CN108277176A

CN108277176A - Alkalescent xylanase and the application of a kind of basophilic streptomycete and its generation

Info

Publication number: CN108277176A
Application number: CN201810096053.6A
Authority: CN
Inventors: 刘森林; 周鹏; 陈伟钊
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2018-01-31
Filing date: 2018-01-31
Publication date: 2018-07-13
Anticipated expiration: 2038-01-31
Also published as: CN108277176B

Abstract

The present invention discloses alkalescent xylanase and the application of a kind of basophilic streptomycete and its generation.The basophilic streptomycete is the production alkalescent xylanase bacterial strain screened from the soil of mangrove.The invention also discloses the alkalescent xylanase that above-mentioned basophilic streptomycete generates, which there is pH wide adaptation ranges, heat-resisting, alkali, metal ion and surfactant etc. to be suitable for the excellent enzymatic property of the industry such as papermaking, can be applied in the industry such as papermaking.The basophilic streptomyces of the present invention can cultivate under highly alkaline conditions in alkaline-resisting extreme microorganism, have the characteristics of can effectively inhibiting living contaminants, being conducive to large-scale fermenting and producing.

Description

Alkalescent xylanase and the application of a kind of basophilic streptomycete and its generation

Technical field

The invention belongs to biotechnology, more particularly to the alkalescent xylanase of a kind of basophilic streptomycete and its generation and Using.

Background technology

Xylan is the hemicellulose by β-Isosorbide-5-Nitrae key connection as unit of xylopyranose, rich in broad leaf tree and mostly In number annual plant body.And hemicellulose content in nature accounts for the 30% of biomass, is only second to cellulose.

Zytase is the enzyme system general name at xylo-oligosaccharide or wood oligose, including a variety of xylans by xylan catalyzing hydrolysis Restriction endonuclease and excision enzyme are distributed widely in bacterium, fungi, yeast, actinomyces, ruminant tumor gastric, snail, crustacean, land In ground plant tissue and various invertebrates, wherein the neutrality of bacterial origin or alkalescent xylanase have higher alkali resistance And thermal stability.Zytase has a wide range of applications, and in paper industry, can be applied to association with pulp bleaching, deinking；It is raising Expect in industry, zytase can reduce chyme stickiness caused by Wheat-Based Diets Water soluble pentosan and increase, to improve digestion Enzyme is to the functioning efficiency of substrate, while zytase may act on insoluble non-starch polysaccharide, is crushed plant cell wall, and discharge Go out nutriment；It is making wine and is being applied in food service industry also widely, if zytase is added in flour, face can be improved Retentiveness, stability and the tolerance to excessive fermentation of group improve and suddenly rise performance into stove, increase the volume that baking is wrapped below, Improve crumb texture, reduce the rate of ageing of bread, extends shelf life.

According to catalysis domain amino acid sequence and the similitude of structure, zytase is divided into 10 family of glycoside hydrolase more Or the 11st family.In addition, the 5th family, the 7th family, the 8th family or the 43rd family zytase also have been reported that.Most of wood Dextranase belongs to two large family of F/10 and G/11.G/11 zytases are mostly single domain, and relative molecular mass is smaller, catalysis production Oligosaccharide is more in object, and monosaccharide is less, and the optimum temperature of enzyme is 50~60 DEG C, and the space structure of enzyme is in " right hand is partly held Shape ".F/10 zytases then contain compared with Multidomain, not only have catalyst structure domain, but also also cellulose binding domain (Cellulose Binding Domain:CBD), relative molecular mass is larger, and monosaccharide is more in catalysate, most suitable action temperature Degree is 60~80 DEG C, and the space structure of enzyme is in " bowl-shape ".Compared with G/11 families, F/10 families zytase is in high temperature resistant, resistance to Sour and alkaline-resisting etc. more superiority.In terms of codon preference, the codon of F/10 xylanase genes is with " A " base Based on, and G/11 families are based on " G/C " base.Currently, industrially directly there is also some problems using zytase：Stablize Property is poor, substrate specificity is low, service life is short and of high cost etc..As a kind of microorganism formulation, zytase is to temperature, humidity There is certain requirement with acid-base value.In order to adapt to the needs of different feeds processing technology and application, need to often screen heat safe Strain, or using genetic engineering and the attribute of protein engineering means transformation zytase, to meet various factors to xylan The requirement of enzyme.The zytase produced at this stage is the zytase of originated from fungus, and the Optimun pH of these enzymes is universal Less than 5.5.In addition, zytase specificity is low, direct utilizing status is poor, therefore should also further investigate substrate kind and quality The relationship of concentration and zytase dosage inquires into the effect between different enzyme preparations, improves the substrate specificity of zytase.

Invention content

In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of basophilic streptomycete Streptomyces sp.WMN-3.The basophilic streptomycete can generate a kind of alkalescent xylanase.

Another object of the present invention is to provide the alkalinity that above-mentioned basophilic streptomycete Streptomyces sp.WMN-3 are generated Zytase.The enzyme has pH wide adaptation ranges, and it is industrial that heat-resisting, alkali, metal ion and surfactant etc. are suitable for papermaking etc. Excellent enzymatic property.

It is still another object of the present invention to provide the applications of above-mentioned basophilic streptomycete and its alkalescent xylanase of generation.

The purpose of the invention is achieved by the following technical solution：

A kind of basophilic streptomycete is the production alkalescent xylanase bacterial strain screened from mangrove forest soil Streptomyces sp.WMN-3.The bacterial strain depositary institution：China typical culture collection center (CCTCC), preservation day Phase：On September 7th, 2017, preservation address：The Chinese Wuhan Wuhan Universitys, deposit number：CCTCC NO：M 2017477.

Colony morphology characteristic：Colony diameter on agar plate is generally 1~1.5 μm, is in rice white, irregular roundness Shape, protrusion, full edge.

The agar plate, formula are agar 20g/L, glucose 10g/L, peptone 5g/L, yeast extract 5g/ L, KH₂PO₄1g/L, MgCl₂0.2g/L, NaCl 50g/L, Na₂CO₃10g/L, pH 9.0；

The Selective agar medium formula of the bacterial strain：Xylan 5.0~8.0g/L, KNO₃1.0g/L, MgSO₄·7H₂O 0.5g/ L, NaCl 10~15g/L, KH₂PO₄1.5g/L, solid medium add 15~20g/L of agar, pH8.0~9.0；Cultivation temperature is 30~37 DEG C.

The alkalescent xylanase that the basophilic streptomycete Streptomyces sp.WMN-3 are generated, is made as follows：

(1) extraction of crude enzyme liquid

Streptomyces sp.WMN-3 strains are connected in liquid selective medium, 48h is cultivated in 37 DEG C, 200rpm, By 48h culture solutions with the rotating speed refrigerated centrifuge 20min of 14000rpm, supernatant is taken；Film is concentrated by ultrafiltration using 3000 dalton will Supernatant is concentrated by ultrafiltration to get crude enzyme liquid, is used for ion-exchange chromatography；

(2) alkalescent xylanase isolates and purifies

(a) anion-exchange chromatography

The medium of ion-exchange chromatography is DEAE Sepharose Fast Flow；Buffer solution system uses pH value for 8.0 Tris-HCl buffer solutions, equilibrium liquid be pH 8.0Tris-HCl buffer solutions, eluent be the 8.0Tris-HCl of pH containing NaCl delay Fliud flushing；100mL media are taken carefully to pour into chromatographic column (specification be Φ 2.6cm × 30cm) so that gel is uniformly and bubble-free；With The equilibrium liquid of 300mL is balanced, until loading (crude enzyme liquid) after A280 stabilizations, peak to be penetrated is collected after occurring and penetrates peak, Dialysis, concentration；

(b) cation-exchange chromatography

Chromatography media is SP Sepharose Fast Flow；Buffer solution system use pH value for 6.0 PBS buffer solution； Dialysis concentration in enzyme activity peak obtained by anion-exchange chromatography, it is added to the layer that the PBS buffer solution for being in advance 6.0 with pH value has balanced It analyses on column, with 60mLh^-1Flow velocity eluted with the PBS buffer solution of the pH6.0 containing NaCl, collect the eluting peak with enzymatic activity, It is concentrated by dialysing, obtains alkalescent xylanase, be placed in 4 DEG C and save backup.

