CN110904075A - Salt-tolerant xylosidase mutant K321D and preparation method and application thereof - Google Patents
Salt-tolerant xylosidase mutant K321D and preparation method and application thereof Download PDFInfo
- Publication number
- CN110904075A CN110904075A CN201911268814.2A CN201911268814A CN110904075A CN 110904075 A CN110904075 A CN 110904075A CN 201911268814 A CN201911268814 A CN 201911268814A CN 110904075 A CN110904075 A CN 110904075A
- Authority
- CN
- China
- Prior art keywords
- mutant
- enzyme
- glu
- leu
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a salt tolerant xylosidase mutant K321D, a preparation method and application thereof, wherein the amino acid sequence of the mutant K321D is obtained by mutating 321 th lysine of wild xylosidase HJ14GH43 into aspartic acid, the sequence is shown as SEQ ID NO.1, and the salt is not NaCl. Compared with the wild enzyme HJ14GH43, the mutant enzyme K321D of the invention has high KCl and Na concentration2SO4And (NH)4)2SO4The stability of the composition is enhanced, the activity of the composition is increased from 48% to 65% by KCl treatment with the concentration of 20.0-30.0%, and the composition is subjected to Na treatment with the concentration of 10.0-30.0%2SO4Treating, wherein the activity is 93-111%; (NH) at a concentration of 20.0-30.0%4)2SO4The activity of the treatment is maintained above 90%, and the treatment method can be applied to industries such as leather making, papermaking and sewage treatment.
Description
Technical Field
The invention relates to a xylosidase mutant, and in particular relates to a salt-tolerant xylosidase mutant K321D and a preparation method and application thereof.
Background
Endoxylanase (endo-1,4- β -D-xylonase, EC3.2.1.8) can randomly cut the main chain skeleton of xylan to generate xylooligosaccharide, xylosidase (β -D-xylosidase, EC 3.2.1.37) can hydrolyze xylooligosaccharide into xylose (Collins et al FEMS microbiological Reviews,2005,29: 3-23.) xylose can be used as raw material for producing ethanol, lactic acid, xylitol, etc. besides xylan, plant glycoproteins and animal in-vivo proteoglycan also contain xylose, which can be degraded by xylosidase (Leszcz. plant and Chemistry,2019,139: 681-690; Takagagagaial. thermal. 1990,265, journal: 854).
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).
However, most enzymes do not have good catalytic activity at high salt concentration due to salting-out action at high salt concentration. 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 salt-tolerant xylosidase mutant K321D, a preparation method and application thereof, the mutant solves the problem that the existing enzyme does not have good stability under high salt concentration, has salt tolerance, and still has good enzyme activity after being treated by high salt concentration.
In order to achieve the aim, the invention provides a salt-tolerant xylosidase mutant K321D, wherein the amino acid sequence of the mutant K321D is obtained by mutating 321 th lysine of wild xylosidase HJ14GH43 into aspartic acid, the sequence of the mutant is shown as SEQ ID NO.1, and the salt is not NaCl.
The invention also provides a gene K321d for encoding the xylosidase mutant K321D, wherein the nucleotide sequence of the gene K321d is shown as SEQ ID NO. 2.
The invention also provides a recombinant vector containing the gene k321 d.
Preferably, the recombinant vector is pEasy-E1.
The invention also provides a recombinant bacterium containing the gene k321 d.
Preferably, the recombinant bacterium employs a host cell comprising: escherichia coli BL 21.
The invention also provides application of the xylosidase mutant K321D in leather making, paper making and sewage treatment.
Preferably, the xylosidase mutant K321D is used for degrading xylan and/or xylosyl-containing material in a salt-containing liquid, the salt concentration is more than 3%, and the salt is not NaCl.
Preferably, the salt comprises: KCl, Na2SO4And (NH)4)2SO4Any one or more than two of them.
