kantlex 40 μ g/mL) overnight incubation, choose mono-clonal and carry out bacterium colony PCR checking, after the bacterium being verified as positive colony is inoculated in and cultivates 8h in LB liquid nutrient medium (containing kantlex 40 μ g/mL), extract plasmid, serve the order-checking of extra large Sani bio tech ltd, sequencing result display is correct, the double-mutant glucose isomerase called after TEGI-W139F/V186T built.
Embodiment 7: the expression of recombination bacillus coli
By the wild-type glucose isomerase TEGI inoculation that builds in embodiment 4 to (containing kantlex 40 μ g/mL) in 50mL LB liquid nutrient medium, 37 DEG C, 150r/min shaking culture is to OD
600=0.8 ~ 1.0; Nutrient solution is inoculated into fresh in the 100mL LB liquid nutrient medium of 40 μ g/mL kantlex with 2% (v/v) inoculum size, 37 DEG C, 150r/min shaking culture is to cell concentration OD
600=0.6 ~ 0.8, add final concentration 0.5mM IPTG, 28 DEG C, 150r/min inducing culture 10h.
After inducing culture 10h, get 20 μ L bacterium liquid, add 20 μ L SDS damping fluid mixing, boiling water bath heating 5min, gets 8 μ L and carries out SDS-PAGE electrophoretic analysis, take empty carrier as contrast, obtain the protein band (see Fig. 9) that a molecular weight is about 50kDa.
Embodiment 8: the enzyme activity determination of glucose isomerase and mutant thereof
By the wild-type glucose isomerase TEGI built in embodiment 4, the single mutant glucose isomerase TEGI-W139F built in embodiment 5, TEGI-V186T, TEGI-V186S, the double-mutant glucose isomerase TEGI-W139F/V186T built in embodiment 6 carries out abduction delivering by the method in embodiment 7 respectively, and this recombinant bacterium of Simultaneously test and mutant bacterium live to the enzyme of D-Glucose.
Enzyme activity determination method is: get induction broth, 4 DEG C, the centrifugal 10min of 8000r/min, is dissolved in by 0.4g bacterial sediment in 20mL deionized water (pH 6.8), and fully mixing suspension thalline, adds final concentration 1mM Co
2+, final concentration 10mM Mg
2+with the D-Glucose of final concentration 100g/L, under 90 DEG C of conditions, react 1h, ice bath 5min termination reaction, get the centrifugal 2min of 1mL reaction solution 8000r/min, get supernatant, after 0.22 μm of membrane filtration, detect the concentration of D-Glucose and D-Fructose with HPLC liquid chromatograph.Analytical column is Hi-Plex Ca ion exchange column (300 × 7.7mm, the U.S., Agilent), Waters 2414 differential refraction detector, Waters 1525 pump, Waters 717 sampler.
Enzyme unit definition alive: at 90 DEG C, under pH 6.8 condition, the enzyme amount that in 1min, catalysis D-Glucose isomery turns to required for 1 μm of olD-fructose is defined as 1U.Representing (U/g) with the enzyme activity unit contained by every g wet thallus than enzyme work of glucose isomerase, the results are shown in Table 1.Glucose isomerase mutant TEGI-W139F/V186T vigor is 2 times of wild-type TEGI, reaches 414.3U/g.
Table 1 glucose isomerase and mutant enzyme vigor thereof
Embodiment 9: the kinetic constant of glucose isomerase and mutant thereof measures
By the wild-type glucose isomerase TEGI built in embodiment 4, the single mutant glucose isomerase TEGI-W139F built in embodiment 5, TEGI-V186T, TEGI-V186S, the double-mutant glucose isomerase TEGI-W139F/V186T built in embodiment 6 carries out abduction delivering by the method in embodiment 7 respectively.After inducing culture 10h, 9000r/min, 10min collected by centrifugation thalline, abandons supernatant, after 0.85% (w/v) physiological saline rinse thalline, and similarity condition collected by centrifugation thalline.Get a certain amount of thalline, dissolve thalline, after shaken well in the ratio of 100g thalline/L physiological saline, ultrasonication, 12000r/min, 20min are centrifugal, supernatant is crude enzyme liquid, through ni-sepharose purification, collects and obtains pure enzyme liquid for catalytic substrate glucose assays kinetic constant.Reaction system is as follows: D-Glucose initial substrate concentration is 9 ~ 72g/L deionized water, with magnesium salts and cobalt salt for auxiliary agent (10mmol/L deionized water and 1mmol/L deionized water), take deionized water as reaction medium, 90 DEG C, reaction after 10 minutes under 150r/min condition, by reaction solution separation and purification, go out kinetic constant by the growing amount the Fitting Calculation of D-Fructose, the results are shown in Table 2.Result display double-mutant TEGI-W139F/V186T increases to substrate avidity, and catalytic efficiency is 1.88 times of wild-type.