The molecular weight of albumen of the alkalescent xylanase is about 39KD, and Rate activity reaches 131.2IU/mL；Optimal pH is 7~9 Left and right, in the ranges of pH6~11, enzymatic activity is positively retained at 80% or more；Optimal reactive temperature is 50~60 DEG C or so；Have Resistance to Co²⁺、Mn²⁺、Zn²⁺、Ca²⁺、Li⁺、Fe³⁺、Ag⁺、Al³⁺、Mg²⁺The superperformance of equal metal ions, but Cu²⁺To alkaline xylan The enzymatic activity of enzyme has certain inhibiting effect；SDS and chelating agent EDTA influences the enzymatic activity of alkalescent xylanase little.

The basophilic streptomycete Streptomyces sp.WMN-3 and its alkalescent xylanase of generation are in industry such as papermaking In application.

The present invention has the following advantages and effects with respect to the prior art：

(1) the Streptomyces sp.WMN-3 that present invention screening obtains belong to alkaline-resisting extreme microorganism, can be high-alkali Property under the conditions of (pH9.0) cultivate, there is the characteristics of can effectively inhibiting living contaminants, being conducive to large-scale fermenting and producing.

(2) Rate activity for the alkalescent xylanase that basophilic streptomycete Streptomyces sp.WMN-3 of the present invention are generated reaches 131.2IU/mL；Optimal pH is 7~9 or so, and in the ranges of pH6~11, enzymatic activity is positively retained at 80% or more；Optimal reaction Temperature is 50~60 DEG C or so；Stablize in 50 DEG C or less enzymatic activitys.With resistance to Co²⁺、Mn²⁺、Zn²⁺、Ca²⁺、Li⁺、Fe³⁺、Ag⁺、 Al³⁺、Mg²⁺The superperformance of equal metal ions, but Cu²⁺There is certain inhibiting effect to the enzymatic activity of alkalescent xylanase；SDS The enzymatic activity of alkalescent xylanase is influenced with chelating agent EDTA little.

Description of the drawings

Fig. 1 is the result of transparent circle caused by Streptomyces sp.WMN-3 bacterium grow on 1% Congo red tablet Figure.

The formula of the 1% Congo red tablet is：Congo red 10g/L, xylan 8.0g/L, KNO₃1.0g/L MgSO₄·7H₂O 0.5g/L, NaCl 15g/L, KH₂PO₄1.5g/L, solid medium add agar 15~20g/L, pH 9.0.

Fig. 2 is xylan canonical plotting.

Fig. 3 is the SDS-PAGE electrophoresis result figures isolated and purified；Wherein：Swimming lane M is marker, public from Fermentas Department；Swimming lane 1 is the destination protein of purifying.

Fig. 4 is the result figure of the optimal reaction pH of alkalescent xylanase WMN-3.

Fig. 5 is the result figure of the optimal reactive temperature (DEG C) of alkalescent xylanase WMN-3.

Fig. 6 is the result figure of the pH stability of alkalescent xylanase WMN-3.

Fig. 7 is the result figure of the temperature stability of alkalescent xylanase WMN-3.

Fig. 8 is Streptomyces sp.WMN-3 bacterium genome dna electrophoresis figures；Wherein：Swimming lane M is takara companies DL15000Marker；Swimming lane 1 is genomic DNA.

Fig. 9 is the electrophoretogram of alkalescent xylanase WMN-3 gene conserved sequence PCR results；Wherein：Swimming lane M is takara The DL5000Marker of company；Swimming lane 1 is WMN-3 gene conserved DNA sequences.

Figure 10 is the electrophoretogram of the upstream PCR results of the conserved DNA sequences of alkalescent xylanase WMN-3 genes；

Wherein：Swimming lane M is the DL5000Marker of takara companies；Swimming lane 1 is the conserved DNA sequences of WMN-3 genes Upstream PCR results.

Figure 11 is the electrophoretogram of the downstream PCR result of the conserved DNA sequences of alkalescent xylanase WMN-3 genes；

Wherein：Swimming lane M is the DL5000Marker of takara companies；Swimming lane 1 is the conserved DNA sequences of WMN-3 genes Downstream PCR result.

Specific implementation mode

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

The experimental method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system Make the experiment condition proposed by manufacturer.

1 bacterial strain of embodiment is separately cultured

1, enrichment culture

Screening obtains production alkalescent xylanase bacterial strain from the soil of mangrove.1g soil samples are weighed, the nothing of 100mL is added to It in bacterium water, fully shakes up, bacteria suspension is made, 1mL bacteria suspensions is taken to be linked into the 5mL enriched mediums for bacterium of having gone out in advance respectively, 37 DEG C, 200rpm enrichment cultures 48h.

Enriched medium：Xylan 8.0g/L, peptone 10g/L, NaCl 15g/L, KH₂PO₄1.5g/L, Na₂HPO₄· 12H₂O9.0g/L, MgSO₄·7H₂O 2.0g/L, pH 9.0.

2, tablet primary dcreening operation

Bacterium solution 10 after enrichment culture is diluted step by step again, it is 10 to take extension rate respectively^-3、10^-5、10^-7Bacteria suspension 0.5mL is coated on the alkalinity selection culture medium flat plate containing xylan, and 37 DEG C of culture 48h choose the larger single bacterium colony of transparent circle Enter alkaline slant medium, repeatedly repeatedly, until being determined as pure bacterium, uses slant medium preservation original strain.

Selective agar medium：Xylan 8.0g/L, KNO₃1.0g/L, MgSO₄·7H₂O 0.5g/L, NaCl 15g/L, KH₂PO₄1.5g/L, solid medium add agar 15~20g/L, pH9.0；Cultivation temperature is 37 DEG C.

Slant medium：Glucose 10g/L, peptone 5g/L, yeast extract 5g/L, KH₂PO₄1g/L, MgCl₂ 0.2g/L, NaCl 50g/L, Na₂CO₃10g/L, pH 9.0.

3, the selection result of alkalescent xylanase bacterial strain is produced

The present invention is from the soil sample of selection, by the Congo red plate screening containing xylan, it was found that one can generate The bacterial strain (such as Fig. 1) of apparent transparent circle.3% wheat bran fermentation medium (wheat bran 3.0g/L, peptone 10g/L, NaCl 15g/L, KH₂PO₄1.5g/L, Na₂HPO₄·12H₂O 9.0g/L, MgSO₄·7H₂O 2.0g/L, pH 9.0) 37 DEG C, 200rpm fermentation trainings After supporting 7 days, crude enzyme liquid enzyme activity is measured, it is found that its activity is up to 131.2IU/mL.Show it with higher enzymatic productivity.It utilizes The 16S rDNA sequences of the PCR amplification bacterial strain, to GENBANK on be compared, comparison result is shown, the 16S rDNA of the bacterial strain Sequence and the 16S rDNA sequences of other more streptomycetes have 99% similitude, are tentatively judged as streptomycete (Streptomyces sp.), is named as Streptomyces sp.WMN-3.

