CN109207457B - Endo-xylanase and application thereof in production of xylobiose - Google Patents

Abstract

The invention relates to the field of biochemical engineering, in particular to a protein RSipoEnXyn10A and application thereof as endo-xylanase in the production of xylobiose. The protein RSipoEnXyn10A is a protein shown in the following 1) or 2): 1) a protein consisting of an amino acid sequence shown in a sequence 3 in a sequence table; 2) protein which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3 in the sequence table, has the same function and is derived from the protein 1). The invention obtains a gene for coding endo-xylanase from the genome sequence of streptomyces IpomoeaeSipoEnXyn10AThe gene can be expressed in a host cell to produce the protein RSipoEnXyn10A，The protein RSipoEnXyn10A is used as endo-xylanase for producing xylobiose, the endo-xylanase is the endo-xylanase which has the properties of releasing the xylobiose as a main product by hydrolyzing xylan of agricultural and forestry residues at high temperature and neutrality, and has important significance for producing the xylobiose.

Description

Endo-xylanase and application thereof in production of xylobiose

Technical Field

The invention relates to the field of biochemical engineering, in particular to endoxylanase and application thereof in production of xylobiose, and specifically relates to a protein RSipoEnXyn10A and application thereof serving as endoxylanase in production of xylobiose by taking agriculture and forestry residues as raw materials.

Background

Xylo-oligosaccharides are linear oligomers of xylose linked by β - (1,4) xylosidic bonds, and molecules with a degree of polymerization of 2 to 20 xylose are generally considered to be Xylo-oligosaccharides, and molecules with a degree of polymerization greater than 20 are considered to be xylans (Carvalho AFA, de Oliva Neto P, da Silva DF, store GM (2013) Xylo-oligosaccharides from lignocellulosic materials: Chemical structures, health resins and production by Chemical and enzymatic hydrolysis. Food Res 51: 75-85.). Xylo-oligosaccharides are not digestible by the human body, so xylo-oligosaccharides are called non-digestible oligosaccharides (NDOs). Xylo-oligosaccharides are very useful functional foods, and xylo-oligosaccharides with different polymerization degrees have different functions. The xylo-oligosaccharide with relatively long polymerization degree (more than 7) can be used as dietary fiber. Xylo-oligosaccharides with a relatively short degree of polymerization (2 to 7) have multiple functions: firstly, it can be exclusively utilized by the probiotic bifidobacteria in the human intestinal tract to promote intestinal health (Gull Lo n P, Moura P, Esteves MP, Girio FM, Dom i nguez H, et al. (2008) Assessment on the nutritional availability of xylooligosaccharide from rice husks by biological bacteria, J Agrric Food Chem 56: 7482-; (iii) promoting calcium absorption and lipid metabolism (Grootaert C, Delcour JA, Courtin CM, Broekaert WF, Verstraete W, et al, (2007) microbiological metabolism and predictive potential of arabinyloxy-oligosaccharides in the human intestine. Trends Food Sci Tech 18: 64-71.); anti-inflammatory, anti-allergic and anti-oxidative, skin and cardiovascular health and immune function promoting (Mendis M, Leclerc E, Simsek S (2016) Arabidopsis, gut microbiota and immunity. Carbohyd Polym 139: 159-; (iv) use as substitute sweetener for diabetic patients without causing elevation of blood sugar level (Carvalho AFA, de Oliva Neto P, da Silva DF, Pastore GM (2013) Xylo-oligosaccharides from lignocellulosic materials Chemical structures, Chemical letters and production by Chemical and enzymatic hydrolizations, Food Res Int 51: 75-85). Of all xylo-oligosaccharides, xylobiose is the xylo-oligosaccharide most rapidly utilized by intestinal probiotics, with the most pronounced prebiotic effect and therefore of most interest (Moura P, Barata R, Carvalheiro F, G i rio F, Loureiro-Dias MC, et al (2007) In vision transfer of xylo-oligosaccharides from corn oligosaccharides autolysis byBifidobacterium and Lactobacillus strains. LWT-Food Sci Technol 40: 963–972.）。

Xylobiose is obtained by hydrolysis of xylan. Xylan is a plant polysaccharide having only one main chain formed by connecting xylose by beta- (1,4) xyloside bonds, and part of xylose residues on the main chain are modified by L-arabinose, D-glucose, D-galactose, D-mannose, D-glucuronic acid and D-galacturonic acid. The sugars used to modify xylose residues vary due to differences in plant origin (Deutschmann R, Dekker RFH (2012) From plant bioglass to bio-based chemicals: last definitions in xylan research. Biotechnol Adv 30: 1627-. Xylan is the second largest polysaccharide of Plant cell walls, has a xylan content of 20-30% in the secondary cell walls of dicotyledonous plants and 20-40% and 40-50% in the primary cell walls and the secondary cell walls of monocotyledonous plants, respectively (Scheller HV, Ulvskov P (2010) Hemicelluloses. Annu Rev Plant Biol 61: 263-289), so that forestry and agricultural residues can be used for producing xylobiose.

The agricultural residues refer to organic substances discarded in agricultural production, and mainly comprise straws or other residual parts of starch crops in grain production; forestry residues refer to organic substances discarded in forestry production, mainly bamboo chips in bamboo product production and mainly wood chips in board production. Since agroforestry residues are readily available, shelf-stable, and their use in the production of xylobiose makes it possible to change waste into valuable and to reduce environmental pollution caused by incineration, it is worth studying their use in the production of xylobiose (Carvalho AFA, de Oliva Net P, de Silva DF, store GM (2013) Xylo-oligosaccharides from lignocellulosic materials: Chemical structures, health resins and products by Chemical and enzymatic hydrolytics. Food Res Int 51: 75-85.).

There are 3 methods for using agroforestry residues for the production of xylobiose, chemical hydrolysis, autohydrolysis and enzymatic hydrolysis, respectively. The chemical hydrolysis method needs to use high-concentration chemicals (such as strong acid and strong alkali), so that the method has the defects of environmental unfriendliness and high requirements on instruments and equipment; the self-hydrolysis method needs to use high temperature (generally far higher than 100 ℃) and high pressure, so the defects of high energy consumption and high requirements on instruments and equipment exist; the enzymatic hydrolysis process is carried out under mild conditions and is therefore an environmentally friendly and low energy consuming process (Mano MCR, Neri-Numa IA, da Silva JB, Paulino BN, Pessoa MG, et al (2018) Oligosaccharide biotechnology: an aproach of biological recycling on the index. Appl Microbiol Biotechnology 102: 17-37.). The enzymatic hydrolysis process is mainly divided into three steps of treatment of raw materials, hydrolysis of enzymes and recovery and purification of products (oxygen DO, Ahring BK (2012), The potential for enzymatic hydrolysis from The microbial fraction of biological through biological processes: xylo-oligosaccharide (XOS), Arabinooligosaccharide (AOS), and Methanooligosaccharide (MOS). Carbohydrar Res 360: 84-92.).

Xylanases are a generic term for all enzymes that hydrolyze xylan, including those that hydrolyze the backbone and those that hydrolyze to remove modifying groups from xylose residues in the backbone (Juuru V, Wu JC (2012) Microbial xylanases: Engineering, production and industrial applications. Biotechnol Adv 30: 1219-. Among all xylanases, Endo-xylanases (EC: 3.2.1.8) hydrolyze the beta- (1,4) xylosidic bonds between any two xylose residues in the backbone in a random manner, and are thus the most important enzymes in the process for producing xylobiose, even with this enzyme alone (Subramaniyan S, Prema P (2002) Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnology 22: 33-64.).