Table 2 glucose isomerase and mutant enzyme kinetic constant thereof
Embodiment 10: glucose isomerase and mutant catalysis thereof are prepared D-Fructose optimum temperuture and measured
According to the result of embodiment 8 and embodiment 9, with wild-type TEGI in contrast, the condition being prepared by double-mutant TEGI-W139F/V186T catalysis to D-Fructose is explored.By the wild-type glucose isomerase TEGI built in embodiment 4, the double-mutant TEGI-W139F/V186T built in embodiment 6 carries out abduction delivering by the method in embodiment 7, in 4 DEG C, the centrifugal 10min of 9000r/min, collect wet thallus as conversion enzyme, take D-Glucose as substrate, carry out conversion reaction and prepare D-Fructose.Concrete operations are as follows: transform in bottle at 50mL and add 20mL deionized water (pH 6.8), the D-Glucose of 0.4g wet thallus, 2g, final concentration 10mM Mg successively
2+with final concentration 1mM Co
2+, respectively 50 ~ 100 DEG C, react 2h under 150r/min condition, get 1mL reaction solution 8000r/min centrifugal, after 0.22 μm of membrane filtration, detect the concentration of D-Glucose and D-Fructose with HPLC liquid chromatograph.Analytical column is Hi-Plex Ca ion exchange column (300 × 7.7mm, the U.S., Agilent), Waters 2414 differential refraction detector, Waters 1525 pump, Waters 717 sampler.Transformation efficiency defines: amount (m) × 100% of D-Fructose amount (the m)/substrate D-Glucose of generation.The results are shown in Table 3.As shown in Table 3, the glucose isomerase of expression can react under the hot conditions of 80 ~ 100 DEG C, and when temperature of reaction is at 90 DEG C, transformation efficiency is the highest, and wild-type is 50.3%, and double-mutant TEGI-W139F/V186T is 50.5%.
Table 3 differential responses temperature is on the impact of transformation efficiency
Embodiment 11: glucose isomerase and mutant thermal stability determination thereof
By the wild-type glucose isomerase TEGI built in embodiment 4, the double-mutant TEGI-W139F/V186T built in embodiment 6 carries out abduction delivering by the method in embodiment 7, in 4 DEG C, the centrifugal 10min of 9000r/min, collect wet thallus as conversion enzyme, take D-Glucose as substrate, carry out conversion reaction and prepare D-Fructose.Concrete operations are as follows: transform in bottle at 50mL and add different soaking time (0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 9h, 24h) wet thallus, the D-Glucose of 1g, final concentration 10mM Mg at 10mL deionized water (pH 6.8), 0.2g 90 DEG C successively
2+with final concentration 1mM Co
2+, 90 DEG C, react 10min under 150r/min condition, get 1mL reaction solution 8000r/min centrifugal, detect the concentration of D-Glucose and D-Fructose with HPLC liquid chromatograph after 0.22 μm of membrane filtration and calculate remnant enzyme activity, the results are shown in Figure 10.Analytical column is Hi-Plex Ca ion exchange column (300 × 7.7mm, the U.S., Agilent), Waters 2414 differential refraction detector, Waters 1525 pump, Waters 717 sampler.Enzyme unit definition alive: at 90 DEG C, under pH 6.8 condition, the enzyme amount that in 1min, catalysis D-Glucose isomery turns to required for 1 μm of ol D-Fructose is defined as 1U.As shown in Figure 10, after the glucose isomerase mutant of expression is incubated 24h under the hot conditions of 90 DEG C, remnant enzyme activity is 68%, is 1.21 times of wild-type glucose isomerase.
