CN107586767B - Heat-resistant endo-xylanase EpXYN1, and coding gene and application thereof - Google Patents

Abstract

The invention discloses a high-temperature-resistant endo-xylanase EpXYN1, a coding gene and application thereof, wherein the amino acid sequence of the high-temperature-resistant endo-xylanase EpXYN1 is shown in SEQ ID No. 1. The high-temperature-resistant endo-xylanase EpXYN1 disclosed by the invention is a novel endo-xylanase cloned from eupenicillium minuteness, the optimum temperature is 75 ℃, the optimum pH is 5.5, the high temperature of 65 ℃ and the good pH stability can be endured, and a hydrolysate of xylan is mainly xylobiose, so that the high-temperature-resistant endo-xylanase has a good application prospect in industrial application. As a novel xylanase, the xylanase can be widely applied to the industries of biomass energy, food, feed, papermaking, medicine, health care and the like.

Description

Heat-resistant endo-xylanase EpXYN1, and coding gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a high-temperature-resistant endo-xylanase EpXYN1, and a coding gene and application thereof.

Background

Heterogeneous xylan is one of main components of the hemicellulose, and is composed of a main chain formed by connecting xylose through β -1,4 glycosidic bonds and side chains containing different substituents, the important step of industrial utilization of the hemicellulose is to degrade the heterogeneous xylan into fermentable monosaccharides, xylooligosaccharides or other chemicals through an enzymatic hydrolysis way, the complete degradation of the heterogeneous xylan requires the synergistic action of a main chain enzyme (such as endo-xylanase and xylosidase) and a side chain enzyme system, the endo-xylanase (endo- β -1,4-xylanase, EC 3.2.1.8) is the most key component in the xylanase system, can hydrolyze β -1, 4-glycosidic bonds of main chain molecules of the xylan, and hydrolyze the xylan into oligosaccharides such as xylobiose or xylooligosaccharide.

To date, most of the reported xylanases are mesophilic enzymes, with an optimum activity temperature between 50-55 ℃ and are difficult to tolerate high temperature conditions of 65 ℃ or above. As high-temperature environment exists in many industrial production links, the development of high-temperature resistant xylanase meets the market demand.

Disclosure of Invention

The purpose of the invention is as follows: in view of the disadvantages of the prior art, the object of the present invention is to provide a thermostable endo-xylanase EpXYN1 from Penicillium microfine Eupenicillus parvum 4-14. It is still another object of the present invention to provide a gene encoding the above-mentioned xylanase. Another object of the present invention is to provide a recombinant vector comprising the above gene. Another object of the present invention is to provide the use of the high temperature resistant endo-xylanase.

The technical scheme is as follows: in order to achieve the purpose of the invention, the invention adopts the technical scheme that:

a heat-resistant endo-xylanase EpXYN1 has an amino acid sequence shown in SEQ ID NO. 1.

The base sequence of the coding gene of the heat-resistant endo-xylanase EpXYN1 is shown in SEQ ID NO. 2.

An expression vector or an expression system containing the coding gene of the heat-resistant endo-xylanase EpXYN 1.

The coding gene of the heat-resistant endo-xylanase EpXYN1 is applied to expression of the heat-resistant endo-xylanase EpXYN 1.

The application of the heat-resistant endo-xylanase EpXYN1 in enzymolysis of hemicellulose.

The application of the heat-resistant endo-xylanase EpXYN1 in enzymolysis of xylan.

Has the advantages that: compared with the prior art, the invention clones a new encoding gene of the heat-resistant endo-xylanase EpXYN1 from the Penicillium microfine (Eupenicillus parvum)4-14, and obtains pure enzyme by carrying out recombinant expression on the encoding gene through pichia pastoris. The recombinant xylanase EpXYN1 has high-temperature (75 ℃) catalytic capability and excellent temperature stability and pH stability, and degradation products of various xylans mainly comprise xylobiose and long-chain xylooligosaccharide. The heat-resistant endo-xylanase EpXYN1 has the advantages of high specific activity (reduced cost), high enzymolysis efficiency (high substrate solubility at high temperature), high stability, difficulty in contaminating mixed bacteria and the like, and has important application prospects in the fields of biomass energy, feed, papermaking, food, medicine, health care and the like.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of the recombinant xylanase EpXYN 1; in the figure, M: marker; EpXYN 1: a purified xylanase;