The basophilic streptomycete, entitled Streptomyces sp.WMN-3, the bacterial strain depositary institution：Chinese Typical Representative is trained Support object collection (CCTCC), preservation date：On September 7th, 2017, preservation address：The Chinese Wuhan Wuhan Universitys, preservation are compiled Number：CCTCC NO：M 2017477.

Compared with current industrial enzyme bacterial strain, Streptomyces sp.WMN-3 Pseudomonas, can in alkaline-resisting extreme microorganism (pH9.0) is cultivated under highly alkaline conditions, and living contaminants can effectively be inhibited by having, and be conducive to the spy of large-scale fermenting and producing Point.

The 16S rDNA sequences such as SEQ ID No of the Streptomyces sp.WMN-3 bacterial strains：Shown in 1, length For 1540bp.

The alkalescent xylanase that 2 basophilic streptomycete Streptomyces sp.WMN-3 of embodiment are generated isolates and purifies

(1) material

1, strain

Basophilic streptomycete is the production alkalescent xylanase bacterial strain Streptomyces screened from the soil of mangrove sp.WMN-3(CCTCC NO：M 2017477).

2, culture medium

3, main agents

Ion-exchange chromatography media DEAE Sepharose Fast Flow, SP-sepharose Fast Flow cations Displacement chromatography medium is purchased from GE companies.Acrylamide (acrylamide), N, N- methylene diacrylamides are by Serva imports point Dress.Archaeal dna polymerase, DNA molecular amount are labeled as precious biotech firm product；Protein markers are Fermentas (MBI) company Product；Ethylenediamine tetra-acetic acid (EDTA), ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) (EGTA), guanidine hydrochloride, glycine, Triton X- 100 remaining equal reagent are that domestic analysis is pure.PCR primer synthesis is completed by Shanghai life work, and determined dna sequence is by Invitrogen Company completes；Gene extracts kit is Oemga Products.

4, instrument

Thermal cycler PCR instruments are Applied Biosystems By Life Technologies companies, DNA And protein electrophoresis system is Bio-rad Products；Thermomixer comfort temperature controls shaker, liquid-transfering gun, desk-top centrifugation Machine is Eppendorf Products；Low-temperature and high-speed centrifuge is Sigma Products；Dolphin-DOC gel imaging systems are U.S.'s WEALTEC Products；Constant-temperature table and thermostat water bath are WAGEN Products.

(2) experimental method

1, the extraction of crude enzyme liquid

2, enzyme activity determination method

It takes the Tris-HCl buffer solutions of 1mL pH=9 to prepare 1% xylan solution, 100 μ L is added through appropriate diluted Above-mentioned enzyme solution, the oscillating reactions 15min at 40 DEG C after terminating reaction, add rapidly 1mL DNS (3,5- dinitrosalicylic acid) solution Then boiling water bath 10min adds 2mL deionized waters, measure reduced sugar in 540nm, and deduct blank test measured value.By above-mentioned item The enzyme amount per minute by needed for substrate 1 μm of ol xylan of generation is defined as an enzyme activity international unit under part, with IU/mL tables Show.

3, xylan standard curve

Accurately weigh anhydrous xylan 1g and be settled to 100mL, then take respectively 1% standard xylan liquid 0.25,0.5, 0.75,1,1.25,1.5mL is settled to 100mL, then above-mentioned each 1.1mL of various concentration solution is taken (separately to set a pipe in test tube respectively 1.1mL distilled water is taken to compare), respectively add 1mL DNS, boil colour developing 10min, the absorbance surveyed at 540nm after cooling (is surveyed 3 times It is averaged).Using absorbance as ordinate, corresponding standard xylan solution sugar content is abscissa, draws standard curve (as schemed 2)。

4, SDS- polyacrylamide gel electrophoresises

SDS- polyacrylamide gel electrophoresises according to a conventional method, separation gel, concentration gum concentration are respectively 12% and 5%, electricity Pole buffer solution is pH8.3Tris-Gly buffer solutions, coomassie brilliant blue staining.

5, alkalescent xylanase isolates and purifies

(a) anion-exchange chromatography

The medium of ion-exchange chromatography is DEAE Sepharose Fast Flow；Buffer solution system uses pH value for 8.0 Tris-HCl buffer solutions, equilibrium liquid be pH 8.0Tris-HCl buffer solutions, eluent be the 8.0Tris-HCl of pH containing NaCl delay Fliud flushing；100mL DEAE Sepharose Fast Flow (GE) are taken carefully to pour into the chromatographic column that specification is Φ 2.6cm × 30cm (GE) in so that gel uniformly and bubble-free；It is balanced with the equilibrium liquid of 300mL, until loading (thick enzyme after A280 stabilizations Liquid), peak to be penetrated is collected after occurring and penetrates peak, dialysis, concentration.

(b) cation-exchange chromatography

Chromatography media is SP Sepharose Fast Flow；Buffer solution system use pH value for 6.0 PBS buffer solution； Dialysis concentration in enzyme activity peak obtained by anion-exchange chromatography, it is added to the layer that the PBS buffer solution for being in advance 6.0 with pH value has balanced It analyses on column, with 60mLh^-1Flow velocity eluted with the PBS buffer solution of the pH6.0 containing NaCl, collect the eluting peak with enzymatic activity, It is concentrated by dialysing, obtains the pure enzyme solution of alkalescent xylanase, be placed in 4 DEG C and save backup.

(3) experimental result

With SDS-PAGE electrophoresis technique determinings, the results are shown in Figure 3, the visible destination protein band apparent together of swimming lane 1, substantially Reach and isolates and purifies requirement.After electrophoresis gel image scanning is carried out with GelDoc2000 (Bio-Rad).Software is carried with scanning system Quantity One carry out molecular weight determination, and the molecular weight of albumen for measuring alkalescent xylanase is about 39KD.

The zymologic property research of 3 alkalescent xylanase of embodiment

(1) experimental method

1, optimal reaction pH and pH stability

Take the pure enzyme solution of the alkalescent xylanase of the acquisition of appropriate embodiment 2,1% xylan for being separately added into different pH value molten Liquid measures enzyme activity according to a conventional method.Meanwhile pure enzyme solution being placed in respectively under the conditions of different predetermined pHs and preserving 30min, then press Conventional method measures its enzyme activity under the conditions of pH 9.0.

2, optimal reactive temperature and temperature stability research

It takes the pure enzyme solution of the alkalescent xylanase of the acquisition of appropriate embodiment 2 to be respectively placed under condition of different temperatures to react 20min measures its enzyme activity.Meanwhile pure enzyme solution being respectively placed under different temperature conditions (20 DEG C~70 DEG C) heat preservations Then 10min reacts 20min in 40 DEG C and measures its enzyme activity.

3, the influence of metal ion, surfactant and metallo-chelate to enzyme activity

The pure enzyme solution of the alkalescent xylanase of the acquisition of appropriate embodiment 2 is taken to be respectively placed in the various metals of final concentration of 10mM Ion and 0.2%, 0.5%EDTA, 0.01%, in 0.05%SDS (dodecyl sodium sulfate), 30 DEG C preserve 10min after press More solito measures its enzyme activity.

