CN103194434A - Novel sulfolobus solfataricus trehalose hydrolase, gene of hydrolase, recombinant expression vector containing gene, and recombinant bacterium, and preparation of hydrolase - Google Patents

Abstract

The invention discloses sulfolobus solfataricus trehalose hydrolase, a gene of the hydrolase, a recombinant expression vector containing the gene, and a recombinant bacterium, and preparation of t hydrolase. The hydrolase has an amino acid sequence shown in SEQ ID NO:3, and can be obtained from the gene including the amino acid sequence shown in SEQ ID NO:4 or a mutant of the gene or a complementary sequence thereof by expression. The hydrolase MTsase disclosed by the invention is high in optimal reaction temperature and thermal stability and low in optimal pH, reduces the contamination risk, improves the production stability, and obviously improves the efficiency for producing trehalose from reducing starch hydrolysate by joint action with pullulanase. The gene of expressing the hydrolase is obtained by the method; the MTSase can be produced by a gene recombination technology; the enzyme expression quantity is high; the preparation efficiency is obviously improved; the cost is obviously reduced; and the production cost of the trehalose is also reduced.

Description

The preparation of novel malt oligosaccharide based mycose lytic enzyme, enzyme gene, the recombinant expression vector that contains this gene and recombinant bacterial strain and enzyme

Technical field

The invention belongs to genetically engineered and enzyme engineering field, be specifically related to malt oligosaccharide based mycose lytic enzyme and expressing gene thereof, contain recombinant expression vector and the recombinant bacterial strain of this gene, and utilize the recombinant bacterial strain that contains this gene to prepare the method for this enzyme.

Background technology

Trehalose (Trehalose) be a kind of by two glucose molecules by the hemiacetal hydroxyl with the nonreducing sugar of α-1,1 glycosidic link combination, molecular formula is C ₁₂H ₂₂O ₁₁, relative molecular weight is 378.33.Its structural formula is as shown in Figure 1:

The molecular structure of Fig. 1 trehalose

Trehalose extensively is present in bacterium, yeast, fungi, algae, insect and the plant.It has nonspecific provide protection to organism and biomacromolecule.This non-specific provide protection is mainly reflected in, and when biomass cells was in that hunger, drying, high temperature, cryogenic freezing, radiation, high osmotic pressure and toxic reagent etc. are various coerces environment, intracellular trehalose content rose rapidly, thus protection life itself.

Ectogenic trehalose has nonspecific provide protection to organism and biomacromolecule equally, and this unique biological character of trehalose makes it be widely used in fields such as medicine, food, makeup.

The production technique of trehalose is divided into three kinds at present:

(1) extraction method

Yeast is (hunger, high temperature, height ooze, high pressure) growth under rugged environment, can accumulate a large amount of trehaloses in the yeast body, accounts for 15% of dry cell weight greatly.Therefore the method for extracting in the yeast body is adopted in the production of initial trehalose.In yeast cell, because trehalose belongs to intracellular product, syntheticly be subjected to the influence of external environmental condition bigger, and the separation and Extraction difficulty, thus lower with the output of this method production trehalose, and the fermentation level that commercialization is produced is 0.4-0.6%, cost is higher, and this kind production method trehalose is expensive.

(2) single enzyme process

Nineteen ninety-five, the former biochemical institute of Japanese woods from Pimelobacter, PsceuclomonusWith ThermusIn three microorganism belonging to genus separation and purification a kind of amylase-trehalose synthase, it can transform maltose is trehalose, and has applied for patent CN1106065A.After this China R﹠D institution has also found trehalose synthase in succession and has carried out gene engineering expression from different bacteriums, for example CN200410013007.3, CN200910114318.1, CN200910007259.8, CN201010614814.6, CN201010614832.4, CN201210160403.3, CN201210011457.3, wherein the Thermus trehalose synthase has 80 ℃ thermostability.Along with temperature of reaction raises, trehalose synthase transforms maltose and produces the transformation efficiency of trehalose and can descend, and 40 ℃ of maltose can reach 80%, 60 ℃ of transformation efficiency to the transformation efficiency of trehalose and be about 60%.Also do not utilize at present the report of this technology scale operation trehalose.

(3) double-enzyme method

The former biochemical institute of woods had found novel alga sugar synthetic enzyme-malt oligosaccharide based mycose synthetase (malt ooligosyl tehalose synthase in 1994, MTSase) and lytic enzyme (maltooligosyl trehalose hydrolase, MTHase), two enzymes cooperate with starch hydrolyzates and produce trehalose, at first MTSase acts on starch hydrolyzates, the terminal trehalose unit that forms of starch hydrolyzates, MTHase cuts away terminal trehalose then, forms the starch hydrolyzates of a part trehalose and few two glucosyl groups.

Be raw material with the reductibility starch hydrolyzates at present, it is most economical operational path that trehalose is produced in two enzymatic conversions, still has following problem:

(1) patent CN99123896.6 provides MTSase and the MTHase of 50 ℃ of optimum temperutures and optimal pH 6.0, and transformation efficiency can reach 80%, and the former biochemical institute of present Japanese woods has utilized this technology to realize scale operation.50 ℃ of two easy microbiological contaminations of enzymatic conversion, starch hydrolyzates needs Pullulanase to take off a processing in addition.Commodity Pullulanase optimum temperuture is 55-60 ℃ at present, optimal pH 5.0-5.5, and vigor is lower during pH6.0, and efficient is lower when causing Pullulanase and the common converted starch of two enzymes to produce trehalose, and transformation time reaches more than 48 h.

(2) document Production of Trehalose from Starch by Thermostable Enzymes from Sulfolo bus acidocaldarius(DOI:10.1002/star.19970490107) reported that the sulfolobus acidocaldarius can produce MTSase and the MTHase of 75 ℃ of optimum temperutures and optimal pH about 5.5.Though this pair enzyme has higher optimal reactive temperature and lower optimal pH, this pair of bibliographical information enzyme activity is all very low.

Summary of the invention

The invention provides a kind of malt oligosaccharide based mycose lytic enzyme and (be called for short MTHase, down together), this enzyme has higher optimal reactive temperature and thermostability, improved the stability of producing trehalose, have identical optimal pH with Pullulanase, significantly improved the efficient of itself and Pullulanase combination producing trehalose.

The present invention also provides a kind of gene of this lytic enzyme, the recombinant expression vector that contains this gene and recombinant bacterial strain of expressing, and this gene expression amount height significantly reduces the enzyme cost, has also further reduced the cost of preparation trehalose.

The present invention also provides the method for a kind of MTHase of preparation, efficiently expresses by gene recombination technology and obtains MTHase, greatly reduces the cost of MTHase.

In order to obtain the good MTSase of characteristic and MTHase, the contriver extensively screens the microorganism with MTSase and MTHase activity from soil and in the existing bacterium storehouse.Through a large amount of screening operations, the contriver filter out a strain oxidation Arthrobacter ( Arthrobacter oxydans) TL-3, it has MTSase and MTHase activity.Identify that through separation and purification MTSase and MTHase have 60 ℃ of optimal reactive temperatures and optimal pH 5.3-5.5, comparing with MTHase with existing MTSase is novel MTSase and the MTHase that has more the industrial applications prospect.The contriver continues research, by gene clone technology obtained the to encode Nucleotide of this enzyme, has further obtained corresponding aminoacid sequence.The contriver in the DNA importing escherichia coli host with coding MTSase and MTHase, has obtained to efficiently express the method for MTSase and MTHase by gene recombination technology.

The concrete technical scheme of the present invention is as follows:

A kind of malt oligosaccharide based mycose lytic enzyme (being called for short MTHase, down together) is characterized in that it has the aminoacid sequence shown in the SEQ ID NO:3.

MT reconnaissance Hase has following characteristic:

(1) effect

Specific effect obtains a part trehalose and the non-reducing sugar that has lacked two glucosyl groups in the non-reducing sugar of trehalose as terminal units;

(2) molecular weight

Be about 65000Da according to sodium lauryl sulphate-polyacrylamide gel electrophoresis determining molecular weight;

(3) optimum temperature

As pH5.3 incubation 60 min, 60 ℃ of optimum temperatures;

(4) the suitableeest action pH

As 60 ℃ of incubation 60 min, optimal pH is 5.3;

(5) thermostability

When bathing 60 min in the pH5.3 temperature, stable down up to 70 ℃;

(6) pH stability

When bathing 60 min in 60 ℃ of temperature, stable under pH4.5-6.5.