FIG. 2 is a graph showing the results of pH optima for the recombinant xylanase EpXYN 1;

FIG. 3 is a graph showing the results of the optimum temperature for the recombinant xylanase EpXYN 1;

FIG. 4 is a graph showing the results of pH stability of the recombinant xylanase EpXYN 1;

FIG. 5 is a graph showing the results of temperature stability of the recombinant xylanase EpXYN 1;

FIG. 6 is a graph showing the results of thin layer chromatography analysis of various xylan hydrolysates with the recombinant xylanase EpXYN 1; in the figure, a, hydrolysis product analysis of beech xylan; b, analyzing hydrolysate of birch xylan; c, analyzing hydrolysate of oat xylan; d, analysis of hydrolysate of xylooligosaccharide (xylodi-to xylohexaose).

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The materials and reagents used in the following examples are as follows:

the strain and the vector are Penicillium microfine 4-14 (Eupenicillus parvum 4-14), which is preserved in China center for type culture Collection, and the preservation number is CCTCC No. M2015404. Pichia pastoris (Pichia pastoris KM71H) and the expression vector pPICZ α B are purchased from Invitrogen company, and Escherichia coli DH5 α and the gene manipulation plasmid pEASY-Blunt are purchased from Promega company.

Enzymes and other biochemical reagents: restriction enzymes, DNA polymerases, ligases and dNTPs were purchased from TaKaRa; oat xylan, birch xylan and beech xylan were purchased from Sigma; others are made in China (all can be purchased from common biochemical agents).

LB culture medium: peptone 10g, Yeast extract 5g, NaCl 10g, distilled water to 1000mL, natural pH (about 7). On the basis of the solid medium, 1.5% (w/v) agar was added.

PDA culture medium: 200g of potato, 20g of glucose and 15g of agar powder, sterilizing at high temperature and high pressure,

YPD medium: 10g Yeast Extract, 20g peptone, dissolved in 900mL water, autoclaved, and 100mL sterilized 20% glucose was added.

BMGY medium: 10g of Yeast Extract and 20g of peptone were dissolved in 700mL of water, autoclaved, cooled to room temperature, and then 100mL of 1M potassium phosphate buffer (pH 6) (autoclaved), 100mL of 10 XYNB (filter sterilized), 2mL of 500 XB (20mg of biotin dissolved in 100mL of water, filter sterilized), 100mL of 10 GY (100mL of glycerol dissolved in 900mL of water autoclaved), mixed well and stored at 4 ℃.

BMMY: 10g of Yeast Extract, 20g of peptone in 700mL of water, autoclaving at elevated temperature, cooling to room temperature, adding 100mL of 1M potassium phosphate buffer pH 6 (autoclaving), 100mL of 10 XYNB (filter sterilization), 2mL of 500 XB (20mg of biotin in 100mL of water, filter sterilization), and 100mL of 10 XM (5% methanol), mixing well and storing at 4 ℃.

The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

EXAMPLE 1 cloning of the Gene encoding the thermostable endo-xylanase EpXYN1

Fungus culture and total RNA extraction: get about 10⁹Spores of the Penicillium microfine 4-14 strain were inoculated into 50mL of PDA liquid medium and cultured at 37 ℃ and 180rpm for 4 days. 1mL of the culture was inoculated into a flask of solid fermentation medium (L.Long, D.Ding, Z.Han, H.ZHao, Q.Lin, S.Ding, Thermotolorant hemicellulytic and cellulolytic enzymes from Eupenicillus paravum 4-14display high efficiency microbial enzyme of bacterial acid from peak 434. J.appl.Microbiol.121(2016) 422-. Rinsing white mycelium with sterile water, and draining water with filter paperAnd (3) quickly freezing by using liquid nitrogen, and storing at-70 ℃. Total RNA from the cells was extracted using a TransZolTM Plant kit (TransGen, Beijing).