The invention also provides a preparation method of the xylosidase mutant K321D, which is characterized by comprising the following steps:
connecting the gene k321d 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 the xylosidase mutant K321D, and recovering and purifying the expressed xylosidase mutant K321D.
The salt-tolerant xylosidase mutant K321D, the preparation method and the application thereof solve the problem that the existing enzyme does not have good stability under high salt concentration, and have the following advantages:
compared with the wild enzyme HJ14GH43, the mutant enzyme K321D of the invention has high KCl and Na concentration2SO4And (NH)4)2SO4The stability in (b) is enhanced. After being treated by KCl with the concentration of 20.0-30.0% (w/v) for 60min, the activity of the wild enzyme HJ14GH43 is about 30% remained, while the activity of the mutant enzyme K321D is increased from 48% to 65%; passing through 10.0-30.0% (w/v) of Na2SO4After 60min of treatment, the activity of a wild enzyme HJ14GH43 is 47-78%, and the activity of a mutant enzyme K321D is 93-111%; after a reaction of 20.0-30.0% (w/v) of (NH)4)2SO4After 60min of treatment, the activity of HJ14GH43 is reduced from 79% to 38%, K321D is kept stable, and the activity is maintained above 90%.
Therefore, the xylosidase mutant K321D with tolerance under high salt concentration can be applied to industries such as leather making, paper making, sewage treatment and the like.
Drawings
FIG. 1 is an SDS-PAGE analysis of the wild-type enzyme HJ14GH43 and the mutant enzyme K321D.
FIG. 2 shows the stability of the purified wild enzyme HJ14GH43 and the mutant enzyme K321D in NaCl.
FIG. 3 shows the stability of the purified wild enzyme HJ14GH43 and the mutant enzyme K321D in KCl.
FIG. 4 shows the purified wild enzyme HJ14GH43 and mutant enzyme K321D in Na2SO4Stability in (1).
FIG. 5 shows the purified wild enzyme HJ14GH43 and mutant enzyme K321D in (NH)4)2SO4Stability 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 used in the experimental examples of the present invention were 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 biochemicals pNP (p-nitrophenyl) and pNPX (p-nitrophenyl- β -d-xylopyranoside) were obtained from Sigma, all other reagents being made in China (all available from general biochemicals).
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 the purchased 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; synthesizing a gene K321d (SEQ ID NO.2) of a mutant enzyme K321D;
the synthesized sequences of the coding gene hJ14GH43 and the coding gene K321d are respectively connected with an expression vector pEasy-E1 to obtain an expression vector containing hJ14GH43 and K321d, and the connection products are respectively transformed into escherichia coli BL21(DE3) to obtain recombinant strains respectively expressing a wild enzyme HJ14GH43 and a mutant enzyme K321D.
Example 2 preparation of the wild enzyme HJ14GH43 and the mutant enzyme K321D
The recombinant strains containing hJ14GH43 and k321d were inoculated in LB (containing 100. mu.g mL) at an inoculum size of 0.1% respectively- 1Amp) in culture, 37Rapidly oscillating at the temperature of 16 h.
Then, the activated bacterial suspension was inoculated into fresh LB (containing 100. mu.g mL) at an inoculum size of 1%-1Amp) culture solution, performing rapid shaking culture for about 2-3 h, OD600After reaching 0.6-1.0, IPTG (isopropyl- β -D-thiogalactoside) with a final concentration of 0.1mM was added for induction, and the shaking culture was continued at 20 ℃ for about 20 hours.
Centrifuging at 12000rpm for 5min, collecting thallus, suspending the thallus in Tris-HCl buffer solution with pH of 7.0, and ultrasonically breaking the thallus in low temperature water bath.
Centrifuging the crude enzyme solution concentrated in the cells at 12,000rpm for 10min, sucking the supernatant, and respectively carrying out affinity elution and elution on the target protein by using Nickel-NTAAgarose (Nickel NTA agarose resin) 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 K321D (M: protein Marker; W: HJ14GH43) shows that the wild enzyme HJ14GH43 and the mutant enzyme K321D are both expressed in Escherichia coli, and after purification, the products are single bands.