A kind of gene of expressing MT reconnaissance Hase, this gene has the nucleotide sequence shown in the SEQ ID NO:4, perhaps contain by the nucleotide sequence shown in the SEQ ID NO:4 and replace the formed nucleotide sequence of one or more bases because of the merger of genetic codon but this mutant nucleotide sequence does not change by the coded aminoacid sequence of Nucleotide shown in the SEQ ID NO:4, perhaps contain the complementary nucleotide sequence of above-mentioned two kinds of nucleotide sequences.MTHase of the present invention can the efficient acquisition by expressing this gene.

The present invention has also obtained containing the recombinant expression vector of MTHase expressing gene; Described recombinant expression vector is preferably made up by expression vector pET24a (+) and MTHase expressing gene and forms.

The present invention has also obtained containing the recombinant bacterial strain of MTHase expressing gene.Described recombinant bacterial strain is changed over to make up in the intestinal bacteria by the recombinant expression vector that contains the MTHase expressing gene and forms; Described intestinal bacteria are preferably e. coli bl21.

Prepare the method for malt oligosaccharide based mycose lytic enzyme, it is characterized in that: the recombinant bacterial strain that contains the MTHase expressing gene by cultivation makes.

Aforesaid method can efficiently obtain MTHase, its step comprises: with recombinant bacterial strain in the LB liquid nutrient medium that contains 0.10-0.30 mMol/L kantlex 34-37 ℃ cultivate 13-16 h after, be cooled to 27-30 ℃, add final concentration and be the lactose of the IPTG of 0.1-0.3 mMol/L or 0.3-0.5 mMol/L as inductor, at 27-30 ℃ of following inducing culture 12-18 h, somatic cells is collected in centrifugal or filtration then; With somatic cells fragmentation, centrifugal collection supernatant liquor, must contain the crude enzyme liquid of MTHase.

From the oxidation Arthrobacter ( Arthrobacter oxydans) the another kind of enzyme that obtains of TL-3 screening---malt oligosaccharide based mycose synthetase (be called for short MTSase, down with) has following characteristic:

(1) effect

Can act on glucose polymerization degree greater than 3 reductibility starch hydrolyzates, form trehalose as the non-reducing sugar of terminal units;

(2) molecular weight

Be about 85000Da according to sodium lauryl sulphate-polyacrylamide gel electrophoresis determining molecular weight;

(3) optimum temperature

As pH5.5 incubation 60 min, 60 ℃ of optimum temperatures;

(4) the suitableeest action pH

As 60 ℃ of incubation 60 min, optimal pH is 5.5;

(5) thermostability

When bathing 60 min in the pH5.5 temperature, stable down up to 65 ℃;

(6) pH stability

When bathing 60 min in 60 ℃ of temperature, stable under pH4.8-6.3;

From the characteristic of MT reconnaissance Sase as can be seen, MTSase also has and the higher optimal reactive temperature of MTHase basically identical and lower optimal pH, also can improve the stability of producing trehalose.MTSase has the aminoacid sequence shown in the SEQ ID NO:1, can have the gene of the nucleotide sequence shown in the SEQ ID NO:2 and efficiently acquisition by expression.

The invention discloses the gene of a kind of novel MTHase, expression this kind of enzyme, contain the gene of this kind of enzyme recombinant expression vector and recombinant bacterial strain, prepare the method for this kind of enzyme, on the basis of the gene of having known enzyme and nucleotide sequence, can obtain the nucleotide sequence of gene and the aminoacid sequence of enzyme, for example pcr amplification technology by prior art.In addition, related vector, host cell, restriction endonuclease, reagent etc. all are disclosed contents in the prior art, and those skilled in the art can obtain easily.

Technical scheme of the present invention compared with prior art has the following advantages:

(1) novel MTHase has higher optimal reactive temperature and thermostability among the present invention, have optimal reactive temperature and 70 ℃ of thermostabilitys of 60 ℃, two enzyme converted starch hydrolyzates are produced in the trehalose process, are difficult for microbiological contamination, reduce the microbiological contamination risk, improved the stability that trehalose is produced.

(2) novel MTHase has lower optimal pH among the present invention, react at acidic conditions pH5.3 left and right sides optimum, has identical optimum pH with Pullulanase, be more suitable for and Pullulanase synergy starch hydrolyzates is produced trehalose, significantly improved the efficient that itself and Pullulanase combined action reductibility starch hydrolyzates are produced trehalose.

(3) the present invention obtains expressing the gene of MTHase, adopt gene recombination technology to make up genetic engineering bacterium and produce MTHase, yeast culture is simple, MTHase expression amount height, the enzyme preparation efficiency significantly improves, the enzyme cost significantly reduces, and has reduced the production cost of trehalose, for trehalose has been established solid cost basis in the widespread use in fields such as food, medicine, makeup.

Description of drawings

Content of the present invention is easier clearly to be understood in order to make, and below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein:

Fig. 1 displays temperature is to the influence of malt oligosaccharide based mycose synthetase activity.

Fig. 2 shows the influence of the malt oligosaccharide based mycose synthetase activity of pH.

Fig. 3 displays temperature is to the influence of malt oligosaccharide based mycose synthetase stability.

Fig. 4 shows the influence of the malt oligosaccharide based mycose synthetase stability of pH.

Fig. 5 is malt oligosaccharide based mycose synthetase recombinant plasmid restriction map, and slightly black solid line shows it is the nucleotide sequence of coding malt oligosaccharide based mycose synthetase.

Fig. 6 displays temperature is to the influence of malt oligosaccharide based mycose hydrolytic enzyme activities.

Fig. 7 shows the influence of the malt oligosaccharide based mycose hydrolytic enzyme activities of pH.

Fig. 8 displays temperature is to the influence of malt oligosaccharide based mycose lytic enzyme stability.

Fig. 9 shows the influence of the malt oligosaccharide based mycose lytic enzyme of pH stability.

Figure 10 is malt oligosaccharide based mycose lytic enzyme recombinant plasmid restriction map, and slightly black solid line shows it is the nucleotide sequence of coding malt oligosaccharide based mycose lytic enzyme.

Embodiment

According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand that the described concrete processing condition of embodiment, material proportion and result thereof only are used for explanation the present invention, and should also can not limit the present invention described in claims.

No specified otherwise, enzyme biopsy survey and enzyme unit definition alive is as follows in following examples:

(1) malt oligosaccharide based mycose synthetase (MTSase) enzyme activity determination and definition

Maltopentaose is dissolved in the citrate buffer solution of 100 mMol/L pH 5.5, is made into 20% solution.Get this solution of 100 mL, add 1 mL MTSase enzyme liquid, 60 ℃ of reaction 10 min boil 10 min termination reactions in 100 ℃ of boiling water.After the conversion fluid cooling, regulate pH to 4.2, add 0.1 mL glucase (Novozymes Company's product), 60 ℃ of saccharification 24 h, the content of trehalose in the conventional liquid chromatogram measuring saccharification liquid.MTSase catalysis maltopentaose generates maltose pentasaccharides base trehalose, and saccharifying enzyme can hydrolyzing alpha-1, the 4-glycosidic link, and can not hydrolyzing alpha-1, the 1-glycosidic link is so only contain trehalose and glucose in the solution after the saccharification.Therefore, the mole growing amount of final trehalose namely equals the amount of the maltopentaose base trehalose mole of MTSase catalysis generation.The enzyme of MTSase unit alive (U) is defined as per 1 min conversion maltopentaose and generates the required enzyme amount of 1 mMol maltopentaose base trehalose.

(2) malt oligosaccharide based mycose lytic enzyme (MTHase) enzyme activity determination and definition

Maltopentaose is dissolved in the citrate buffer solution of 100 mMol/L pH 5.5, is made into 20% solution.Get this solution of 100 mL, add 200 U MTSase enzyme liquid, 60 ℃ of reaction 5 h boil 10 min termination reactions in 100 ℃ of boiling water.After treating the solution cooling, regulate pH 5.3, add 1 mLMTHase enzyme liquid, 60 ℃ of reaction 10 min boil 10 min termination reactions in 100 ℃ of boiling water.HPLC measures the content of trehalose in the conversion fluid.The enzyme of MTHase unit alive (U) is defined as per 1 min hydrolysis maltopentaose base trehalose and generates the required enzyme amount of 1 mMol trehalose.