Cloning genes: taking a proper amount of total RNA, and carrying out reverse transcription reaction by taking an easy script One-Step gDNA Removal and cDNAsynthesis SuperMix kit (TransGen, Beijing) and oligo (dT) as primers to obtain cDNA. And performing conventional PCR reaction by using the obtained cDNA as a template and primers enXyn1_ f1 (5'-ttgccgtccacgctctatc-3') and enXyn1_ r1 (5'-acagcctacgaggaattaacaac-3') to obtain a target gene fragment. Further, the target gene fragment was cloned into the vector pEASY-Blunt (TransGen, Beijing) and sequence analysis was performed by Shanghai Boshang Biotechnology Ltd.

Finally, a full-length fragment containing the gene of the endo-xylanase EpXYN1 was obtained. Sequencing results show that the total length of the endo-xylanase EpXYN1 gene is 1212bp, the DNA sequence is shown in SEQ ID NO.2, the sequence of the expressed protein (endo-xylanase EpXYN1) is shown in SEQ ID NO.1, the reading frame of the expressed protein comprises 404 amino acids, and the first 22 amino acids are signal peptides. Protein homology comparison shows that the mature protein belongs to a member of glycoside hydrolase family 10, the theoretical molecular weight of the mature protein is 40.75kDa, and the theoretical isoelectric point (pI) is 4.97.

Example 2 Pichia expression and purification of the thermostable endo-xylanase EpXYN1

Specific primers enXyn1_ f2 and enXyn1_ r2 for synthesizing endoxylanase Epxyn1 were designed respectively as follows:

enXyn1_f2：5′-agagaggctgaagctgaattctcaggcctggatacagcggca-3′；

enXyn1_r2：5′-gagatgagtttttgttctagatcagtgatggtgatggtgatgcagacattgagagtacca-3′。

the pair of primers is used for amplifying an endoxylanase EpXYN1 gene fragment without a signal peptide from a plasmid containing the endoxylanase EpXYN1 gene. The amplified target gene fragment is subjected to gel cutting purification and passes through HieffClone^TMOne Step Cloning Kit (Yeasen, Shanghai) and EcoRI-XbaI digested plasmid pPICZ α B are subjected to homologous recombination to obtain gene expression plasmid pPIC-EpXyn 1. recombinant plasmid pPIC-EpXyn1 is linearized by restriction endonuclease SacI and then electrically recombinedAfter transfer into Pichia pastoris KM71H, YPD plates containing 1M sorbitol and 100. mu.g/mL Zeocin (Invitrogen) antibiotics were used for screening of positive clones. Inoculating the screened positive clones into a test tube containing 5mLYPD and 100 μ g/mL antibiotic Zeocin, performing shake culture at 28 deg.C and 200rpm, transferring the bacterial liquid into 30mL BMGY culture solution respectively after 20h, performing shake culture at 28 deg.C and 200rpm until OD is reached₆₀₀After reaching 4.0, the thalli are collected by centrifugation, transferred into 25mL BMMY culture solution for 28 ℃, shake-bed culture is carried out at 200rpm, methanol is supplemented every 24 hours, the concentration of the methanol is 0.8% (v/v), and crude enzyme solution is collected by centrifugation after continuous culture is carried out for 5 days. The crude enzyme solution was first filled into dialysis bags and dialyzed at 4 ℃ for 24 hours with gentle stirring in phosphate buffer pH 8.0. The enzyme solution was purified according to the method of Ni-NTA Agarose (Qiagen). Protein quantification was performed using a BCA assay kit (Thermo Tech, USA). The purified protein was detected by SDS-PAGE. The result is shown in figure 1, and the recombinant xylanase is expressed in pichia pastoris, is a single band after purification, and has a molecular weight close to 52.5 kDa. The actual molecular weight is larger than the theoretical molecular weight and is caused by glycosylation.

