CN109797142A

CN109797142A - A kind of degradation pulullan generates glycosidase and its encoding gene and the application of single panose

Info

Publication number: CN109797142A
Application number: CN201910163389.4A
Authority: CN
Inventors: 张桂敏; 王美星; 胡慧贞; 卢争辉; 巫攀; 马延和
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2019-03-05
Filing date: 2019-03-05
Publication date: 2019-05-24
Anticipated expiration: 2039-03-05
Also published as: CN109797142B

Abstract

The present invention provides glycosidase and its encoding gene and applications that a kind of degradation pulullan generates single panose, which includes amino acid sequence shown in SEQ ID NO.1.Glycosidase of the invention has many advantages, such as that enzyme activity is high, pH tolerance is good, can be widely applied to the enzymatic hydrolysis of cyclodextrin (alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin), pulullan, soluble starch.Most of all, the product of glucosides enzyme hydrolysis pulullan of the invention is single panose, there is huge application potential in the industry.

Description

It is a kind of degradation pulullan generate single panose glycosidase and its encoding gene and Using

Technical field

The invention belongs to technical field of molecular biology, generate single Pan in particular to a kind of degradation pulullan The glycosidase and its encoding gene of sugar and application.

Background technique

The trisaccharide that panose is made up of a maltose unit and a glucose unit α -1,6- glucosides key connection. Panose can be widely used for food industry, anti-color fading agent, food antioxidant as food colour.The main application of panose is mouth Product, including food and beverage are taken, because this carbohydrate cannot be fermented by oral microorganism, and in addition to as sweet tea Its fairly effective preventing decayed tooth, therefore its sweetener that can be used as anti-caries tooth are also assigned except taste agent.Panose can also be used for promoting The proliferation of Exiguobacterium sp microorganism belonging to genus in human body intestinal canal, to be considered as prebiotics, for stimulating beneficial microbe (such as lactic acid Bacillus and Bifidobacterium) growth, and inhibit the growth of unwanted microorganism such as Escherichia coli and salmonella, to human body Enteron aisle has potential beneficial effect.

In conclusion it is very significant that hydrolysis pulullan, which generates the research of panose, and hydrolysate is single The novel enzyme of panose be even more that there is industrial huge application potential.

Summary of the invention

The purpose of the present invention is to provide a kind of glycosidase and its encoding gene and its in hydrolysis cyclodextrin, (α-ring is pasted Essence, beta-cyclodextrin, gamma-cyclodextrin), pulullan, the application in soluble starch.

In order to achieve the object of the present invention, inventor is finally obtained as follows by a large number of experiments research and unremitting effort Technical solution:

A kind of glycosidase, the enzyme have amino acid sequence shown in SEQ ID NO.1 in sequence table.

It should be noted that glycosidase provided by the present invention can hydrolyze cyclodextrin (alpha-cyclodextrin, beta-cyclodextrin, γ- Cyclodextrin), pulullan, soluble starch, and the product for hydrolyzing pulullan is single panose, and hydrolysis efficiency is significant. Amino acid sequence shown in SEQ ID NO.1 is made of 589 amino acid residues.

In order to make above-mentioned glucosides zymoprotein convenient for purifying, the amino for the protein that can be formed in above-mentioned amino acid sequence End or carboxyl terminal connect upper label as shown in Table 1.

The sequence of 1 label of table

Label	Residue	Amino acid sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tagII	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned protein can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.Above-mentioned egg The encoding gene of white matter can also be by that will lack, replace, be inserted into or add one in amino acid sequence shown in SEQ ID NO.1 To several and keep original enzymatic activity, or connect the amino acid sequence of label shown in table 1 and further obtain.

A kind of encoding gene of glycosidase, gene coding have the albumen of amino acid sequence shown in SEQ ID NO.1 Matter.

Further, the encoding gene of the glycosidase has nucleotide sequence shown in SEQ ID NO.2.SEQ ID Nucleotide sequence shown in NO.2 is made of 1770 nucleotide.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing any of the above-described encoding gene also belong to Protection scope of the present invention.

The present invention provides a kind of recombinant vector, which includes the encoding gene of above-mentioned glycosidase.Specifically, institute State recombinant vector be any of the above-described encoding gene insertion is set out carrier (such as: what multiple cloning sites pET28a) obtained Recombinant expression carrier.The recombinant expression carrier of the gene can be contained with existing expression vector establishment.Use the gene structure It, can be plus any enhanced promoter or composing type starting before its transcription initiation nucleotide when building recombinant expression carrier Son, they can be used alone or are used in combination with other promoters；In addition, being carried using gene constructed recombinant expression of the invention When body, enhancer, including translational enhancer or transcriptional enhancer also can be used, it is close that these enhancer regions can be ATG starting Numeral or neighboring region initiation codon etc., but must be identical as the reading frame of coded sequence, to guarantee the correct of entire sequence Translation.The source of the translation control signal and initiation codon be it is extensive, can be natural, be also possible to synthesis. Translation initiation region can come from transcription initiation region or structural gene.