Embodiment 12: divalent-metal ion is on the impact of glucose isomerase and mutant thereof
By the wild-type glucose isomerase TEGI built in embodiment 4, the double-mutant TEGI-W139F/V186T built in embodiment 6 carries out abduction delivering by the method in embodiment 7, in 4 DEG C, the centrifugal 10min of 9000r/min, collect wet thallus as conversion enzyme, take D-Glucose as substrate, carry out conversion reaction and prepare D-Fructose.Concrete operations are as follows: transform in bottle at 50mL and add 10mL deionized water (pH 6.8), the various divalent-metal ion (Mg of the D-Glucose of 0.2g wet thallus, 1g, 10mM successively
2+, Co
2+, Mn
2+, Zn
2+, Ni
2+, Cu
2+, Ca
2+, Ba
2+and Fe
2+), 90 DEG C, react 10min, to add 10mM Mg under 150r/min condition
2+with 1mM Co
2+in contrast, get 1mL reaction solution 8000r/min centrifugal, detect the concentration of D-Glucose and D-Fructose with HPLC liquid chromatograph after 0.22 μm of membrane filtration and calculate relative enzyme and live, the results are shown in Table 4.Analytical column is Hi-Plex Ca ion exchange column (300 × 7.7mm, the U.S., Agilent), Waters 2414 differential refraction detector, Waters 1525 pump, Waters 717 sampler.Enzyme unit definition alive: at 90 DEG C, under pH 6.8 condition, the enzyme amount that in 1min, catalysis D-Glucose isomery turns to required for 1 μm of ol D-Fructose is defined as 1U.As shown in Table 4, Mg
2+, Co
2+live impact comparatively greatly to the enzyme of glucose isomerase and mutant thereof, and wherein, mutant is to Ca
2+tolerance be greater than wild-type glucose isomerase.
The different divalent-metal ion of table 4 is on the impact of glucose isomerase and mutant thereof
ND:the activity is not detectable.
Embodiment 13: the suitableeest Co of D-Fructose is prepared in glucose isomerase and mutant catalysis thereof
2+concentration determination
According to the result of embodiment 12, to Co
2+concentration measures.According to the result of embodiment 10, temperature of reaction is set to 90 DEG C, Co is set
2+final concentration be 0.1 ~ 5mM, other conditions are with embodiment 10.The results are shown in Table 5.From result, Co
2+when concentration is 1 ~ 2mM, the transformation efficiency of wild-type TEGI and double-mutant TEGI-W139F/V186T, all more than 50%, wherein, works as Co
2+when concentration is 1mM, wild-type TEGI transformation efficiency reaches and is up to 52.5%; Work as Co
2+when concentration is 2mM, double-mutant TEGI-W139F/V186T transformation efficiency reaches and is up to 53.0%.
The different Co of table 5
2+concentration is on the impact of transformation efficiency
Embodiment 14: the suitableeest Mg of D-Fructose is prepared in glucose isomerase enzyme catalysis
2+concentration determination
According to the result of embodiment 10 and embodiment 13, temperature of reaction is set to 90 DEG C, Co
2+final concentration be set to 2mM with the optimal concentration of double-mutant TEGI-W139F/V186T, Mg
2+final concentration be 5 ~ 25mM, other conditions are with embodiment 10.The results are shown in Table 6.From result, Mg
2+when concentration is 15mM, the transformation efficiency of wild-type TEGI and double-mutant TEGI-W139F/V186T reaches the highest, and wherein the transformation efficiency of double-mutant TEGI-W139F/V186T reaches more than 55%.
The different Co of table 6
2+concentration is on the impact of transformation efficiency
Embodiment 15: the suitableeest concentration of substrate that D-Fructose is prepared in glucose isomerase and mutant catalysis thereof measures
According to the result of embodiment 10, embodiment 13 and embodiment 14, temperature of reaction is set to 90 DEG C, Co
2+final concentration be 2mM, Mg
2+final concentration be 15mM, the concentration of substrate D-Glucose is respectively 50 ~ 500g/L, and other conditions are with embodiment 10.The results are shown in Table 7.From result, the suitableeest concentration of substrate is 100g/L.