Example 3 establishment of method for measuring Activity of Heat-resistant endo-xylanase EpXYN1

And (3) measuring the content of reducing sugar by using Beechwood xylan as a substrate and adopting a Somogyi-Nelson method, and calculating the enzyme activity. 1.5mL of the reaction system: 1.0mL of 50mM sodium citrate buffer pH 5.0, 400. mu.L of 0.5% (w/v) substrate, gently mixed and then preheated for 10min, followed by addition of 100. mu.L of crude enzyme solution (pre-diluted to different gradient times) and reaction time of 10min at 75 ℃. And then sequentially taking out, adding 500 mu L of Somogyi Reagent solution, putting into a 99 ℃ water bath kettle for 15min, cooling to room temperature, adding 0.5mL of Nelson Reagent, reacting for 20min, centrifuging for 10min, absorbing 200 mu L of the mixture onto an ELISA plate, and placing the ELISA plate in an ELISA reader for measuring the absorbance (OD value) of a sample at the wavelength of 520 nm. Meanwhile, xylose solutions with different concentrations were prepared, and the OD of each xylose solution was measured by the above-described method (Somogyi-Nelson method)₅₂₀And (6) reading. Taking the concentration of xylose solution as abscissa and OD₅₂₀The readings are plotted on the ordinate, and a standard curve is drawn. The standard curve is: y is 0.0136 x-0.0592; wherein y is OD₅₂₀Reading, x is xyloseConcentration (. mu.g/mL). Xylanase activity in the samples was calculated according to the standard curve. One xylanase activity unit (U) refers to the amount of enzyme required to produce 1. mu. mol xylose per minute. The xylanase activity calculation formula is as follows:

wherein X is the xylose content (μ g) calculated according to the xylose standard curve; m is the molar mass of xylose (150 g/mol); c is enzyme solution volume (mL); t is enzymolysis time (min); n is the dilution factor of the enzyme solution.

Example 4 determination of the Properties of the thermostable Endochromase EpXYN1

1) Optimum pH and optimum temperature of heat-resistant endo-xylanase EpXYN1

And (3) optimum pH determination: the enzyme solution purified in example 2 was assayed for enzyme activity at 75 ℃ using beech xylan as a substrate in a wide range of buffers (B.L. Turner, Variation in pH optima of hydrolyticenzyme activities in tropical tract in soil soils, appl.environ.Microbiol.2010,76(19):6485-93) at pH 3.0 to 9.5, respectively. The results are shown in FIG. 2, which shows that EpXYN1 has an optimum pH of 5.5.

Optimum temperature measurement: the enzyme activity was measured in 50mM sodium citrate buffer pH5.5 with beechwood xylan as substrate at 30-90 ℃ respectively. The relative enzyme activities at each temperature were calculated with the highest enzyme activity as 100%. The results show that: the xylanase EpXYN1 has an optimum reaction temperature of 75 ℃ and 90% activity at 80 ℃ (FIG. 3).

2) Determination of pH stability and determination of thermal stability of Heat-resistant endo-xylanase EpXYN1

Tolerance determination of pH: the pure enzyme solutions prepared in example 2 were stored at 4 ℃ for 12 hours under different pH conditions (extensive buffer pH 3.0-12.0). The enzyme activity of each treated sample was determined under optimum conditions using beechwood xylan as a substrate. The relative enzyme activity of each treatment was calculated using the untreated enzyme solution as a control. As shown in figure 4, the enzyme is treated under the conditions of pH9 and 10, and the residual enzyme activity can reach more than 90 percent; the residual enzyme activity can reach more than 80 percent after the treatment under the conditions of pH3 and 4; the residual enzyme activity can reach more than 70 percent after the treatment under the condition of pH 5-8.

And (3) measuring the temperature stability: the enzyme solution with the same enzyme amount is placed in a set temperature (at 65 ℃, 70 ℃ and 75 ℃) for heat preservation for 0-90 minutes, and then placed on ice. The activity of each sample was measured at optimum temperature and pH using zelkova xylan as substrate. The relative enzyme activity of the treated samples was calculated as 100% of the enzyme activity of the untreated samples. The results show that: the xylanase EpXYN1 has good heat resistance; 84% activity can be maintained after 90min treatment at 65 ℃; 74% activity was also retained by treatment at 70 ℃ for 30min (FIG. 5).