The process for producing xylobiose requires an endoxylanase with high temperature, neutrality and high yield of xylobiose from residues of agriculture and forestry, since high temperature prevents loss of xylobiose due to microbial growth (Bhala, Bansal N, Kumar S, Bischoff KM, Sani RK (2013) Improved lipid metabolism to biologicals with thermo-bacterial and thermo-stable enzymes, BioResourcer Technol 128: 751-759.); the high temperature can enable the high-temperature endoxylanase to exert the highest enzyme activity, thereby improving the hydrolysis efficiency, shortening the production time, improving the equipment utilization rate and avoiding additional instrument and equipment for cooling (Viikari L, Alapuren M, Puranen T, Vehmaper ä J, Siika-aho M (2007) thermoplastic enzymes In lignocellulose Hydrolysis, In Biofuels spring-Verlag Berlin Heidelberg 108: 121-145.); strong acid and strong alkaline environments not only cause loss of xylobiose by spontaneous hydrolysis to xylose, but also corrode production equipment (Courtin CM, Swennen K, Verjans P, Delcour JA (2009) Heat and pH stability of preventive arabinoxylo oligosaccharides, xylo oligosaccharides and fructooligosaccharides, Food Chem 112: 831-; the higher The proportion of xylobiose in The hydrolysate, The lower The difficulty and cost of purification and The higher The probiotic nutritional and sweetener value of The resulting xylobiose product (Otieno DO, Ahring BK (2012), The functional for oligosaccharide production from The carbohydrate fraction of polysaccharides (XOS), Arabinooligosaccharide (AOS), and Methanooligosaccharide (MOS) Carbohydr Res 360: 84-92.).

Some high temperature, neutral endoxylanases have been reported for the production of xylo-oligosaccharides by hydrolysis of agroforestry residues in laboratory tests, but their hydrolysis products do not contain mainly xylobiose. For example, endoxylanase Xyn10CD18 obtained from cow dung compost has optimum action temperature and pH value of 75 ℃ and 7.0, respectively, but the hydrolysis pretreated corn cob releases xylose as the main component, xylo-oligosaccharide as the minor component, and xylobiose as the lower component (Sun MZ, Zheng HC, Meng LC, Sun JS, Song H, Bao YJ, Pei HS, Yan Z, Zhang XQ, Zhang JS, Liu YH, Lu FP (2015) Direct cloning, expression of a thermostable xylase gene from the microbial DNA of a sugar production and enzymatic purity of xylo polysaccharides from sugar sugars, biotechnological Lett 37: 1877, 1886.); the optimum working temperature and pH of endoxylanase rMxyl obtained from hot spring bottom mud are 80 ℃ and 7.0, respectively, but the product released from bagasse after hydrolysis pretreatment is not xylobiose as the main component, but also contains a lot of xylotriose, xylotetraose and xylooligosaccharide with higher degree of polymerization (Verma D, Kawarabayasi Y, Miyazaki K, Satyananayana T (2013) Cloning, expression and characterization of a novel alkaline and thermostable xylosidase encoding (Mxyl) obtained from purified free composition-sometric company, PLoS ONE 8: 459e 5252.).

Disclosure of Invention

The invention aims to provide a protein RSipoEnXyn10A and application thereof as endo-xylanase in the production of xylobiose, wherein the endo-xylanase is the endo-xylanase which takes xylobiose released by high-temperature, neutral and hydrolyzed agricultural and forestry residues xylan as a main product and has important significance for the production of the xylobiose.

The protein RSipoEnXyn10A provided by the invention is a protein shown in the following 1) or 2):

1) a protein consisting of an amino acid sequence shown in a sequence 3 in a sequence table;

2) protein which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3 in the sequence table, has the same function and is derived from the protein 1). The substitution and/or deletion and/or addition of one or more amino acid residues is the substitution and/or deletion and/or addition of no more than 10 amino acid residues in the 53 th to 382 th positions of the sequence 3 in the sequence table.

The invention also aims to provide a gene for coding the protein RSipoEnXyn10A, wherein the nucleotide sequence of the gene is shown as a sequence 2 in a sequence table.

An expression vector containing the protein RSipoEnXyn10A gene is a vector suitable for expression in escherichia coli.

The protein RSipoEnXyn10A gene of the invention was inserted into pET30a (+) vector.

The protein RSipoEnXyn10A gene (namely the gene shown in the sequence 1 in the sequence table) is inserted into a pET30a (+) vectorEcoRI andXhoi between the restriction enzyme sites.

The vector is the expression vector of any one of the preceding claims.

A strain for producing protein RSipoEnXyn10A is prepared by constructing recombinant expression vector from nucleotide sequence of protein RSipoEnXyn10A gene (gene shown in sequence 1 in sequence table), and converting into Escherichia coliEscherichia coliStrain BL21 (DE 3);

a strain for producing protein RSipoEnXyn10A is prepared by the following steps:

extracting Streptomyces Ipomoeae by PCR methodStreptomyces ipomoeaeThe CGMCC 4.1381 genome DNA is used as a template for PCR amplification, and the obtained PCR product with the sequence 1 in the sequence table is inserted into a prokaryotic expression vector pET30a (+) to obtain a recombinant expression vector pET30a (+) -RSipoEnXyn10AAnd transformed into Escherichia coliEscherichia coliThe recombinant expression strain containing the recombinant expression vector for producing the protein RSipoEnXyn10A is obtained after strain BL21 (DE 3).

The sequences of the primers used for PCR amplification are as follows:

the upstream primer is 5' -CCGGAATTCGTGCAGCCGGCCTCCGCCCAT-3' (containing DNA restriction enzyme)EcoThe cleavage site GAATTC of RI),

the downstream primer is 5' -CCGCTCGAGCCGCCCCGCGATCAACCCCTC-3' (containing DNA restriction enzyme)XhoThe cleavage site of I CTCGAG),

using the extracted genomic DNA of Streptomyces Ipomoeae as a template, 2X Phanta was used^®Performing PCR amplification by using a Master Mix reagent, wherein the reaction system comprises the following steps:

the overall sequence of the 5 steps in the PCR reaction program is from (i) to (v), the specific sequence is that the (i) step is operated only for 1 time, the (ii), the (iii) and the (iv) step are operated in sequence to form a cycle, the (iv) step is performed after 35 cycles are continued, and the (iv) step is operated only for 1 time.

The protein RSipoEnXyn10A is used as endo-xylanase in the production of xylobiose, particularly in the production of xylobiose by using agriculture and forestry residues as raw materials, and particularly in the production of xylobiose by using alkali extracts of corncobs, cassava stems, phyllostachys pubescens and cedarwood as raw materials.