Substratum is formed (W/V):1% peptone, 0.5% yeast extract paste, 1% NaCl, pH7.0 ± 0.2.

Substratum is formed (W/V):2% peptone, 0.5% yeast extract paste, 0.05% NaCl, 0.0186% KCl, 0.095% MgCl ₂, 0.36% glucose, pH7.0 ± 0.2.

The used technology of the present invention, for example pcr amplification technology, design of primers technology, vector construction technology, engineering bacteria constructing technology, detection technique, electrophoretic technique etc. are the technology of comparative maturity in the genetically engineered, and those skilled in the art can be according to existing techniques in realizing.Used equipment or reagent, carrier, enzyme etc. if no special instructions, all can obtain in market in operating process.

The screening of malt oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose lytic enzyme (MTHase)

Embodiment 1 yeast culture

Through a large amount of screenings, select oxidation Arthrobacter (Arthrobacter oxydans) TL-3 with MTSase and MTHase excellent activity, this bacterial strain is stored in the US mode bacterial classification and collects center (ATCC), deposit number: 14358.The substratum of this bacterial strain consists of: peptone 10 g/L, yeast extract 30 g/L, glucose 10 g/L, MgSO ₄0.06 g/L, KH ₂PO ₄2.13 g/L, K ₂HPO ₄3H ₂O 16.43 g/L, pH 7.0.121 ℃ of heat sterilization 15 min.

The yeast culture process is: after the substratum cooling, from slant strains, get two rings A. Oxydans1000 mL triangular flasks of the above-mentioned substratum of 200 mL are equipped with in the inoculation of TL-3 thalline, 220 rpm, and 24 h are cultivated in 38 ℃ of concussions.

Embodiment 2 MTSase separation and purification and enzymatic property research

Embodiment 2-1 MTSase separation and purification

(1) bacterial cell disruption

Carry out yeast culture by method among the embodiment 1, obtain 10 liters of cultures, with centrifugal 20 min of 8000 rpm, gather in the crops 150 g wet thallus, be suspended in the damping fluid ultrasonication thalline under the condition of ice bath again.Damping fluid consists of 0.2 Mol/L citrate buffer solution, pH5.5.Centrifugal after the ultrasonication (10000 r/min, 20 min) get supernatant liquor, get crude enzyme liquid.

(2) ammonium sulfate precipitation

Ammonium sulfate precipitation and dialysis are all carried out in ice bath, slowly adding ammonium sulfate in crude enzyme liquid makes saturation ratio reach 40 %, 4 ℃ of placements are spent the night, centrifugal 20 min of 12000 r/min then, continuing slowly to add ammonium sulfate in the supernatant liquor makes saturation ratio reach 60 %, recentrifuge, collecting precipitation also is dissolved in the 0.025 Mol/L potassium phosphate buffer (pH5.8), is the dialysis tubing dialysis desalination of 10000 Da with molecular weight cut-off.

(3) DEAE-Sepharose anion-exchange chromatography

Ammonium sulfate precipitation and dialysis enzyme liquid are later carried out DEAE-Sepharose (16 mMol/L * 350 mm) anion-exchange chromatography, sample-loading buffer A is 0.025 Mol/L potassiumphosphate (pH=5.8) damping fluid, elution buffer B is that buffer A+1Mol/L NaCl. carries out linear gradient elution with the 0-100% buffer B, be in charge of the collection elutriant, detect enzyme and live.

(4) SP Fast Flow strong cation exchange chromatography

The enzyme liquid that contains enzyme activity that the DEAE-Sepharose anion-exchange chromatography obtains is dialysed, concentrated, carry out SP Fast Flow strong cation exchange chromatography, sample-loading buffer C is 0.025 Mol/L potassiumphosphate (pH=5.8) damping fluid, and elution buffer D is damping fluid C+1 Mol/L NaCl.With 0%-25% damping fluid D linear gradient elution, collect active peak, concentrate.The pure enzyme of concentration carries out gel permeation chromatography, uses molecular sieve Sephacryl S-300(Mr:1 * 10 ⁴-1.5 * 10 ⁶) enzyme is carried out the molecular weight demarcation.The molecular sieve standard substance are respectively: Thyroglobulin (669 kD), Ferritin (440 kD), Aldolase (158 kD), BSA (67 kD), VB12(1.382 kD).

The MTSase purification result sees Table 1:

Sample is the wall scroll band through electrophoresis detection behind the SP Fast Flow cation-exchange chromatography, makes logMr Rf molecular weight standard curve, and contrast Marker molecular weight calculates the MTSase molecular weight and is about 85 kDa.

Embodiment 2-2 MTSase zymologic property

(1) optimum temperuture of MTSase

Carry out enzyme reaction 30 ℃ of-80 ℃ of scopes, survey consistently with the enzyme biopsy except temperature exoenzyme reactions steps and condition, measure the MTSase enzyme activity, the temperature when enzyme work is the highest is optimum temperuture.The result shows that the MTSase optimum temperature is 60 ℃, sees accompanying drawing 1.

(2) optimal pH of MTSase

Maltopentaose is dissolved in the damping fluid (concentration is 0.025 Mol/L) of different pH values, and the same enzyme activity determination of other conditions is measured enzyme activity, and the pH when the MTSase vigor is the highest is optimum pH.The result shows that the MTSase optimum pH is 5.5, sees accompanying drawing 2.

(3) thermostability of MTSase

Enzyme liquid is preserved 60 min respectively in 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ water-baths, carrying out the enzyme biopsy then surveys, be contrast with the enzyme enzyme activity that liquid is surveyed that is untreated, calculate the relative enzyme activity of MTSase, enzyme activity (%)=enzyme liquid residual enzyme vigor (U/mg)/enzyme liquid enzyme activity (U/mg) is untreated in the processing back relatively.The result shows that MTSase sees accompanying drawing 3 stable down up to 65 ℃.

(4) the pH stability of MTSase

Maltopentaose is dissolved in the damping fluid (concentration is 0.025 Mol/L) of different pH values, in 60 ℃ of water-baths, preserve 60 min, carrying out the enzyme biopsy then surveys, be contrast with the enzyme enzyme activity that liquid is surveyed that is untreated, calculate the relative enzyme activity of MTSase, enzyme activity (%)=enzyme liquid residual enzyme vigor (U/mg)/enzyme liquid enzyme activity (U/mg) is untreated in the processing back relatively.The result shows that MTSase is stable under pH4.8-6.3, sees accompanying drawing 4.

Embodiment 3 MTHase separation and purification and enzymatic property research

Embodiment 3-1 MTHase separation and purification

(1) bacterial cell disruption

Carry out yeast culture by method among the embodiment 1, obtain 10 liters of cultures with the centrifugal 20min of 8000rpm, results 150g wet thallus is suspended in the damping fluid ultrasonication thalline under the condition of ice bath again.Damping fluid consists of 0.2Mol/L citrate buffer solution, pH5.3.Centrifugal after the ultrasonication (12000 r/min, 20 min) get supernatant liquor, get crude enzyme liquid.

(2) ammonium sulfate precipitation

Ammonium sulfate precipitation and dialysis are all carried out in ice bath, slowly adding ammonium sulfate in crude enzyme liquid makes saturation ratio reach 40%, 4 ℃ of placements are spent the night, centrifugal 20 min of 12000 r/min then, continuing slowly to add ammonium sulfate in the supernatant liquor makes saturation ratio reach 60%, recentrifuge, collecting precipitation also is dissolved in the 0.025 Mol/L potassium phosphate buffer (pH5.8), is the dialysis tubing dialysis desalination of 10000 Da with molecular weight cut-off.

(3) DEAE-Sepharose anion-exchange chromatography

Ammonium sulfate precipitation and dialysis enzyme liquid are later carried out DEAE-Sepharose (16 mMol/L * 350 mm) anion-exchange chromatography, sample-loading buffer A is 0.025 Mol/L potassiumphosphate (pH=5.5) damping fluid, elution buffer B is that buffer A+1Mol/L NaCl. carries out linear gradient elution with the 0-100% buffer B, be in charge of the collection elutriant, detect enzyme and live.

