阅读说明：本技术 一种具有高浓度木糖、醇和盐耐受性的木糖苷酶Xyl21及其编码基因和应用 (Xylosidase Xyl21 with high-concentration xylose, alcohol and salt tolerance, and coding gene and application thereof ) 是由 李中媛 张同存 陈世恒 赵军旗 于 2019-07-26 设计创作，主要内容包括：本发明涉及一种具有高浓度木糖、醇和盐耐受性的木糖苷酶Xyl21,其氨基酸序列为SEQ ID NO.1。本发明木糖苷酶Xyl21的最适温度为45℃,该酶在45℃处理1h后的剩余酶活为80％,最适pH为5.5,在pH5.0-6.5范围内酶活都保持在80％以上的相对酶活性,具有良好的木糖耐受性,具有良好的乙醇耐受性,对高浓度盐离子具有很好的耐受性。本发明木糖苷酶Xyl21新颖、可耐受高浓度木糖、乙醇和盐,上述性质使木糖苷酶Xyl21作为一种新型的酶制剂,可以广泛的应用于能源、饲料和食品等工业。(The invention relates to xylosidase Xyl21 with high-concentration xylose, alcohol and salt tolerance, and the amino acid sequence of the xylosidase is SEQ ID NO. 1. The xylosidase Xyl21 has the optimum temperature of 45 ℃, the residual enzyme activity of 80 percent after being treated for 1 hour at 45 ℃, the optimum pH value of 5.5, the relative enzyme activity of more than 80 percent of the enzyme activity in the pH range of 5.0-6.5, good xylose tolerance, good ethanol tolerance and good tolerance to high-concentration salt ions. The xylosidase Xyl21 is novel and can tolerate high-concentration xylose, ethanol and salt, and the properties enable xylosidase Xyl21 to be used as a novel enzyme preparation and be widely applied to industries such as energy, feed, food and the like.)

1. Xylosidase Xyl21 having a high concentration of xylose, alcohol and salt tolerance, characterized in that: the amino acid sequence is SEQ ID NO. 1.

2. xylosidase Xyl21 with high concentration xylose, alcohol and salt tolerance according to claim 1, characterized in that: the xylosidase Xyl21 belongs to intermediate-temperature neutral partial acid xylosidase, the optimum temperature is 45 ℃, the residual enzyme activity of the xylosidase after the xylosidase is treated at 45 ℃ for 1 hour is 80%, the optimum pH is 5.5, and the enzyme activity is kept at more than 80% of relative enzyme activity within the pH range of 5.0-6.5; metal ion resistance and xylose, ethanol and salt tolerance; the mature xylosidase Xyl21 has a theoretical molecular weight of 58.43kDa and an isoelectric point of 5.72.

3. Xylosidase Xyl21 with high concentration xylose, alcohol and salt tolerance according to claim 1 or 2, characterized in that: the xylosidase Xyl21 was isolated from the soil environment.

4. A gene encoding xylosidase Xyl21 having high-concentration xylose, ethanol and salt tolerance according to any one of claims 1 to 3, characterized in that: the nucleotide sequence is SEQ ID NO. 2.

5. a recombinant vector comprising the gene encoding xylosidase Xyl21 having high-concentration xylose, ethanol and salt tolerance according to claim 4.

6. A recombinant vector pET28a (+) -Xyl21 comprising the gene encoding xylosidase Xyl21 having high xylose, ethanol and salt tolerance according to claim 4.

7. A recombinant strain comprising the gene encoding xylosidase Xyl21 having high-concentration xylose, ethanol and salt tolerance according to claim 4.

8. A method of preparing xylosidase Xyl21 having high concentrations of xylose, ethanol and salt tolerance as claimed in any one of claims 1 to 3, characterized in that: the method comprises the following steps:

Cloning a new GH39 family xylosidase gene Xyl21 from an alkaline soil metagenome by means of Touch-down PCR and Tail-PCR; inserting xylosidase gene Xyl21 between restriction sites of expression vector to connect its nucleotide sequence with expression regulation sequence to obtain recombinant vector; transforming the recombinant vector into a host cell to obtain a recombinant strain;

Secondly, culturing recombinant strains, and inducing and recombining xylosidase Xyl21 encoding gene expression with high-concentration xylose, ethanol and salt tolerance;

the expressed xylosidase Xyl21 with high concentration of xylose, ethanol and salt tolerance was recovered and purified.

9. Use of xylosidase Xyl21 as claimed in any one of claims 1 to 3 having a high concentration of xylose, ethanol and salt tolerance as an enzyme preparation.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to xylosidase Xyl21 with high-concentration xylose, alcohol and salt tolerance, and a coding gene and application thereof.

background

hemicellulose is a major constituent of plants and can be converted into more valuable products by degradation with biological enzyme systems. The main component of hemicellulose is xylan, which is a heterogeneous polysaccharide connected by 1, 4-glycosidic bonds and provided with multiple substituents. Its complete degradation is a complex process in which xylosidases, which are key enzymes in the degradation of xylan, exogenously hydrolyze xylan from its non-reducing ends to ultimately produce xylose. However, excessive xylose as an end product in the industrial reaction process can generate feedback inhibition effect on xylosidase, thereby inhibiting the activity of xylosidase, and finally leading to the activity loss. Xylosidase from different sources generally has low xylose tolerance, and especially xylosidase from fungal source can only tolerate 2-10mM xylose. The existing method for removing the inhibition is to eliminate the influence of the inhibitor xylose by adding the dosage of enzyme or an ultrafiltration method, but the cost of industrial production is undoubtedly increased, so that the xylosidase with high-concentration xylose tolerance can improve the production efficiency and reduce the cost; meanwhile, in practical industrial application, xylosidase with the characteristic of tolerating high-concentration ethanol can improve the production efficiency of ethanol, and xylosidase with the characteristic of tolerating high-concentration salt ions can be applied to the food processing industry with high salt content such as salty cookies, bread and marine products and the treatment of sewage, so that the development of xylosidase with high-concentration xylose, ethanol and salt tolerance is urgently needed.