Experiments prove that the invention uses streptomyces Ipomoeae as a raw materialStreptomyces ipomoeaeA gene encoding a putative protein was found in the genomic sequence of CGMCC 4.1381SipoEnXyn10AThe gene can be expressed in a host cell to produce the protein RSipoEnXyn10A，The protein RSipoEnXyn10A is used as endo-xylanase in the production of xylobiose. Enzyme activity correlation detection is carried out on the endo-xylanase, the optimum action pH value of the enzymatic reaction is 6.5, and the optimum action temperature value is 75-80 ℃. The specific activities of the endo-xylanase RSipoEnXyn10A on beech xylan and arabinoxylan under the optimal action conditions (pH 6.5, 75 ℃) were 197.75 + -1.42U/mg and 627.30 + -15.11U/mg protein, respectively.

The endo-xylanase RSipoEnXyn10A is an endo-xylanase which has the properties of high temperature, neutrality and hydrolysis of agricultural and forestry residues, namely release of xylobiose as a main product, is not possessed by the reported endo-xylanase and has important significance for the production of the xylobiose.

Drawings

FIG. 1 is an agarose gel electrophoresis of genomic DNA of Streptomyces Ipomoeae CGMCC 4.1381;

FIG. 2 is an agarose gel electrophoresis of the PCR product of a nucleotide sequence encoding a putative protein in the Streptomyces Ipomoeae CGMCC 4.1381 genome;

FIG. 3 shows a recombinant expression vector pET30a (+)-RSipoEnXyn10AThe construction diagram of (1);

FIG. 4 is an SDS-PAGE analysis of the purified recombinant endo-xylanase RSipoEnXyn 10A;

FIG. 5 is an HPLC profile of purified recombinant endo-xylanase RSipoEnXyn10A hydrolyzed beech xylan;

FIG. 6 is the pH optimum curve of purified recombinant endo-xylanase RSipoEnXyn 10A;

FIG. 7 is the optimum temperature profile of purified recombinant endo-xylanase RSipoEnXyn 10A;

FIG. 8 is a pH tolerance curve of the purified recombinant endo-xylanase RSipoEnXyn 10A;

FIG. 9 is a temperature tolerance curve of the purified recombinant endo-xylanase RSipoEnXyn 10A;

FIG. 10 is an HPLC chromatogram of the product of purified recombinant endoxylanase RSipoEnXyn10A hydrolysis of corncob alkali extract;

FIG. 11 is an HPLC chromatogram of the product of the hydrolysis of cassava stalk alkali extract by purified recombinant endo-xylanase RSipoEnXyn 10A;

FIG. 12 is an HPLC chromatogram of the product of the purified recombinant endoxylanase RSipoEnXyn10A hydrolysis of phyllostachys pubescens alkali extract;

FIG. 13 is an HPLC chromatogram of the product of hydrolysis of a cedarwood alkali extract by the purified recombinant endoxylanase RSipoEnXyn 10A.

Detailed Description

Unless otherwise specified, the experimental methods used in all the examples are conventional methods, the materials and reagents used are commercially available, and the percentages (%) are mass percentages. The quantitative tests in all examples were set up in triplicate and results presented were either mean or mean ± standard deviation.

1. Strain, vector, restriction enzyme, antibiotic and kit

Streptomyces IpomoeaeStreptomyces ipomoeaeCGMCC 4.1381 was purchased from China General Microbiological Culture Collection Center (CGMCC). Restriction enzymeEcoRⅠ(catalog No. 1040S) andXhoi (catalog # 1094S) was purchased from TaKaRa, Liaoning, China. T4 DNA ligase (catalog number 2011A) was purchased from TaKaRa Inc. (Liaoning, China). Escherichia coli (Escherichia coli) Chemically competent cells of strains Trans 1T1 and BL21 (DE 3) were purchased from Hopkinson (Beijing, China) under the catalog numbers CD501-02 and CD601-02, respectively. The expression vector pET30a (+) was purchased from Merck, Darmstadt, Germany, under catalog number 69909-3. Kanamycin sulfate (Kanamycin A sulfate) (Cat. K8020-5 g), IPTG (isopropyl-. beta. -D-thiogalactoside) (Cat. I8070) were purchased from Solarbio corporation, Beijing, China. 2X Phanta^®Master Mix kit (catalog number P511-02) and Green Taq Mix kit (catalog number P131-01) were purchased from Novozam, Jiangsu, China. The DNA purification and recovery kit (catalog No. DP 214-03) and the plasmid petiole kit (catalog No. DP 103-02) were purchased from Tiangen corporation (Beijing, China). The rapid extraction kit of genome DNA (catalog number SK 8230) is purchased from Biotechnology engineering, Inc. (Shanghai, China). The BCA kit (catalog # 23225) was purchased from Pierce, Inc. (Rockford, USA). Ni-Agarose Resin filler (catalog number CW 0010) was purchased from Kangshu century corporation (Beijing, China).

2. Polysaccharides and oligosaccharides

Beech xylan (catalog number P-XYLNBE-10G), arabinoxylan (catalog number P-waxrs), β -barley glucan (catalog number P-BGBL), mannan (catalog number P-MANIV), lichenin (catalog number P-LICHN), xyloglucan (catalog number P-XYGLN), chitin (catalog number P-CHITN), starch (catalog number P-AMYL), polygalacturonic acid (catalog number P-PGACT) was purchased from Megazyme corporation (Wicklow, Ireland). Sodium carboxymethylcellulose (catalog No. C-5678) was purchased from Sigma. Xylobiose (catalog No. O-XBI), xylotriose (catalog No. O-XTR), xylotetraose (catalog No. O-XTE) were purchased from Megazyme Inc. (Wicklow, Ireland).

3. Culture medium

The culture of the streptomyces Ipomoeae uses an improved Gao's I culture medium, and the formula is (g/L): 20 parts of soluble starch, 2 parts of beef extract, 5 parts of peptone and KNO ₃ 1，K₂HPO₄•3H₂O 0.5，MgSO₄•7H₂O 0.5，NaCl 0.5，FeSO₄•7H₂O0.01, pH 7.2-7.4. The solid culture medium for the flat plate is a liquid culture medium, and 3% agar powder is added.

An LB culture medium is used for culturing the escherichia coli, and the formula is (g/L): yeast extract 5, tryptone 10, sodium chloride 10, pH 7.0. The solid culture medium for the flat plate is a liquid culture medium, and 3% agar powder is added.

4. Determination of enzyme Activity of endo-xylanase

The enzyme activity is determined by a DNS method, and the protein content is determined by a BCA method. The specific description is as follows:

(1) enzyme activity assay

Making standard xylose curve

A xylose standard solution of 1 mg/mL was prepared with deionized water, and 9 xylose solutions of 400. mu.L continuous concentration were prepared in a 1.5 mL centrifuge tube using the standard solution and deionized water, respectively at 0 mg/mL (i.e., deionized water itself), 0.125 mg/mL, 0.25 mg/mL, 0.375 mg/mL, 0.5 mg/mL, 0.625 mg/mL, 0.75 mg/mL, 0.875 mg/mL, and 10 mg/mL, with three replicates for each sample concentration. Adding 800 mu L of DNS reagent into all samples, uniformly mixing, carrying out boiling water bath for 5 minutes, placing in tap water, cooling to room temperature, adding 200 mu L of each sample into a 96-well serum plate, and measuring the light absorption value at 540 nm by using a microplate reader. In Excel software, a scatter plot was made with the xylose concentration on the horizontal axis (X-axis) and the light absorption value on the vertical axis (Y-axis), and a trend line was added to obtain a standard curve of Y =3.5321X-0.0325 (correlation coefficient R)²0.999).