(4) SP Fast Flow strong cation exchange chromatography

The enzyme liquid that contains enzyme activity that the DEAE-Sepharose anion-exchange chromatography obtains is dialysed, concentrated, carry out SP Fast Flow strong cation exchange chromatography, sample-loading buffer C is 0.025 Mol/L potassiumphosphate (pH=5.5) damping fluid, and elution buffer D is damping fluid C+1 Mol/L NaCl.With 0%-25% damping fluid D linear gradient elution, collect active peak, concentrate.The pure enzyme of concentration carries out gel permeation chromatography, uses molecular sieve Sephacryl S-300(Mr:1 * 10 ⁴-1.5 * 10 ⁶) enzyme is carried out the molecular weight demarcation.The molecular sieve standard substance are respectively: Thyroglobulin (669 kD), Ferritin (440 kD), Aldolase (158 kD), BSA (67 kD), VB12(1.382 kD).

The MTHase purification result sees Table 2:

Sample is the wall scroll band through electrophoresis detection behind the SP Fast Flow cation-exchange chromatography, makes logMr Rf molecular weight standard curve, and contrast Marker molecular weight calculates the MTHase molecular weight and is about 65 kDa.

Embodiment 3-2 MTHase zymologic property

(1) optimum temperuture of MTHase

Carry out enzyme reaction 30 ℃ of-80 ℃ of scopes, survey consistently with the enzyme biopsy except temperature exoenzyme reactions steps and condition, measure the MTHase enzyme activity, the temperature when enzyme work is the highest is optimum temperuture.The result shows that the MTHase optimum temperature is 63 ℃, sees accompanying drawing 6.

(2) optimal pH of MTHase

Maltopentaose is dissolved in the damping fluid (concentration is 0.025 Mol/L) of different pH values, and the same enzyme activity determination of other conditions is measured enzyme activity, and the pH when the MTHase vigor is the highest is the optimal pH value.The result shows that MTHase is the suitableeest to be 5.3, sees accompanying drawing 7.

(3) thermostability of MTHase

Enzyme liquid is preserved 60 min respectively in 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ water-baths, carrying out the enzyme biopsy then surveys, be contrast with the enzyme enzyme activity that liquid is surveyed that is untreated, calculate the relative enzyme activity of MTHase, enzyme activity (%)=enzyme liquid residual enzyme vigor (U/mg)/enzyme liquid enzyme activity (U/mg) is untreated in the processing back relatively.The result shows that MTHase sees accompanying drawing 8 stable down up to 70 ℃.

(4) the pH stability of MTHase

Maltopentaose is dissolved in the damping fluid (concentration is 0.025 Mol/L) of different pH values, in 60 ℃ of water-baths, preserve 60 min, carrying out the enzyme biopsy then surveys, be contrast with the enzyme enzyme activity that liquid is surveyed that is untreated, calculate the relative enzyme activity of MTHase, enzyme activity (%)=enzyme liquid residual enzyme vigor (U/mg)/enzyme liquid enzyme activity (U/mg) is untreated in the processing back relatively.The result shows that MTHase is stable under pH 4.5-6.5, sees accompanying drawing 9.

The clone of gene and expression

Embodiment 4 total DNA extraction

A.oxydansTL-3 presses and cultivates 18 h among the embodiment 1, the centrifugal collection thalline of 8000 rpm, and the Genonic DNA Purification Kit operation instruction that provides according to Takara Dalian company is extracted genomic dna then.

Embodiment 5 MTSase gene clones, expression

(1) MTSase gene clone

Design of primers:According to the Arthrobacter of having reported on the Genbank Arthrobacter sp. the gene order of the MTSase in Q36 source, use Vector NTI software design primer:

Upstream primer: 5 '-CC CATATGGGATGACGCACACCTACCCT-3 ' underscore is Nde I enzyme is cutThe site;

Downstream primer: 5 '-TT GCGGCCGCAAAGTCAGCGACTGCTGC-3 ' underscore is Not I enzyme is cutThe site.

The amplification system of (polymerase chain reaction): with A.oxydansThe TL-3 genome is template, the amplifying target genes fragment, and condition is as follows: genomic dna 2 μ L, each 2 μ L of upstream and downstream primer, dNTP 4 μ L, 10 * Taq damping fluid, 5 μ L, TaqEnzyme (precious biotechnology (Dalian) company limited) 1 μ L, ddH ₂O 34 μ L;

The PCR response procedures is: 94 ℃ of pre-sex change 2 min; 96 ℃ of sex change 30 s, 59 ℃ of annealing 1 min then, 72 ℃ are extended 1 min, circulate 25 times; Last 72 ℃ are extended 10 min;

The product order-checking:Product verifies that with 1% agarose gel electrophoresis amplification obtains the dna fragmentation about 2300 bp, cuts glue and reclaims, gene sequencing shows that fragment is the open reading frame of 2331 bp, shown in SEQ ID NO:2, the protein of being made up of 776 amino acid of encoding is shown in SEQ ID NO:1.NCBI sequence alignment analysis revealed, this albumen belong to amylase family and Arthrobacter sp. the MTSase aminoacid sequence in Q36 source has 75% similarity.

Genetic expression

Restriction enzyme digestion:At first use The Nde IRestriction enzyme (precious biotechnology (Dalian) company limited) carries out enzyme to PCR product and pET24a (+) in above-mentioned (1) respectively and cuts processing, and the enzyme system of cutting is: DNA 5 μ L, The Nde I5 μ L, 10 * H damping fluid, 10 μ L, ddH ₂O 80 μ L, cumulative volume 100 μ L, the product after enzyme is cut are precious biotech firm dna fragmentation purification kit purifying through Dalian.Behind the purifying, use respectively again The Not IRestriction enzyme (precious biotechnology (Dalian) company limited) enzyme is cut, and the endonuclease reaction system is: DNA 5 μ L, The Not I5 μ L, 10 * H damping fluid, 10 μ L, 0.1%BSA(bovine serum albumin) 10 μ L, 0.1%TritonX-100 10 μ L, ddH ₂O 60 μ L, cumulative volume 100 μ L, the product after enzyme is cut are precious biotech firm dna fragmentation purification kit purifying through Dalian.

Connect:Connect with T4 ligase enzyme (precious biotechnology (Dalian) company limited) behind the double digestion product purification, the ligation system is: enzyme is cut the PCR product 8 μ L of purifying, and enzyme is cut pET24a (+) the 8 μ L of purifying, T4 ligase enzyme 2 μ L, 10 * T4 damping fluid, 2 μ L.37 ℃ connect 2 h, obtain recombinant plasmid pET24a (+)-MTSase behind the purifying, see Fig. 5.

Transform: E.coliThe BL21(intestinal bacteria) the host bacterium is cultivated 12 h in the LB liquid nutrient medium, move in the fresh LB liquid nutrient medium by 5% inoculum size, cultivate 2 h for 37 ℃, getting 1 mL nutrient solution adds in the 1.5 mL centrifuge tubes, centrifugal 5 min(4 of 5000 rpm ℃), the supernatant liquor that inclines adds with 0.1 ice-cooled Mol/L CaCl ₂500 μ L, concussion is even, and 5000 rpm low-temperature centrifugations, 5 min add 500 μ L CaCl again ₂, concussion is even, and 5000 rpm low-temperature centrifugations, 5 min collect thalline, add 200 μ L CaCl ₂Shake up, as competent cell.Drawing 5 μ L recombinant plasmid pET24a (+)-MTSase adds in the 100 μ L competent cells, ice bath 30 min, 42 ℃ of water-bath 90 s, rapidly centrifuge tube is transferred in the ice bath, cooling 1-2 min adds in the 1 mL SOC substratum then, behind 37 ℃ of cultivation 45 min, get 200 μ L and coat the LB solid culture primary surface that contains 0.15 mMol/L kantlex, cultivate 12-16h to single bacterium colony appearance, preserve single bacterium colony in the LB culture medium slant for 37 ℃.