The present invention also provides a kind of transformant, which includes above-mentioned recombinant vector.Transformant can be recombinant bacterium, For example, any of the above-described encoding gene insertion is set out the weight that the multiple cloning sites of carrier (such as: pET28a carrier) obtain Group expression vector is converted to e. coli bl21 (DE3), obtains recombinant bacterium.

The present invention also provides a kind of primer pair, for expand above-mentioned glycosidase encoding gene overall length and its any piece Section.Such as: the sequence of primer pair is as shown in SEQ ID NO.3 and SEQ ID NO.4.

It is any of the above-described protein, any of the above-described encoding gene, any of the above-described recombinant expression carrier, described Any one in expression cassette, transgenic cell line or recombinant bacterium is in degradation cyclodextrin (alpha-cyclodextrin, beta-cyclodextrin, γ-ring paste Essence), pulullan, soluble starch these types substrate application also belong to protection scope of the present invention.

It during concrete application, can adopt in the following method: with cyclodextrin (alpha-cyclodextrin, beta-cyclodextrin, γ- Cyclodextrin), pulullan, soluble starch be substrate, preferably under conditions of neutral ph, using glycosidase to cyclodextrin (α-ring Dextrin, beta-cyclodextrin, gamma-cyclodextrin), pulullan, soluble starch digested.

Therefore, the answering in hydrolysis cyclodextrin, pulullan or/and soluble starch the present invention also provides above-mentioned glycosidase It is chosen from the followings more than either one or two with, the cyclodextrin: alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin.

Significantly, since the product of above-mentioned glucosides enzyme hydrolysis pulullan is single panose, hydrolysis efficiency is aobvious It writes, therefore it is the application in single product panose that the present invention also provides above-mentioned glycosidases in hydrolysis pulullan.

Finally, the present invention provides a kind of method digested using above-mentioned glycosidase, method includes the following steps: Using cyclodextrin, pulullan or/and soluble starch as substrate, in pH5.4-9.0 (further preferably 5.6-8.0, then into one Step be preferably 6.0-7.5), temperature be 20-40 DEG C (preferably 30 DEG C) reaction system under the conditions of, using glycosidase to ring paste Essence, pulullan or/and soluble starch are digested.

Compared with prior art, zymoprotein provided by the invention has to cyclodextrin (alpha-cyclodextrin, beta-cyclodextrin, γ-ring Dextrin), the biological degradation activity of pulullan and soluble starch, and the amino of the albumen and other reported glycosidases Acid sequence is compared, and similitude is not more than 65%, belongs to a kind of completely new glycosidase.The most suitable substrate of glycosidase of the invention is α-ring Dextrin, most suitable natural substrate is pulullan, and has active higher characteristic under conditions of neutral ph, while it is in different pH Under stability it is preferable.

In addition, glycosidase provided by the invention has significant progress, major embodiment is gone back in the following areas:

(1) glycosidase of the invention is using alpha-cyclodextrin as substrate, and optimum temperature is 30 DEG C, optimal pH 7.0.The enzyme is in temperature Degree still has 80% or more enzyme activity in the range of being 20-40 DEG C, with 60% or more in the range of pH is 5.6-8.0 Enzyme activity.After the enzyme keeps 16h under conditions of pH is 7.0-12.0, still there is 50% or more enzymatic activity, specific good pH Tolerance.The enzyme is a neutral enzymatic, does not need to provide extreme environment and play a role.

(2) glycosidase of the invention have to cyclodextrin (alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin), pulullan, The biological degradation activity of soluble starch, but degradation capability difference, it is as a result as follows: will dilution enzyme solution respectively in same concentrations Enzyme activity determination is carried out under the conditions of the different substrates of (mass fraction 1%).With alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, general It is respectively 851.9U/mg, 388.4U/mg, 94.8U/mg, 62.4U/ that the blue sugar in Shandong, soluble starch, which survey opposite enzyme activity by substrate, mg,29.3U/mg.It can be seen that the enzyme is to alpha-cyclodextrin activity highest, other take second place.Therefore, glycosidase of the present invention is most Suitable substrate is alpha-cyclodextrin, and most suitable natural substrate is pulullan.

(3) product of glycosidase hydrolyzing natural substrates pulullan of the invention is single panose.And panose is in industry In it is very widely used, including as the anti-color fading agent of food colour, food antioxidant, anti-caries tooth sweetener, prebiotics. Therefore, the application of this enzyme industrially has very big potentiality.