The formulation of the DNS reagent is described in the literature (Xiaoan L, Wang F, Luo X, Feng YL, Feng JX (2015) Purification and characterization of a high by efficient calcium-independent α-amylase from Talaromyces pinophilus 1-95. PLoS ONE 10：e0121531.）。

② measurement of enzyme activity

The enzyme activity determination reaction is divided into a reaction group and a control group. The reaction group is that 350 mu L of buffer solution containing 0.5% beech xylan is added into a 1.5 mL centrifuge tube (the pH value is set according to different experimental requirements in the invention), 50 mu L of enzyme solution with the concentration of 0.01 g/L is added after preheating for 3 minutes in a water bath kettle with constant temperature (the temperature is set according to different experimental requirements in the invention), and the mixture is rapidly mixed. After the reaction was incubated for 10 minutes, 800. mu.L of DNS reagent was added to terminate the reaction and the reaction was incubated for 5 minutes in a boiling water bath to develop color sufficiently. The control group was prepared by inactivating the enzyme solution in boiling water bath for 5min, and treating with the same method as the reaction group. After the reaction group and the control group were placed in tap water and cooled to room temperature, 200. mu.L of the test solution was added to a 96-well serum plate, and the light absorption value at 540 nm was measured with a microplate reader. And subtracting the light absorption value of the inactivated enzyme solution control group from the light absorption value of the inactivated enzyme solution group, substituting the obtained difference value into a xylose standard curve to calculate the amount of reducing sugar generated in the reaction system, and further calculating to obtain the enzyme activity. The activity unit (U) of an endoxylanase is defined as: under the set assay conditions, the amount of enzyme required to hydrolyze xylan liberate 1. mu. mol reducing sugars per minute.

Enzyme specific activity is defined as: the enzyme activity per mg of protein (U/mg) under the set measurement conditions.

(2) Determination of protein content

Protein was determined using the BCA method (Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using biochemical analysis, 150: 76-85). The determination method was performed according to the product instructions. The horizontal axis (X axis) of the calf serum protein concentration and the vertical axis (Y axis) of the 562 nm light absorption value were used as scattergrams, and the standard curve of Y =0.9074X +0.0521 (correlation coefficient R) was obtained by adding the trend line²0.9939).

Example 1 GeneSipoEnXyn10AObtained by

(1) Extraction of streptomyces Ipomoeae genome DNA

Inoculating streptomyces Ipomoeae to the improved Gao's I solid culture medium plate, and standing and culturing at 28 deg.C for 5 days to obtain fresh viable spore. Spores were inoculated into Gao's I liquid medium (50 mL in a 250 mL-size triangular flask), and after shaking culture at 28 ℃ and 180 rpm for 2 days, the grown mycelia were used for extraction of genomic DNA.

And (3) extracting the genomic DNA of the streptomyces Ipomoeae by using the genomic DNA rapid extraction kit, and operating according to the product use instruction. The DNA solution obtained by the extraction was subjected to agarose gel electrophoresis at a concentration of 0.8%, and the results are shown in FIG. 1. The sample in lane 1 of FIG. 1 is a 1 kb ladder containing 7 DNA fragments, 15, 10, 7.5, 5, 2.5, 1 and 0.25 kb from large to small; the sample in lane 2 is genomic DNA of Streptomyces Ipomoeae. A band is evident above 15 kb in lane 2, indicating that the genomic DNA of S.Ipomoeae has been successfully extracted.

(2) Genes, genesSipoEnXyn10AObtained by

Designing a pair of primers for PCR amplification of genesSipoEnXyn10AThe upstream primer is 5' -CCGGAATTCGTGCAGCCGGCCTCCGCCCAT-3' (containing DNA restriction enzyme)EcoCleavage site GAATTC of RI), and the downstream primer is 5' -CCGCTCGAGCCGCCCCGCGATCAACCCCTC-3' (containing DNA restriction enzyme)XhoI cleavage site CTCGAG), 2X Phanta was used with the extracted Streptomyces Ipomoeae genomic DNA as a template^®Performing PCR amplification by using a Master Mix reagent, wherein the reaction system comprises the following steps:

the overall sequence of the 5 steps in the PCR reaction program is from (i) to (v), the specific sequence is that the (i) step is operated only for 1 time, the (ii), the (iii) and the (iv) step are operated in sequence to form a cycle, the (iv) step is performed after 35 cycles are continued, and the (iv) step is operated only for 1 time.

The PCR product was subjected to agarose gel electrophoresis at a concentration of 0.8%, and the results are shown in FIG. 2. The sample in lane 1 of FIG. 2 is a 1 kb DNA ladder containing 14 bands of 10, 8, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75, 0.5 and 0.25 kb in size; the sample in lane 2 is geneSipoEnXyn10AThe PCR product of (1). The electrophoresis result shows that lane 2 has a band at about 1 kb, and the length of the band matches the length of the expected product. The PCR product is sent to Beijing Okkomy to determine the DNA sequence thereof, and the determination result shows that the fragment is a target gene and the nucleotide sequence thereof is sequence 1.

Example 2 functional verification of the recombinant protein RSipoEnXyn10A

One, the recombinant expression vector pET30a (+) -RSipoEnXyn10AConstruction of

The vector pET30a (+) was used with restriction enzymeEcoRI andXhothe product of the double digestion is recovered by a DNA purification recovery kit and then subjected to 0.8% agarose gel electrophoresis, and only one 5.4 kb long carrier band is detected in the lane (FIG. 3A, the sample in lane 1 is a 1 kb DNA ladder containing 14 DNA fragments from large to small, 10, 8, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75, 0.5 and 0.25 kb, respectively; the sample in lane 2 is detected by restriction endonucleaseEcoRI andXhoi the empty vector pET30a (+) is subjected to double digestion and then is recovered by a DNA purification and recovery kit).

The PCR product having the sequence shown in SEQ ID No. 1 obtained in example 1 was subjected to 0.8% agarose gel electrophoresis, and the gel piece with the band was excised and recovered by a DNA purification recovery kit. Recovering the product and using restriction enzymeEcoRI andXhoand I, carrying out double enzyme digestion, and recovering a double enzyme digestion product by using a DNA purification and recovery kit. The recovered product was subjected to 0.8% agarose gel electrophoresis to obtain an exogenous DNA band of about 1 kb (FIG. 3B, lane 1 sample is 1 kb DNA ladder containing 14 DNA fragments of 10, 8, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75, 0.5 and 0.25 kb from large to small; lane 2 sample is obtained by restriction enzymeEcoRI andXhoi is shown in sequence 1And (3) carrying out electrophoresis on the PCR product, then recycling, carrying out double enzyme digestion, and then recycling to obtain the product).