3) Specific activity and kinetic constant determination of heat-resistant endoxylanase EpXYN1

The specific activity and kinetic constants of the recombinant xylanase EpXYN1 were determined at 75 ℃ in 50mM sodium citrate buffer, pH 5.5. EpXYN1 specific activity was measured by the method described in example 3, and the enzyme activity per enzyme (mg) was calculated. Determination of enzyme kinetic constants activity assays were performed at different concentrations of substrate (0.125-2.5 mg/mL). Wherein the substrates are respectively: zelkova xylan, birch xylan and oat xylan.

Data on enzyme kinetic constants the kinetic constants of the enzyme were calculated using nonlinear regression analysis with Graphpad Prism 5.0 software. The results show that: the specific activities of EpXYN1 on beech xylan, birch xylan and oat xylan as substrates were 384.42U/mg, 486.71U/mg and 372.16U/mg, respectively; k_m0.86mg/mL, 1.00mg/mL and 2.19mg/mL, respectively; v_max664.80, 694.50 and 711.30 mu mol/mg, respectively; k_catAre respectively 554.00s^-1、578.75s^-1And 592.75s^-1(Table 1).

TABLE 1 specific enzyme Activity and kinetic constants of recombinant xylanase EpXYN1

Substrate	Specific activity (U/mg)	Km(mg/mL)	Vmax(μmol/mg)	Kcat(s-1)
					Beech xylan	384.42±29.42	0.86±0.10	664.80±31.01	554.00±25.84
Birch xylan	486.71±37.72	1.00±0.07	694.50±16.63	578.75±13.86
					Oat xylan	372.16±20.64	2.19±0.37	711.30±59.50	592.75±49.58

Note: k of EpXYN1_catThe value was calculated as its actual molecular weight of 53 kDa.

4) Effect of Metal ions or chemical reagents on the Activity of thermostable endo-xylanase EpXYN1

To 50mM sodium citrate buffer, 1 or 5mM MgCl was added, respectively₂Or FeCl₃Or MnCl₂Or CoCl₂Or ZnCl₂Or CuCl₂Or CaCl₂Or NiSO₄0.1 or EDTA or SDS, and the xylanase EpXYN1 was assayed under optimal reaction conditions using zelkoxyglycan as a substrate. Relative enzyme activity was calculated for each treatment as a control for the treatment group. The results show that: ca²⁺、Zn²⁺、Mg²⁺、Cu²⁺、Co²⁺、Mn²⁺Or Ni²⁺The effect on the enzyme activity is not significant at low concentration (1mM), and the enzyme activity can be reduced in a small range at high concentration (5 mM). Fe³⁺Can obviously inhibit the activity of enzyme at high concentration (5 mM). The addition of EDTA (1mM) resulted in an approximately 17% increase in enzyme activity. SDS had no significant effect on the enzyme activity at low concentration (1mM), but was able to greatly inhibit the enzyme activity at high concentration (5mM) (Table 2).

TABLE 2 Effect of Metal ions or chemical reagents on the Activity of the recombinant xylanase EpXYN1

Example 5 analysis of the product of the degradation of xylan by the thermostable endo-xylanase EpXYN1