Detailed description of the invention

Fig. 1 is the SDS-PAGE electrophoresis before and after glycosidase Amy117 protein purification.

The result that the activity with temperature that Fig. 2 is glycosidase Amy117 changes.

The result that the activity that Fig. 3 is glycosidase Amy117 changes with pH.

The stability of the activity that Fig. 4 is glycosidase Amy117 at different temperatures.

Stability of the activity that Fig. 5 is glycosidase Amy117 at different pH.

Fig. 6 is the product measurement that glycosidase Amy117 hydrolyzes pulullan.

Specific embodiment

The principle and features of the present invention will be described below with reference to the accompanying drawings, and illustrated embodiment is served only for explaining the present invention, It is not intended to limit the scope of the present invention.Experimental method used in following embodiments is conventional side unless otherwise specified Method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The preparation and purification of embodiment 1, albumen and gene

1, the preparation of template DNA can be carried out by following two mode respectively:

(1) extraction of Alkaliphilic bacillus Bacillus sp.703 total DNA.Using Alkaliphilic bacillus Bacillus Sp.703 takes its 20 grams of fresh wet thallus, is suspended from 10 milliliters of 50mM Tris buffers (pH8.0), be added a small amount of lyase bacterium and 8 milliliters of 0.25mM EDTA (PH8.0), 37 DEG C of placement 20min after mixing；The SDS that 2 milliliters of mass fractions are 10% is added later, 55 DEG C of placement 5min are respectively extracted with isometric phenol, chloroform primary respectively；2 times of volume second are added in the supernatant solution for taking last time Alcohol recycles DNA, is washed respectively with 70% and dehydrated alcohol；Precipitating is dissolved in 0.5 milliliter of TE buffer (pH8.0,10mM Tris, 1mM EDTA), 3 μ L of 10mg/mL RNase is added.37 DEG C keep the temperature one hour, are respectively extracted with isometric phenol, chloroform respectively primary；Supernatant 2 times of volume ethanols are added in solution, recycle DNA, are washed respectively with 70% and dehydrated alcohol, are dried in vacuo, use deionized water dissolving.

The information reference document of Alkaliphilic bacillus Bacillus sp.703: Lu Zhenghui, He Jiaheng, Zhang Guimin, one plants of alkali Property amylase producing strains separation and enzymatic property analysis, Agriculture of Anhui science, 2013,41 (7): 2857-2859.

(2) DNA fragmentation of nucleotide sequence shown in artificial synthesized SEQ ID NO.2.

2, the amplification of gene order

According to nucleotide sequence design primer shown in SEQ ID NO.2 to as follows:

Forward primer: 5 '-CGCGGATCCCAAAAGGAAGCCATTTATCACCGTC-3 ', as shown in SEQ ID NO.3；

Reverse primer: 5 '-CCGCTCGAGTTAAAGCTTCTTTAAAACAATGGCTTGTT-3 ', such as SEQ ID NO.4 institute Show；

The underscore part of forward primer is the restriction enzyme site of BamHI, and the underscore part of reverse primer is XhoI digestion Site.

The synthesis of primer is completed in Sangon Biotech (Shanghai) Co., Ltd..

Using the total DNA of Alkaliphilic bacillus Bacillus sp.703 as template, PCR amplification is carried out with the primer pair of design.

PCR reaction system:

10 × buffer	5μL
		dNTP	4μL
ExTaq archaeal dna polymerase	0.5μL
		Forward primer	1μL
Reverse primer	1μL
		Template	0.5μL
Water	38μL

PCR reaction condition: 94 DEG C of initial denaturation 5min, then 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 30 circulations, last 72 DEG C of extensions 10min.

The agarose gel electrophoresis that PCR product mass fraction is 0.7% detects yield and specificity, and is purified with DNA Kit (ultrathin centrifugal column type, the production of Tiangeng company) purifying.The PCR product of purifying is sequenced, the results showed that PCR product Sequence include and being named as amy117DNA segment 1-1770 shown in SEQ ID NO.2.

3, the building of recombinant expression carrier

1) by above-mentioned sequencing correct PCR product BamHI and XhoI double digestion, agarose electrophoresis recycles digestion products.

2) plasmid pET28a (Cat.N069864-3, Novogen) is used into BamHI and XhoI double digestion, agarose electrophoresis is returned Receive digestion products.

3) digestion products of the digestion products of step 1) and step 2) are attached, the electroporated large intestine bar of connection product The LB plate containing 50 μ g/mL kanamycins is coated on after bacterium DH5 α, 37 DEG C are incubated overnight, by obtained transformant with above-mentioned Forward primer and reverse primer carry out bacterium colony PCR, screen the recombinant bacterium containing amy117 gene, extract the plasmid of recombinant bacterium, Carry out sequence verification, the results showed that, amy117DNA segment is inserted between BamHI the and XhoI restriction enzyme site of pET28a, it should Segment includes the 1st to 1770 nucleotide from 5 ' ends of SEQ ID NO.2, and direction of insertion is correct, which is ordered Entitled pET28a-amy117.