The recovered double-restriction enzyme gene fragment and the recovered double-restriction enzyme vector pET30a (+) are connected by using T4 DNA ligase (performed according to the instruction), and the connection product is used for chemically transforming escherichia coli (the method is performed by the instruction)Escherichia coli) Competent cells of the strain Trans 1T1, resulting in a transformed strain containing the unverified vector. The unverified vector of the transformed strain was extracted using a plasmid miniprep kit, and PCR amplification was performed using the unverified vector as a template and the T7 forward primer 5'-TAATACGACTCACTATAGGG-3' and the reverse primer 5'-TGCTAGTTATTGCTCAGCGG-3' according to the system and procedure described in example 1. The amplified product was subjected to 0.8% agarose gel electrophoresis to obtain a band of about 1.2 kb in length. The size of the band minus the length of the vector-bearing partial sequence is very similar to the length of the foreign DNA as described in the vector specification (FIG. 3C, the sample in lane 1 is a 1 kb DNA ladder containing 14 DNA fragments, 10, 8, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75, 0.5 and 0.25 kb from large to small, respectively; the sample in lane 2 is amplified using the extracted unverified vector as a template). Restriction enzyme for vector verified by PCR amplificationEcoRI andXhothe double restriction enzyme digestion was performed, and the product of the double restriction enzyme digestion was subjected to 0.8% agarose gel electrophoresis to obtain an about 5.4 kb pET30a (+) vector band and an about 1 kb exogenous DNA band, indicating that the vector verified by PCR amplification contained the expected size of the exogenous gene (FIG. 3D, the sample in lane 1 was a 1 kb DNA ladder containing 14 DNA fragments from the large to the small of 10, 8, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.75, 0.5 and 0.25 kb, respectively; the sample in lane 2 was an unverified vector restriction enzyme for restriction enzymesEcoRI andXhoproduct obtained by double enzyme digestion of I). Will contain recombinant vectors that have been verified by PCR amplification and double digestionEscherichia coliThe Trans 1T1 transformed strain is sent to Beijing Okkomy company, and is sequenced by using a T7 upstream primer 5'-TAATACGACTCACTATAGGG-3' and a T5'-TGCTAGTTATTGCTCAGCGG-3' downstream primer, and the sequencing result shows that the inserted sequence is sequence 1, and the recombinant expression vector is successfully constructed.

In the case of using the vector pET30a (+)EcoRI andXhowhen the restriction enzyme cutting site expresses the foreign gene, the promoter carried by a vector pET30a (+) is used, so that 52 amino acid residues are additionally added at the N terminal of the recombinant protein; the C-terminus of the recombinant protein was increased by 8 amino acid residues due to the use of the terminator from the vector (for details, see the commercial description of vector pET30a (+). The sequence from the start codon to the terminator on the vector is a novel gene encoding a recombinant protein, and the gene encoding the recombinant protein and the original gene are convenient to useSipoEnXyn10AThe successfully constructed recombinant expression vector was named pET30a (+) -RSipoEnXyn10AThe gene encoding the recombinant protein was namedRSipoEnXyn10AThe encoded recombinant protein is named RSipoEnXyn10A ("R" is the initial letter of the English word recombiant, meaning "recombinant"). GeneRSipoEnXyn10AThe complete sequence of the encoded recombinant protein RSipoEnXyn10A is shown in a sequence 2, and the complete amino acid sequence of the encoded recombinant protein RSipoEnXyn10A is shown in a sequence 3.

The full length of the sequence 2 is 1173 bp. In the sequence 2, the 1 st to 156 th bases are the sequences carried by the vector pET30a (+) itself (the 1 st to 3 rd bases are the initiation codon for expression of the inserted gene in the vector pET30a (+) and the 151 th to 156 th bases are the initiation codons for expression of the inserted geneEcoRI enzyme cutting site), the 157-th and 1146-th bases areSipoEnXyn10AThe gene sequence, the sequence of the vector pET30a (+) at the position 1147-1173 (the sequence at the position 1147-1152)XhoI, the site of the enzyme digestion I, the site 1171-1173 is a stop codon). Positions 1-52 of the sequence 3 are coded by a base sequence carried by the carrier pET30a (+) per se (bases 1-156 of the sequence 2), wherein positions 2-7 are 6 histidines (coded by bases 4-21 of the sequence 2) for facilitating purification by nickel column packing; from 53 to 382, areSipoEnXyn10AThe gene code (base 157-1146 of the sequence 2); the 383-position 390 th site is encoded by the base sequence carried by the pET30a (+) itself (1147-position 1170 base of the sequence 2), wherein the 385-position 390 th site is 6 histidines (encoded by the 1153-position 1170 base of the sequence 2) which are convenient for purification by nickel column packing. Sequence 3 was calculated on-line (https:// web. expasy. org/protparam /), andthe theoretical molecular weight of the recombinant protein RSipoEnXyn10A is 43.46 kDa.

II, obtaining the recombinant protein RSipoEnXyn10A

The recombinant expression vector pET30a (+) -RSipoEnXyn10ATransformation of E.coli by chemical transformation (Escherichia coli) Expression of chemically competent cells of strain BL21 (DE 3) according to the description, recombinant expression strains containing recombinant expression vectors were obtained. Meanwhile, the empty vector pET30a (+) without the foreign gene is transformed into escherichia coli by a chemical transformation method (Escherichia coli) Expression of the chemically competent cells of strain BL21 (DE 3) to obtain recombinant expression strains containing empty vectors for comparative experiments.

OD of recombinant expression strain containing recombinant expression vector or empty vector in culture solution₆₀₀At 0.8, IPTG (isopropyl-. beta. -D-thiogalactoside) was added to a final concentration of 1 mM for induction, and the recombinant protein was expressed for 16 hours in a constant temperature shaker set at 20 ℃ and 100 rpm. After induction, the culture broth was centrifuged at 6000 Xg for 5 minutes to obtain recombinant expression strain cells. The cells were resuspended using a suspension (i.e., the starting buffer for purification of recombinant proteins using nickel column packing) having the following formulation: NaH₂PO₄50 mM, NaCl 300 mM, imidazole 10 mM, pH 8.0. The cells in suspension were disrupted using sonication with parameters: the operation at 250 Hz for 4 seconds and the pause for 6 seconds form a cycle, 150 cycles are carried out, and the suspended cells are kept at low temperature by ice water in the whole process to prevent the heat inactivation of the protein. After the disruption is finished, the disruption solution is used for measuring the activity of xylanase, and as a result, the disruption solution obtained by the recombinant expression strain containing the vector pET30a (+) has no activity of xylanase, but contains the recombinant expression vector pET30a (+) -RSipoEnXyn10AThe obtained breaking liquid of the recombinant expression strain has xylanase activity, which indicates that the induction expression is successful.

The disruption solution was centrifuged at 6000 Xg for 5 minutes to remove precipitates, and the supernatant was subjected to an affinity chromatography purification of the recombinant protein RSipoEnXyn10A using a 0.45 μm aqueous phase filtration membrane to remove impurities, followed by using Ni-Agarose Resin packing, available from Kangji corporation. The used eluent is initial buffer solution containing 500 mM imidazole, and the initial buffer solution and the elution buffer solution are mixed according to different proportions to obtain buffer solutions containing imidazole with different concentrations for eluting the recombinant protein with different strengths. The purification steps are as follows:

(1) adding 10 mL of Ni-Agarose Resin filler into the distributed empty column, and adding 5 times of column volume of deionized water to flow through the filler after the liquid for preservation is drained, thereby achieving the purpose of thorough cleaning;

(2) adding 2 times of column volume of initial buffer solution to balance the filler after the deionized water is drained;

(3) pouring the filler into a centrifuge tube filled with 100 mL of the supernatant containing the recombinant protein RSipoEnXyn10A, burying the centrifuge tube in ice, and rapidly oscillating on a shaker for 1 hour to achieve the purpose of full adsorption;

(4) transferring the supernatant together with the filler into an empty column to ensure that the liquid flows out naturally;

(5) washing the filler with 5 times of column volume of initial buffer solution to make the liquid flow out naturally;

(6) the packing was washed with 5 column volumes of 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM imidazole, respectively, and the eluates were collected and stored at 4 ℃ for further use.