(2) experimental result

1, optimal reaction pH

Appropriate pure enzyme solution is taken, 1% xylan solution of different pH value is separately added into, measures enzyme activity according to a conventional method, is tied Fruit is as shown in Figure 4.From fig. 4, it can be seen that the optimal pH of WMN-3 enzymes is 8 or so, it is a typical alkalescent xylanase.

2, optimal reactive temperature

Pure enzyme solution is respectively placed under condition of different temperatures and reacts 20min, measures its enzyme activity, as a result such as Fig. 5.By Fig. 5 As it can be seen that the optimal reactive temperature of WMN-3 enzymes is 55 DEG C.

3, pH stability

To study the pH stability of WMN-3 albumen, pure enzyme solution is placed in respectively under the conditions of different predetermined pHs and is preserved 30min, then its enzyme activity is measured under the conditions of pH 9.0 according to a conventional method.As a result show the enzyme pH 5~9 (such as Fig. 6) In range, enzymatic activity can keep 60% or more.It can be seen that the alkalescent xylanase enzyme has relatively by force under weak acid and weak basic condition Stability.

4, temperature stability research

Pure enzyme solution is respectively placed under different temperature conditions (20 DEG C~70 DEG C) heat preservation 30min, then in 40 DEG C of reactions 20min measures its enzyme activity.As a result (such as Fig. 7) shows that 65 DEG C of enzyme or less has better stability, shows WMN-3 zymoproteins For excellent heat resistance alkalescent xylanase, good application prospect is shown in terms of production application.

5, the influence of metal ion, surfactant and metallo-chelate to enzyme activity

The WMN-3 zymoproteins that purifying obtains are respectively placed in each metal ion species of final concentration of 10mM, mass percent 0.2%, 0.5%EDTA, 0.01%, in 0.05%SDS, 30 DEG C preserve 10min after conventionally measure its enzyme activity, tie Fruit is as shown in table 1.The result shows that WMN-3 zymoproteins are in 10mM Co²⁺、Mn²⁺、Zn²⁺、Ca²⁺、Li⁺、Fe³⁺、Ag⁺、Al³⁺、Mg²⁺ Enzyme activity in equal metal ions is held in 90% or more, the superperformance with resistant to many metal ion, but Cu²⁺Have to enzyme activity Certain inhibiting effect.SDS and chelating agent EDTA influences WMN-3 enzymatic activitys little.As it can be seen that WMN-3 enzymes have resistant to many gold The good characteristic for belonging to ion and surfactant, meets the basic demand of the industrial enzymes such as papermaking.

1 metal ion of table, the influence of surfactant and metallo-chelate to enzyme activity

Embodiment 4

(1) material

1, strain

Basophilic streptomycete is the production alkalescent xylanase bacterial strain Streptomyces screened from the soil of mangrove sp.WMN-3(CCTCC NO：M 2017477).E.coli TOP10F ' are purchased from Invitrogen companies；

2, carrier.E. coli cloning vector pMD18-T is purchased from Dalian treasured biotech firm, coli expression carrier pET- 28a (+) (Novagen, KanR) is purchased from Novagen companies.

3, culture medium

(1) Selective agar medium：Xylan 8.0g/L, KNO₃1.0g/L, MgSO₄·7H₂O 0.5g/L, NaC1 15g/L, KH₂PO₄1.5g/L, solid medium add agar 15~20g/L, pH 9.0；

(2) LB culture mediums：Tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, solid medium add agar 9.0,121 DEG C of high pressure sterilization 20min of 15~20g/L, pH.

4, main agents

Archaeal dna polymerase, DNA molecular amount are labeled as precious biotech firm product；Lysozyme(DNase RNase non- Detected,>70,000U/mg), it is that work bioengineering is given birth in Shanghai that one-step method, which quickly prepares competent cell kit (SSCS), Products.PCR primer synthesis is completed by Shanghai life work, and determined dna sequence is completed by Invitrogen companies；Gene extraction examination Agent box, PCR product fragment purification kit, plastic recovery kit, plasmid extraction kit are Oemga Products.

5, instrument

Thermal cycler PCR instruments are Applied Biosystems By Life Technologies companies, DNA Electrophoresis system is Bio-rad Products；Spectrophotometer and micro-spectrophotometer produce for Pharmacia Biotech companies Product；Thermomixer comfort temperature controls shaker, liquid-transfering gun, desk centrifuge are Eppendorf Products；Low temperature is high Fast centrifuge is Sigma Products；Dolphin-DOC gel imaging systems are U.S.'s WEALTEC Products；Constant-temperature table And thermostat water bath is WAGEN Products.

(2) experimental method and result

1, the extraction of genomic DNA

Thalline were collected by centrifugation after 37 DEG C, 200r/min shaking flask culture Streptomyces sp.WMN-3 bacterium, 48h, then Genome DNA is extracted using The E.Z.N.A Bacterial DNA Kit kits.Its basic step is as follows：

(a) culture medium inoculated Streptomyces sp.WMN-3 bacterium are put into temperature control shaking table, in 37 DEG C, 200rpm shaking flasks Taking-up packing is spare after cultivating 36h.

(b) by 3mL Streptomyces sp.WMN-3 bacterium solutions, 8000rpm centrifuges 10min at ambient temperature；

(c) liquid is discarded supernatant, precipitation is retained, 180 μ L TE buffer is then added, thalline is resuspended, add 20 μ L Water-bath 10min under the conditions of 30 DEG C of 50mg/mL lysozyme solns；

(d) 8000rpm centrifuges 5min at ambient temperature after the completion of water-bath, removes supernatant, and 200 μ L BTL are added Buffer, of short duration vortex；

(e) 25~40mg beades, high speed vortex 5min is added；

(f) 25 μ L Proteinase K Solutions, of short duration vortex, then 55 DEG C of water-bath 60min is added；

(g) 5 μ L RNaseA solution are added, overturns repeatedly, is then placed at room temperature for 5min；

(h) 12000rpm centrifuges 5min under room temperature, draws in supernatant to another clean centrifuge tube, avoids inhaling To precipitation；

(i) 220 μ L BDL buffer, of short duration vortex, then 65 DEG C of water-bath 10min is added；

(j) 220 μ L absolute ethyl alcohols are added, then mixed liquor is transferred to and is cased with 2.0mL collecting pipes by high speed vortex 20s In Akibaiin Column, 12000rpm centrifuges 1min；

(k) liquid in collecting pipe is discarded, the HB buffer of 500 μ L are added, then 10000rpm centrifuges 1min；

(l) liquid in collecting pipe is discarded, the DNA Wash buffer of 700 μ L are added, then 10000rpm is centrifuged 1min；

(m) the DNA Wash buffer that 700 μ L are added are repeated, then 10000rpm centrifuges 1min；

(n) liquid in collecting pipe is discarded, then 2min is centrifuged to eliminate remaining Wash buffer with 12000rpm；

(h) Akibaiin Column are put into a clean EP pipe, then in Akibaiin Column films The Elution Buffer of 50~100 μ L are added at centre, are placed at room temperature for 2min, 10000rpm centrifugations 1min extracts DNA.

Extraction product is detected using 1% agarose gel electrophoresis.The results are shown in Figure 8, it can be seen that one more Apparent band, DNA Ladder Marker maximum bands used are 15,000bp, and genome band is in Marker maximum bands On, illustrate that its size is more than 15,000bp, meets Genome Size requirement.