Hydrolysis of different xylans (beechwood xylan, birch xylan or oat xylan) or xylooligosaccharides (xylobiose to xylohexaose, Qingdao Bozhihui Biotech limited, Qingdao) with purified recombinant xylanase EpXYN 1. The hydrolysis products were each analyzed for their composition by Thin Layer Chromatography (TLC). The hydrolysis process of xylan is as follows: 2mL of sodium citrate buffer (pH 5.0,50mM) was added 10mg of beechxylan or 20mg of birch xylan or 20mg of oat xylan or 2mg of xylooligosaccharide and 25. mu.g of purified EpXYN1, and reacted at 75 ℃ for 24 hours. The reaction was terminated by boiling water bath for 10 min. The hydrolysate was centrifuged at 5,000 Xg for 10min and 3. mu.l of the supernatant was spotted onto a thin layer chromatography plate TLC Silica gel 60F₂₅₄glass plates (Merck, Whitehouse Station, NJ, USA) and developing in acetonitrile-ethyl acetate-isopropanol-water (17:5:11:10, v/v/v) (B. -D.Amel, B.Nawel, B.Khelix, G.Mohammed, J.Manon, K. -G.Salima, N.Farida, H.hocine, O.Bernard, C.Jean-Luc, F.Marie-Laure, Charactration of irradiated thermostable xylanEnzyme from calcium algeneresensisp. nov.strain TH7C1T, carbohy. Res.419(2016) 60-68.) the TLC plate was air dried for 10min, sprayed with methanol-sulfuric acid mixture (9:1, v/v) containing 0.2% orcinol, baked at 85 ℃ for 5min, and the product was observed (H.Liao, S.Sun, P.Wang, W.et., A new acid end- β -1,4-xylanase from Penicillium oxidatum: cloning, purification, and antibiotics inter the antibiotic of cellulose oxidase, J.Ind.Microbiol.Biotech.41 (7) (2014)1071, 1083) and analyzed by TLC of xylose and xylose-xylose oligosaccharides (2014) as standard disaccharide-xylose disaccharide from xylose.

The results show that: the main products of the hydrolysis of three xylans by the recombinant xylanase EpXYN1 are xylobiose, xylotriose and long-chain xylooligosaccharide, respectively, with very little xylose (FIGS. 6A-C); the hydrolysis products of all xylo-oligosaccharides were xylobiose and a very small amount of xylose (fig. 6D). The xylanase is suitable for producing xylooligosaccharide products such as xylobiose, xylotriose and the like.

Sequence listing

<110> Nanjing university of forestry

<120> heat-resistant endo-xylanase EpXYN1, and coding gene and application thereof

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<213> Penicillium microfine (Eupenicillium parvum)

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Met Val Tyr Leu Ser Ala Arg Thr Leu Ala Leu Val Ala Cys Val Leu