The protein purity in the eluate was determined by SDS-PAGE (Laemmli UK. 1970. Cleavage of structural proteins during the analysis of the head of bacterial proteins T4. Nature 227: 680-685), and the results are shown in FIG. 4. Lanes 1 and 5 in FIG. 4 are protein molecular weight markers comprising 7 bands, 97, 66.5, 45, 35, 25, 18.4 and 14.4 kDa from top to bottom; lane 2 is the disruption solution obtained from the recombinant expression strain containing the empty vector pET30a (+), and lane 3 is the disruption solution containing the recombinant vector pET30a (+) - RSipoEnXyn10A Lane 4 shows the eluate of 200 mM imidazole. As can be seen from FIG. 4, a more prominent protein band is observed in the sample in lane 3 than in the sample in lane 2, which is of very high purity in lane 4 and corresponds to a molecular weight of about 44 kDa, compared to the on-line calculation (https:// webrotparam /) the theoretical molecular weight of 43.46 kDa of the resulting recombinant protein RSipoEnXyn10A, indicating successful purification of the recombinant protein RSipoEnXyn10A according to the above described method.

The purified recombinant protein RSipoEnXyn10A was concentrated by a centrifugal ultrafiltration tube with a molecular weight cutoff of 10 kDa, then desalted and concentrated by adding deionized water, and desalted and concentrated by repeatedly adding deionized water 3 times to obtain desalted and concentrated RSipoEnXyn10A, which was used in experiments to determine all the enzymatic properties described below in this specification.

Thirdly, the enzymological properties of the recombinant protein RSipoEnXyn10A

1. Mode for hydrolyzing xylan by recombinant protein RSipoEnXyn10A

Dissolving beech xylan in 0.1M citric acid-Na₂HPO₄(pH 6.5) in a buffer at a concentration of 1%. The dosage of RSipoEnXyn10A is 0.8 mg per gram of xylan, and the xylan is hydrolyzed at 70 ℃ for 0, 30 min, 2 h, 4 h, 12 h, 24 h and 48 h. The products released by hydrolysis were detected by HPLC using Agilent 1260 definition liquid chromatograph (California, USA), Hypersil NH₂An amino column (column No. E2918007, alligat instruments ltd, alligat, china) and an Alltech ELSD 2000ES evaporative light scattering detector (illinois, usa). The sample amount of the hydrolysate sample was 20 μ L, the column temperature of the amino column was 30 ℃, and the mobile phase was acetonitrile: water =75:25 (volume ratio), flow rate 1 mL/min, gas drift tube temperature 90 ℃, gas velocity 2L/min. The results are shown in FIG. 5.

FIG. 5A is a standard sample containing xylose, xylobiose, xylotriose, xylotetraose, all at a concentration of 1 g/L; FIG. 5B, C, D, E, F, G, H shows hydrolysates obtained by hydrolysis for 0, 30 min, 2 h, 4 h, 12 h, 24 h and 48 h, respectively. From this figure it can be seen that RSipoEnXyn10A hydrolyzes beech xylan to release xylobiose, xylotriose and xylotetraose, and as the hydrolysis time is prolonged, the content of xylobiose and xylotriose gradually increases until the final product is mainly xylobiose and a small amount of xylose and xylotriose. This hydrolysis pattern corresponds to the pattern of endonucleases (Juuru V, Wu JC (2012) Microbial xylanases: Engineering, production and industrial applications. Biotechnol Adv 30: 1219-1227.), so RSipoEnXyn10A is an endoxylanase.

2. Optimum action pH value of recombinant protein RSipoEnXyn10A

Separately preparing buffers with pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, and 11.0 containing 0.5% xylan, wherein 0.1M citric acid-Na is used at pH of 3.0-7.0₂HPO₄Buffer, pH 6.0-8.0 using 0.1M Na₂HPO₄-NaH₂PO₄The buffer solution, Tris-HCl buffer 0.1M at pH 7.0-9.0 and glycine-NaOH buffer 0.1M at pH 8.5-11.0. The desalted and concentrated RSipoEnXyn10A is diluted to 0.01 g/L, and the enzyme activity of the RSipoEnXyn10A under different pH values is measured at 70 ℃ according to the method. The highest enzyme activity obtained by determination is set as 100%, and the enzyme activity under other pH values is compared with the highest enzyme activity and converted into relative enzyme activity. The pH value is taken as an X axis, the relative enzyme activity is taken as a Y axis for plotting, and the result is shown in figure 6, which shows that the optimum action pH value of RSipoEnXyn10A is 6.5.

3. Optimum action temperature of recombinant protein RSipoEnXyn10A

With 0.1M citric acid-Na at pH 6.5₂HPO₄Preparing 0.5% (W/V) xylan solution with buffer solution, diluting the desalted and concentrated RSipoEnXyn10A to 0.01 g/L, and determining the enzyme activity of RSipoEnXyn10A at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C and 95 deg.C according to the above method for determining enzyme activity. The highest enzyme activity is set as 100%, and the enzyme activity at other temperatures is converted into relative enzyme activity compared with the highest enzyme activity. The temperature is taken as an X axis, the relative enzyme activity is taken as a Y axis for plotting, and the result is shown in figure 7, which shows that the optimum action temperature value of RSipoEnXyn10A is 75-80 ℃.

4. pH tolerance of the recombinant protein RSipoEnXyn10A

0.1M buffer pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11.0 was prepared, wherein 0.1M citric acid-Na was used at pH 3.0-7.0₂HPO₄Buffer, pH 6.0-8.0 using 0.1M Na₂HPO₄-NaH₂PO₄In a buffer solution, 0.1M Tris-HCl buffer solution was used at pH 7.0 to 9.0, and 0.1M glycine-NaOH buffer solution was used at pH 8.5 to 11.0, RSipoEnXyn10A was stored in buffer solutions of different pH values (and the concentration of RSipoEnXyn10A was 0.01 g/L), and after standing at 4 ℃ for 24 hours, the residual enzyme activities in the respective pH buffer solutions were measured at pH 6.5 and 75 ℃. The enzyme activity of the preserved enzyme solution which is not treated as above is taken as 100 percent, and the enzyme activity of the preserved enzyme solution at each pH value is converted into relative enzyme activity. The results are shown in FIG. 8, where the pH value is plotted on the X-axis and the relative enzyme activity is plotted on the Y-axis. The results show that the relative enzyme activity of RSipoEnXyn10A is above 90% in the pH range of 4.0-11.0, and the tolerance is good in the pH range.

5. Temperature tolerance of the recombinant protein RSipoEnXyn10A

Desalting and concentrating RSipoEnXyn10A with 0.1M citric acid-Na pH 6.5₂HPO₄Diluting buffer solution (containing 0.5% xylan, W/V) to concentration of 0.01 g/L, respectively maintaining at different temperatures (65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C) for 15 min, 30 min, 45 min and 1 hr, immediately cooling with ice water after the temperature maintaining time is over, and measuring enzyme activity at pH 6.5 and 75 deg.C. The enzyme activity of the enzyme solution which is not treated as above is set as 100%, and the residual enzyme activity after heat preservation is converted into relative enzyme activity. The results are shown in FIG. 9, where the temperature is plotted on the X-axis and the relative enzyme activity is plotted on the Y-axis. The results show that RSipoEnXyn10A has good stability below 65 ℃.