EXAMPLE 3 determination of the Properties of the purified wild enzyme HJ14GH43 and the mutant enzyme K321D
The activity of the purified wild enzyme HJ14GH43 and the mutant enzyme K321D was determined by the pNP method, which was 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 break down the substrate per minute to produce 1. mu. mol pNP.
1. Stability of purified wild enzyme HJ14GH43 and mutant enzyme K321D 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 pNPX is used as a substrate and reacted for 10min, and the enzymological properties of the purified HJ14GH43 and the mutant enzyme K321D are measured.
As shown in FIG. 2, the stability results of the purified wild enzyme HJ14GH43 and the mutant enzyme K321D in NaCl show that the wild enzyme HJ14GH43 and the mutant enzyme K321D are not stable in NaCl, and 20-44% of the activity of the wild enzyme HJ14GH43 and 9-18% of the activity of the mutant enzyme K321D are remained after the wild enzyme HJ14GH43 and the mutant enzyme K321D are treated by 3.0-30.0% (w/v) NaCl for 60 min.
2. Stability of purified wild enzyme HJ14GH43 and mutant enzyme K321D 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 pNPX is used as a substrate and reacted for 10min, and the enzymological properties of the purified HJ14GH43 and the mutant enzyme K321D are measured.
As shown in FIG. 3, for the stability results of the purified wild enzyme HJ14GH43 and the mutant enzyme K321D in KCl, it was shown that the stability of the wild enzyme HJ14GH43 and the mutant enzyme K321D in KCl were different, after being treated with 3.0-10.0% (w/v) KCl for 60min, the activity of the wild enzyme HJ14GH43 was reduced from 114% to 56%, the activity of the mutant enzyme K321D was reduced from 79% to 43%, and after being treated with 20.0-30.0% (w/v) KCl for 60min, the activity of the wild enzyme HJ14GH43 was about 30% remained, while the activity of the mutant enzyme K321D was increased from 48% to 65% (FIG. 3).
3. Purified wild enzyme HJ14GH43 and mutant enzyme K321D in Na2SO4Stability in
Placing the purified enzyme solution in 3.0-30.0% (w/v) Na2SO4The enzyme solution was treated at 20 ℃ for 60min in an aqueous solution, and then the enzyme reaction was carried out at pH7.0 and 20 ℃ with an untreated enzyme solution as a control. The pNPX is used as a substrate and reacted for 10min, and the enzymological properties of the purified HJ14GH43 and the mutant enzyme K321D are measured.
As shown in FIG. 4, the wild enzyme HJ14GH43 and the mutant enzyme K321D were purified in Na2SO4The stability results in (1) show that the wild enzyme HJ14GH43 and the mutant enzyme K321D are in Na2SO4Has different stability in the middle through 3.0-30.0% (w/v) Na2SO4After 60min of treatment, the enzyme activity of the wild enzyme HJ14GH43 is basicallyThe enzyme activity is reduced, 47-86% of the enzyme activity is remained, the enzyme activity of the mutant enzyme K321D is reduced firstly and then increased, and the enzyme activity is increased from 64% to 111% at most.
4. Purified wild enzyme HJ14GH43 and mutant enzyme K321D are in (NH)4)2SO4Stability in
Placing the purified enzyme solution in 3.0-30.0% (w/v) (NH)4)2SO4The enzyme solution was treated at 20 ℃ for 60min in an aqueous solution, and then the enzyme reaction was carried out at pH7.0 and 20 ℃ with an untreated enzyme solution as a control. The pNPX is used as a substrate and reacted for 10min, and the enzymological properties of the purified HJ14GH43 and the mutant enzyme K321D are measured.