through searching, the following patent publications related to the patent application of the invention are found:

Xylosidase Xyl43B with high xylose tolerance, a gene and application thereof (CN103275955A) provides xylosidase Xyl43B derived from Humicola sp.L8, the amino acid sequence of which is shown in SEQ ID No.1, and the invention provides a coding gene Xyl43B for coding the xylosidase. The xylosidase has the following properties: high xylose tolerance (Ki value 292mM), optimal pH7.0, pH5.5-10.0, optimal temperature 50 deg.C, and good thermal stability at 50 deg.C. As a novel enzyme preparation, the xylanase can be widely used in food, feed, energy industry and the like.

By contrast, xylosidase Xyl21 of the present invention is resistant to ethanol and salts in addition to xylose, and thus has a wider range of applications.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide xylosidase Xyl21 with high-concentration xylose, alcohol and salt tolerance, a coding gene and application thereof, wherein xylosidase Xyl21 has high-concentration xylose, alcohol and salt tolerance, can be used as a novel enzyme preparation, and can be widely applied to industries such as energy, feed, food and the like.

The technical scheme adopted by the invention for solving the technical problems is as follows:

Xylosidase Xyl21 with high concentration xylose, alcohol and salt tolerance has amino acid sequence of SEQ ID No. 1.

Moreover, the xylosidase Xyl21 belongs to medium-temperature neutral partial acid xylosidase, the optimum temperature is 45 ℃, the residual enzyme activity of the xylosidase after being treated for 1 hour at 45 ℃ is 80%, the optimum pH is 5.5, and the enzyme activity is kept at more than 80% of relative enzyme activity within the pH range of 5.0-6.5; metal ion resistance and xylose, ethanol and salt tolerance; the mature xylosidase Xyl21 has a theoretical molecular weight of 58.43kDa and an isoelectric point of 5.72.

Further, the xylosidase Xyl21 was isolated from the soil environment.

the gene encoding xylosidase Xyl21 having high xylose, ethanol and salt tolerance as described in the above, having the nucleotide sequence of SEQ ID NO. 2.

A recombinant vector comprising the gene encoding xylosidase Xyl21 having high concentrations of xylose, ethanol and salt tolerance as described above.

The recombinant vector pET28a (+) -Xyl21 comprising the gene encoding xylosidase Xyl21 having high xylose, ethanol and salt tolerance as described above.

A recombinant strain comprising the gene encoding xylosidase Xyl21 having high concentrations of xylose, ethanol and salt tolerance as described above.

A method of preparing xylosidase Xyl21 having high xylose, ethanol and salt tolerance as described above, comprising the steps of:

Cloning a new GH39 family xylosidase gene Xyl21 from an alkaline soil metagenome by means of Touch-down PCR and Tail-PCR; inserting xylosidase gene Xyl21 between restriction sites of expression vector to connect its nucleotide sequence with expression regulation sequence to obtain recombinant vector; transforming the recombinant vector into a host cell to obtain a recombinant strain;

Secondly, culturing recombinant strains, and inducing and recombining xylosidase Xyl21 encoding gene expression with high-concentration xylose, ethanol and salt tolerance;

The expressed xylosidase Xyl21 with high concentration of xylose, ethanol and salt tolerance was recovered and purified.

use of xylosidase Xyl21 with high concentrations of xylose, ethanol and salt tolerance as described above as an enzyme preparation.

The invention has the advantages and positive effects that:

1. the optimum temperature of xylosidase Xyl21 is 45 ℃, the residual enzyme activity of the xylosidase after being treated for 1 hour at 45 ℃ is 80%, the optimum pH is 5.5, the enzyme activity is kept at more than 80% of the relative enzyme activity within the pH range of 5.0-6.5, the xylosidase has good xylose tolerance, and the inhibition constant (Ki) of the xylosidase to xylose is 1100 mmol/L; has good ethanol tolerance, and maintains 156.34% and 32.14% relative enzyme activity under ethanol conditions with the concentration of 10% and 20% (v/v); has good tolerance to high-concentration salt ions, and still maintains 128.13 percent and 83.45 percent of relative activity under the conditions of 1.2M and 3.0M NaCl. Compared to other GH39 xylosidases, Xyl21 has better xylose, ethanol and salt tolerance. The xylosidase Xyl21 is novel and can tolerate high-concentration xylose, ethanol and salt, and the properties enable xylosidase Xyl21 to be used as a novel enzyme preparation and be widely applied to industries such as energy, feed, food and the like.