6. Substrate specificity of the recombinant protein RSipoEnXyn10A

Respectively preparing 0.1M Na containing 0.5% (W/V) beech xylan, beta-barley glucan, chitin, lichenin, mannan, sodium carboxymethylcellulose, polygalacturonic acid, starch, arabinoxylan, and xyloglucan₂HPO₄-NaH₂PO₄Buffer solution, under the optimal action condition (pH 6.5, 75 ℃), the enzyme activity of the recombinant protein RSipoEnXyn10A (0.01 g/L) on different substrates is measured. The results are shown in Table 1, RSipoEnXyn10A has enzymatic activity only on xylan, RSipoEnXyn10A on beech xylan andthe specific activity of the arabinoxylan is 197.75 +/-1.42U/mg and 627.30 +/-15.11U/mg respectively.

7. Recombinant protein RSipoEnXyn10A hydrolysis of agroforestry residues to produce xylobiose

The agricultural residues used in the invention are corncobs and cassava straws, and the forestry residues are phyllostachys pubescens and cedar. Various residues were collected as follows: corn cob (Zea maysL.) collected from the western campus vegetable market of Guangxi university, corn kernels were shaved off for freshly picked corn, and the roots of the corn kernels were cut off with a kitchen knife; cassava (Manihot esculentaCrantz) the straws are collected from agricultural experiment bases of Guangxi university, and leaves and peels are removed; phyllostachys pubescens (A)Phyllostachys heterocycla) The scraps are collected from bamboo stick factories in Firmiana city in Guangxi, and are residues of stalk parts used for producing barbecued bamboo sticks; fir wood chips were collected from the board mill of the firmicutus city, guangxi as the residue after the trunks were used to produce boards.

Collecting various residues, oven drying at 60 deg.C to constant weight, pulverizing with small plant tissue pulverizer, and separating with 60 mesh sieve to obtain powder for preparing alkali extract. 10g of the residue was pulverized and suspended in 100 mL of 8% sodium hydroxide, and after standing at 30 ℃ for 12 hours, the suspension was centrifuged at 8000 Xg for 5 minutes, and the supernatant was separated. Adjusting the pH value of the supernatant to 6.0 with concentrated hydrochloric acid, adding 4 times volume of anhydrous ethanol, mixing, and standing the mixture at 4 deg.C for 12 hr to precipitate completely. Centrifuging the mixture at 4 deg.C and 8000 Xg for 5min, discarding supernatant, oven drying the precipitate at 60 deg.C to constant weight, grinding, and separating with 60 mesh sieve to obtain alkali extract for hydrolysis of RSipoEnXyn10A to obtain xylooligosaccharide.

Dissolving the alkali extract in 0.1M citric acid-Na₂HPO₄(pH 6.5) in the buffer, the concentration was 1% (W/V). RSipoEnXyn10A was hydrolyzed at 70 deg.C for 0, 30 min, 2 h, 4 h, 12 h, 24 h and 48 h at an amount of 0.8 mg per gram of base extract. The enzymes were inactivated by boiling the hydrolysate samples in a water bath for 5 minutes at each hydrolysis time point, and the enzyme concentration was increasedAnd detecting the products released by hydrolysis by using the HPLC. The content of each component in the product is calculated by a peak area method.

The invention extracts alkali extracts from 10g of corncob, cassava stalk, phyllostachys pubescens and fir wood respectively to obtain 3.71, 2.20, 1.60 and 0.28 g of alkali extracts, and obtains xylobiose 1.123, 0.083, 0.229 and 0.027 g after being hydrolyzed by RSipoEnXyn10A for 48 hours (Table 2).

From table 3, it can be seen that xylobiose is the only xylooligosaccharide detected after hydrolyzing the corncob, cassava stalk, phyllostachys pubescens, and cedrine extracts, and the ratio of xylobiose to total sugars (the sum of xylose, xylobiose, xylotriose, and xylotetraose) is as high as 85.99%, 87.76%, 86.99%, and 84.52%, so that RSipoEnXyn10A is an excellent enzyme for producing xylobiose (table 3).

The HPLC patterns of the product changes in the course of the enzymatic hydrolysis are shown in FIGS. 10, 11, 12, and 13, wherein each figure specifically is:

FIG. 10 shows the HPLC test results of RSipoEnXyn10A hydrolyzed corncob alkali extract. FIG. 10A shows a standard sample containing xylose, xylobiose, xylotriose and xylotetraose at a concentration of 1 g/L; fig. 10B, C, D, E, F, G, H shows hydrolyzates at 0 min, 30 min, 2 h, 4 h, 12 h, 24 h, 48 h, respectively.

Fig. 11 shows HPLC detection results of RSipoEnXyn10A hydrolysis of cassava stalk alkali extract. FIG. 11A is a standard sample containing xylose, xylobiose, xylotriose, xylotetraose, all at a concentration of 1 g/L; fig. 11B, C, D, E, F, G, H shows hydrolyzates at 0 min, 30 min, 2 h, 4 h, 12 h, 24 h, 48 h, respectively.

FIG. 12 shows the HPLC detection results of RSipoEnXyn10A hydrolyzed phyllostachys pubescens alkali extract. FIG. 12A is a standard sample containing xylose, xylobiose, xylotriose, xylotetraose, all at a concentration of 1 g/L; fig. 12B, C, D, E, F, G, H shows hydrolyzates at 0 min, 30 min, 2 h, 4 h, 12 h, 24 h, 48 h, respectively.

FIG. 13 shows HPLC test results of RSipoEnXyn10A hydrolyzed cedrine extract. FIG. 13A is a standard sample containing xylose, xylobiose, xylotriose, xylotetraose, all at a concentration of 1 g/L; fig. 13B, C, D, E, F, G, H shows hydrolyzates at 0 min, 30 min, 2 h, 4 h, 12 h, 24 h, 48 h, respectively.

Sequence listing

<110> Guangxi university

<120> endo-xylanase and application thereof in production of xylobiose

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 990

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gtgcagccgg cctccgccca tggcccgtcc ctgcgggcgc tggccgaccg ggcgggcgta 60

cgcatcggca cggccgtcga catggcggcg ctggcggacg acacgacgta ccggagaacg 120

acggcccgcg agttcaactc ggtgaccgcc gagaacgtca tgaagtggga gtcggtggag 180

ccgcagcgcg gcgtgtacga ctggaagccg gccgacgacc tggtccgcta cgcgcgggcc 240

cacggccagg tggtccgggg ccacaccctg gtctggcaca gccagctgcc gggctggttg 300

acgtcggggg tggcggacgg gtcgatcgac gcgacggagc tgcggggcat cctgcgggac 360

cacatcacca ccgaggtgaa gcggtacaag gggcggatcc agcagtggga cgtggtgaac 420

gaggtcttcg aggaggacgg cagcctgcgg aactcgatct ggctgcagca gctcggcccg 480

tcgtacatcg ccgacgcctt ccgctgggcc cacgccgccg accccagggc caagctcttc 540

ctcaacgact acaacgtgga gggcgtcaac gcgaagtcca cggcgtacta cgaactcgcc 600

aagcggctga gggcggaggg tgtgccggtg cagggcttcg gcatacaggg gcatctggcg 660

atccagtacg gcttcccggg gcaggtcgcg gagaacctgg cgcgtttcga ggcgctgggg 720

atgcagacgg cgttcacgga ggtggacgtg cggatgctgc tgccggtgga cgaggcgaag 780

ctggcgaccc aggcgtcgta cttccggagg ctgctggacg cctgcctcgg cacccggagc 840

tgcaggtcct tcaccgcgtg gggctacacg gaccggtact cgtgggttcc gggggtgttc 900

gaggggcagg gcgcggccac gcccatggac gaggggtacg ggcggaagcc ggcgtatggg 960

aagctgcggg aggggttgat cgcggggcgg 990

<210> 2

<211> 1173

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgcaccatc atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa 60