4, the preparation of engineering bacteria

By the electroporated e. coli bl21 of plasmid pET28a-amy117 (DE3) (Cat.N0 CD601, Quan Shi King Company) It is coated on the LB plate containing 50 μ g/mL kanamycins afterwards, 37 DEG C are incubated overnight, and obtain containing plasmid pET28a-amy117's Engineering bacteria is denoted as BL21/pET28a-amy117.

PET28a-amy117 is replaced with pET28a, is converted e. coli bl21 (DE3), step is same as above, and is contained The recombinant bacterium of pET28a, as control bacterium.The positive restructuring bacterium that pET28a is successfully transferred to BL21 (DE3) is denoted as BL21/ pET28a。

5, the expression and purifying of target protein

His60 Ni Superflow resin purification column is purchased from TaKaRa company, catalog number 635660.

GE HiTrap Desalting purification column is purchased from GE Healthcare company, and catalog number is respectively 17- 1408-01。

Positive restructuring bacterium BL21/pET28a-amy117 prepared by above-mentioned steps 4 is incubated to that is mould containing 50 μ g/mL cards In the LB culture medium of element, 37 DEG C of culture 3h；OD₆₀₀When=0.7, IPTG to its final concentration 0.5mM in LB culture medium is added, 18 DEG C are gone to continue to cultivate 16h.

Thalline were collected by centrifugation under the conditions of 3800rpm, 15min, be suspended in PBS solution (50mM Tris-HCl, pH7.4, 0.5M NaCl) in, ultrasonication (60w, the 10min in ice bath；Ultrasonic 1s stops 2s), 12000rpm is centrifuged 10min later Cell fragment is removed, supernatant is taken；Supernatant is crossed into His60Ni Superflow resin purification column, is rushed with 5mL ultrapure water It washes, then with 10mL solution A (50mM Na₂HPO₄-NaH₂PO₄, pH7.0,25mM imidazoles) and rinsing, finally use 5mL solution B (50mM Na₂HPO₄-NaH₂PO₄, pH7.0,500mM imidazoles) and elution, collect eluent.Then by eluent desalting column GE HiTrap Desalting carries out desalting processing, with solution C (50mM Na₂HPO₄-NaH₂PO₄, pH7.0) and it is eluted, it is pure to obtain Amy117 Enzyme solution.

Control bacterium prepared by step 4 is cultivated and purified using identical step, obtained solution is as control enzyme Liquid.

The molecular weight of the Amy117 albumen of SDS-PAGE electrophoresis showed purifying is about 70kDa, meets theoretical implications 70kDa.As a result as shown in Figure 1, in Fig. 1, swimming lane M expression Protein Marker (180,130,100,70,55,40,35,25, 15kDa)；Swimming lane 1 indicates that e. coli bl21/pET28a-amy117 breaks the supernatant after bacterium；Swimming lane 2 indicates that Ni-NTA column is pure Change and the Amy117 albumen after GE Desalting desalting column desalination.The experiment of control group has been carried out simultaneously, but compares bacterium not Obtain destination protein.

Embodiment 2 verifies protein function by substrate of alpha-cyclodextrin

Enzyme-activity unit is defined as under determination condition, discharges enzyme amount required for 1 μm of ol reduced sugar per minute as one Enzyme-activity unit U.

(1) optimum temperature

With the 50mM Na of pH7.0₂HPO₄-NaH₂PO₄Buffer dilutes the pure enzyme solution of Amy117 in the step 5 of embodiment 1, Enzyme activity determination is carried out with the enzyme solution after dilution.Enzyme solution after dilution is denoted as dilution enzyme solution.

Solution A composition: by 50mM, pH7.0Na₂HPO₄-NaH₂PO₄Buffer and alpha-cyclodextrin solution composition；Substrate α-ring Concentration of the dextrin in solution A is 1g/100mL (mass fraction 1%).

Experimental group: determination of activity reaction system is 0.5mL, dilutes enzyme solution by 0.45mL solution A and 0.05mL；Reaction system PH value be 7.0；After reaction system incubates 30min in specific range of temperatures (4-80 DEG C), boiling water bath 5min terminates reaction, adds Enter 0.5mL dinitrosalicylic acid solution (DNS), then boiling water bath 5min, measures OD after cooling rapidly₅₄₀。

As a result as shown in Figure 2.Fig. 2 shows that glycosidase Amy117 has the activity of degradation alpha-cyclodextrin.In 30 DEG C of conditions Under, glycosidase Amy117 has highest enzymatic activity, is 851.9U/mg albumen；By the suction of the enzyme activity reaction system at a temperature of this Light value is as relative activity 100%, the light absorption value of the light absorption value of enzyme activity reaction system and this highest enzyme activity system at a temperature of other Ratio as relative activity.60% or more activity is all had under the conditions of 20-40 DEG C.