The different D-Glucose concentration of table 7 is on the impact of transformation efficiency
Embodiment 16: D-Fructose is prepared in glucose isomerase and mutant catalysis thereof
According to the result of embodiment 10, embodiment 13, embodiment 14 and embodiment 15, by the wild-type glucose isomerase TEGI built in embodiment 4, the double-mutant TEGI-W139F/V186T built in embodiment 6 carries out abduction delivering by the method in embodiment 7, in 4 DEG C, the centrifugal 10min of 9000r/min, collect wet thallus as conversion enzyme, take D-Glucose as substrate, carry out conversion reaction and prepare D-Fructose.Concrete operations are as follows: transform in bottle at 50mL and add 10mL deionized water (pH 6.8), the D-Glucose of 0.2g wet thallus, 1g, final concentration 15mM Mg successively
2+with final concentration 2mM Co
2+, 90 DEG C, react 5h under 150r/min condition, get 1mL reaction solution 8000r/min centrifugal, detect the concentration of D-Glucose and D-Fructose with HPLC liquid chromatograph after 0.22 μm of membrane filtration and calculate transformation efficiency, the results are shown in Figure 11.Analytical column is Hi-Plex Ca ion exchange column (300 × 7.7mm, the U.S., Agilent), Waters 2414 differential refraction detector, Waters 1525 pump, Waters 717 sampler.As shown in Figure 11, wild-type TEGI needs more than 3h just can reach balance, and glucose isomerase mutant TEGI-W139F/V186T can make catalyzed reaction reach balance in 1.5h, and transformation efficiency, higher than 53%, is up to 55.4%.
From above-mentioned experimental result, the recombination bacillus coli that glucose isomerase mutant TEGI-W139F/V186T gene transformation intestinal bacteria of the present invention obtain is had stronger malaga sugar isomerase ability, can directly to contain the somatic cells of enzyme for carrying out bioconversion reaction in enzyme source.Using glucose isomerase mutant as conversion enzyme, take D-Glucose as substrate, can carry out bioconversion reaction and generate D-Fructose under high temperature (90 DEG C), its transformation efficiency reaches 55.4%, will be beneficial to the scale operation of HFCS-55.
Claims (10)
1. a glucose isomerase mutant, is characterized in that described mutant the 139th of aminoacid sequence shown in SEQ ID NO.2 and/or the 186th amino acids is carried out single mutation or two sudden change to obtain.
2. glucose isomerase mutant as claimed in claim 1, is characterized in that described mutant is for one of following: the tryptophane of aminoacid sequence shown in SEQ ID NO.2 the 139th is sported phenylalanine by (1); (2) be Serine by the valine mutation of aminoacid sequence shown in SEQ ID NO.2 the 186th; (3) be Threonine by the valine mutation of aminoacid sequence shown in SEQ ID NO.2 the 186th; (4) tryptophane of aminoacid sequence shown in SEQ ID NO.2 the 139th is sported phenylalanine, be Threonine by the valine mutation of aminoacid sequence shown in SEQ ID NO.2 the 186th simultaneously.
3. the recombination engineering bacteria built by glucose isomerase mutant described in claim 1 or 2.
4. glucose isomerase mutant described in a claim 1 prepares the application in D-Fructose in the isomerization of catalysis D-Glucose, it is characterized in that described being applied as: the wet thallus obtained through inducing culture with the recombination engineering bacteria containing glucose isomerase mutant encoding gene is for enzyme source, take D-Glucose as substrate, with magnesium salts and cobalt salt for auxiliary agent, take deionized water as reaction medium, 50-100 DEG C, react under 150r/min condition, after reacting completely, by reaction solution separation and purification, obtain D-Fructose; Described wet thallus consumption is 20g/L deionized water, and described magnesium salts final concentration is 5 ~ 25mmol/L deionized water, and cobalt salt final concentration is 0.1 ~ 5mmol/L deionized water, and described initial substrate concentration is 50 ~ 500g/L deionized water.