2. The xylosidase Xyl21 is obtained by cloning from a saline-alkali soil metagenome, and xylosidase Xyl21 is a novel xylosidase gene of a GH39 glycoside hydrolase family, and can be successfully expressed in escherichia coli BL21(DE3) in a heterologous manner.

drawings

FIG. 1 shows a diagram of the purification of Xyl21 recombinant protein according to the present invention; wherein, 1 is unpurified protein, 2 is purified recombinant protein Xyl 21;

FIG. 2 is a diagram showing the optimum pH of recombinant xylosidase Xyl21 in the present invention;

FIG. 3 is a graph showing the pH stability of recombinant xylosidase Xyl21 in the present invention;

FIG. 4 is a temperature optimum diagram of recombinant xylosidase Xyl21 according to the present invention;

FIG. 5 is a graph showing the thermostability of recombinant xylosidase Xyl21 in the present invention;

FIG. 6 is a graph showing the effect of metal ions and chemicals on the activity of recombinant xylosidase Xyl21 in the present invention;

FIG. 7 is a graph showing the effect of different concentrations of salt ions on the activity of recombinant xylosidase Xyl21 in the present invention;

FIG. 8 is a graph showing the effect of various concentrations of monosaccharides on the enzymatic activity of recombinant xylosidase Xyl 21;

FIG. 9 is a graph showing the effect of various concentrations of alcohol on the enzymatic activity of recombinant xylosidase Xyl21 in the present invention;

FIG. 10 is a graph showing the analysis of the hydrolysate of recombinant xylosidase Xyl21 in the present invention; wherein, 1: 1% beech xylan; 2: 1% of corncob; 3: 1% birchwood xylan; 4: 1% wheat arabinoxylan; x1: a xylose standard; x2: xylobiose standards; x3 is xylotriose standard product; 5: xyl21 hydrolyzes zelkova; 6: xyl21 hydrolyzed corncobs; 7: xyl21 hydrolyzes birchwood xylan; 8: xyl21 hydrolyzed wheat arabinoxylan; 9: xyl21 hydrolyzes xylobiose; 10: xyl21 hydrolyzes xylotriose, the concentrations being given by mass.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

xylosidase Xyl21 having high concentration xylose, alcohol and salt tolerance and having the amino acid sequence of SEQ ID No. 1: MEKIMISRDNNIPFNKHWKKCIGTGRLGLALQKEYVDHLERLQQDIGFDYIRGHGLFHEDIGIYKEVDVGGTPTPFYNFTYIDRIFDTFLQNNLRPFVELGFMPKKLASGTQTIFYWEGNVTPPAEEKKWEELIYHTVIHFVERYGVEEVLQWPFEVWNEPNLSNFWENADKQAYFQLYKITAKTIKSIHPDLNVGGPAICGGTDEWITDFLHFCHKEQVPVDFISRHAYTSKPPKKKTPDYYYQDLADPNDMLTQLTSVKKLIHDTPYPDLPFHITEYNTSYSPINPIHDTNYNAAYLGKILSHAGDIVSSFSYWTFSDVFEEFDIPRAPFHGGFGLMALHAIRKPTYHLFSFFNKLGDTCLYKDDTMIVTKHRDNTISIVAWNLIHDKGENHDKTITLSIPFSSDDVFLYREITNEHHANPWRVWKQMGRPRFPSKEQITLLKSCDIPFIQTDKLMTENKLLTYQLTMAKNEVTLLHFSDIIDETNTYIGLDDSLLTSY

wherein the enzyme gene codes 501 amino acids and a stop codon, and the sequence is free of a signal peptide and an intron, so that the mature xylosidase Xyl21 has the theoretical molecular weight of 58.43kDa and the isoelectric point of 5.72.

the xylosidase Xyl21 belongs to medium-temperature neutral partial acid xylosidase, the optimum temperature is 45 ℃, the residual enzyme activity of the xylosidase after the xylosidase is treated for 1 hour at 45 ℃ is 80%, the optimum pH is 5.5, and the enzyme activity is kept at more than 80% of relative enzyme activity within the pH range of 5.0-6.5; has better metal ion resistance and very good xylose, ethanol and salt tolerance.

a coding gene of the xylosidase Xyl21, specifically, the nucleic acid sequence of the gene is shown in SEQ ID NO. 2:

ATGGAGAAAATAATGATTTCTAGAGATAACAATATCCCGTTTAATAAACATTGGAAAAAGTGTATTGG AACCGGTCGACTAGGCTTAGCTTTACAAAAAGAGTATGTGGATCATCTTGAACGTCTACAACAAGATATTGGTTTT GATTATATTAGAGGACATGGACTCTTTCATGAAGACATCGGCATTTATAAAGAAGTAGACGTAGGTGGCACTCCTA CTCCTTTCTATAATTTTACATATATTGATAGAATTTTCGATACGTTTCTTCAGAATAATTTACGTCCATTTGTTGA GTTAGGCTTTATGCCCAAAAAGCTTGCTTCCGGTACCCAAACTATCTTTTATTGGGAAGGTAATGTCACCCCTCCG GCTGAGGAAAAGAAGTGGGAAGAGCTCATCTATCATACAGTAATCCATTTTGTAGAAAGATACGGGGTTGAGGAAG TGTTACAGTGGCCGTTTGAAGTTTGGAATGAACCTAACCTATCCAACTTCTGGGAGAACGCTGATAAACAAGCTTA TTTTCAACTCTACAAAATTACTGCAAAAACAATTAAATCTATTCATCCAGATTTAAACGTTGGGGGCCCAGCAATT TGTGGTGGTACTGATGAGTGGATAACTGATTTTCTCCACTTTTGTCACAAAGAGCAAGTACCCGTTGATTTTATAA GTCGACATGCTTATACGTCTAAGCCTCCAAAGAAAAAAACGCCTGACTATTATTATCAAGACTTGGCCGATCCTAA CGACATGCTTACTCAACTTACGAGTGTAAAAAAATTAATTCATGACACACCTTATCCTGACCTTCCATTTCACATT ACCGAATACAATACATCTTATAGCCCAATCAATCCTATTCATGATACGAACTATAATGCTGCGTACCTAGGCAAAA TCTTAAGCCATGCTGGAGACATTGTGTCTTCTTTTTCCTATTGGACATTTAGTGACGTTTTTGAAGAGTTTGATAT TCCACGTGCCCCTTTCCATGGAGGCTTCGGGTTAATGGCACTACACGCTATTCGGAAACCAACTTATCATTTGTTT TCATTCTTTAACAAATTAGGTGATACTTGTCTTTACAAAGACGATACAATGATCGTCACTAAGCATCGAGATAATA CCATTTCCATTGTGGCTTGGAACTTAATACATGACAAAGGAGAAAATCATGACAAAACAATCACGCTCTCTATTCC GTTTTCTTCTGATGACGTCTTTTTGTACAGAGAGATTACTAACGAACATCATGCAAACCCTTGGCGTGTTTGGAAG CAGATGGGAAGACCACGCTTTCCATCGAAAGAACAAATAACACTATTAAAATCATGTGACATTCCTTTCATTCAAA CAGATAAACTGATGACAGAAAATAAGCTTCTAACTTATCAATTGACGATGGCAAAAAATGAAGTTACTTTACTACA TTTTTCAGACATTATAGATGAAACCAACACCTATATCGGGCTCGACGACAGTCTGCTCACATCTTATTAA。