Control group: above-mentioned experiment is carried out to compare the albumen (being denoted as control enzyme solution) of bacterium BL21/pET28a acquisition, as a result not Pipe is under the conditions of which temperature, activity of the control enzyme solution all without degradation alpha-cyclodextrin.

Experiment sets 3 repetitions, as a result unanimously.

(2) optimal pH

Dilution enzyme solution in following each group is the Amy117 in the step 5 with the buffer dilution embodiment 1 in each group What pure enzyme solution obtained.

Experimental group: determination of activity reaction system be 0.5mL, respectively by 0.45mL solution B (B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12 and B13) and 0.05mL dilution enzyme solution composition.

The composition of solution B 1: 50mM Na₂HPO₄-NaH₂PO₄Buffer and substrate alpha-cyclodextrin composition；Alpha-cyclodextrin is molten Concentration in liquid B1 is 1g/100mL (mass fraction 1%)；The pH value of solution B 1 is 5.0.

The composition of solution B 2: it is identical as the composition of solution B 1, the difference is that the pH value of solution B 2 is 5.4.

The composition of solution B 3: it is identical as the composition of solution B 1, the difference is that the pH value of solution B 3 is 5.6.

The composition of solution B 4: it is identical as the composition of solution B 1, the difference is that the pH value of solution B 4 is 6.0.

The composition of solution B 5: it is identical as the composition of solution B 1, the difference is that the pH value of solution B 5 is 6.5.

The composition of solution B 6: it is identical as the composition of solution B 1, the difference is that the pH value of solution B 6 is 7.0.

The composition of solution B 7: it is identical as the composition of solution B 1, the difference is that the pH value of solution B 7 is 7.5.

The composition of solution B 8: it is identical as the composition of solution B 1, the difference is that the pH value of solution B 8 is 8.0.

The composition of solution B 9: it is identical as the composition of solution B 1, the difference is that by 50mM Na₂HPO₄-NaH₂PO₄Buffer replaces It is changed to 50mM Tris-HCl buffer.The pH value of solution B 9 is 8.0.

The composition of solution B 10: it is identical as the composition of solution B 1, the difference is that by 50mM Na₂HPO₄-NaH₂PO₄Buffer Replace with 50mM Tris-HCl buffer.The pH value of solution B 10 is 9.0.

The composition of solution B 11: it is identical as the composition of solution B 1, the difference is that by 50mM Na₂HPO₄-NaH₂PO₄Buffer Replace with 50mM glycine-NaOH buffer.The pH value of solution B 11 is 9.0.

The composition of solution B 12: it is identical as the composition of solution B 1, the difference is that by 50mM Na₂HPO₄-NaH₂PO₄Buffer Replace with 50mM glycine-NaOH buffer.The pH value of solution B 12 is 9.5.

The composition of solution B 13: it is identical as the composition of solution B 1, the difference is that by 50mM Na₂HPO₄-NaH₂PO₄Buffer Replace with 50mM glycine-NaOH buffer.The pH value of solution B 13 is 10.0.

By reaction system after 30 DEG C of incubation 30min, boiling water bath 5min terminates reaction, and 0.5mL dinitrosalicylic acid is added Solution (DNS), then boiling water bath 5min, measures OD after cooling rapidly₅₄₀。

Experiment is set to be repeated three times.

As a result as shown in Figure 3.Glycosidase Amy117 all has hydrolyzing alpha-ring paste under conditions of pH is between 5.0 to 10.0 The activity of essence.

Fig. 3 shows that glycosidase Amy117 has highest enzymatic activity under the conditions of pH7.0.With the suction of this highest enzymatic activity system Light value is made as relative activity 100%, the light absorption value of other reaction systems and the ratio of the light absorption value of this highest enzymatic activity system For respective relative activity.There is 60% or more enzyme activity in the range of pH is 5.6-8.0.

Control group: above-mentioned experiment is carried out to compare the albumen (being denoted as control enzyme solution) of bacterium BL21/pET28a acquisition, as a result not Pipe is under the conditions of which pH, activity of the control enzyme solution all without degradation alpha-cyclodextrin.

Experiment sets 3 repetitions, as a result unanimously.