5. apply as claimed in claim 4, it is characterized in that the preparation method in described enzyme source is: the LB liquid nutrient medium containing final concentration 40 μ g/mL kantlex will be seeded to containing the recombination engineering bacteria of glucose isomerase mutant encoding gene, 37 DEG C, 150r/min is cultured to OD
600=0.8 ~ 1.0, obtain seed liquor; Seed liquor is proceeded in the LB liquid nutrient medium containing final concentration 40 μ g/mL kantlex with the inoculum size of volumetric concentration 2%, in 37 DEG C, cultivate OD under 150r/min condition
600=0.4 ~ 0.8, add final concentration 0.5mM IPTG, 28 DEG C, 150r/min inducing culture 10h, obtain induction broth, induction broth is centrifugal, collect wet thallus, be enzyme source.
6., for building a glucose isomerase for glucose isomerase mutant described in claim 1, it is characterized in that the aminoacid sequence of described glucose isomerase is for shown in SEQ ID NO.2.
7. to encode the gene of glucose isomerase described in claim 6, it is characterized in that the nucleotides sequence of described gene is classified as shown in SEQ ID NO.1.
8. the recombination engineering bacteria built by glucose isomerase described in claim 6.
9. glucose isomerase described in a claim 6 prepares the application in D-Fructose in the isomerization of catalysis D-Glucose, it is characterized in that described being applied as: the wet thallus obtained through fermentation culture with the recombination engineering bacteria containing colt Glucose Isomerase Gene is for enzyme source, take D-Glucose as substrate, with magnesium salts and cobalt salt for auxiliary agent, take deionized water as reaction medium, 50-100 DEG C, react, after reacting completely under 150r/min condition, by reaction solution separation and purification, obtain D-Fructose; Described wet thallus consumption is 20g/L deionized water, and described magnesium salts final concentration is 5 ~ 25mmol/L deionized water, and cobalt salt final concentration is 0.1 ~ 5mmol/L deionized water, and described initial substrate concentration is 50 ~ 500g/L deionized water.
10. apply as claimed in claim 8, it is characterized in that the preparation method in described enzyme source is: the LB liquid nutrient medium containing final concentration 40 μ g/mL kantlex will be seeded to containing the recombination engineering bacteria of glucose isomerase gene, 37 DEG C, 150r/min is cultured to OD
600=0.8 ~ 1.0, obtain seed liquor; Seed liquor is proceeded in the LB liquid nutrient medium containing final concentration 40 μ g/mL kantlex with the inoculum size of volumetric concentration 2%, in 37 DEG C, cultivate OD under 150r/min condition
600=0.4 ~ 0.8, add final concentration 0.5mM IPTG, 28 DEG C, 150r/min inducing culture 10h, obtain induction broth, induction broth is centrifugal, collect wet thallus, be enzyme source.
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| CN111440785A (en) * | 2020-02-28 | 2020-07-24 | 浙江工业大学 | Method for immobilizing glucose isomerase-containing cells by using modified diatomite |
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| CN113481257A (en) * | 2021-07-09 | 2021-10-08 | 东北师范大学 | Application of novel recombinase in synthesis of fructose derivative containing alpha- (1,4) glycosidic bond |
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| CN108034649A (en) * | 2018-01-04 | 2018-05-15 | 浙江工业大学 | A kind of glucose isomerase mutant and its application |
| CN108034649B (en) * | 2018-01-04 | 2020-08-21 | 浙江工业大学 | Glucose isomerase mutant and application thereof |
| CN111440785A (en) * | 2020-02-28 | 2020-07-24 | 浙江工业大学 | Method for immobilizing glucose isomerase-containing cells by using modified diatomite |
| CN111440785B (en) * | 2020-02-28 | 2022-07-15 | 浙江工业大学 | Method for immobilizing glucose isomerase-containing cells by using modified diatomite |
| CN113151240A (en) * | 2021-05-21 | 2021-07-23 | 浙江工业大学 | Glucose isomerase, mutant, coding gene and application thereof |
| CN113481257A (en) * | 2021-07-09 | 2021-10-08 | 东北师范大学 | Application of novel recombinase in synthesis of fructose derivative containing alpha- (1,4) glycosidic bond |
| CN113481257B (en) * | 2021-07-09 | 2022-11-29 | 东北师范大学 | Application of recombinase in synthesis of fructose derivative containing alpha- (1, 4) glycosidic bond |
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