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CN110846298B - Sodium sulfate-resistant xylosidase mutant T326D and its preparation and use - Google Patents

Sodium sulfate-resistant xylosidase mutant T326D and its preparation and use Download PDF

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CN110846298B
CN110846298B CN201911268839.2A CN201911268839A CN110846298B CN 110846298 B CN110846298 B CN 110846298B CN 201911268839 A CN201911268839 A CN 201911268839A CN 110846298 B CN110846298 B CN 110846298B
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leu
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CN110846298A (en
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周峻沛
黄遵锡
张蕊
李娜
韩楠玉
唐湘华
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Yunnan Normal University
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/26—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof
    • C02F2103/28—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof from the paper or cellulose industry

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Abstract

The invention discloses a sodium sulfate-resistant xylosidase mutant T326D, and a preparation method and application thereof, wherein the amino acid sequence of the mutant T326D is obtained by mutating threonine at 326 th site of wild xylosidase HJ14GH43 into aspartic acid, and the sequence is shown as SEQ ID NO. 1. The mutant enzyme T326D of the invention is at high Na concentrations compared to the wild enzyme HJ14GH432SO4The stability in (b) is enhanced. Passing through 10.0-30.0% (w/v) of Na2SO4After 60min of treatment, the activity of the wild enzyme HJ14GH43 is 47-78%, and the activity of the mutant enzyme T326D is 101-120%. Therefore, the sodium sulfate-resistant xylosidase mutant T326D can be applied to industries such as leather making, papermaking and sewage treatment.

Description

Sodium sulfate-resistant xylosidase mutant T326D and preparation and application thereof
Technical Field
The invention relates to a xylosidase mutant, in particular to a sodium sulfate-resistant xylosidase mutant T326D and preparation and application thereof.
Background
Xylose is present in xylan, plant glycoproteins, and proteoglycans in animals, xylosidase (. beta. -D-xylosidase, EC 3.2.1.37) can hydrolyze xylooligosaccharide into xylose (Collins et al FEMS Microbiology Reviews,2005,29: 3-23.), and can cleave xylose from plant glycoproteins and proteoglycans in animals (Leszczczczuk et al. plant Physiology and Biochemistry,2019,139: 681-690; Takagaki et al. the Journal of Biological Chemistry,1990,265: 854-860.). Xylose can be used as raw material for producing ethanol, lactic acid, xylitol, etc.
Salts can have a large influence on the properties of the enzyme. In neutral salts, salting-out of the enzyme occurs, resulting in a large proportion of the enzyme not having good catalytic activity at high salt concentrations. Salt is widely found in nature and in various manufacturing practices including sewage, washing, tanning, food, paper, and the like. The enzyme with salt tolerance can be better applied to the biotechnology field of high-salt environment, for example, sodium sulfate needs to be added in the leather softening process, and xylanase is added in the process, so that the effects of promoting the loosening of leather fibers and improving the softness, hand feeling and physical and mechanical properties of finished leather can be achieved (for example, an animal leather fiber loosening method based on the xylanase effect disclosed in Chinese patent ZL 201710574969.3).
Therefore, in order to make the enzyme have better applicability in the biotechnology field of high salt environment, it is required to improve the stability of the enzyme in salt.
Disclosure of Invention
The invention aims to provide a sodium sulfate-resistant xylosidase mutant T326D, and preparation and application thereof, wherein the mutant solves the problem that the existing enzyme does not have good stability in a sodium sulfate-containing liquid, and still has good enzyme activity after being treated by high-concentration sodium sulfate.
In order to achieve the aim, the invention provides a sodium sulfate-resistant xylosidase mutant T326D, wherein the amino acid sequence of the mutant T326D is obtained by mutating threonine at position 326 of wild xylosidase HJ14GH43 into aspartic acid, and the sequence is shown as SEQ ID NO. 1.
The invention also provides a gene T326d for encoding the xylosidase mutant T326D, wherein the nucleotide sequence of the gene T326d is shown as SEQ ID NO. 2.