Identify:Above-mentioned single colony inoculation in the LB liquid nutrient medium that contains 0.15 mMol/L kantlex, is cultivated 15 h for 37 ℃, be cooled to 27 ℃, add final concentration and be the IPTG of 0.2 mMol/L as inductor, centrifugal results somatic cells behind 27 ℃ of inducing culture 14 h.The conventional ultrasonication of cell process, the centrifugal collection supernatant liquor of 8000 rpm, the enzyme biopsy is surveyed and is shown that every milliliter of fermented liquid can produce MTSase 28.7 U, illustrates that MTSase expresses successfully, and compares with original bacterium, and yield of enzyme has improved 89.7 times.

Embodiment 6 MTHase gene clones, expression

(1) MTHase gene clone

Design of primers:According to the Arthrobacter of having reported on the Genbank Arthrobacter sp. the gene order of the MTHase in Q36 source, use Vector NTI software design primer:

Upstream primer: 5 '-CC CATATGGGATGAGTACGCCAGTGTCC-3 ' underscore is Nde I enzyme is cutThe site;

Downstream primer: 5 '-TT GCGGCCGCAAGTGAGAGGTGGACAGACG-3 ' underscore is Not I enzyme is cutThe site.

The amplification system of (polymerase chain reaction): with A.oxydansThe TL-3 genome is template, the amplifying target genes fragment, and condition is as follows: genomic dna 2 μ L, each 2 μ L of upstream and downstream primer, dNTP 4 μ L, 10 * Taq damping fluid, 5 μ L, TaqEnzyme 1 μ L, ddH ₂O 34 μ L;

The PCR response procedures is: 94 ℃ of pre-sex change 2 min; 96 ℃ of sex change 30 s, 58 ℃ of annealing 1 min then, 72 ℃ are extended 1 min, circulate 25 times; Last 72 ℃ are extended 10 min;

The product order-checking:Product verifies that with 1% agarose gel electrophoresis amplification obtains the dna fragmentation about 1800 bp, cuts glue and reclaims, gene sequencing shows that fragment is the open reading frame of 1797 bp, shown in SEQ ID NO:4, the protein of being made up of 598 amino acid of encoding is shown in SEQ ID NO:3.NCBI sequence alignment analysis revealed, this albumen belong to amylase family and Arthrobacter sp. the MTHase aminoacid sequence in Q36 source has 79% similarity.

Genetic expression

Restriction enzyme digestion:At first use The Nde IRestriction enzyme carries out enzyme to PCR product and pET24a (+) in above-mentioned (1) respectively and cuts processing, and the enzyme system of cutting is: DNA 5 μ L, The Nde I5 μ L, 10 * H damping fluid, 10 μ L, ddH ₂O 80 μ L, cumulative volume 100 μ L, the product after enzyme is cut are precious biotech firm dna fragmentation purification kit purifying through Dalian.Behind the purifying, use respectively again The Not IDigestion with restriction enzyme, the endonuclease reaction system is: DNA 5 μ L, The Not I5 μ L, 10 * H damping fluid, 10 μ L, 0.1%BSA 10 μ L, 0.1%TritonX-100 10 μ L, ddH ₂O 60 μ L, cumulative volume 100 μ L, the product after enzyme is cut are precious biotech firm dna fragmentation purification kit purifying through Dalian.

Connect:Connect with the T4 ligase enzyme behind the double digestion product purification, the ligation system is: enzyme is cut the PCR product 8 μ L of purifying, and enzyme is cut pET24a (+) the 8 μ L of purifying, T4 ligase enzyme 2 μ L, 10 * T4 damping fluid, 2 μ L.37 ℃ connect 2 h, obtain recombinant plasmid pET24a (+)-MTHase behind the purifying, see Figure 10.

Transform: E.coliBL21 host bacterium is cultivated 12 h in the LB liquid nutrient medium, move in the fresh LB liquid nutrient medium by 5% inoculum size, cultivate 2 h, get 1 mL nutrient solution and add in the 1.5 mL centrifuge tubes for 37 ℃, centrifugal 5 min(4 of 5000 rpm ℃), the supernatant liquor that inclines adds with 0.1 ice-cooled Mol/L CaCl ₂500 μ L, concussion is even, and 5000 rpm low-temperature centrifugations, 5 min add 500 μ L CaCl again ₂, concussion is even, and 5000 rpm low-temperature centrifugations, 5 min collect thalline, add 200 μ L CaCl ₂Shake up, as competent cell.Drawing 5 μ L recombinant plasmid pET24a (+)-MTHase adds in the 100 μ L competent cells, ice bath 30 min, 42 ℃ of water-bath 90 s, rapidly centrifuge tube is transferred in the ice bath, cooling 1-2 min adds in the 1 mLSOC substratum then, behind 37 ℃ of cultivation 45 min, get 200 μ L and coat the LB solid culture primary surface that contains 0.15 mMol/L kantlex, cultivate 12-16 h to single bacterium colony appearance, preserve single bacterium colony in the LB culture medium slant for 37 ℃.

Identify:Above-mentioned single colony inoculation in the LB liquid nutrient medium that contains 0.15 mMol/L kantlex, is cultivated 15 h for 37 ℃, be cooled to 28 ℃, add final concentration and be the IPTG of 0.2 mMol/L as inductor, centrifugal results somatic cells behind 28 ℃ of inducing culture 16 h.The conventional ultrasonication of cell process, the centrifugal collection supernatant liquor of 8000 rpm, the enzyme biopsy is surveyed and is shown that every milliliter of fermented liquid can produce MTHase 86.2 U, and the recombinant expressed success of MTHase is described, and compares with original bacterium, and yield of enzyme has improved 70.4 times.

Embodiment 7 MTSase preparation

MTSase reorganization bacterium (contains recombinant plasmid pET24a (+)-MTSase's E.coliThe BL21 intestinal bacteria) cultivate 15 h 37 ℃ of LB liquid nutrient mediums that contains 0.30 mMol/L kantlex, be cooled to 26 ℃, add final concentration and be the lactose of 0.3 mMol/L as inductor, 26 ℃ of inducing culture 18 h after-filtration results somatic cells.Cell is through conventional ultrasonication, and centrifugal 20 min of 8000 rpm collect supernatant liquor as crude enzyme liquid, and the enzyme biopsy is surveyed and shown that every milliliter of fermented liquid can produce MTSase 30.4 U.

Embodiment 8 MTHase preparation

Embodiment 8-1 MTHase preparation

MTHaseThe reorganization bacterium (contains recombinant plasmid pET24a (+)-MTHase's E.coliThe BL21 intestinal bacteria) cultivate 16 h 34 ℃ of LB liquid nutrient mediums that contains 0.10 mMol/L kantlex, be cooled to 27 ℃, add final concentration and be the IPTG of 0.1 mMol/L as inductor, the centrifugal results somatic cells of 8000 rpm behind 27 ℃ of inducing culture 18h.Cell is through conventional ultrasonication, and centrifugal 20 min of 8000 rpm collect supernatant liquor as crude enzyme liquid, and the enzyme biopsy is surveyed and shown that every milliliter of fermented liquid can produce MTHase 84.7 U.

Embodiment 8-2 MTHase preparation

MTHaseThe reorganization bacterium is cultivated 13 h 37 ℃ of LB liquid nutrient mediums that contains 0.2 mMol/L kantlex, is cooled to 30 ℃, adds final concentration and be the IPTG of 0.3 mMol/L as inductor, 30 ℃ of inducing culture 12 h after-filtration results somatic cells.Cell is through conventional high-pressure homogenization crusher machine, and centrifugal 20 min of 8000 rpm collect supernatant liquor as crude enzyme liquid, and the enzyme biopsy is surveyed and shown that every milliliter of fermented liquid can produce MTHase 81.2 U.

Embodiment 8-3 MTHase preparation

MTHaseThe reorganization bacterium is cultivated 15h 37 ℃ of LB liquid nutrient mediums that contains 0.3 mMol/L kantlex, is cooled to 28 ℃, adds final concentration and be the lactose of 0.3 mMol/L as inductor, 28 ℃ of inducing culture 16h after-filtration results somatic cells.Cell is through conventional ultrasonication, and the centrifugal 20min of 8000 rpm collects supernatant liquor as crude enzyme liquid, and the enzyme biopsy is surveyed and shown that every milliliter of fermented liquid can produce MTHase 90.2 U.