2, TouchDown PCR clone xylanase gene conserved sequence

(1) 5.0 Software for Design upstream and downstream primers of PrimerPremier are used.It is protected according to known 10 family zytase Keep sequence design upstream degenerate primer WMN-3JBUP (5'-CTCTGGAAGCCNAYNCMRTSNA-3') and downstream degenerate primer WMN-3JBDOWN (5'-GACTGGGAYGTNGTNAAYGA-3'), sends to synthesis.

(2) the PCR Amplification Kit kits for using Takara, using genomic DNA as template, according to as follows Reaction system prepares PCR reaction solution：

Then Touchdown PCR reactions are carried out according to the following conditions：

PCR product is detected using 1% agarose gel electrophoresis.The results are shown in Figure 9.In swimming lane 1, there is one The band that size is about 250bp meets known 10 family's xylanase gene conserved sequence sizes, so being carried out to the band Recycling.

3, Touchdown PCR products recycle

PCR product recycling uses Gel Extraction Kit (100) D2500-01 that Omega companies produce, main Want that steps are as follows：

(1) Ago-Gel containing target DNA is cut out in the UV lamp, and the liquid of gel surface is exhausted with paper handkerchief, this When should be noted that as possible excision without target DNA part gel, reduce gel volume, improve the DNA rate of recovery, be put into later EP pipe In.(DNA not being exposed under ultraviolet lamp for a long time when glue is cut in attention, to prevent DNA damage)

(2) blob of viscose is shredded.The blob of viscose thawing time for the step (3) that can speed operations after blob of viscose chopping, improve the recycling of DNA Rate.Blob of viscose weight is weighed, blob of viscose volume is calculated.When calculating blob of viscose volume, calculated with 1mg=1 μ L, be added into blob of viscose etc. The blob of viscose melting liquid Binding Buffer of amount.

(3) 50 DEG C of 7~15min of dissolving, ensure that colloidal sol is complete, otherwise can influence subsequent recovery.

(4) colloidal sol is put into chromatographic column and (is no more than 700 μ L), 12,000rpm centrifugation 1min outwell the liquid in collecting pipe Body.

(5) the Binding Buffer, 12,000rpm centrifugation 1min that 300 μ L are added outwell the liquid in collecting pipe.

(6) SPW of 700 μ L is added, 12,000rpm centrifugation 1min outwell the liquid in collecting pipe.

(7) step (6) is repeated.

(8) by void column 13,000rpm centrifuges 2min, to remove the ethyl alcohol of remaining, otherwise can seriously affect returning for follow-up DNA It receives.

(9) collecting pipe is discarded, changes clean EP pipes into, the adsorbed film toward adsorption column middle is carefully added into 15 μ L Elution Buffer, are stored at room temperature 3min, and 13,000rpm centrifugation 2min are not stored in if in -20 DEG C of refrigerators immediately.

4, prepared by competent cell

The preparation of competent cell uses the Competent Cell Prepatation Kit kits of TaKaRa companies It prepares, steps are as follows：

(1) use transfer needle picking Escherichia coli in the LB solids training containing ampicillin (AMP, final concentration of 100 μ g/mL) It supports and is classified scribing line on base, be advisable with that single bacterium colony can occur, 37 DEG C are incubated overnight.

(2) picking colony is placed in the conical flask of the LB liquid medium containing 20mL, 37 DEG C, 220rpm cultures.

(3) OD values are measured, when OD600 values reach 0.35~0.5, places and stops culture in ice, if OD values exceed this model Enclose the transformation efficiency that will not ensure that competent cell.

(4) take the above-mentioned bacterium solutions of 1mL in 1.5mL EP pipes, 4,000rpm, 4 DEG C of centrifugation 5min abandon supernatant (on removing as possible Clearly).

(5) the Solution A being pre-chilled in 100 μ L ice are added in each EP pipes, gently springing EP pipes make precipitation suspend, Forbid acutely vibrating, 4,000rpm, 4 DEG C of centrifugation 5min abandon supernatant (removing supernatant as possible).

(6) the Solution B being pre-chilled in 100 μ L ice are added in each EP pipes, gently springing EP pipes make precipitation suspend, Forbid acutely vibrating.Prepared by competent cell completes, if not using immediately, is placed in -80 DEG C of preservations.

5, the connection, conversion and sequencing of Touchdown PCR products

(1) 100 μ L competent cell suspensions are taken from -80 DEG C of refrigerators, make its defrosting in ice.

(2) by PCR product, 1.5 μ L pMD18-T (Simple Vector) and the 4 μ L Solution I of 4.5 μ L recycling Mixing in 100 μ L competent cells is added in invertase (production of TaKaRa companies), and 16 DEG C of water-baths connect 3h.

(3) the recombinant DNA solution being added after connection gently shakes up, and places 30min on ice.

90s is placed in (4) 42 DEG C of water-baths, is immediately placed in cooled on ice 15min later.

(5) 1mL LB liquid mediums (being free of ampicillin) are added into pipe, 37 DEG C, 250rpm oscillation trainings after mixing 1h is supported, bacterium is made to restore normal growth state, the antibiotics resistance gene (Ampr) encoded with expression plasmid.

(6) it takes the above-mentioned bacterium solutions 6 of 1mL, 000rpm to centrifuge 5min, removes about 1mL supernatants, remaining mixing.Take 100 μ L coatings Half an hour is placed in the screening flat board containing ampicillin, facing up, is cultured completely after base absorbs after bacterium solution and is inverted training Foster ware, 37 DEG C of cultures 16~for 24 hours.

(5) single bacterium colony grown is selected with oese is put in 1mL LB culture mediums (1 μ L ampicillins of addition, final concentration For 100 μ g/mL) in, 37 DEG C, 250rpm shake bacterium until bacterium solution become cloudy (4~10h), sequencing is sent in every group of selection 1.

Sequencing obtains the sequencing result of Streptomyces sp.WMN-3 xylanase gene conserved sequences, and sequence is such as Under：

(a) nucleotide sequence such as SEQ ID No：(243bp) shown in 2.

(b) NCBI nucleotide sequence comparisons result

The nucleotides sequence is listed on ncbi database, (BLAST) is compared, find the conserved sequence be all 10 The zytase (Streptomyces davawensis strain JCM 4913) of race has 83% higher similitude, can Gene where inferring the conserved sequence of clone belongs to 10 family's xylanase genes.

6, TAIL-PCR clones the upstream and downstream gene of xylanase gene conserved sequence

(1) TAIL-PCR be in order to obtain Streptomyces sp.WMN-3 xylanase genes conserved sequence upstream and The gene in downstream uses 5.0 Software for Design specific primers of PrimerPremier and random primer.According to Touchdown Obtained conserved sequence is sequenced in PCR product, separately designs the upstream and downstream specific primer of 2 groups of 20bp or so, and every group of upstream is special Specific primer (Upstream primer, abbreviation USP) and downstream specific primer (Downstream primer, abbreviation DSP) Respectively there is three-wheel of 3 nested primers for TAIL-PCR to react.Design 7 couples of random primer (Arbitrary simultaneously Degenerate primer, abbreviation AD).Conserved sequence and primer sequence are shown in Table 2.After primer sends to synthesis, you can carry out next Step experiment.