The invention also provides a recombinant vector containing the gene t326 d.
Preferably, the recombinant vector is pEasy-E1.
The invention also provides a recombinant bacterium containing the gene t326 d.
Preferably, the recombinant bacterium employs a host cell comprising: escherichia coli BL 21.
The invention also provides application of the xylosidase mutant T326D in leather making, papermaking and sewage treatment.
Preferably, the xylosidase mutant T326D is used for the degradation of xylan and/or xylosyl-containing substances in sodium sulphate-containing liquids.
Preferably, the concentration of the sodium sulfate is 3.0-30.0%.
The invention also provides a preparation method of the xylosidase mutant T326D, which comprises the following steps:
connecting the gene t326d with an expression vector to obtain a recombinant vector; transforming the recombinant vector into a host cell to obtain a recombinant strain; culturing the recombinant strain, inducing expression of xylosidase mutant T326D, and recovering and purifying the expressed xylosidase mutant T326D.
The sodium sulfate-resistant xylosidase mutant T326D, the preparation method and the application thereof solve the problem that the existing enzyme does not have good stability in sodium sulfate-containing liquid, and have the following advantages:
the mutant enzyme T326D of the invention is at high Na concentrations compared to the wild enzyme HJ14GH432SO4The stability in (b) is enhanced. Passing through 10.0-30.0% (w/v) of Na2SO4After 60min of treatment, the activity of the wild enzyme HJ14GH43 is 47-78%, and the activity of the mutant enzyme T326D is 101-120%. Therefore, the sodium sulfate-resistant xylosidase mutant T326D can be applied to industries such as leather making, papermaking and sewage treatment.
Drawings
FIG. 1 shows the results of SDS-PAGE analysis of the wild-type enzyme HJ14GH43 and the mutant enzyme T326D.
FIG. 2 shows the stability results of the purified wild enzyme HJ14GH43 and the mutant enzyme T326D in NaCl.
FIG. 3 shows the stability results of purified wild enzyme HJ14GH43 and mutant enzyme T326D in KCl.
FIG. 4 shows the purified wild enzyme HJ14GH43 and mutant enzyme T326D in Na2SO4Stability results in (1).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental materials and reagents in the experimental examples of the invention are as follows:
bacterial strain and carrier: escherichia coli BL21(DE3) and expression vector pEasy-E1 were purchased from Beijing Quanyujin Biotechnology Ltd;
enzymes and other biochemical reagents: pNP (p-nitrophenyl) and pNPX (p-nitrophenyl-beta-d-xylopyranoside) were purchased from Sigma, and others were made from reagents (all available from general Biochemical Co.);
LB culture medium: peptone 10g, Yeast extract 5g, NaCl 10g, distilled water to 1000mL, natural pH (about 7). On the basis of the solid medium, 2.0% (w/v) agar was added.
The molecular biological experiments which are not specifically described in the following experimental examples are carried out by referring to the specific methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruke, or according to kits and product instructions.
Experimental example 1 construction and transformation of expression vector
Synthesizing a coding gene hJ14GH43 of the wild xylosidase HJ14GH43 according to a xylosidase nucleotide sequence KY391885(SEQ ID NO.4) recorded by GenBank; a gene T326d (SEQ ID NO.2) encoding the mutant enzyme T326D was also synthesized.
Respectively connecting the synthesized nucleotide sequences of xylosidase and mutant enzyme T326D with an expression vector pEasy-E1 to obtain an expression vector containing hJ14GH43 and T326d, respectively transforming the connection products into escherichia coli BL21(DE3) to obtain recombinant strains respectively expressing wild enzyme HJ14GH43 and mutant enzyme T326D.
EXAMPLE 2 preparation of the wild enzyme HJ14GH43 and the mutant enzyme T326D
The recombinant strains containing hJ14GH43 and t326d were inoculated in LB (containing 100. mu.g mL) at an inoculum size of 0.1% respectively- 1Amp) in the culture medium, the mixture was rapidly shaken at 37 ℃ for 16 hours.