Embodiment 8-4 MTHase preparation

The reorganization bacterium is cultivated 15 h 35 ℃ of LB liquid nutrient mediums that contains 0.15 mMol/L kantlex, is cooled to 27 ℃, adds final concentration and be the lactose of 0.5 mMol/L as inductor, the centrifugal results somatic cells of 8000 rpm behind 27 ℃ of inducing culture 14 h.Cell is through conventional ultrasonication, and centrifugal 20 min of 8000 rpm collect supernatant liquor as crude enzyme liquid, and the enzyme biopsy is surveyed and shown that every milliliter of fermented liquid can produce MTHase 87.5 U.

Embodiment 9 MTSase and the MTHase application in trehalose is produced

Embodiment 9-1 MTSase and the MTHase application in trehalose is produced

The starch milk of mass concentration 27.5% is transferred pH5.8, and every liter of reaction solution adds Novi's letter alpha-amylase (Novozymes Company, down with) of 0.3mL, and 95 ℃ of reaction 25 min can obtain the DE value and be 8.3 starch hydrolyzates, 132 ℃ of enzyme 5 min that go out.

The DE value is 8.3 reductibility starch hydrolyzatess, 1 L, adds 300 U MTSase, 300 U MTHase, 1.0 mL Novi letter Pullulanase, and following 60 ℃ of reaction 29.5 h of pH5.4 condition, the transformation efficiency of trehalose can reach 85.4%.

Embodiment 9-2 MTSase and the MTHase application in trehalose is produced

The starch milk of mass concentration 27.5% is transferred pH5.4, and every liter of reaction solution adds Novi's letter alpha-amylase of 0.5 mL, and 94 ℃ of reaction 25 min can obtain the DE value and be 7.6 starch hydrolyzates, 132 ℃ of enzyme 7 min that go out.

The DE value is 7.6 reductibility starch hydrolyzatess, 1 L, adds 100 U MTSase, 900 U MTHase, 0.6 mL Novi letter Pullulanase, and following 65 ℃ of reaction 32 h of pH5.3 condition, the transformation efficiency of trehalose can reach 83.7%.

Above-described embodiment only is for example clearly is described, and is not the restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here need not also can't give all embodiments exhaustive.And the apparent variation of being extended out thus or change still are among the protection domain of the invention.

＜110〉Shandong Tianli Pharmaceutical Co., Ltd.

＜120〉preparation of novel malt oligosaccharide based mycose lytic enzyme, enzyme gene, the recombinant expression vector that contains this gene and recombinant bacterial strain and enzyme

〈160〉4

〈210〉1

〈211〉776

〈212〉PRT

＜213〉the oxidation Arthrobacter ( Arthrobacter oxydans)

〈400〉1

Met Ser Thr Pro Val Ser Thr Tyr Arg Leu Glu Ile Arg Lys Gly Phe

5 10 15

Thr Leu Phe Asp Ala Ala Glu Lys Val Leu Tyr Leu Lys Ser Ile Gly

20 25 30

Val Asp Trp Val Tyr Leu Ser Pro Ile Leu Thr Ala Glu Gln Gly Ser

35 40 45

Asp His Gly Tyr Asp Val Thr Asp Pro Ser Ala Val Asp Pro Glu Arg

50 55 60

Gly Gly Pro Glu Gly Leu Leu Ala Leu Ser Ser Ala Ala Arg Glu His

65 70 75 80

Gly Met Gly Leu Leu Met Asp Ile Leu Pro Asn His Val Cys Val Ala

85 90 95

Thr Pro Val Gln Asn Pro Trp Trp Ser Ser Leu Leu Lys Glu Gly Arg

100 105 110

Arg Ser Arg Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu Gly Gly

115 120 125

Gly Lys Val Arg Leu Thr Lys Leu Gly Thr Asp Glu Asp Leu Asp Gln

130 135 140

Leu Glu Ile Lys Asp Gly Glu Leu Arg Tyr Tyr Asp His Arg Phe Pro

145 150 155 160

Leu Ala Glu Gly Thr Tyr Ser Glu Arg Asp Ser Pro Gln Glu Val His

165 170 175

Ala Arg Gln His Tyr Glu Leu Met Asp Trp Arg Arg Ala Asp Thr Glu

180 185 190

Leu Asn Tyr Arg Arg Phe Phe Ala Val Thr Thr Leu Asp Gly Ile Arg

195 200 205

Val Glu Met Pro Ala Val Phe Glu Glu Ala His Ala Glu Val Gly Arg

210 215 220

Trp Phe Pro Glu Gly Leu Val Asp Gly Leu Arg Val Asp His Pro Asp

225 230 235 240

Gly Leu Ala Asp Pro Ala Gly Tyr Leu Arg Trp Leu Gln Asp Leu Thr

245 250 255

Gly Gly Ala Tyr Val Leu Val Glu Lys Ile Leu Glu Pro Gly Glu Val

260 265 270

Leu Pro Ala Asn Phe Ala Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu

275 280 285

Ala Asp Val Asp Arg Val Phe Val Asp Pro Ala Gly Glu Gln Pro Leu

290 295 300

Asp Ala Leu Asp Ala Ala Leu Arg Gly Ala Pro Glu Ala Ala Asp Tyr

305 310 315 320

Ala Glu Met Ile Pro Gly Thr Lys Arg Leu Ile Ala Asp Gly Ile Leu

325 330 335

Arg Ser Glu Val Leu Arg Leu Ala Arg Leu Val Pro Glu Ser His Gly

340 345 350

Leu Ala Leu Glu Glu Ala Ala Asp Ala Ile Ala Glu Ile Ile Ala Ser

355 360 365

Phe Pro Val Tyr Arg Ser Tyr Leu Pro Thr Gly Ala Glu Val Leu Lys

370 375 380

Glu Ala Ser Glu Ser Ala Ala Glu Asp Arg Pro Asp Leu Ala Val Ala

385 390 395 400

Ile Gly Thr Leu Leu Pro Leu Leu Leu Asp Pro Ala Asn Arg Ile Ala

405 410 415

Val Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu

420 425 430

Asp Thr Ala Phe Tyr Arg Tyr Thr Arg Leu Gly Thr Leu Thr Glu Val

435 440 445

Gly Ala Glu Pro Thr Glu Phe Ala Val Tyr Pro Glu Glu Phe His Asp

450 455 460

Arg Met Arg Arg Arg Gln Asp Glu Leu Pro Leu Ser Met Thr Thr Met

465 470 475 480

Ser Thr His Asp Thr Lys Arg Ser Glu Asp Ala Arg Ala Arg Ile Ser

485 490 495

Val Ile Ala Glu Leu Pro Gly Glu Trp Ala Ala Thr Leu Asp Thr Leu

500 505 510

Arg Lys Leu Ala Pro Ile Pro Asp Gly Pro Tyr Glu His Leu Leu Trp

515 520 525

Gln Ala Ile Val Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Cys

530 535 540

Tyr Ala Glu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Lys Trp Thr

545 550 555 560

Asp Pro Asp Glu Asp Phe Glu Tyr Arg Val Lys Ala Ala Val Asp Ala

565 570 575

Val Phe Asp Asp Ala Gly Val Ala Arg Val Val Glu Gly Leu Val Ala

580 585 590

Arg Ile Asp Ala Leu Ala Ala Ser Asn Ser Leu Ala Ala Lys Leu Val

595 600 605

Gln Leu Thr Met Pro Gly Val Pro Asp Gly Tyr Gln Gly Ser Glu Phe

610 615 620

Trp Glu Arg Ser Leu Thr Asp Pro Asp Asn Arg Arg Pro Val Asp Phe

625 630 635 640

Ala Ile Arg Arg Ala Glu Leu Ala Arg Ile Asp Ala Gly Thr Leu Pro

645 650 655

Ala Ser Gly Thr Glu Ala Ser Lys Leu Leu Ala Thr Thr Arg Ala Leu

660 665 670

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

675 680 685

Pro Ala Thr Gly Ala Ala Ala Gly Leu Leu Leu Gly Phe Tyr Arg Gly

690 695 700

Thr Ala Asp Gly Thr Pro Gly Ala Leu Thr Leu Ala Thr Arg Leu Pro

705 710 715 720

Ala Gly Leu Glu Ala Gly Gly Gly Trp Arg Asp Thr Val Ile Glu Leu

725 730 735

Asn Ala Ala Met Tyr Asp Glu Leu Thr Gly Ala Gly Phe Gly Thr Val

740 745 750

Ala Val Lys Ile Ala Asp Ile Phe Arg Thr Phe Ala Val Ala Pro Leu

755 760 765

Val Pro Gln Thr Gly Gly Glu Ser

770 775

〈210〉2

〈211〉2331

〈212〉DNA

＜213〉oxidation Arthrobacter (Arthrobacter oxydans)