accgctgctg ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac 120

gacgacaagg ccatggctga tatcggatcc gaattcgtgc agccggcctc cgcccatggc 180

ccgtccctgc gggcgctggc cgaccgggcg ggcgtacgca tcggcacggc cgtcgacatg 240

gcggcgctgg cggacgacac gacgtaccgg agaacgacgg cccgcgagtt caactcggtg 300

accgccgaga acgtcatgaa gtgggagtcg gtggagccgc agcgcggcgt gtacgactgg 360

aagccggccg acgacctggt ccgctacgcg cgggcccacg gccaggtggt ccggggccac 420

accctggtct ggcacagcca gctgccgggc tggttgacgt cgggggtggc ggacgggtcg 480

atcgacgcga cggagctgcg gggcatcctg cgggaccaca tcaccaccga ggtgaagcgg 540

tacaaggggc ggatccagca gtgggacgtg gtgaacgagg tcttcgagga ggacggcagc 600

ctgcggaact cgatctggct gcagcagctc ggcccgtcgt acatcgccga cgccttccgc 660

tgggcccacg ccgccgaccc cagggccaag ctcttcctca acgactacaa cgtggagggc 720

gtcaacgcga agtccacggc gtactacgaa ctcgccaagc ggctgagggc ggagggtgtg 780

ccggtgcagg gcttcggcat acaggggcat ctggcgatcc agtacggctt cccggggcag 840

gtcgcggaga acctggcgcg tttcgaggcg ctggggatgc agacggcgtt cacggaggtg 900

gacgtgcgga tgctgctgcc ggtggacgag gcgaagctgg cgacccaggc gtcgtacttc 960

cggaggctgc tggacgcctg cctcggcacc cggagctgca ggtccttcac cgcgtggggc 1020

tacacggacc ggtactcgtg ggttccgggg gtgttcgagg ggcagggcgc ggccacgccc 1080

atggacgagg ggtacgggcg gaagccggcg tatgggaagc tgcgggaggg gttgatcgcg 1140

gggcggctcg agcaccacca ccaccaccac tga 1173

<210> 3

<211> 390

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser

1 5 10 15

Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp

20 25 30

Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile

35 40 45

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

50 55 60

Ala Leu Ala Asp Arg Ala Gly Val Arg Ile Gly Thr Ala Val Asp Met

65 70 75 80

Ala Ala Leu Ala Asp Asp Thr Thr Tyr Arg Arg Thr Thr Ala Arg Glu

85 90 95

Phe Asn Ser Val Thr Ala Glu Asn Val Met Lys Trp Glu Ser Val Glu

100 105 110

Pro Gln Arg Gly Val Tyr Asp Trp Lys Pro Ala Asp Asp Leu Val Arg

115 120 125

Tyr Ala Arg Ala His Gly Gln Val Val Arg Gly His Thr Leu Val Trp

130 135 140

His Ser Gln Leu Pro Gly Trp Leu Thr Ser Gly Val Ala Asp Gly Ser

145 150 155 160

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

165 170 175

Glu Val Lys Arg Tyr Lys Gly Arg Ile Gln Gln Trp Asp Val Val Asn

180 185 190

Glu Val Phe Glu Glu Asp Gly Ser Leu Arg Asn Ser Ile Trp Leu Gln

195 200 205

Gln Leu Gly Pro Ser Tyr Ile Ala Asp Ala Phe Arg Trp Ala His Ala

210 215 220

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

225 230 235 240

Val Asn Ala Lys Ser Thr Ala Tyr Tyr Glu Leu Ala Lys Arg Leu Arg

245 250 255

Ala Glu Gly Val Pro Val Gln Gly Phe Gly Ile Gln Gly His Leu Ala

260 265 270

Ile Gln Tyr Gly Phe Pro Gly Gln Val Ala Glu Asn Leu Ala Arg Phe

275 280 285

Glu Ala Leu Gly Met Gln Thr Ala Phe Thr Glu Val Asp Val Arg Met

290 295 300

Leu Leu Pro Val Asp Glu Ala Lys Leu Ala Thr Gln Ala Ser Tyr Phe

305 310 315 320

Arg Arg Leu Leu Asp Ala Cys Leu Gly Thr Arg Ser Cys Arg Ser Phe

325 330 335

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

340 345 350

Glu Gly Gln Gly Ala Ala Thr Pro Met Asp Glu Gly Tyr Gly Arg Lys

355 360 365

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

370 375 380

His His His His His His

385 390

Claims (10)

1. A protein RSipoEnXyn10A, characterized in that: the amino acid sequence of the protein RSipoEnXyn10A is shown as a sequence 3 in a sequence table.

2. A gene encoding the protein RSipoEnXyn10A according to claim 1, wherein: the nucleotide sequence of the gene is shown as a sequence 2 in a sequence table.

3. A recombinant expression vector comprising the gene of claim 2.

4. The recombinant expression vector of claim 3, wherein: the expression vector is suitable for expression in Escherichia coli.

5. The recombinant expression vector of claim 4, wherein: the gene described in sequence 1 in the sequence table is inserted into pET30a (+) vector.

6. The recombinant expression vector of claim 5, wherein: the gene described in the sequence 1 in the sequence table is inserted into pET30a (+) vectorEcoRI andXhoi between the restriction enzyme sites.

7. A strain producing the protein RSipoEnXyn10A, characterized in that: the nucleotide sequence of the gene described in sequence 1 in the sequence table is constructed into a recombinant expression vector and then is transformed into escherichia coliEscherichia coliStrain BL21 (DE 3).

8. The strain producing the protein RSipoEnXyn10A according to claim 7, wherein: the vector is the recombinant expression vector of any one of claims 3 to 5.

9. The strain producing the protein RSipoEnXyn10A according to claim 7 or 8, comprising the steps of: extracting Streptomyces Ipomoeae by PCR methodStreptomyces ipomoeaeThe CGMCC 4.1381 genome DNA is used as a template for PCR amplification, and the obtained PCR product with the sequence 1 in the sequence table is inserted into a prokaryotic expression vector pET30a (+) to obtain a recombinant expression vector pET30a (+) -RSipoEnXyn10AAnd transformed into Escherichia coliEscherichia coliStrain BL21 (DE 3) to obtain recombinant expression strain containing recombinant expression vector for producing the protein RSipoEnXyn 10A.

10. Use of the protein RSipoEnXyn10A of claim 1 as an endoxylanase in the production of xylobiose.

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