1 5 10 15

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

20 25 30

Gly Lys Val Tyr Phe Gly Thr Ala Thr Asp Asn Pro Glu Leu Thr Asp

35 40 45

Thr Ala Tyr Val Lys Gln Leu Ser Asn Thr Asp Asp Phe Gly GlnIle

50 55 60

Thr Pro Gly Asn Ser Gln Lys Trp Asp Ala Thr Glu Pro Ser Gln Asn

65 70 75 80

Ser Phe Thr Phe Thr Asn Gly Asp Val Val Ala His Leu Ala Glu Gly

85 90 95

Asn Gly Gln Lys Leu Arg Cys His Asn Leu Val Trp His Asn Gln Leu

100 105 110

Pro Asn Trp Val Thr Ser Gly Ser Trp Thr Asn Glu Thr Leu Leu Ala

115 120 125

Ala Met Lys Asn His Ile Thr Asn Val Val Thr His Tyr Lys Gly Gln

130 135 140

Cys Tyr Ala Trp Asp Val Val Asn Glu Ala Leu Asn Glu Asp Gly Thr

145 150 155 160

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

165 170 175

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

180 185 190

Tyr Asn Asp Tyr Asn Ile Glu Tyr Ala Gly Ala Lys Ala Thr Gly Ala

195 200 205

Gln Arg Ile Val Lys Leu Ile Gln Ser Tyr Gly Ala Lys Ile Asp Gly

210 215 220

Val Gly Leu Gln Ser His Phe Ile Val Gly Ser Thr Pro Ser Gln Ser

225 230 235 240

Ala Gln Ala Ser Asn Met Ala Ala Phe Thr Ala Leu Gly Val Asp Val

245 250 255

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

260 265 270

Leu Leu Ala Gln Gln Lys Thr Asp Tyr His Ser Thr Val Ala Ala Cys

275 280 285

Val Gln Thr Ala Arg Cys Val Gly Val Thr Ile Trp Asp Trp Thr Asp

290 295 300

Lys Tyr Ser Trp Val Pro Ser Thr Phe Ser Gly Gln Gly Ala Ala Cys

305 310 315 320

Pro Trp Asp Glu Asn Phe Gln Lys Lys Pro Ala Tyr Asp Gly Ile Leu

325 330 335

Ser Ala Leu Gly Gly Ser Ala Ser Ala Thr Ser Thr Ser Thr Ala Gly

340 345 350

Gln Ser Ala Ser Thr Thr Ser Thr Ser Gly Ser Gly Gly Ser Thr Ala

355 360 365

Val Ala Gln His Trp Gly Gln Cys Gly Gly Gln Gly Trp Thr Gly Ala

370 375 380

Thr Ser Cys Ala Thr Gly Tyr Ile Cys Thr Phe Val Asn Asp Trp Tyr

385 390 395 400

Ser Gln Cys Leu

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<213> Penicillium microfine (Eupenicillium parvum)

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caagcatcag gcctggatac agcggcagta gccatcggaa aggtctactt cggcacggcc 120

accgataacc ccgagttgac ggacactgcc tatgtcaagc agctcagcaa cacggatgac 180

tttggtcaaa tcacaccggg aaactcgcag aagtgggatg cgacagagcc atcgcagaac 240

tcgtttacct tcacgaacgg agatgtcgtc gcccacctcg cggagggtaa tggccagaaa 300

ttgcggtgcc ataatctggt ctggcataat cagctaccca actgggtcac cagtggctcg 360

tggaccaacg aaacactcct tgcggcaatg aagaaccaca ttaccaatgt ggtcacccat 420

tataaaggac agtgttatgc ctgggatgtt gtcaacgaag cactcaacga agatggaacc 480

taccgagaca gcatattcta ccaaaccatc ggcgaagcct acatccccat tgccttcaaa 540

acagccgcag ccgcagatcc aaccgtcaag ctctactaca acgactacaa catcgaatac 600

gccggcgcca aggcgacagg cgcacaacga atcgtcaagc taatccagtc ctatggagcg 660

aagatcgacg gcgtcggtct ccaatcccac ttcatcgtcg gcagtacccc gagccagagt 720

gcgcaggcga gcaacatggc agccttcact gctctaggtg ttgacgttgc catcaccgag 780

ttggatatcc ggatgacgct accatcgacg gatgcccttt tggcccagca gaagacggac 840

taccatagta ctgtcgctgc ctgcgtgcag acagctcgct gcgtcggcgt cacgatctgg 900

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ccctgggatg agaatttcca gaagaaaccc gcttacgatg ggatcctttc tgcccttggg 1020

ggtagtgctt cggcgacgtc gacttctact gcgggtcaga gtgcctcgac tacttctact 1080

tctggttcag gtggctcgac ggctgttgct cagcattggg gtcagtgtgg tggacagggt 1140

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tctcaatgtc tgtga 1215

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ttgccgtcca cgctctatc 19

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<213> enXyn1_ r1 sequence (Artificial)

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acagcctacg aggaattaac aac 23

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agagaggctg aagctgaatt ctcaggcctg gatacagcgg ca 42

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<213> enXyn1_ r2 sequence (Artificial)

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gagatgagtt tttgttctag atcagtgatg gtgatggtga tgcagacatt gagagtacca 60

Claims (6)

1. A heat-resistant endo-xylanase EpXYN1 has an amino acid sequence shown in SEQ ID NO. 1.

2. The coding gene of the heat-resistant endo-xylanase EpXYN1 of claim 1, the base sequence of which is shown in SEQ ID NO. 2.

3. An expression vector or expression system comprising the gene encoding the thermotolerant endo-xylanase EpXYN1 of claim 2.

4. The use of the encoding gene of the heat-resistant endo-xylanase EpXYN1 of claim 2 for expressing the heat-resistant endo-xylanase EpXYN 1.

5. The use of the thermostable endo-xylanase EpXYN1 of claim 1 for the enzymatic hydrolysis of hemicellulose.

6. Use of the thermostable endo-xylanase EpXYN1 of claim 1 in the enzymatic hydrolysis of xylan.

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