〈400〉2

atgagtacgc cagtgtccac ttaccggttg gagatccgca agggtttcac cctgttcgac 60

gccgccgaga aggtcctgta cctcaaatcg atcggcgttg actgggtcta cctgtcgccc 120

atccttacgg cggaacaggg ctccgaccac ggctacgacg tgaccgatcc ctccgccgtc 180

gaccccgagc gtggtggccc ggaagggctg ctggcactgt ccagtgccgc ccgcgagcac 240

ggcatgggcc tgctgatgga catcttgccc aaccacgtgt gcgtcgcgac cccggtgcag 300

aacccctggt ggtcatcgct gctgaaggaa ggccgtcggt cgcggtacgc ggaagcgttc 360

gacgtcgatt gggacttggg cggcggcaag gtccggctca cgaagctggg caccgacgag 420

gacctggacc agctggagat caaggacggg gaactccggt actacgacca ccgattcccg 480

ctcgctgagg gaacctactc cgagagggac tccccacagg aggtacacgc ccgccagcac 540

tacgagctca tggactggcg ccgcgccgac accgagctca actaccgccg cttcttcgcg 600

gtgaccacgt tggacggcat ccgggtggag atgccggcgg tcttcgaaga agcccacgcc 660

gaagttggcc gctggttccc cgaaggcctg gtggacggtc tgcgcgtcga ccacccggac 720

ggcctggccg accccgcagg ctacctgagg tggctccagg acctcactgg cggcgcctac 780

gtcctggtgg agaagatcct ggaacccggc gaggtgctgc cggccaactt cgcctgcgag 840

ggcaccacag ggtacgacgc tctcgccgac gtggaccggg tgttcgtgga tcctgccggg 900

gagcagcccc tggacgcact ggacgcggcg ttgcggggcg ctcccgaggc cgctgactac 960

gcggagatga tccccggcac caagcggctg atcgccgacg gcatcctccg ctccgaggtg 1020

ctgcggctgg ccaggctggt tccggaatcc cacggtctgg cgctggagga agcagccgac 1080

gctatcgccg agatcatcgc ttccttcccg gtctaccgca gctacctgcc caccggggcc 1140

gaggtgctga aggaagccag cgagtctgcc gcagaagacc gtccggacct cgccgtcgca 1200

atcgggacac tccttccgct gctgttggat ccggccaatc gcattgccgt caggttccag 1260

cagacctccg gcatggtgat ggccaagggt gtggaagaca ccgcgttcta ccgctacacc 1320

cgccttggca cgctgaccga ggtgggcgca gaacccaccg agttcgccgt gtacccggag 1380

gaattccacg accggatgcg ccggcggcag gacgagctgc cgctgtccat gaccaccatg 1440

tccacgcacg ataccaagcg cagcgaggac gcccgggcac ggatctccgt gatcgccgag 1500

ctgccggggg agtgggcagc caccctggac actctccgga agctggctcc catcccggac 1560

ggcccctacg agcacctgct gtggcaggcc atcgtgggcg cctggcctgc cagccgggaa 1620

cggctccagt gctacgcgga gaaggccgcc cgagaagccg gcaactccac gaagtggacc 1680

gacccggacg aggacttcga gtaccgggtg aaggcggccg tggatgcggt attcgacgac 1740

gccggcgtgg ccagggtggt ggagggcctc gttgcacgca tcgatgcctt agcggcctcc 1800

aactccctcg ccgcgaagct ggtccagctg accatgcccg gcgtccccga cggctaccaa 1860

ggcagcgagt tctgggaacg gtcgttgact gatcccgata accgccggcc cgtcgacttc 1920

gcaatccggc gagccgagct ggccaggatc gacgccggaa cgttgcccgc gtcgggcacg 1980

gaagccagca agctccttgc caccacacgg gcgctccgcc tgcggcggga ccggccggaa 2040

ctcttcgagg gctaccgccc ggtcccggcc acgggcgctg cagccgggct cctgctcgga 2100

ttctaccgcg gcacggcgga cggcacgccc ggggcactga ctctggcaac gcggctgcct 2160

gcgggcctgg aagccggcgg cggctggcgc gataccgtca tcgaacttaa tgctgcaatg 2220

tatgacgaac tgaccggtgc cggcttcgga acggtggcag tgaagattgc cgacatattc 2280

cggacgttcg ccgttgcgcc gctggtgccg cagacaggag gagagtcatg a 2331

〈210〉3

〈211〉598

〈212〉PRT

＜213〉oxidation Arthrobacter (Arthrobacter oxydans)