(3) enzyme heat stability

With the 50mM Na of pH7.0₂HPO₄-NaH₂PO₄Buffer dilutes the pure enzyme solution of Amy117 in the step 5 of embodiment 1, Enzyme activity determination is carried out by substrate of alpha-cyclodextrin with the enzyme solution after dilution.Enzyme solution after dilution is denoted as dilution enzyme solution.

Dilution enzyme solution is placed 30 minutes in 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C of water-baths respectively, measures the residual activity of enzyme.Knot Fruit is as shown in Figure 4: enzyme is relatively stable in 20 DEG C to 30 DEG C of enzyme activity, handles at 40 DEG C and loses within 30 minutes 40% activity, at 50 DEG C Processing has completely lost activity in 30 minutes.The hydrolysis temperature of glycosidase Amy117 of the present invention is 20-40 DEG C, preferably 20-30℃。

(4) pH tolerance

Enzyme solution will be diluted respectively under the conditions of 5.0,6.0,7.0,8.0,9.0,10.0,11.0,12.0,13.0 pH, temperature After placing 16h under the conditions of 4.0 DEG C, remnant enzyme activity is measured by substrate of alpha-cyclodextrin.As a result as shown in Figure 5: enzyme is in pH 7.0- Still 50% or more opposite enzyme activity is remained under the conditions of 12.0.Illustrate that the enzyme has good pH tolerance.

(5) substrate specificity

Dilution enzyme solution is subjected to enzyme activity determination under the conditions of the different substrates of same concentrations (mass fraction 1%) respectively, Substrate is respectively alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, pulullan, soluble starch.

Opposite enzyme activity point is surveyed using alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, pulullan, soluble starch by substrate It Wei not 851.9U/mg, 388.4U/mg, 94.8U/mg, 62.4U/mg, 29.3U/mg.It can be seen that the enzyme is living to alpha-cyclodextrin Than highest, other take second place property.Therefore, the most suitable substrate of glycosidase Amy117 of the present invention is alpha-cyclodextrin, most suitable natural substrate For pulullan.

Embodiment 3 verifies protein function by substrate of pulullan

Dilution enzyme solution in following each group is the 50mM Na with pH7.0₂HPO₄-NaH₂PO₄Buffer dilutes embodiment 1 What the pure enzyme solution of Amy117 in step 5 obtained.

Substrate solution composition: by 50mM, pH7.0Na₂HPO₄-NaH₂PO₄Buffer and pulullan composition；The general Shandong of substrate Concentration of the blue sugar in substrate solution is 1g/100mL (mass fraction 1%).

Experimental group: using pulullan as substrate (mass fraction 1%), determination of activity reaction system is 0.5mL, by 0.45mL substrate solution and 0.05mL dilute enzyme solution；The pH value of reaction system is 7.0；By reaction system in 30 DEG C of incubation 30min Afterwards, boiling water bath 5min terminates reaction, is added 0.5mL dinitrosalicylic acid solution (DNS), then boiling water bath 5min, cooling rapidly After measure OD₅₄₀。

Experiment is set to be repeated three times, as a result unanimously.

As the result is shown under the conditions of 30 DEG C, pH7.0, there is certain enzyme activity by substrate of pulullan.

Embodiment 4, the product measurement for hydrolyzing pulullan

Determination step: determination of activity reaction system is 0.5mL, dilutes enzyme solution by 0.45mL substrate solution and 0.05mL；Instead The pH value for answering system is 7.0；By reaction system after 30 DEG C of incubation 30min, boiling water bath 5min terminates reaction, uses efficient liquid phase Chromatography-Evaporative light scattering detector (HPLC-ELSD) carries out the measurement of product, with maltotriose, panose, Isomaltotriose in measurement As standard items (standard concentration 10mg/mL).

Experiment is set to be repeated three times.

As a result as shown in Figure 6: under the conditions of 30 DEG C, pH7.0, the product that glycosidase Amy117 hydrolyzes pulullan is single Panose.

The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Sequence table

<110>Hubei University

<120>a kind of degradation pulullan generates glycosidase and its encoding gene and the application of single panose

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 589

<212> PRT

<213>Alkaliphilic bacillus 703 (Bacillus sp. 703)