Then, the activated bacterial suspension was inoculated into fresh LB (containing 100. mu.g mL) at an inoculum size of 1%-1Amp) culture solution, rapidly shaking and culturing for about 2-3 h (OD)6000.6-1.0) was reached, induction was carried out by adding IPTG at a final concentration of 0.1mM, and shaking culture was continued at 20 ℃ for about 20 hours.
Centrifugation was carried out at 12000rpm for 5min to collect the cells. After the cells were suspended in an appropriate amount of Tris-HCl buffer (pH7.0), the cells were disrupted by ultrasonication in a low-temperature water bath.
And centrifuging the crude enzyme solution concentrated in the cells at 12,000rpm for 10min, sucking a supernatant, and respectively carrying out affinity elution and elution on the target protein by using Nickel-NTA Agarose and 0-500 mM imidazole to obtain the purified target protein.
As shown in FIG. 1, SDS-PAGE analysis of the wild enzyme HJ14GH43 and the mutant enzyme T326D (M: protein Marker; W: HJ14GH43) shows that the wild enzyme HJ14GH43 and the mutant enzyme T326D are both expressed in E.coli, and after purification, the products are single bands.
Experimental example 3 determination of the Properties of the purified wild enzyme HJ14GH43 and the mutant enzyme T326D
The activity of the purified wild enzyme HJ14GH43 and mutant enzyme T326D was determined by the pNP method as follows:
dissolving pNPX in a buffer solution to make the final concentration of the pNPX be 2 mM; the reaction system contains 50 mu L of proper enzyme solution and 450 mu L of 2mM substrate; preheating substrate at reaction temperature for 5min, adding enzyme solution, reacting for a proper time, and adding 2mL of 1M Na2CO3The reaction was terminated and the released pNP was measured at 405nm after cooling to room temperature; 1 enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate per minute to produce 1. mu. mol pNP.
1. Stability of purified wild enzyme HJ14GH43 and mutant enzyme T326D in NaCl
The purified enzyme solution was placed in 3.0-30.0% (w/v) NaCl aqueous solution, treated at 20 ℃ for 60min, and then subjected to enzymatic reaction at pH7.0 and 20 ℃ with untreated enzyme solution as a control. The enzymatic properties of the purified HJ14GH43 and the mutant enzyme T326D were determined by reaction for 10min using pNPX as a substrate.
As shown in FIG. 2, the stability results of the purified wild enzyme HJ14GH43 and the mutant enzyme T326D in NaCl show that the wild enzyme HJ14GH43 and the mutant enzyme T326D are not stable in NaCl, 20-44% of the activity of the wild enzyme HJ14GH43 is remained after the wild enzyme HJ14GH43 and the mutant enzyme T326 GH D are treated by 3.0-30.0% (w/v) NaCl for 60min, and 9-16% of the activity of the mutant enzyme T326D is remained after the wild enzyme HJ14GH43 and the mutant enzyme T326 GH D are treated by 5.0-25.0% (w/v) NaCl for 60 min.
2. Stability of purified wild enzyme HJ14GH43 and mutant enzyme T326D in KCl
The purified enzyme solution was placed in a 3.0-30.0% (w/v) KCl aqueous solution, treated at 20 ℃ for 60min, and then subjected to enzymatic reaction at pH7.0 and 20 ℃ with untreated enzyme solution as a control. The enzymatic properties of the purified HJ14GH43 and the mutant enzyme T326D were determined by reaction for 10min using pNPX as a substrate.
As shown in FIG. 3, for the stability results of the purified wild enzyme HJ14GH43 and the mutant enzyme T326D in KCl, the stability of the wild enzyme HJ14GH43 and the mutant enzyme T326D in KCl are relatively similar, after being treated with KCl of 3.0-30.0% (w/v) for 60min, the activity of the wild enzyme HJ14GH43 is reduced from 114% to 28%, and after being treated with KCl of 5.0-25.0% (w/v) for 60min, the activity of the mutant enzyme T326D is remained 34-85%.