Table 2Streptomyces sp.WMN-3 xylanase genes conserved sequences and the primer for TAIL-PCR

(2) TAIL-PCR cloned upstreams gene

Using the PCR Amplification Kit kits of Takara, using genomic DNA as template, according to following anti- System is answered to prepare PCR reaction solution：

Then the reaction of first round TAIL-PCR is carried out according to the following conditions：

The second wheel TAIL-PCR reaction systems same first round, but the PCR product ddH that the first round is reacted₂O dilutes It is used as template DNA, USP1 to be changed to USP2 after 100 times, reaction condition is as follows：

The third round TAIL-PCR reaction systems same first round, but the PCR product ddH that the second wheel is reacted₂O dilutes It is used as template DNA, USP2 to be changed to USP3 after 100 times, reaction condition is as with the second wheel reaction.After reaction, using 1% Agarose gel electrophoresis is detected third round PCR product.

TAIL-PCR is carried out by 3 nested upstream specific primers and random primer AD3, is cloned The upstream gene of Streptomyces sp.WMN-3 xylanase gene conserved sequences, PCR product electrophoretogram such as Figure 10 institutes Show.It can be seen from fig. 10 that by 3 wheel PCR, the apparent band occurred at this time only has one, and size is about 600bp, is met Target gene size requirements primarily determine as conserved sequence upstream gene.It recycles, connect, convert and is sequenced by PCR product.

By sequencing, Streptomyces sp.WMN-3 xylanase gene conserved sequence upstream genes sequencing result is such as Under (728bp)：Its nucleotide sequence such as SEQ ID No：Shown in 3.

(3) TAIL-PCR cloned downstreams gene

With the clone of upstream gene, it is specific that corresponding upstream specific primer is changed to downstream for reaction system, reaction condition Primer.After reaction, third round PCR product is detected using 1% agarose gel electrophoresis.

TAIL-PCR is carried out by 3 nested downstream specific primers and random primer AD3, is cloned The downstream gene of Streptomyces sp.WMN-3 xylanase gene conserved sequences, PCR product electrophoretogram such as Figure 11 institutes Show.It can be seen from fig. 11 that by 3 wheel PCR, the apparent band occurred at this time only has one, and size is about 750bp, is met Target gene size requirements primarily determine as conserved sequence downstream gene.It recycles, connect, convert and is sequenced by PCR product.

By sequencing, Streptomyces sp.WMN-3 xylanase gene conserved sequence downstream genes sequencing result is such as Under (711bp)：Its nucleotide sequence such as SEQ ID No：Shown in 4.

7, Streptomyces sp.WMN-3 xylanase gene full length sequences are obtained

The sequencing result for the conserved sequence upstream and downstream gene cloned according to TAIL-PCR is spliced, and is obtained The complete enzyme gene sequence of Streptomyces sp.WMN-3 zytases.Its nucleotide sequence such as SEQ ID No：Shown in 5.

Alkalescent xylanase gene order is a complete open reading frame (ORF), and the open reading frame is close to originate Numeral ATG is started and is terminated with terminator codon TAA, includes 1476 nucleotide altogether.Wherein, preceding 75 nucleotide is signal peptide Coded sequence.

The amino acid sequence of alkalescent xylanase gene code such as SEQ ID No：Shown in 6.

It analyzes to obtain through 2.2 softwares of DNAssist Version, alkalescent xylanase gene open reading frame codes 491 A amino acid, wherein preceding 25 amino acid is the signal peptide of gene code, in maturase protein secretion in Gly²⁵Site quilt Excision.Therefore, ripe zymoprotein, amounts to 466 amino acid, and theoretical molecular weight (MWt) is 50.23kD.

According to the sequencing results combination zymoprotein in the diversity judgement of molecular weight and enzymatic property etc., alkalinity wood is poly- Carbohydrase gene WMN-3 is a newfound inscribe β -1,4- xylanase genes.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications, Equivalent substitute mode is should be, is included within the scope of the present invention.

Sequence table

<110>Shenzhen University

<120>Alkalescent xylanase and the application of a kind of basophilic streptomycete and its generation

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1540

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>The 16S rDNA sequences of Streptomyces sp. WMN-3 bacterial strains

<400> 1

taccggtacg cgcggatctt ccagagattt acggctacct tgttacgact tcgtcccaat 60

cgccagtccc acctttgacg attccctccc acaaggggtt gggccaccgg cttcgggtgt 120

taccgacttt cgtgacgtga caggcggtgt gtacaaggcc cgggaacgta ttcaccgcag 180

caatgctgat ctgcgattac tagcgactcc gacttcatgg ggtcgagttg cagaccccaa 240

tccgaactga gaccggcttt ttgagattcg ctccaccttg cggtatcgca gctcattgta 300

ccggccgttg cagcacgtgt gcagcccaag acataagggg catgatgact tgacgtcgtc 360

cccaccttcc tccgagttga ccccggcagt ttcctgtgag tccccatcac cccgaaaggc 420

atgctggcaa cacagaacaa gggttgcgct cgttgcggga cttaacccaa catctcacga 480

cacgagctga cgacagccat gcaccacctg tacaccgacc acaagggggc acccatctct 540

ggatgtttcc ggtgtatgtc aagccttggt aaggttcttc gcgttgcgtc gaattaagcc 600

acatgctccg ccgcttgtgc gggcccccgt caattccttt gagttttagc cttgcggccg 660

tactccccag gcggggaact taatgcgtta gctgcggcac ggacgacgtg gaatgtcgcc 720

cacacctagt tcccaacgtt tacggcgtgg actaccaggg tatctaatcc tgttcgctcc 780

ccacgctttc gctcctcagc gtcagtatcg gcccagagat ccgccttcgc caccggtgtt 840

cctcctgata tctgcgcatt tcaccgctac accaggaatt ccgatctccc ctaccgaact 900

ctagcctgcc cgtatcgaat gcagacccgg ggttaagccc cgggctttca catccgacgt 960

gacaagccgc ctacgagctc tttacgccca ataattccgg acaacgctcg caccctacgt 1020

attaccgcgg ctgctggcac gtagttagcc ggtgcttctt ctgcaggtac cgtcacttgc 1080

gcttcttccc tgctgaaaga ggtttacaac ccgaaggccg tcatccctca cgcggcgtcg 1140

ctgcatcagg ctttcgccca ttgtgcaata ttccccactg ctgcctcccg taggagtctg 1200

ggccgtgtct cagtcccagt gtggccggtc gccctctcag gccggctacc cgtcgtcgcc 1260

ttggtaggcc attaccccac caacaagctg ataggccgcg ggctcatcct gcaccgccgg 1320

agctttccac acattcacta tgcagtgatg tgtcgtatcc ggtattagac cccgtttcca 1380

gggcttgtcc cagagtgcag ggcagattgc ccacgtgtta ctcacccgtt cgccactaat 1440

ccaccccgaa gggcttcatc gtccgacttg catgtgttaa ccacgccgcc agcgttcgtc 1500

ctgagcagga tcaaactcta atcgtcgaac ggcaggcctc 1540

<210> 2

<211> 243

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>The nucleotide sequence of alkalescent xylanase WMN-3 gene conserved sequences

<400> 2

tgggacgtcg taaacgaagc tttcgagggc gacgggactc gccgccagtc tgtcttccag 60

cgggtgcttg gtgacggata catcgaagag gctttccgtg ctgcccgtgc cgctgacccg 120

tcagctcagc tatgcattaa cgactactca acggactgga tcaacgcgaa gtccacggcc 180

atctacaact tggtgaagga cttcaaggaa cgcggtgtcc ccatgcactg tatcggcttc 240

cag 243

<210> 3

<211> 728

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>The nucleotide sequence of alkalescent xylanase WMN-3 gene conserved sequence upstream genes