<400> 1

Gln Lys Glu Ala Ile Tyr His Arg Pro Lys Asn Glu Phe Ala Tyr Ala

1 5 10 15

Tyr Asn Glu Arg Asp Leu His Ile Arg Leu Lys Thr Lys Lys Asp Asp

20 25 30

Val Glu Glu Val Glu Leu Leu Tyr Gly Asp Pro Tyr Glu Trp Asp Lys

35 40 45

Glu Thr Gly Trp Met Phe Gln Thr Glu Lys Met Met Lys Thr Gly Ser

50 55 60

Asp Asp Leu Phe Asp Tyr Trp Phe Lys Glu Val Ser Pro Pro Phe Arg

65 70 75 80

Arg Leu Arg Tyr Gly Phe Arg Leu Arg Ser Lys Asn Glu Cys Leu Thr

85 90 95

Tyr Thr Glu Arg Gly Phe Tyr Glu Asn Ser Pro Ala Asp Ala Gly Phe

100 105 110

Tyr Phe Cys Phe Pro Phe Leu Asn Glu Val Asp Val Phe Ala Pro Pro

115 120 125

Glu Trp Val Lys Asp Thr Val Trp Tyr Gln Ile Phe Pro Glu Arg Phe

130 135 140

Ala Asn Gly Asp Lys Ser Leu Asn Pro Asp Gly Thr Leu Pro Trp Gly

145 150 155 160

Ser Thr Ser Pro Thr Pro Thr Asn Phe Phe Gly Gly Asp Leu Val Gly

165 170 175

Ile Glu Asn His Leu Asp Tyr Leu Ser Gln Leu Gly Ile Ser Gly Ile

180 185 190

Tyr Leu Thr Pro Ile Phe Lys Ala His Ser Asn His Lys Tyr Asp Thr

195 200 205

Ile Asp Tyr Met Glu Ile Asp Pro Gln Phe Gly Thr Lys Asn Thr Phe

210 215 220

Lys His Phe Val Lys Ala Cys His Ala Arg Gly Ile Arg Val Met Leu

225 230 235 240

Asp Ala Val Phe Asn His Ser Gly Tyr Tyr Phe Glu Pro Phe Gln Asp

245 250 255

Val Leu Lys Asn Gly Lys Asp Ser Asn Tyr Arg Glu Trp Phe His Ile

260 265 270

His Glu Phe Pro Ile Thr Thr Ile Glu Glu Ala Asp Gly Asp Val Arg

275 280 285

Pro Asn Tyr Asp Ala Phe Ala Phe Val Pro Thr Met Pro Lys Leu Asn

290 295 300

Thr Glu His Pro Glu Val Lys Glu Tyr Leu Leu Asn Val Ala Ala Tyr

305 310 315 320

Trp Ile Arg Glu Phe Asp Ile Asp Gly Trp Arg Leu Asp Val Ala Asn

325 330 335

Glu Ile Asp His Ser Phe Trp Arg Glu Phe Arg Lys Val Val Lys Asp

340 345 350

Ile Lys Pro Asp Val Tyr Ile Leu Gly Glu Ile Trp His Asp Ser Met

355 360 365

Pro Trp Leu Leu Gly Asp Gln Phe Asp Ala Val Met Asn Tyr Pro Val

370 375 380

Thr Asn Ala Ile Ile Asp Phe Ala Gly Lys His Ser Ile Asn Ser Lys

385 390 395 400

Ala Phe Ile Glu Gln Phe Thr Lys Leu Leu His Met Tyr Pro Arg Ser

405 410 415

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

420 425 430

Ile Leu Thr Leu Ser Ala Asn Asp Glu Asp Leu Val Lys Leu Gln Leu

435 440 445

Leu Leu Gln Phe Ser Leu Pro Gly Thr Pro Cys Ile Tyr Tyr Gly Asp

450 455 460

Glu Ile Gly Met Thr Gly Gly Ala Asp Pro Gly Cys Arg Glu Cys Met

465 470 475 480

Val Trp Asp Glu Glu Lys Gln Asn Gln Pro Leu Phe Lys Tyr Val Gln

485 490 495

Gln Leu Ile Gln Leu Arg Thr Gln Ser Glu Ala Phe Gly Thr Arg Gly

500 505 510

Asp Leu Thr Phe Val Pro Thr Asn Leu Ala Ser Pro Leu Leu Ser Tyr

515 520 525

Ile Lys Val Thr Lys Asn Glu Gln Leu Leu Phe Val Ile Asn Ala Glu

530 535 540

Arg Glu Ser His Glu Phe Glu Leu Pro Asp Glu Phe Ile Asn Leu Pro

545 550 555 560

Thr Lys Glu Leu Phe Ser Glu Gln Val Met Glu Pro Ser Ala Lys Ile

565 570 575

Arg Ile Gly Pro Lys Gln Ala Ile Val Leu Lys Lys Leu

580 585

<210> 2

<211> 1770

<212> DNA

<213>Alkaliphilic bacillus 703 (Bacillus sp. 703)