3. Purified wild enzyme HJ14GH43 and mutant enzyme T326D were in Na2SO4Stability in
Placing the purified enzyme solution in 3.0-30.0% (w/v) Na2SO4Treating in water solution at 20 deg.C for 60min, and performing enzymatic reaction at pH7.0 and 20 deg.CUntreated enzyme solution was used as a control. The enzymatic properties of the purified HJ14GH43 and the mutant enzyme T326D were determined by reaction for 10min using pNPX as a substrate.
As shown in FIG. 4, the wild enzyme HJ14GH43 and the mutant enzyme T326D were purified in Na2SO4The stability results in (1) show that the wild enzyme HJ14GH43 and the mutant enzyme T326D are in Na2SO4Has different stability in the middle through 3.0-30.0% (w/v) Na2SO4After the treatment for 60min, the enzyme activity of the wild enzyme HJ14GH43 is basically in a descending trend, 47-86% of the enzyme activity is remained, the enzyme activity of the mutant enzyme T326D is in a descending and then ascending trend, and the enzyme activity can be increased from 49% to 120%.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Sequence listing
<110> university of Yunnan Master
<120> sodium sulfate-resistant xylosidase mutant T326D and preparation and application thereof
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Met Lys Ile Thr Asn Pro Val Leu Lys Gly Phe Asn Pro Asp Pro Ser
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Ile Cys Arg Val Gly Glu Asp Tyr Tyr Met Ala Val Ser Thr Phe Glu
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Trp Phe Pro Gly Val Gln Ile Tyr His Ser Lys Asp Leu Val His Trp
35 40 45
Arg Leu Ala Ala Arg Pro Leu Gln Lys Thr Ser Gln Leu Asp Met Lys
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Gly Asn Pro Asp Ser Gly Gly Val Trp Ala Pro Cys Leu Ser Tyr Ala
65 70 75 80
Asp Gly Gln Phe Trp Leu Ile Tyr Ser Asp Ile Lys Val Val Asp Gly
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Pro Phe Lys Asp Gly His Asn Tyr Leu Val Thr Ala Ser Glu Val Asp
100 105 110
Gly Asp Trp Ser Glu Pro Ile Leu Leu Asn Ser Ser Gly Phe Asp Pro
115 120 125
Ser Leu Phe His Asp His Ser Gly Lys Lys Tyr Val Leu Asn Met Leu
130 135 140
Trp Asp His Arg Glu Lys His His Ser Phe Ala Gly Ile Ala Leu Gln
145 150 155 160
Glu Tyr Ser Val Ala Glu Lys Lys Leu Ile Gly Gln Arg Lys Val Ile
165 170 175
Phe Lys Gly Thr Pro Ile Lys Leu Thr Glu Ala Pro His Leu Tyr His
180 185 190
Ile Gly Asp Tyr Tyr Tyr Leu Leu Thr Ala Glu Gly Gly Thr Arg Tyr
195 200 205
Glu His Ala Ala Thr Ile Ala Arg Ser Ser His Ile Glu Gly Pro Tyr
210 215 220
Glu Val His Pro Asp Asn Pro Ile Val Ser Ala Phe His Val Pro Glu
225 230 235 240
His Pro Leu Gln Lys Cys Gly His Ala Ser Ile Val Gln Thr His Thr
245 250 255
Asn Glu Trp Tyr Leu Ala His Leu Thr Gly Arg Pro Ile Gln Ser Ser