〈400〉3

Met Thr His Thr Tyr Pro Arg Gln Ala Ala Ile Pro Val Leu Gly Pro

5 10 15

Ala Arg Tyr Asp Val Trp Ser Pro Asn Ala Gly Ser Val Ser Leu Leu

20 25 30

Ser Gly Gln Glu Arg Tyr Ala Met Gln His Arg Ala Glu Ser Gly Leu

35 40 45

Glu Glu Ala Asp Trp Arg Thr Ala Pro Asp Ala Pro Ala Asp Gly Glu

50 55 60

Val Asp Tyr Gly Tyr Arg Leu Asp Val Asp Asn His Pro Leu Pro Asp

65 70 75 80

Arg Arg Ser Arg Arg Val Pro Glu Gly Val His Asp Leu Ser Arg Thr

85 90 95

Phe Asp Pro Ala Ala Tyr Ala Trp Lys Asp Ala Ala Trp Lys Gly Thr

100 105 110

Glu Leu Thr Gly Ala Val Ile Tyr Glu Leu His Leu Gly Thr Phe Thr

115 120 125

Pro Lys Gly Thr Leu Asp Ala Ala Ser Gly Lys Leu Gly Tyr Leu Ala

130 135 140

Asp Leu Gly Ile Asp Phe Met Glu Leu Leu Pro Val Asn Ala Phe Asn

145 150 155 160

Gly Thr His Asn Trp Gly Tyr Asp Gly Val Gln Trp Tyr Ala Val His

165 170 175

Glu Ala Tyr Gly Cys Pro Glu Ala Tyr Glu Arg Leu Val Asp Ala Ala

180 185 190

His Ala Ala Gly Leu Gly Val Ile Gln Asp Val Val Tyr Asn Arg Leu

195 200 205

Gly Ile Ser Gly Asn Tyr Leu Pro Gln Phe Gly Thr Tyr Leu Lys Gln

210 215 220

Cys Asp Gly Asn Thr Trp Ser Asp Ser Val Asn Leu Asp Gly Pro Gly

225 230 235 240

Ser Asp Val Val Arg Gln Tyr Ile Ile Asp Asn Leu Ala Met Trp Leu

245 250 255

Arg Asp Tyr Arg Val Asp Gly Leu Arg Leu Asp Ser Val His Ala Leu

260 265 270

Asn Asp Glu Arg Ala Val His Ile Leu Glu Asp Leu Val Ala Leu Gly

275 280 285

Asp Ala Val Ser Thr Glu Ala Gly Leu Pro Gln Thr Leu Ile Ala Glu

290 295 300

Ser Asp Leu Asn Ile Pro Arg Leu Leu Tyr Gln Arg Val Ala Asn Gly

305 310 315 320

Tyr Gly Leu Glu Glu Gln Cys Ser Asp Asp Phe Asp His Thr Val His

325 330 335

Ala Lys Val Thr Gly Glu Thr Thr Gly Tyr Tyr Ser Asp Phe Glu Ser

340 345 350

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

355 360 365

Arg Tyr Ser Arg Phe Arg Asp Arg His His Gly Arg Pro Ile Asn Ala

370 375 380

Ser Leu Val Thr Pro Ala Ala Leu Val Val Cys Tyr Gln Asn His Asp

385 390 395 400

Gln Ile Gly Asn Arg Ala Thr Arg Asp Arg Leu Ser Gln Ser Leu Pro

405 410 415

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

420 425 430

Thr Pro Met Leu Leu Met Val Glu Glu Tyr Gly Ala Thr Thr Arg Trp

435 440 445

Gln Phe Phe Thr Ser His Ala Glu Pro Glu Leu Val Met Ala Thr Glu

450 455 460

Glu Gly Arg Ile Lys Glu Phe Gln Arg Ile Gly Trp Asp Arg Ala Val

465 470 475 480

Val Pro Asp His Gln Asp Pro Glu Thr Phe Cys Arg Tyr Lys Leu Asn

485 490 495

Trp Asp Glu Ala Ala Met Gly Asp His Ala Arg Leu Leu Glu Leu Tyr

500 505 510

His Ser Leu Thr Ala Leu Arg Arg Tyr His Gln Glu Leu Thr Glu Leu

515 520 525

Gly Phe Gly Glu Thr Glu Leu Ala Phe Asp Glu Asp Ser Gly Trp Leu

530 535 540

Arg Phe Ser Arg Gly Arg Val Gln Leu Leu Leu Asn Phe Ser Glu Gln

545 550 555 560

Leu Val Ser Leu Asp Gly Ala Gly Ser Ala Leu Gln Leu Ala Thr Asp

565 570 575

Asp Ala Val Arg Leu Asp Gly Glu Ser Ala Glu Leu Gly Arg Leu Ser

580 585 590

Ala Ala Val Val Ser Asp

595

〈210〉4

〈211〉1797

〈212〉DNA

＜213〉oxidation Arthrobacter (Arthrobacter oxydans)

〈400〉4

atgacgcaca cctaccctcg gcaagccgcg ataccagtcc tggggcccgc tcgctacgac 60

gtctggtcgc cgaacgctgg atccgtgtcg ctgctgtccg gccaggagcg ctacgccatg 120

cagcaccggg ccgagtccgg gctggaggaa gccgactgga ggacggcacc ggacgcgcct 180

gccgacggcg aagtggacta cggctaccgg ctcgacgtcg acaaccaccc gctgccggac 240

cgccggtccc gccgcgtgcc cgaaggagtc cacgacctgt cccggacgtt cgatcccgcc 300

gcgtacgcgt ggaaggacgc cgcctggaag ggcacggaac tcaccggcgc ggtcatctac 360

gaactccacc tgggcacatt cacgccgaag ggcacgctgg acgcggcctc cgggaagctt 420

ggctacctgg cggatctggg catcgacttc atggagctac tgccggtcaa cgcgttcaac 480

ggcactcaca actggggcta cgacggcgtg cagtggtacg ccgtccacga ggcctacggc 540

tgccctgaag cgtacgagcg gctcgttgac gcagcgcatg cggccgggct tggcgtgatc 600

caggatgtgg tctacaaccg cctcggcatc agcggcaatt acctgccgca gttcggcacg 660

tacctgaaac agtgcgacgg taacacctgg agcgactccg tcaacctcga cgggccaggc 720

tcggacgtgg tccgccagta catcatcgac aacctcgcca tgtggctccg tgattaccgg 780

gtggacggtc tgcgcctcga ctccgtgcat gcgctgaacg acgagcgtgc ggtgcacatc 840

ctcgaagacc tcgtggcgct gggtgacgcg gtctccaccg aggccggcct cccccaaacg 900

ctgatcgcag agtcagacct caacatcccg cgcctgctct atcagcgtgt cgccaacggg 960

tacggcctgg aagagcagtg cagcgacgac ttcgaccaca ccgtccacgc aaaagttacc 1020

ggcgaaacca ccgggtatta cagcgacttc gagtcgttgg ccgtcctagc caaggtactc 1080

caagacggct tcctccacga cagcaggtac tccaggttcc gcgaccgtca ccacggaagg 1140

cccatcaacg cctcgctggt aacgcctgcg gcgctggtgg tgtgctacca gaaccacgat 1200

cagataggca accgtgccac gcgggacagg ctctcgcagt cactgcccta cgggcagttg 1260

gccctggctg cggtacttac gctgacgtcg ccgttcacgc ccatgctgct catggtggag 1320

gaatacgggg ccaccacacg gtggcagttc ttcacctcgc acgcggaacc ggagctcgtc 1380

atggccaccg aggagggccg catcaaggaa ttccagcgca tcgggtggga tcgcgcagtc 1440

gtgcccgatc accaggaccc cgaaaccttc tgccggtaca agctgaactg ggacgaggcc 1500

gccatgggtg accacgctcg cctgctcgag ctgtaccatt cgctcaccgc gctgcggcgc 1560

taccaccagg agctcaccga actgggtttc ggggagacgg agctggcgtt cgacgaggac 1620

tccggctggc tacggttcag ccggggtcga gtgcagctgc tgctcaactt ctcagaacag 1680

ctcgtgagct tggacggtgc aggctcggcc ctgcagctgg ccaccgacga cgcagtccgg 1740

ctagacggtg agagtgcgga actcggtcgg ctgagcgccg ccgtcgtcag cgactga 1797

Claims (10)

1. a malt oligosaccharide based mycose lytic enzyme is characterized in that: have the aminoacid sequence shown in SEQ ID NO:3.

2. malt oligosaccharide based mycose lytic enzyme according to claim 1 is characterized in that: have following characteristic:

(1) optimum temperature

As pH5.3 incubation 60 min, 60 ℃ of optimum temperatures;

(2) the suitableeest action pH

As 60 ℃ of incubation 60 min, optimal pH is 5.3;

(3) thermostability

When bathing 60 min in the pH5.3 temperature, stable down up to 70 ℃;

(4) pH stability

When bathing 60 min in 60 ℃ of temperature, stable under pH4.5-6.5.

3. malt oligosaccharide based mycose lytic enzyme according to claim 1 and 2 is characterized in that: got by following any one genetic expression:

Gene 1: the gene that contains the nucleotide sequence shown in the SEQ ID NO:4;

Gene 2: contain by the nucleotide sequence shown in the SEQ ID NO:4 and replace the gene of the formed nucleotide sequence of one or more bases because of the merger of genetic codon, the nucleotide sequence of this gene does not change by the coded aminoacid sequence of nucleotide sequence shown in the SEQ ID NO:4;

Gene 3: the gene that contains complementary nucleotide sequence in gene 1 or the gene 2.

4. gene of expressing claim 1 or 2 described malt oligosaccharide based mycose lytic enzymes.

5. gene according to claim 4 is characterized in that: contain following any one nucleotide sequence:

Nucleotide sequence 1: the nucleotide sequence shown in SEQ ID NO:4;

Nucleotide sequence 2: replace the formed nucleotide sequence of one or more bases by the nucleotide sequence shown in the SEQ ID NO:4 because of the merger of genetic codon, this nucleotide sequence does not change by the coded aminoacid sequence of nucleotide sequence shown in the SEQ ID NO:4;

Nucleotide sequence 3: the complementary nucleotide sequence of nucleotide sequence 1 or nucleotide sequence 2.

6. the recombinant expression vector that contains the gene of claim 4 or 5 described malt oligosaccharide based mycose lytic enzymes; Described recombinant expression vector is preferably formed by expression vector pET24a (+) and claim 5 or 6 the gene constructed of described malt oligosaccharide based mycose lytic enzyme.

7. the recombinant bacterial strain that contains the gene of claim 4 or 5 described malt oligosaccharide based mycose lytic enzymes.

8. recombinant bacterial strain according to claim 7 is characterized in that: the described recombinant expression vector of claim 6 is changed over to make up in the intestinal bacteria form; Described intestinal bacteria are preferably e. coli bl21.

9. a method for preparing the malt oligosaccharide based mycose lytic enzyme is characterized in that: make by cultivating claim 7 or 8 described recombinant bacterial strains.

10. method according to claim 9, it is characterized in that: specifically may further comprise the steps: with recombinant bacterial strain in the LB liquid nutrient medium that contains 0.10-0.30 mMol/L kantlex 34-37 ℃ cultivate 12-15 h after, be cooled to 26-29 ℃, add final concentration and be the lactose of the IPTG of 0.1-0.3 mMol/L or 0.3-0.5 mMol/L as inductor, at 26-29 ℃ of following inducing culture 12-18 h, somatic cells is collected in centrifugal or filtration then; With somatic cells fragmentation, centrifugal collection supernatant liquor, must contain the crude enzyme liquid of malt oligosaccharide based mycose lytic enzyme.

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