The invention can clone a new GH39 family xylosidase gene Xyl21 from metagenome DNA of saline-alkali soil (39 degrees 11 '67' N, 117 degrees 73 '43' E) in new Tianjin coastal areas by utilizing Touch-down PCR and Tail-PCR, and the whole sequence analysis result of DNA shows that the total length of the structural gene of xylosidase Xyl21 is 1506 bp.

A BLAST alignment of the sequence of xylosidase Xyl21 and the deduced amino acid sequence in GenBank showed 63% amino acid sequence identity to the GH39 family xylosidase gene from Alicyclobacillus ferrooxydans, indicating that Xyl21 is a novel xylosidase.

a recombinant vector containing the xylosidase gene Xyl21 was named pET28a (+) -Xyl 21. The xylosidase gene of the present invention is inserted between appropriate restriction sites of an expression vector so that its nucleotide sequence is operably linked to an expression control sequence. As a most preferred embodiment of the present invention, it is preferred that the xylosidase gene of the present invention is inserted between EcoR I and Not I restriction sites on the plasmid pET28a (+) such that the nucleotide sequence is located downstream of and under the control of the T7 promoter to give a recombinant E.coli expression plasmid pET28a (+) -Xyl 21.

A recombinant strain comprising the above xylosidase, preferably the strain is Escherichia coli, preferably recombinant strain BL21/Xyl 21.

a method for preparing a xylosidase gene Xyl21, comprising the steps of:

1) Transforming host cells by using the recombinant vector to obtain a recombinant strain;

2) Culturing the recombinant strain, and inducing the expression of the recombinant xylosidase;

3) The expressed xylosidase Xyl21 was recovered and purified.

Preferably, the host cell is an Escherichia coli cell, and preferably, the recombinant Escherichia coli expression plasmid is transformed into Escherichia coli (Escherichia coli) BL21 to obtain a recombinant strain BL21/Xyl 21.

The xylosidase Xyl21 with high xylose, ethanol and salt tolerance was isolated from the soil environment.

specifically, the relevant steps are as follows:

Test materials and reagents:

1. Bacterial strain and carrier: the invention separates a new xylosidase gene Xyl21 from GH39 family (xylosidase) obtained from soil environment. Coli (Escherichia coli) DH5 alpha, BL21(DE3) strains were stored in the laboratory, pET-28a (+) plasmid was stored in the laboratory, and pMD-19T was purchased from Takara Bio Inc.

2. Enzymes and other biochemical reagents: LATaq was purchased from TaKaRa; p-nitrophenyl-D-xylopyranoside (pNPX), zelkoxysan, birch xylan, corn cob, wheat arabinoxylan were purchased from Sigma; other chemical reagents are purchased from Jiangtian chemical technology limited company in Tianjin city and are analytically pure; isopropyl thio-D-galactoside (IPTG) and a plasmid miniprep kit are purchased from Beijing Soilebao; the bacterial genome extraction kit is purchased from Tiangen company; EcoRI, Not I restriction enzymes and BCA protein quantification kits were purchased from Thermo Scientific.

3. Culture medium:

(1) LB culture medium: 1% NaCl, 0.5% yeast extract, 1% peptone;

(2) LB solid medium: 2% agar powder is added into LB liquid culture medium.

Specifically, the following description is provided: the molecular biology experiments not specifically described in the following steps were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions.

cloning of xylosidase encoding Gene Xyl21

The metagenome DNA from the saline-alkali soil (39 degrees 11 '67' N, 117 degrees 73 '43' E) of the new Tianjin coastal region is extracted by adopting a bacterial genome extraction kit (Tiangen company), and the specific operation is shown in the specification.

Degenerate primers Xyl39F, Xyl39R were designed based on the conserved region sequences (EVWNEPN and SYWTFSD) of the GH39 family xylosidase gene.

And (3) amplifying the conserved region by using Touch-down PCR by using the environmental metagenome DNA as a template. The PCR reaction parameters are as follows: denaturation at 94 deg.C for 5 min; then denaturation at 94 ℃ for 30sec, annealing at 48-42 ℃ for 30sec (0.5 degree reduction per cycle), and extension at 72 ℃ for 40sec for 12 cycles; denaturation at 94 ℃ for 30sec, annealing at 45 ℃ for 30sec, extension at 72 ℃ for 40sec, and heat preservation at 72 ℃ for 10min after 28 cycles. An approximately 499bp fragment was obtained, recovered, ligated with the PMD-19T vector and sequenced.

Specific primers for the upstream and downstream were designed based on the conserved region sequence of Xyl21 (Table 1), and the full length of the gene sequence was obtained by 3 Tail-PCR. The design direction is an unknown region direction needing amplification, the position of sp2 is designed to be inside sp1, and sp3 is located inside sp 2. The distance between every two primers is not strictly specified, the length of the primers is generally 22-30 nt, and the annealing temperature is 60-65 ℃. And they were named 21-1u1,21-1u2,21-1u3, 21-2u1, 21-2u2, 21-2u3 (upstream specific primers), 21-1d1,21-1d2,21-1d3, 21-2d1, 21-2d2, 21-2d3, 21-3d1, 21-3d2, 21-3d1 (downstream specific primers), respectively.

TABLE 1 xylosidase Xyl21TAIL-PCR specific primers

And (3) taking the conserved region sequence obtained by amplification as a template, obtaining a flanking sequence of a known gene sequence through TAIL-PCR, respectively purifying and recovering the cloned upstream and downstream fragments, connecting the upstream and downstream fragments with pMD-19T for sequencing, and splicing the upstream and downstream fragments with the sequence correct to be sequenced with the conserved region sequence through vector NTI software. After splicing, the full-length xylosidase gene of the GH39 family is finally obtained. The Xyl21 xylosidase gene has a full length of 1506bp, encodes 501 amino acids and a stop codon, and has no signal peptide and intron. The molecular weight of the mature protein is predicted to be 58.43KDa, and the isoelectric point is predicted to be 5.72. The Xyl21 gene sequence has been submitted to the NCBI database (KU 353562).

Preparation of di-and recombinant xylosidase Xyl21

The expression vector pET28a (+) was subjected to double digestion (EcoR I + NotI), and at the same time, the gene Xyl21 coding for xylosidase was subjected to double digestion (EcoR I + NotI), and the gene fragment coding for mature xylosidase was excised and ligated to the expression vector pET28a (+) to obtain a recombinant plasmid pET28a (+) -Xyl21 containing xylosidase gene Xyl21, which was transformed into E.coli DH5 α to obtain recombinant E.coli strain DH5 α/Xyl 21. The recombinant plasmid is transformed into Escherichia coli BL21 to obtain recombinant Escherichia coli strain BL21/Xyl 21.

the BL21 strain containing the recombinant plasmid is taken and inoculated in 5mL LB liquid culture medium containing antibiotic (100 mug/mL), cultured at 37 ℃ for 12h, the bacterial liquid cultured for 12h is inoculated in 100mL LB culture medium containing antibiotic (100 mug/mL) according to the inoculum size of 1%, when cultured at 37 ℃ until OD600 is 0.6-0.8, IPTG with the final concentration of 1mM is added to carry out induction expression at 25 ℃ for 20h, the bacterial strain is collected and centrifuged at 8000r/min for 10min, the culture medium is poured off, standard bacterial suspension of 50mLPBS (pH7.4) is added to each 1.5g wet bacterial strain, the ultrasonic disruption is carried out, and the bacterial strain after the ultrasonic disruption is centrifuged at 12000r/min for 15 min. Purifying the supernatant crude enzyme solution with nickel affinity column, eluting target protein with imidazole (10mmol/L-500mmol/L) with different concentrations, collecting eluates with different concentration gradients, and performing enzyme activity determination and SDS-PAGE protein electrophoresis detection on the eluates. After being purified by a nickel column, SDS-PAGE results show that the recombinant xylosidase is expressed in escherichia coli. The results showed that electrophoretic purity was achieved and the purified protein was approximately 58.43kDa in size (FIG. 1).

Activity analysis of xylosidase

The specific method comprises the following steps: mu.L of 2mM pNPX substrate was mixed with 150. mu.L of citric acid-disodium hydrogenphosphate buffer, 100. mu.L of enzyme solution (2U/mL) was added, the reaction was allowed to proceed at 37 ℃ for 10min, 1.5mL of 1M Na2CO3 was added to terminate the reaction, and the OD405 value was measured using a spectrophotometer to calculate the amount of p-nitrophenol as a product. 1 xylosidase activity unit (U) is defined as the amount of enzyme required to decompose p-nitrophenyl-beta-D-xyloside (pNPX) substrate to 1. mu. mol p-nitrophenol (pNP) per minute under the given corresponding reaction conditions.

Determination of Properties of Tetraxylosidase Xyl21

1. the optimum pH and pH stability of recombinant xylosidase Xyl21 were determined as follows:

The purified recombinant xylosidase from step two was subjected to enzymatic reaction at different pH to determine its optimum pH. Using pNPX as substrate and a final concentration of 2mM, 250. mu.L of substrate was taken and 150. mu.L of the corresponding buffer was added. The buffer gradient of the buffer solution was different: 100mM citric acid-Na 2HPO4(pH 3.0-8.0), 100mM glycine-NaOH (pH 9.0-12.0). Preheating the mixed solution of the substrate and the buffer solution for 5min under the reaction condition of 37 ℃, adding 100 mu L of enzyme solution (2U/ml), uniformly mixing and accurately reacting for 10min, adding 1.5ml of Na2CO3 to terminate the reaction, and cooling to room temperature and then determining the OD405 value. As shown in FIG. 2, the results showed that the recombinase had an optimum pH of 5.5 when pNPX was used as the substrate, and the enzyme activities were maintained at 80% or more in the pH range of 5.0 to 6.5.

in order to study the pH stability of the enzyme, the purified enzyme was diluted with buffers of different pH, treated at 37 ℃ for 1h, diluted 10-fold with a buffer of optimum pH, and the residual enzyme activity was measured in a buffer of optimum pH and at optimum temperature. As shown in FIG. 3, the results show that the xylosidase activity is stable within the pH range of 5-7.0, and the relative residual enzyme activity is over 70%.

2. the method for measuring the optimal temperature and the thermal stability of the recombinant xylosidase comprises the following steps:

the optimum temperature of xylosidase is determined by measuring the enzyme activity of the purified enzyme at different temperatures (10-70 ℃) in a buffer system of citric acid-disodium hydrogen phosphate buffer (pH 5.5). The results of the enzyme reaction optimum temperature measurement are shown in FIG. 4, which indicates that the recombinant enzyme activity optimum temperature is 45 ℃ and belongs to a medium-temperature enzyme, but Xyl21 still has activity under a low-temperature condition, the relative enzyme activities at 10 ℃ and 20 ℃ are 19% and 32% respectively, and the relative enzyme activity at a high-temperature condition, such as 60 ℃, is 25%, indicating that the reaction temperature range of the enzyme is wide.

The thermal stability is determined by pretreating the diluted recombinant enzyme solution (2U/ml) in a buffer solution of pH5.5 at 45 deg.C, 50 deg.C and 55 deg.C for 1h, sampling at different time points (0, 1,2, 5, 10, 20, 30, 60min), determining the residual enzyme activity at the optimum temperature and pH, using the untreated enzyme as a control, and cooling to room temperature to determine the absorbance of OD 405. The enzyme thermal stability test shows that the residual enzyme activity of Xyl21 is more than 80% after being treated at 45 ℃ for 1h, and the enzyme is relatively stable. However, the thermal stability at 50 ℃ and 55 ℃ is lower than that at 45 ℃, the enzyme activity is almost completely lost after the treatment at 50 ℃ for 5min and the treatment at 55 ℃ for 2min, and the results are shown in FIG. 5.

3. The Km value of the recombinant xylosidase is determined as follows:

And (3) determining a kinetic constant: preparing pNPX with the concentration of 0.2mmol/L to 2mmol/L, respectively using the pNPX with different concentrations as substrates, measuring the enzyme activity under the pNPX with different concentrations in a buffer solution system of citric acid-disodium hydrogen phosphate (pH5.5) at 45 ℃, calculating Km and Vmax according to the Mie's equation, and measuring three times in parallel in each experiment. The Km, Vmax and Kcat values of recombinase Xyl21 were determined to be 1.71mmol/L, 17.6. mu. mol/min/mg and 19.04s-1, respectively.

4. The effect of different metal ions and chemicals on the activity of Xyl21 enzyme was determined as follows:

different metal ions with a final concentration of 5mmol/L, chemical reagents (Mn2+, K +, Ca2+, Cu2+, Fe3+, Ni +, Mg2+, Li +, Fe2+, Zn2+, Co2+, SDS, EDTA-Na2, mercaptoethanol) and NaCl with different final concentrations (0.6mol/L, 1.2mol/L, 1.8mol/L, 2.4mol/L, 3.0mol/L, 3.6mol/L, 4.0mol/L) are added into a standard reaction system, and the influence of the chemical reagents on the enzyme activity is researched. The enzyme activity was measured at 45 ℃ and pH5.5, using as a control only the corresponding metal ions, chemical reagents and salt ions without adding a suitably diluted enzyme solution.

The result is shown in fig. 6, when pNPX is used as a substrate, when different metal ions with a final concentration of 5mmol/L are added into a reaction system, Fe2+ has a certain inhibition effect on the enzyme activity, the residual enzyme activity is 74%, when other metal ions are added, the enzyme activity of Xyl21 is hardly affected, and more than 85% of residual enzyme activity is maintained, which indicates that Xyl21 has better resistance to most of metal ions. In the presence of SDS, the enzyme activity was completely lost. However, metal ions and reagents such as Co2+, Li +, K +, Ca2+, EDTA-Na2, mercaptoethanol and the like have different degrees of promoting effects on enzyme activity, wherein the activating effect of Ca2+ is 124.29% most remarkably. It is presumed that the reason for this may be that Ca2+ can activate the catalytically active site of xylosidase and can prevent it from being inactivated by denaturation under high temperature conditions.

Salt ion tolerance test results as shown in fig. 7, low concentrations of salt ions can enhance the relative stability of the enzyme. Xyl21 still retained more than 80% of enzyme activity under the condition of 3M salt ion. The residual enzyme activity is more than 40 percent under the condition of the existence of 3.6mol/L salt ions.

5. monosaccharide tolerance of recombinant xylosidase

Monosaccharides at final concentrations of 0.6, 1.2 and 2.25M were added to the standard reaction system and reacted with substrate (pNPX) under optimal reaction conditions. Fig. 8 shows that when the enzyme is in a low-concentration monosaccharide environment, the relative activity of the enzyme is affected to different degrees, wherein the activity of glucose on the enzyme is improved most obviously, namely 281.32%, the relative activity of fructose on the enzyme is improved, namely 111.77%, but galactose has a certain inhibition effect on the enzyme activity, and the residual enzyme activity is 86.47%. With increasing monosaccharide concentration, the activation of the enzyme by glucose is reduced, and the influence of the remaining two fructose and galactose on the activity of the enzyme is almost kept unchanged.

The low-concentration xylose has certain activation effect on the enzyme, the activity of the enzyme is correspondingly inhibited along with the increase of the final concentration of the xylose, and when the final concentration of the xylose is 1.5M, the relative activity of the enzyme is more than 50 percent. Xylose solutions of final concentrations of 0.5, 0.8, 1, 1.25 and 1.6M were added to the standard reaction system, and pNPX of final concentrations of 2mmol/L and 3mmol/L, respectively, was used as a substrate, and the enzyme activity of Xyl21 was measured under the optimum reaction conditions (45 ℃ C., pH5.5) and its Ki value was calculated. The experimental results show that the Ki value of Xyl21 is 1100mmol/L, and the high xylose tolerance ensures that Xyl21 can not be inactivated due to excessive accumulation of xylose in the reaction process, thereby reducing the production cost.

6. Alcohol tolerance of recombinant xylosidase

The effect of lower alcohol on the activity of beta-xylosidase is shown in fig. 9, and the result shows that the activity of enzyme is correspondingly improved when the enzyme is in the condition of low-concentration alcohol (methanol, ethanol, isopropanol, isoamyl alcohol), when the addition amount of lower alcohol is 20% (v/v), the remaining alcohol except isoamyl alcohol and methanol has the inhibition effect on the activity of enzyme, and the relative enzyme activity is remained below 40%; when isoamyl alcohol was added at 30% (v/v), the activity of Xyl21 enzyme was not significantly affected. The recombinant beta-xylosidase Xyl21 is shown to have better tolerance to lower alcohols.

The effect of ethanol on Xyl21 activity is shown in FIG. 9, and ethanol tolerance indicates that enzyme activity is increased when the enzyme is exposed to low concentrations of ethanol, and 154.18% of relative enzyme activity is maintained at 10% (v/v) ethanol. The relative activity of the enzyme is reduced along with the increase of the ethanol concentration, when the addition amount of ethanol is 15% (v/v), the activity of the enzyme is inhibited, is 87.20% of the original enzyme activity, and still maintains 32.36% of the relative enzyme activity under the ethanol condition of 20% (v/v) concentration, which indicates that the recombinant beta-xylosidase Xyl21 has better tolerance to ethanol.

8. Analysis of hydrolysate of recombinant xylosidase

since Xyl21 had the activity of hydrolyzing pNPX, Xyl21 was further analyzed for its degradation activity on natural glycans as well as oligosaccharides. Preparing a substrate of zelkova xylan, birch xylan, corncob and wheat arabinoxylan with the mass concentration of 1%, adding 40U/mL of purified enzyme solution, reacting at 45 ℃ for 12h, boiling in water bath for 10min for inactivation, centrifuging at 12000rpm for 5min, taking supernatant, carrying out thin-layer chromatography analysis, developing twice in a developing agent (n-butyl alcohol: water: acetic acid (V: V: V: V): 2:1:1), soaking in a color developing agent (acetone: diphenylamine: aniline: phosphoric acid ═ 50mL: 1mL:5mL) for several seconds, and immediately drying in an oven at 90 ℃ for color development. As shown in FIG. 10, Xyl21 showed degradation of all glycan and oligosaccharide substrates, indicating that Xyl21 has broad substrate specificity, but some differences in the degradation capacity for different substrates. The hydrolyzed product is mainly xylose, and the Xyl21 has stronger degradation capability on corncobs, xylobiose and xylotriose as is obvious from the generation amount of the xylose; the degradation effect is most obvious when the corncob is used as a substrate, the content of xylose is obviously increased after recombinase Xyl21 acts on the substrate, and xylobiose and xylotriose are all degraded into monosaccharide. In addition, the degrading activity of Xyl21 on xylooligosaccharide such as xylobiose and xylotriose is higher than that on natural glycan.

although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Sequence listing

<110> Tianjin science and technology university

<120> xylosidase Xyl21 with high-concentration xylose, alcohol and salt tolerance, and coding gene and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 501

<212> PRT

<213> amino acid sequence of xylosidase Xyl21 (Unknown)

<400> 1

Met Glu Lys Ile Met Ile Ser Arg Asp Asn Asn Ile Pro Phe Asn Lys

1 5 10 15

His Trp Lys Lys Cys Ile Gly Thr Gly Arg Leu Gly Leu Ala Leu Gln

20 25 30

Lys Glu Tyr Val Asp His Leu Glu Arg Leu Gln Gln Asp Ile Gly Phe

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Asp Arg Ile Phe Asp Thr Phe Leu Gln Asn Asn Leu Arg Pro