260 265 270
Lys Glu Ser Ile Phe Gln Gln Arg Gly Trp Cys Pro Leu Gly Arg Glu
275 280 285
Thr Ala Ile Gln Lys Leu Glu Trp Lys Asp Gly Trp Pro Tyr Val Val
290 295 300
Gly Gly Lys Glu Gly Thr Leu Glu Val Glu Ala Pro Lys Ile Glu Glu
305 310 315 320
Lys Val Phe Ala Pro Asp Tyr His Thr Val Asp Glu Phe Lys Glu Ser
325 330 335
Thr Leu Asn Arg His Phe Gln Thr Leu Arg Ile Pro Phe Thr Asp Gln
340 345 350
Ile Gly Ser Leu Thr Glu Lys Pro Gln His Leu Arg Leu Phe Gly Arg
355 360 365
Glu Ser Leu Thr Ser Lys Phe Thr Gln Ala Phe Val Ala Arg Arg Trp
370 375 380
Gln Ser Phe Tyr Phe Glu Ala Glu Thr Ala Val Ser Phe Phe Pro Glu
385 390 395 400
Asn Phe Gln Gln Ala Ala Gly Leu Val Asn Tyr Tyr Asn Thr Glu Asn
405 410 415
Trp Thr Ala Leu Gln Val Thr Tyr Asp Glu Glu Leu Gly Arg Thr Leu
420 425 430
Glu Leu Ser Val Cys Gln Asn Leu Ala Phe Ser Gln Pro Leu Thr His
435 440 445
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465 470 475 480
Trp Lys Glu Ile Asp Val Pro Phe Glu Ser Ile His Leu Ser Asp Asp
485 490 495
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Cys Gln Asp Thr Ser Gly Glu Arg Leu Pro Ala Asp Phe His Tyr Phe
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Arg Tyr Glu Glu Thr Asp Glu
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atgaagatta ccaatccagt gctcaaaggg tttaatcctg atccaagtat ttgccgtgta 60
ggagaagatt attatatggc cgtctctaca tttgaatggt ttccaggggt gcaaatttat 120
cattcaaagg atctcgtcca ttggcgtctt gctgcgcgtc cattgcaaaa aacgtcgcag 180
ctggatatga aggggaatcc tgactctggc ggggtatggg cgccgtgctt aagctatgct 240
gatgggcagt tttggcttat ttattcagat atcaaagtag tggatggccc atttaaagac 300
ggtcataatt atttggtcac ggcaagcgag gtggacggcg attggagtga accgatcctg 360
ctcaacagct ctggctttga tccatcttta ttccatgatc acagcgggaa gaaatacgtc 420
ttaaatatgc tgtgggatca tagggaaaag catcattcgt ttgcaggtat tgccttgcag 480
gaatatagtg tggctgaaaa gaagctcatc ggtcaaagga aggtcatttt taaaggcaca 540
ccgattaaac tgacagaagc gccgcatctg tatcatatcg gtgactacta ctatttatta 600
acggcagaag gaggtacccg gtatgagcat gcagcaacga tcgcccggtc ctcgcatatt 660
gaagggcctt atgaggttca tcctgataac ccgattgtaa gtgccttcca tgtgcctgaa 720
catccgcttc aaaaatgcgg gcatgcttca atcgttcaaa cgcatacaaa tgaatggtat 780
ctcgctcatc tcactggccg cccgattcaa tccagcaagg aatcgatttt tcaacagaga 840
gggtggtgcc ctttaggaag agaaacagcg atccaaaagc ttgaatggaa ggatggatgg 900
ccttatgttg taggcggaaa agaggggacg ctagaggttg aagcgccaaa gatcgaagaa 960
aaggtttttg caccagatta tcatacagtc gatgaattta aagaatcaac tctaaataga 1020
cactttcaaa cattaagaat tccgtttacc gatcagattg gttcgttaac ggagaaacct 1080
cagcatttaa ggttattcgg ccgtgaatct ttaacgtcta agtttaccca agcatttgtt 1140
gcaagacgct ggcaaagctt ttattttgaa gcagagacag ctgtttcgtt cttcccagaa 1200
aactttcagc aagccgcagg tcttgtgaat tattataata cggaaaactg gacagcactc 1260