<400> 3

tgctttgccc tggaacgatg actattccac aaaagcggct ctcactgaac ttgcgacagt 60

gatgggggcg caaccagcgt ccacaactga cccaacagat ccgaccgatc caactgaccc 120

aacagatccg accgatccaa ctgatccacc aagcgatgcg gtatgcaccg ttgtaccgca 180

ggtcaactca tggaacacgg gctacaccgc aaacctcact atccgcaaca ctggtgacac 240

tccaatcaac ggatgggagc tcagcatcgg attgccccaa gggcaccaac tcagccaagg 300

ttggtcggcg cagttctcgc agtctggtca aaccctgatc gcttctaacg cagcgtggaa 360

cggaacgtta gcgcccggag ccagcgttga ggttggtttc aacgcaacgc atcaaggaac 420

gcttgggcaa cttggcccat ttgttctcaa cggtactacc tgcaactaac ccacattgag 480

aacggcttct gccgtcctca actcatcgag cggcctcaaa cctccttttc tgaaggtttg 540

aggccgctct cacgatgggg agcagtaacg acctcaaccc tgatttcgac gtaaaacccc 600

atggggagat gcatactgca ttctctcgtc atgcagaaat caccacccta ccggtcggtc 660

gataatcacg atagagcatc gtttcccgca cagtagtaac aaggggtcag ttgtggacgc 720

tggttgcg 728

<210> 4

<211> 711

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>The nucleotide sequence of alkalescent xylanase WMN-3 gene conserved sequence downstream genes

<400> 4

accaagcacc cgctggaaga cagactggcg gcgagtcccg tcgccctcga aagcttcatt 60

cacaacatcc catgctgcaa tgtcacctgc gtagcgtccg gcaacactgt tgatgtggtc 120

aaccatgacg gtccgcaatt cagctgggtc agtgattgac gccgcccact ggggaagctg 180

agagtgccac accagtgtgt ggccgtacac ctcagcgtca ttgtccttgg cgaactgaac 240

gaccgaatca gcccctgccc acgtgaactg cccacgttgt ggctgggttg catcccattt 300

catagcgttt tctgcagtga tcatggaaaa ttctcgttca acgatttgct tgtattgact 360

gtcgctgtcc gctaaatgtg gtgcataagc caccccaaag gtacggccag agcgctccgc 420

agcgtcgcgc agcggctcaa aatcatgccc aataccgggc gaagcttgtg gtgtggcatc 480

ttgcgcagcg ccgggggcgc caaaagccaa cattgcaggg gtgcacagcg ccgcagaaag 540

agctagcgcg cccgtcgcaa ggaacttctt catcacagat ttatccgttc tggcaggttg 600

tgccgttgag ggtgaaggag gtaggggatg aatgtgcccc attgtgggtc ccgttgaacc 660

cgatcgtgac gctggccccg ggggccaccg tccccattcc acgcagcatt c 711

<210> 5

<211> 1476

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>The nucleotide sequence of alkalescent xylanase WMN-3 genes

<400> 5

atgaagaagt tccttgcgac gggcgcgcta gctctttctg cggcgctgtg cacccctgca 60

atgttggctt ttggcgcccc cggcgctgcg caagatgcca caccacaagc ttcgcccggt 120

attgggcatg attttgagcc gctgcgcgac gctgcggagc gctctggccg tacctttggg 180

gtggcttatg caccacattt agcggacagc gacagtcaat acaagcaaat cgttgaacga 240

gaattttcca tgatcactgc agaaaacgct atgaaatggg atgcaaccca gccacaacgt 300

gggcagttca cgtgggcagg ggctgattcg gtcgttcagt tcgccaagga caatgacgct 360

gaggtgtacg gccacacact ggtgtggcac tctcagcttc cccagtgggc ggcgtcaatc 420

actgacccag ctgaattgcg gaccgtcatg gttgaccaca tcaacagtgt tgccggacgc 480

tacgcaggtg acattgcagc atgggatgtt gtgaatgaag ctttcgaggg cgacgggact 540

cgccgccagt ctgtcttcca gcgggtgctt ggtgacggat acatcgaaga ggctttccgt 600

gctgcccgtg ccgctgaccc gtcagctcag ctgtgcatta acgactactc aacggactgg 660

atcaacgcga agtccacggc catctacaac ttggtgaagg acttcaagga acgcggtgtc 720

cccatcgact gtgttggttt ccagtcgcac ttaatcgttg gacaggtccc aactaatttc 780

caacaaaact tgcaacggtt tgtggatctc ggggttgatg ttcgcattac cgaactagat 840

attcgtatgg cgacaccacc aactgccgcg aaccttgcaa cgcaggctga ggactaccgc 900

aaggtattcc aggcctgctg gaacgttgat ggctgcaccg gggtaactat atggggcatc 960

acagatgcct actcttggat accgcaggtg ttcgcaggtg agggtgctgc tttgccctgg 1020

aacgatgact attccacaaa agcggctctc actgaacttg cgacagtgat gggggcgcaa 1080

ccagcgtcca caactgaccc aacagatccg accgatccaa ctgacccaac agatccgacc 1140

gatccaactg atccaccaag cgatgcggta tgcaccgttg taccgcaggt caactcatgg 1200

aacacgggct acaccgcaaa cctcactatc cgcaacactg gtgacactcc aatcaacgga 1260

tgggagctca gcatcggatt gccccaaggg caccaactca gccaaggttg gtcggcgcag 1320

ttctcgcagt ctggtcaaac cctgatcgct tctaacgcag cgtggaacgg aacgttagcg 1380

cccggagcca gcgttgaggt tggtttcaac gcaacgcatc aaggaacgct tgggcaactt 1440

ggcccatttg ttctcaacgg tactacctgc aactaa 1476

<210> 6

<211> 491

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<220>

<223>The amino acid sequence of the protein of alkalescent xylanase WMN-3 gene codes