<400> 2

caaaaggaag ccatttatca ccgtccaaaa aacgaattcg cttatgcgta taatgaaaga 60

gatttacata ttcgtttaaa gacaaaaaaa gatgatgtag aagaggtcga gctcctatac 120

ggagatcctt atgagtggga taaggaaact ggctggatgt ttcaaactga aaagatgatg 180

aaaacgggaa gtgatgacct ttttgattat tggtttaaag aagtctctcc cccttttcgc 240

cgcttaagat atggatttcg ccttcgttca aagaatgagt gtttaacata tacagaacga 300

gggttttatg agaattcgcc agctgatgct ggattttact tttgttttcc ttttttaaat 360

gaggttgacg ttttcgcgcc tccagaatgg gtaaaggaca cagtctggta tcagatcttc 420

cctgaaaggt ttgctaatgg tgataagtct cttaatcctg atggcacctt gccttgggga 480

agtacgtctc ctacaccgac aaatttcttt ggcggcgatc ttgtaggaat tgagaatcat 540

ctagattatc tatcccaatt aggaattagc ggcatctacc tcaccccgat ttttaaagcc 600

cattccaatc ataagtatga cacgattgac tacatggaaa ttgatccaca attcggtact 660

aagaacacgt ttaaacactt tgtcaaagct tgtcatgcta gaggtattcg agtaatgtta 720

gatgctgtat ttaatcactc aggctactat tttgaacctt ttcaagacgt cttaaaaaat 780

gggaaggatt cgaactaccg tgaatggttt catattcatg agtttccaat cactacaata 840

gaagaagctg acggtgacgt gcgtccaaat tatgatgcgt ttgcttttgt tccaacaatg 900

cctaagttaa acacagaaca tccagaggtg aaagaatacc tgctaaacgt agctgcttat 960

tggattcgtg aatttgatat tgacggctgg cgtttagacg ttgcaaatga aattgatcat 1020

tccttttgga gagagttcag gaaagtagta aaagatataa aacctgatgt gtatatatta 1080

ggagagattt ggcatgattc aatgccttgg cttctaggag accaatttga cgcagtaatg 1140

aactatcctg tgactaacgc cattattgac tttgcaggaa aacattcaat taacagtaaa 1200

gcgttcattg aacaatttac aaaattactg catatgtacc ctcgttctgt tcaagaagtt 1260

gcttttaatt tattgggaag tcatgataca ccaaggatat taacgctctc agccaacgat 1320

gaagatctgg ttaagcttca gttgttactc caattttctc tgcctggaac cccttgtatt 1380

tactacggag atgagatcgg tatgactggc ggtgctgacc ctgggtgcag agaatgtatg 1440

gtttgggatg aagagaagca aaaccaacca ttattcaaat atgtacaaca gctcattcaa 1500

cttcgtactc agtccgaagc ctttggcacg agaggtgatt taacgtttgt tccaacaaac 1560

cttgcctccc ccctcctctc ttatataaag gtcacaaaga atgagcagct gttatttgtc 1620

ataaatgcag aaagagaatc tcatgaattc gagttgcctg atgagtttat taacctgcca 1680

acgaaggaat tattttctga gcaggttatg gagccatctg ctaaaatcag gataggtcct 1740

aaacaagcca ttgttttaaa gaagctttaa 1770

<210> 3

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cgcggatccc aaaaggaagc catttatcac cgtc 34

<210> 4

<211> 38

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ccgctcgagt taaagcttct ttaaaacaat ggcttgtt 38

Claims

1. a kind of glycosidase, which is characterized in that the enzyme includes amino acid sequence shown in SEQ ID NO.1.

2. a kind of encoding gene of glycosidase, which is characterized in that gene coding has amino acid sequence shown in SEQ ID NO.1 The protein of column.

3. a kind of encoding gene of glycosidase according to claim 2, which is characterized in that the gene has SEQ ID Nucleotide sequence shown in NO.2.

4. a kind of recombinant vector, which is characterized in that the encoding gene comprising glycosidase described in claim 2 or 3.

5. a kind of transformant, which is characterized in that include recombinant vector as claimed in claim 4.

6. a kind of primer pair, which is characterized in that for expand glycosidase described in claim 2 or 3 encoding gene overall length and Its any segment；It is preferred that the sequence of primer pair is as shown in SEQ ID NO.3 and SEQ ID NO.4.

7. a kind of glycosidase as described in claim 1 answering in hydrolysis cyclodextrin, pulullan or/and soluble starch It is chosen from the followings more than either one or two with, the cyclodextrin: alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin.

8. a kind of glycosidase as described in claim 1 is the application in single product panose in hydrolysis pulullan.

9. a kind of method digested using glycosidase described in claim 1, which is characterized in that this method includes following step It is rapid: using cyclodextrin, pulullan or/and soluble starch as substrate, in pH5.4-9.0, the reaction system that temperature is 20-40 DEG C Under the conditions of, cyclodextrin, pulullan or/and soluble starch are digested using glycosidase.

10. according to the method described in claim 9, temperature is 30 DEG C it is characterized in that, the pH value of enzymatic hydrolysis is 5.6-8.0.