caggtgacat atgatgagga acttggccgc acgcttgaac tatccgtctg tcaaaacctt 1320
gccttttctc agccgttgac acataaaatc atcattcctg acgaggtcac ttatgtctat 1380
ttaaaagtga ccgttcggaa agagacatat aaatattctt attcatttga tcagaaagag 1440
tggaaggaaa ttgatgtacc gtttgaatcc atccatttat ccgatgattt cattcgaggt 1500
gggggttttt ttacaggggc atttgtcggt atgcagtgcc aagatacgag cggcgagcgt 1560
cttcctgctg attttcacta ttttcgctat gaggaaacag acgaataa 1608
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<213> HJ14GH43
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Met Lys Ile Thr Asn Pro Val Leu Lys Gly Phe Asn Pro Asp Pro Ser
1 5 10 15
Ile Cys Arg Val Gly Glu Asp Tyr Tyr Met Ala Val Ser Thr Phe Glu
20 25 30
Trp Phe Pro Gly Val Gln Ile Tyr His Ser Lys Asp Leu Val His Trp
35 40 45
Arg Leu Ala Ala Arg Pro Leu Gln Lys Thr Ser Gln Leu Asp Met Lys
50 55 60
Gly Asn Pro Asp Ser Gly Gly Val Trp Ala Pro Cys Leu Ser Tyr Ala
65 70 75 80
Asp Gly Gln Phe Trp Leu Ile Tyr Ser Asp Ile Lys Val Val Asp Gly
85 90 95
Pro Phe Lys Asp Gly His Asn Tyr Leu Val Thr Ala Ser Glu Val Asp
100 105 110
Gly Asp Trp Ser Glu Pro Ile Leu Leu Asn Ser Ser Gly Phe Asp Pro
115 120 125
Ser Leu Phe His Asp His Ser Gly Lys Lys Tyr Val Leu Asn Met Leu
130 135 140
Trp Asp His Arg Glu Lys His His Ser Phe Ala Gly Ile Ala Leu Gln
145 150 155 160
Glu Tyr Ser Val Ala Glu Lys Lys Leu Ile Gly Gln Arg Lys Val Ile
165 170 175
Phe Lys Gly Thr Pro Ile Lys Leu Thr Glu Ala Pro His Leu Tyr His
180 185 190
Ile Gly Asp Tyr Tyr Tyr Leu Leu Thr Ala Glu Gly Gly Thr Arg Tyr
195 200 205
Glu His Ala Ala Thr Ile Ala Arg Ser Ser His Ile Glu Gly Pro Tyr
210 215 220
Glu Val His Pro Asp Asn Pro Ile Val Ser Ala Phe His Val Pro Glu
225 230 235 240
His Pro Leu Gln Lys Cys Gly His Ala Ser Ile Val Gln Thr His Thr
245 250 255
Asn Glu Trp Tyr Leu Ala His Leu Thr Gly Arg Pro Ile Gln Ser Ser
260 265 270
Lys Glu Ser Ile Phe Gln Gln Arg Gly Trp Cys Pro Leu Gly Arg Glu
275 280 285
Thr Ala Ile Gln Lys Leu Glu Trp Lys Asp Gly Trp Pro Tyr Val Val
290 295 300
Gly Gly Lys Glu Gly Thr Leu Glu Val Glu Ala Pro Lys Ile Glu Glu
305 310 315 320
Lys Val Phe Ala Pro Thr Tyr His Thr Val Asp Glu Phe Lys Glu Ser
325 330 335
Thr Leu Asn Arg His Phe Gln Thr Leu Arg Ile Pro Phe Thr Asp Gln
340 345 350
Ile Gly Ser Leu Thr Glu Lys Pro Gln His Leu Arg Leu Phe Gly Arg
355 360 365
Glu Ser Leu Thr Ser Lys Phe Thr Gln Ala Phe Val Ala Arg Arg Trp
370 375 380
Gln Ser Phe Tyr Phe Glu Ala Glu Thr Ala Val Ser Phe Phe Pro Glu
385 390 395 400
Asn Phe Gln Gln Ala Ala Gly Leu Val Asn Tyr Tyr Asn Thr Glu Asn
405 410 415
Trp Thr Ala Leu Gln Val Thr Tyr Asp Glu Glu Leu Gly Arg Thr Leu
420 425 430
Glu Leu Ser Val Cys Gln Asn Leu Ala Phe Ser Gln Pro Leu Thr His
435 440 445
Lys Ile Ile Ile Pro Asp Glu Val Thr Tyr Val Tyr Leu Lys Val Thr
450 455 460
Val Arg Lys Glu Thr Tyr Lys Tyr Ser Tyr Ser Phe Asp Gln Lys Glu
465 470 475 480
Trp Lys Glu Ile Asp Val Pro Phe Glu Ser Ile His Leu Ser Asp Asp
485 490 495
Phe Ile Arg Gly Gly Gly Phe Phe Thr Gly Ala Phe Val Gly Met Gln
500 505 510
Cys Gln Asp Thr Ser Gly Glu Arg Leu Pro Ala Asp Phe His Tyr Phe
515 520 525
Arg Tyr Glu Glu Thr Asp Glu
530 535
<210> 4
<211> 1608
<212> DNA
<213> KY391885
<400> 4
atgaagatta ccaatccagt gctcaaaggg tttaatcctg atccaagtat ttgccgtgta 60
ggagaagatt attatatggc cgtctctaca tttgaatggt ttccaggggt gcaaatttat 120
cattcaaagg atctcgtcca ttggcgtctt gctgcgcgtc cattgcaaaa aacgtcgcag 180
ctggatatga aggggaatcc tgactctggc ggggtatggg cgccgtgctt aagctatgct 240
gatgggcagt tttggcttat ttattcagat atcaaagtag tggatggccc atttaaagac 300
ggtcataatt atttggtcac ggcaagcgag gtggacggcg attggagtga accgatcctg 360
ctcaacagct ctggctttga tccatcttta ttccatgatc acagcgggaa gaaatacgtc 420
ttaaatatgc tgtgggatca tagggaaaag catcattcgt ttgcaggtat tgccttgcag 480
gaatatagtg tggctgaaaa gaagctcatc ggtcaaagga aggtcatttt taaaggcaca 540
ccgattaaac tgacagaagc gccgcatctg tatcatatcg gtgactacta ctatttatta 600
acggcagaag gaggtacccg gtatgagcat gcagcaacga tcgcccggtc ctcgcatatt 660
gaagggcctt atgaggttca tcctgataac ccgattgtaa gtgccttcca tgtgcctgaa 720
catccgcttc aaaaatgcgg gcatgcttca atcgttcaaa cgcatacaaa tgaatggtat 780
ctcgctcatc tcactggccg cccgattcaa tccagcaagg aatcgatttt tcaacagaga 840
gggtggtgcc ctttaggaag agaaacagcg atccaaaagc ttgaatggaa ggatggatgg 900
ccttatgttg taggcggaaa agaggggacg ctagaggttg aagcgccaaa gatcgaagaa 960
aaggtttttg caccaaccta tcatacagtc gatgaattta aagaatcaac tctaaataga 1020
cactttcaaa cattaagaat tccgtttacc gatcagattg gttcgttaac ggagaaacct 1080
cagcatttaa ggttattcgg ccgtgaatct ttaacgtcta agtttaccca agcatttgtt 1140
gcaagacgct ggcaaagctt ttattttgaa gcagagacag ctgtttcgtt cttcccagaa 1200
aactttcagc aagccgcagg tcttgtgaat tattataata cggaaaactg gacagcactc 1260
caggtgacat atgatgagga acttggccgc acgcttgaac tatccgtctg tcaaaacctt 1320
gccttttctc agccgttgac acataaaatc atcattcctg acgaggtcac ttatgtctat 1380
ttaaaagtga ccgttcggaa agagacatat aaatattctt attcatttga tcagaaagag 1440
tggaaggaaa ttgatgtacc gtttgaatcc atccatttat ccgatgattt cattcgaggt 1500
gggggttttt ttacaggggc atttgtcggt atgcagtgcc aagatacgag cggcgagcgt 1560
cttcctgctg attttcacta ttttcgctat gaggaaacag acgaataa 1608

Claims (10)

1.一种耐硫酸钠的木糖苷酶突变体T326D，其特征在于，该突变体T326D的氨基酸序列由野生木糖苷酶HJ14GH43第326位的苏氨酸突变为天冬氨酸获得，其序列如SEQ ID NO.1所示。1. a sodium sulfate-resistant xylosidase mutant T326D is characterized in that, the amino acid sequence of this mutant T326D is obtained by mutation of the threonine at position 326 of wild xylosidase HJ14GH43 into aspartic acid, and its sequence is as follows: shown in SEQ ID NO.1. 2.一种编码如权利要求1所述的木糖苷酶突变体T326D的基因t326d，其特征在于，该基因t326d的核苷酸序列如SEQ ID NO.2所示。2. A gene t326d encoding the xylosidase mutant T326D according to claim 1, wherein the nucleotide sequence of the gene t326d is shown in SEQ ID NO.2. 3.一种含有如权利要求2所述的基因t326d的重组载体。3. A recombinant vector containing the gene t326d as claimed in claim 2. 4.根据权利要求3所述的重组载体，其特征在于，所述重组载体采用pEasy-E1。4. The recombinant vector according to claim 3, wherein the recombinant vector adopts pEasy-E1. 5.一种含有如权利要求2所述的基因t326d的重组菌。5. A recombinant bacterium containing the gene t326d as claimed in claim 2. 6.根据权利要求5所述的重组菌，其特征在于，所述重组菌采用的宿主细胞包含：大肠杆菌BL21。6 . The recombinant bacteria according to claim 5 , wherein the host cells adopted by the recombinant bacteria comprise: Escherichia coli BL21. 7 . 7.如权利要求1所述的木糖苷酶突变体T326D在制革、造纸和污水处理中的应用。7. The application of the xylosidase mutant T326D as claimed in claim 1 in tanning, papermaking and sewage treatment. 8.根据权利要求7所述的应用，其特征在于，所述木糖苷酶突变体T326D用于在含硫酸钠的液体中对木聚糖或/和含木糖基的物质进行降解。8 . The use according to claim 7 , wherein the xylosidase mutant T326D is used to degrade xylan or/and xylosyl-containing substances in a liquid containing sodium sulfate. 9 . 9.根据权利要求8所述的应用，其特征在于，所述硫酸钠的浓度为3.0～30.0％。9 . The application according to claim 8 , wherein the concentration of the sodium sulfate is 3.0 to 30.0%. 10 . 10.一种如权利要求1所述的木糖苷酶突变体T326D的制备方法，其特征在于，该方法包含：10. A method for preparing a xylosidase mutant T326D as claimed in claim 1, wherein the method comprises: 将如权利要求2所述的基因t326d与表达载体相连接，获得重组载体；将所述重组载体转化宿主细胞，获得重组菌；培养所述重组菌株，诱导木糖苷酶突变体T326D表达，回收并纯化表达的木糖苷酶突变体T326D。The gene t326d as claimed in claim 2 is connected with an expression vector to obtain a recombinant vector; the recombinant vector is transformed into a host cell to obtain a recombinant strain; the recombinant strain is cultured, the expression of the xylosidase mutant T326D is induced, and the The expressed xylosidase mutant T326D was purified.
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Citations (1)

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CN105950586A (en) * 2016-07-15 2016-09-21 云南师范大学 Low temperature xylosidase HJ14GH43 and salt-tolerant mutant thereof

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US7993884B2 (en) * 2006-10-10 2011-08-09 The United States Of America As Represented By The Secretary Of Agriculture Beta-xylosidase for conversion of plant cell wall carbohydrates to simple sugars

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CN105950586A (en) * 2016-07-15 2016-09-21 云南师范大学 Low temperature xylosidase HJ14GH43 and salt-tolerant mutant thereof

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"内切木聚糖酶和木糖苷酶的耐盐性改性研究";刘钰;《中国优秀硕士学位论文全文数据库 基础科学辑》;20180215;全文 *

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