As shown in FIG. 5, the wild enzyme HJ14GH43 and the mutant enzyme K321D were purified at (NH)4)2SO4The stability results in (1) indicate that the wild enzyme HJ14GH43 and the mutant enzyme K321D are in (NH)4)2SO4Has different stability, and is subjected to (NH) of 3.0-15.0% (w/v)4)2SO4After 60min of treatment, the wild enzyme HJ14GH43 is kept stable, and the mutant enzyme K321D has 39-82% of enzyme activity but 20.0-30.0% (w/v) of (NH)4)2SO4After 60min of treatment, the activity of HJ14GH43 is reduced from 79% to 38%, K321D is kept stable, and the activity is maintained above 90%.
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> salt-tolerant xylosidase mutant K321D and preparation method and application thereof
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>535
<212>PRT
<213>Artificial Sequence
<400>1
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
Asp 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>2
<211>1608
<212>DNA
<213>Artificial Sequence
<400>2
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
gatgtttttg 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
<210>3
<211>535
<212>PRT
<213>HJ14GH43
<400>3
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 GluAsp 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 ThrGlu 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 IlePro 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 ValGly 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. A salt-tolerant xylosidase mutant K321D, wherein the amino acid sequence of the mutant K321D is obtained by mutating the 321 st lysine of wild xylosidase HJ14GH43 to aspartic acid, the sequence is shown in SEQ ID NO.1, and the salt is not NaCl.
2. A gene K321d encoding the xylosidase mutant K321D as claimed in claim 1, wherein the nucleotide sequence of the gene K321d is shown in SEQ ID No. 2.
3. A recombinant vector comprising the gene k321d according to claim 2.
4. The recombinant vector according to claim 3, wherein pEasy-E1 is used as the recombinant vector.
5. A recombinant bacterium containing the gene k321d according to claim 2.
6. The recombinant bacterium according to claim 5, wherein the host cell used in the recombinant bacterium comprises: escherichia coli BL 21.
7. Use of the xylosidase mutant K321D according to claim 1 in leather-making, paper-making and sewage treatment.
8. Use according to claim 7, wherein the xylosidase mutant K321D is used for degradation of xylan or/and xylosyl-containing material in saline liquids at a salt concentration of more than 3% and the salt is not NaCl.
9. Use according to claim 8, wherein the salt comprises: KCl, Na2SO4And (NH)4)2SO4Any one or more than two of them.
10. A method for preparing the xylosidase mutant K321D as claimed in claim 1, comprising:
connecting the gene k321d of claim 2 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 the xylosidase mutant K321D, and recovering and purifying the expressed xylosidase mutant K321D.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911268814.2A CN110904075B (en) | 2019-12-11 | 2019-12-11 | Salt-tolerant xylosidase mutant K321D and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911268814.2A CN110904075B (en) | 2019-12-11 | 2019-12-11 | Salt-tolerant xylosidase mutant K321D and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110904075A true CN110904075A (en) | 2020-03-24 |
| CN110904075B CN110904075B (en) | 2021-03-23 |
Family
ID=69824759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911268814.2A Active CN110904075B (en) | 2019-12-11 | 2019-12-11 | Salt-tolerant xylosidase mutant K321D and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110904075B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750015A (en) * | 2019-03-27 | 2019-05-14 | 云南师范大学 | The xylanase mutant and its application that a kind of thermostability improves |
| CN113481185A (en) * | 2021-08-05 | 2021-10-08 | 云南师范大学 | Salt-tolerant beta-galactosidase GalNC2-13 and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950586A (en) * | 2016-07-15 | 2016-09-21 | 云南师范大学 | Low temperature xylosidase HJ14GH43 and salt-tolerant mutant thereof |