<400> 6

Met Lys Lys Phe Leu Ala Thr Gly Ala Leu Ala Leu Ser Ala Ala Leu

1 5 10 15

Cys Thr Pro Ala Met Leu Ala Phe Gly Ala Pro Gly Ala Ala Gln Asp

20 25 30

Ala Thr Pro Gln Ala Ser Pro Gly Ile Gly His Asp Phe Glu Pro Leu

35 40 45

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

50 55 60

Pro His Leu Ala Asp Ser Asp Ser Gln Tyr Lys Gln Ile Val Glu Arg

65 70 75 80

Glu Phe Ser Met Ile Thr Ala Glu Asn Ala Met Lys Trp Asp Ala Thr

85 90 95

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

100 105 110

Gln Phe Ala Lys Asp Asn Asp Ala Glu Val Tyr Gly His Thr Leu Val

115 120 125

Trp His Ser Gln Leu Pro Gln Trp Ala Ala Ser Ile Thr Asp Pro Ala

130 135 140

Glu Leu Arg Thr Val Met Val Asp His Ile Asn Ser Val Ala Gly Arg

145 150 155 160

Tyr Ala Gly Asp Ile Ala Ala Trp Asp Val Val Asn Glu Ala Phe Glu

165 170 175

Gly Asp Gly Thr Arg Arg Gln Ser Val Phe Gln Arg Val Leu Gly Asp

180 185 190

Gly Tyr Ile Glu Glu Ala Phe Arg Ala Ala Arg Ala Ala Asp Pro Ser

195 200 205

Ala Gln Leu Cys Ile Asn Asp Tyr Ser Thr Asp Trp Ile Asn Ala Lys

210 215 220

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

225 230 235 240

Pro Ile Asp Cys Val Gly Phe Gln Ser His Leu Ile Val Gly Gln Val

245 250 255

Pro Thr Asn Phe Gln Gln Asn Leu Gln Arg Phe Val Asp Leu Gly Val

260 265 270

Asp Val Arg Ile Thr Glu Leu Asp Ile Arg Met Ala Thr Pro Pro Thr

275 280 285

Ala Ala Asn Leu Ala Thr Gln Ala Glu Asp Tyr Arg Lys Val Phe Gln

290 295 300

Ala Cys Trp Asn Val Asp Gly Cys Thr Gly Val Thr Ile Trp Gly Ile

305 310 315 320

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

325 330 335

Ala Leu Pro Trp Asn Asp Asp Tyr Ser Thr Lys Ala Ala Leu Thr Glu

340 345 350

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

355 360 365

Asp Pro Thr Asp Pro Thr Asp Pro Thr Asp Pro Thr Asp Pro Thr Asp

370 375 380

Pro Pro Ser Asp Ala Val Cys Thr Val Val Pro Gln Val Asn Ser Trp

385 390 395 400

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

405 410 415

Pro Ile Asn Gly Trp Glu Leu Ser Ile Gly Leu Pro Gln Gly His Gln

420 425 430

Leu Ser Gln Gly Trp Ser Ala Gln Phe Ser Gln Ser Gly Gln Thr Leu

435 440 445

Ile Ala Ser Asn Ala Ala Trp Asn Gly Thr Leu Ala Pro Gly Ala Ser

450 455 460

Val Glu Val Gly Phe Asn Ala Thr His Gln Gly Thr Leu Gly Gln Leu

465 470 475 480

Gly Pro Phe Val Leu Asn Gly Thr Thr Cys Asn

485 490

<210> 7

<211> 22

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>The upstream degenerate primer WMN-3JBUP of conserved sequence

<220>

<221> misc_feature

<222> (12)..(12)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (15)..(15)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is a, c, g, t or u

<400> 7

ctctggaagc cnayncmrts na 22

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>The downstream degenerate primer WMN-3JBDOWN of conserved sequence

<220>

<221> misc_feature

<222> (12)..(12)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (15)..(15)

<223> n is a, c, g, t or u

<400> 8

gactgggayg tngtnaayga 20

<210> 9

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> WMN-3-USP1

<400> 9

actcaacgga ctggatcaac g 21

<210> 10

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> WMN-3-USP2

<400> 10

gatgttcgca ttaccgaac 19

<210> 11

<211> 17

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> WMN-3-USP3

<400> 11

tgctttgccc tggaacg 17

<210> 12

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> WMN-3-DSP1

<400> 12

cgacgattct ctggaagccg 20

<210> 13

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> WMN-3-DSP2

<400> 13

gtcagcggca cgggcagca 19

<210> 14

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> WMN-3-DSP3

<400> 14

accaagcacc cgctggaaga 20

<210> 15

<211> 15

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> AD1

<220>

<221> misc_feature

<222> (5)..(5)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (10)..(10)

<223> n is a, c, g, t or u

<400> 15

agtgnwgwan caacg 15

<210> 16

<211> 16

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> AD2

<220>

<221> misc_feature

<222> (5)..(5)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (10)..(10)

<223> n is a, c, g, t or u

<400> 16

wgtgnagawn cagasa 16

<210> 17

<211> 15

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> AD3

<220>

<221> misc_feature

<222> (2)..(2)

<223> n is a, c, g, t or u

<400> 17

tncsagtwtg gwstt 15

<210> 18

<211> 16

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> AD4

<220>

<221> misc_feature

<222> (5)..(5)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (10)..(10)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (13)..(13)

<223> n is a, c, g, t or u

<400> 18

ctwsntactn ctntgc 16

<210> 19

<211> 15

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> AD5

<220>

<221> misc_feature

<222> (5)..(5)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (10)..(10)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (13)..(13)

<223> n is a, c, g, t or u

<400> 19

tcwgncttan tangt 15

<210> 20

<211> 16

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> AD6

<220>

<221> misc_feature

<222> (5)..(5)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (10)..(10)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (13)..(13)

<223> n is a, c, g, t or u

<400> 20

tgagnagwan stnaga 16

<210> 21

<211> 16

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223> AD7

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, t or u

<220>

<221> misc_feature

<222> (11)..(11)

<223> n is a, c, g, t or u

<400> 21

ngwcsagwga natgaa 16

Claims

1. a kind of basophilic streptomycete, it is characterised in that：Entitled Streptomyces sp.WMN-3 were protected on the 7th in September in 2017 It is hidden in the China typical culture collection center positioned at Wuhan, China Wuhan University, deposit number is CCTCC NO：M 2017477。

2. the Selective agar medium of basophilic streptomycete described in claim 1, it is characterised in that：The formula of the Selective agar medium For：Xylan 5.0~8.0g/L, KNO₃1.0g/L, MgSO₄·7H₂O 0.5g/L, NaCl 10~15g/L, KH₂PO₄ 1.5g/ L, pH8.0~9.0.

3. the Selective agar medium of basophilic streptomycete according to claim 2, it is characterised in that：The Selective agar medium Formula is：Xylan 8.0g/L, KNO₃1.0g/L, MgSO₄·7H₂O 0.5g/L, NaCl 15g/L, KH₂PO₄1.5g/L pH9.0。

4. a kind of preparation method of alkalescent xylanase, it is characterised in that：The alkalescent xylanase is made as follows：

(1) extraction of crude enzyme liquid

Basophilic streptomycete Streptomyces sp.WMN-3 strains described in claim 1 are connected in liquid selective medium, In 37 DEG C, 200rpm cultures 48h supernatant is taken by 48h culture solutions with the rotating speed refrigerated centrifuge 20min of 14000rpm；It utilizes 3000 dalton are concentrated by ultrafiltration film and supernatant are concentrated by ultrafiltration to get crude enzyme liquid, are used for ion-exchange chromatography；

(2) alkalescent xylanase isolates and purifies

(a) anion-exchange chromatography

The medium of ion-exchange chromatography is DEAE Sepharose Fast Flow；Buffer solution system uses pH value for 8.0 Tris-HCl buffer solutions, equilibrium liquid are pH 8.0Tris-HCl buffer solutions, and eluent is the 8.0Tris-HCl bufferings of pH containing NaCl Liquid；Take 100mL media carefully pour into specification be Φ 2.6cm × 30cm chromatographic column in so that gel uniformly and bubble-free；With The equilibrium liquid of 300mL is balanced, and the loading after A280 stablizes, peak to be penetrated is collected after occurring and penetrates peak, dialyses, is dense Contracting；

(b) cation-exchange chromatography

Chromatography media is SP Sepharose Fast Flow；Buffer solution system use pH value for 6.0 PBS buffer solution；The moon Dialysis concentration in enzyme activity peak obtained by ion-exchange chromatography is added to the chromatographic column that the PBS buffer solution for being in advance 6.0 with pH value has balanced On, with 60mLh^-1Flow velocity eluted with the PBS buffer solution of the pH6.0 containing NaCl, collect the eluting peak with enzymatic activity, pass through Dialysis concentration, obtains alkalescent xylanase, is placed in 4 DEG C and saves backup.

5. application of the basophilic streptomycete described in claim 1 in paper industry.