85 90 95

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

100 105 110

Thr Ile Phe Tyr Trp Glu Gly Asn Val Thr Pro Pro Ala Glu Glu Lys

115 120 125

Lys Trp Glu Glu Leu Ile Tyr His Thr Val Ile His Phe Val Glu Arg

130 135 140

Tyr Gly Val Glu Glu Val Leu Gln Trp Pro Phe Glu Val Trp Asn Glu

145 150 155 160

Pro Asn Leu Ser Asn Phe Trp Glu Asn Ala Asp Lys Gln Ala Tyr Phe

165 170 175

Gln Leu Tyr Lys Ile Thr Ala Lys Thr Ile Lys Ser Ile His Pro Asp

180 185 190

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

195 200 205

Thr Asp Phe Leu His Phe Cys His Lys Glu Gln Val Pro Val Asp Phe

210 215 220

Ile Ser Arg His Ala Tyr Thr Ser Lys Pro Pro Lys Lys Lys Thr Pro

225 230 235 240

Asp Tyr Tyr Tyr Gln Asp Leu Ala Asp Pro Asn Asp Met Leu Thr Gln

245 250 255

Leu Thr Ser Val Lys Lys Leu Ile His Asp Thr Pro Tyr Pro Asp Leu

260 265 270

Pro Phe His Ile Thr Glu Tyr Asn Thr Ser Tyr Ser Pro Ile Asn Pro

275 280 285

Ile His Asp Thr Asn Tyr Asn Ala Ala Tyr Leu Gly Lys Ile Leu Ser

290 295 300

His Ala Gly Asp Ile Val Ser Ser Phe Ser Tyr Trp Thr Phe Ser Asp

305 310 315 320

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

325 330 335

Gly Leu Met Ala Leu His Ala Ile Arg Lys Pro Thr Tyr His Leu Phe

340 345 350

Ser Phe Phe Asn Lys Leu Gly Asp Thr Cys Leu Tyr Lys Asp Asp Thr

355 360 365

Met Ile Val Thr Lys His Arg Asp Asn Thr Ile Ser Ile Val Ala Trp

370 375 380

Asn Leu Ile His Asp Lys Gly Glu Asn His Asp Lys Thr Ile Thr Leu

385 390 395 400

Ser Ile Pro Phe Ser Ser Asp Asp Val Phe Leu Tyr Arg Glu Ile Thr

405 410 415

Asn Glu His His Ala Asn Pro Trp Arg Val Trp Lys Gln Met Gly Arg

420 425 430

Pro Arg Phe Pro Ser Lys Glu Gln Ile Thr Leu Leu Lys Ser Cys Asp

435 440 445

Ile Pro Phe Ile Gln Thr Asp Lys Leu Met Thr Glu Asn Lys Leu Leu

450 455 460

Thr Tyr Gln Leu Thr Met Ala Lys Asn Glu Val Thr Leu Leu His Phe

465 470 475 480

Ser Asp Ile Ile Asp Glu Thr Asn Thr Tyr Ile Gly Leu Asp Asp Ser

485 490 495

Leu Leu Thr Ser Tyr

500

<210> 2

<211> 1506

<212> DNA/RNA

<213> nucleotide sequence of encoding Gene of xylosidase Xyl21 (Unknown)

<400> 2

atggagaaaa taatgatttc tagagataac aatatcccgt ttaataaaca ttggaaaaag 60

tgtattggaa ccggtcgact aggcttagct ttacaaaaag agtatgtgga tcatcttgaa 120

cgtctacaac aagatattgg ttttgattat attagaggac atggactctt tcatgaagac 180

atcggcattt ataaagaagt agacgtaggt ggcactccta ctcctttcta taattttaca 240

tatattgata gaattttcga tacgtttctt cagaataatt tacgtccatt tgttgagtta 300

ggctttatgc ccaaaaagct tgcttccggt acccaaacta tcttttattg ggaaggtaat 360

gtcacccctc cggctgagga aaagaagtgg gaagagctca tctatcatac agtaatccat 420

tttgtagaaa gatacggggt tgaggaagtg ttacagtggc cgtttgaagt ttggaatgaa 480

cctaacctat ccaacttctg ggagaacgct gataaacaag cttattttca actctacaaa 540

attactgcaa aaacaattaa atctattcat ccagatttaa acgttggggg cccagcaatt 600

tgtggtggta ctgatgagtg gataactgat tttctccact tttgtcacaa agagcaagta 660

cccgttgatt ttataagtcg acatgcttat acgtctaagc ctccaaagaa aaaaacgcct 720

gactattatt atcaagactt ggccgatcct aacgacatgc ttactcaact tacgagtgta 780

aaaaaattaa ttcatgacac accttatcct gaccttccat ttcacattac cgaatacaat 840

acatcttata gcccaatcaa tcctattcat gatacgaact ataatgctgc gtacctaggc 900

aaaatcttaa gccatgctgg agacattgtg tcttcttttt cctattggac atttagtgac 960

gtttttgaag agtttgatat tccacgtgcc cctttccatg gaggcttcgg gttaatggca 1020

ctacacgcta ttcggaaacc aacttatcat ttgttttcat tctttaacaa attaggtgat 1080

acttgtcttt acaaagacga tacaatgatc gtcactaagc atcgagataa taccatttcc 1140

attgtggctt ggaacttaat acatgacaaa ggagaaaatc atgacaaaac aatcacgctc 1200

tctattccgt tttcttctga tgacgtcttt ttgtacagag agattactaa cgaacatcat 1260

gcaaaccctt ggcgtgtttg gaagcagatg ggaagaccac gctttccatc gaaagaacaa 1320

ataacactat taaaatcatg tgacattcct ttcattcaaa cagataaact gatgacagaa 1380

aataagcttc taacttatca attgacgatg gcaaaaaatg aagttacttt actacatttt 1440

tcagacatta tagatgaaac caacacctat atcgggctcg acgacagtct gctcacatct 1500

tattaa 1506

<210> 3

<211> 20

<212> DNA/RNA

<213> Xyl39F(Unknown)

<400> 3

gaagtntgga aygarccnaa 20

<210> 4

<211> 21

<212> DNA/RNA

<213> Xyl39R(Unknown)

<400> 4

gacgtcrctr aangtccart a 21

<210> 5

<211> 29

<212> DNA/RNA

<213> 21-1u1(Unknown)

<400> 5

gcatggctta agattttgcc taggtacgc 29

<210> 6

<211> 30

<212> DNA/RNA

<213> 21-1u2(Unknown)

<400> 6

gtgaaatgga aggtcaggat aaggtgtgtc 30

<210> 7

<211> 25

<212> DNA/RNA

<213> 21-1u3(Unknown)

<400> 7

gtaccaccac aaattgctgg gcccc 25

<210> 8

<211> 24

<212> DNA/RNA

<213> 21-1d1(Unknown)

<400> 8

cgttgggggc ccagcaattt gtgg 24

<210> 9

<211> 29

<212> DNA/RNA

<213> 21-1d2(Unknown)

<400> 9

gcccagcaat ttgtggtggt actgatgag 29

<210> 10

<211> 28

<212> DNA/RNA

<213> 21-1d3(Unknown)

<400> 10

caagacttgg ccgatcctaa cgacatgc 28