| CN105950592A (en) * | 2016-07-15 | 2016-09-21 | 云南师范大学 | Salt-resistant ethanol-resistant trypsin-resistant xylosidase JB13GH39 and preparation method thereof |
-
2019
- 2019-12-11 CN CN201911268814.2A patent/CN110904075B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950586A (en) * | 2016-07-15 | 2016-09-21 | 云南师范大学 | Low temperature xylosidase HJ14GH43 and salt-tolerant mutant thereof |
| CN105950592A (en) * | 2016-07-15 | 2016-09-21 | 云南师范大学 | Salt-resistant ethanol-resistant trypsin-resistant xylosidase JB13GH39 and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| RUI ZHANG ET AL.: "Biochemical and structural properties of a low-temperature-active glycoside hydrolase family 43 β-xylosidase: Activity and instability at high neutral salt concentrations", 《FOOD CHEMISTRY》 * |
| 刘钰: "内切木聚糖酶和木糖苷酶的耐盐性改性研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750015A (en) * | 2019-03-27 | 2019-05-14 | 云南师范大学 | The xylanase mutant and its application that a kind of thermostability improves |
| CN109750015B (en) * | 2019-03-27 | 2023-05-23 | 云南师范大学 | Xylanase mutant with improved thermostability and application thereof |
| CN113481185A (en) * | 2021-08-05 | 2021-10-08 | 云南师范大学 | Salt-tolerant beta-galactosidase GalNC2-13 and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110904075B (en) | 2021-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108588061B (en) | Low-temperature alkaline pectinase mutant with improved specific enzyme activity and thermal stability | |
| JP6330240B2 (en) | Thermostable β-glucosidase | |
| CN116355881B (en) | Beta-xylosidase mutant D395G with improved acid tolerance and application thereof | |
| CN110904075B (en) | Salt-tolerant xylosidase mutant K321D and preparation method and application thereof | |
| CN116426506B (en) | Beta-xylosidase mutant D259G with improved low-temperature activity and application thereof | |
| JP6689487B2 (en) | Endoxylanase mutant, enzyme composition for decomposing biomass, and method for producing sugar solution | |
| JP6354462B2 (en) | Thermostable xylanase belonging to GH family 10 | |
| KR101919105B1 (en) | A Novel alpha-neoagarobiose hydrolase from Gayadomonas joobiniege G7 and use thereof | |
| CN110904082B (en) | Salt-tolerant xylosidase mutant T326DH328D and preparation and application thereof | |
| CN110846301A (en) | Recombinant chitin deacetylase and preparation method and application thereof | |
| CN112342205B (en) | Salt-tolerant xylosidase mutant T329E and preparation method and application thereof | |
| CN110846298B (en) | Sodium sulfate-resistant xylosidase mutant T326D and preparation and application thereof | |
| CN110904077B (en) | Low-temperature improved xylosidase mutant MutLK10 and preparation and application thereof | |
| KR101994252B1 (en) | Recombinant bacillus subtilis strain producing beta agarase and uses thereof | |
| CN111269903B (en) | Xylanase, gene and application thereof | |
| CN110904078B (en) | Sodium sulfate and ammonium sulfate resistant xylosidase mutant V322R and application thereof | |
| JP6319907B2 (en) | Thermostable β-xylosidase | |
| CN110862976B (en) | Xylosidase mutant K321DH328D with improved salt tolerance and application thereof | |
| CN110862977B (en) | Sodium chloride and potassium chloride resistant xylosidase mutant H328D and application thereof | |
| CN111004789B (en) | Ammonium sulfate-resistant xylosidase mutant V322DH328DT329E | |
| CN110904076B (en) | Potassium chloride-resistant xylosidase mutant K317D and application thereof | |
| KR102017247B1 (en) | Use of neoagarobiose hydrolase-2 from Gayadomonas joobiniege G7 | |
| JP6315807B2 (en) | Super thermostable endoglucanase belonging to GH family 12 | |
| CN116410960B (en) | Beta-xylosidase mutant D41G with cold and pH adaptability improved halophilic suitability and application thereof | |
| KR101359781B1 (en) | Novel β-1,3-1,4-glucanase and Method for Producing Thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |