CN110904076B - Potassium chloride-resistant xylosidase mutant K317D and application thereof - Google Patents

Potassium chloride-resistant xylosidase mutant K317D and application thereof Download PDF

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CN110904076B
CN110904076B CN201911268830.1A CN201911268830A CN110904076B CN 110904076 B CN110904076 B CN 110904076B CN 201911268830 A CN201911268830 A CN 201911268830A CN 110904076 B CN110904076 B CN 110904076B
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glu
leu
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ser
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CN110904076A (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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    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)

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Abstract

The invention relates to the technical field of genetic engineering and protein modification, and discloses a potassium chloride-resistant xylosidase mutant K317D and application thereof, wherein the amino acid sequence of the mutant K317D is shown as SEQ ID NO. 1. The mutant has good stability in high-concentration KCl, and the activity is kept above 68% after the mutant is treated by KCl with the concentration of 3.0-30.0% (w/v) for 60 min. The potassium chloride-resistant xylosidase mutant K317D can be applied to the industries of agriculture, sewage treatment and the like.

Description

Potassium chloride-resistant xylosidase mutant K317D and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, relates to a protein modification technology, and particularly relates to a potassium chloride-resistant xylosidase mutant K317D and application thereof.
Background
Xylan is widely present in plant cell walls, and is a major component constituting plant hemicellulose, accounting for about 20% of the dry weight in higher plants and agricultural wastes. 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 xylose can be used as a carbon source for microorganisms and other organisms, or used as a raw material for producing ethanol, lactic acid, xylitol and the like.
Salt is widely found in nature and in various production practices including agriculture, sewage, washing, tanning, food, paper, and the like. For example, potassium chloride is a fertilizer that is relatively widely used in agricultural planting. However, salts can have a large impact 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. The salt-tolerant xylosidase cannot be applied with a chemical fertilizer at the same time, and is not beneficial to degrading xylooligosaccharide in agricultural waste, so that the cyclic utilization of xylan is reduced, and the soil fertility is further reduced. Therefore, in order to make xylosidase have better applicability in the biotechnology field of high salt environment, it is necessary to improve stability of xylosidase in salt.
Disclosure of Invention
In view of the technical problems, the invention aims to provide potassium chloride-resistant xylosidase mutants K317D and K317D which can be applied to the industries of agriculture, sewage treatment and the like.
In order to achieve the technical purpose, the invention is specifically realized by the following technical scheme:
according to the invention, a potassium chloride-resistant xylosidase mutant K317D is designed by a protein modification technology, wherein the amino acid sequence of the mutant K317D is shown as SEQ ID NO.1, and compared with a xylosidase sequence AQM74402(SEQ ID NO.3) recorded by GenBank, the 317 th amino acid of K317D is aspartic acid, and the 317 th amino acid of AQM74402 is lysine.
The mutant K317D has different stability in different salts: after K317D is treated by 3.0-30.0% (w/v) NaCl for 60min, 29-54% of activity remains; after the mutant is treated by 3.0-30.0% (w/v) KCl for 60min, the activity is remained by 68-106%; the mutant is subjected to Na treatment by 5.0-25.0% (w/v)2SO4After 60min of treatment, 34-78% of activity remains.
The invention provides a coding gene K317d of the potassium chloride resistant xylosidase mutant K317D, and the nucleotide sequence is shown in SEQ ID NO. 2.
Another objective of the invention is to provide a recombinant vector containing a gene encoding xylosidase mutant K317D.
Another object of the present invention is to provide a recombinant bacterium comprising a gene encoding xylosidase mutant K317D.
In addition, the application of the xylosidase mutant K317D in agriculture and sewage treatment is also within the protection scope of the invention.
The preparation method of the potassium chloride resistant xylosidase mutant K317D specifically comprises the following steps:
1) synthesizing a gene K317d (SEQ ID NO.2) of the mutant K317D;
2) connecting the sequence synthesized in the step (1) with an expression vector pEasy-E1 to obtain an expression vector containing k317 d;
3) transforming the ligation product into escherichia coli BL21(DE3) to obtain a recombinant strain expressing the mutant K317D;
4) culturing the recombinant strain, and inducing expression of a xylosidase mutant K317D;
5) the expressed xylosidase mutant K317D was recovered and purified.
The invention has the beneficial effects that:
the mutant enzyme K317D has enhanced stability in KCl at high concentration compared to the wild enzyme HJ14GH 43. After being treated by KCl with 10.0-30.0% (w/v) for 60min, the activity of the wild enzyme HJ14GH43 is reduced from 56% to 28%, and the activity of the mutant enzyme K317D is maintained at about 70%. The potassium chloride-resistant xylosidase mutant K317D can be applied to the industries of agriculture, sewage treatment and the like.
Drawings
Figure 1 is an SDS-PAGE analysis of the wild enzyme HJ14GH43 and the mutant enzyme K317D, wherein M: a protein Marker; w: hJ14GH 43;
FIG. 2 is the stability of the purified wild enzyme HJ14GH43 and the mutant enzyme K317D in NaCl;
FIG. 3 is the stability of the purified wild enzyme HJ14GH43 and the mutant enzyme K317D in KCl;
FIG. 4 shows the purified wild enzyme HJ14GH43 and mutant enzyme K317D in Na2SO4Stability in (1).
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
Experimental materials and reagents in the following examples of the invention:
1. bacterial strain and carrier: escherichia coli BL21(DE3) and expression vector pEasy-E1 were purchased from Beijing Quanyujin Biotechnology, Inc.
2. Enzymes and other biochemical reagents: pNP (p-nitrophenyl) and pNPX (p-nitrophenyl-. beta. -d-xylopyranoside) were purchased from Sigma, while other reagents were made in China (all available from general Biochemical Co.).
3. Culture medium
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.
Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.
EXAMPLE 1 construction and transformation of expression vectors
1) 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 K317d (SEQ ID NO.2) of a mutant enzyme K317D;
2) respectively connecting the sequences synthesized in the step (1) with expression vectors pEasy-E1 to obtain expression vectors containing hJ14GH43 and k317 d;
3) the ligation products were transformed into E.coli BL21(DE3), respectively, to obtain recombinant strains expressing the wild enzyme HJ14GH43 and the mutant enzyme K317D, respectively.
Example 2 preparation of the wild enzyme HJ14GH43 and the mutant enzyme K317D
The recombinant strains containing hJ14GH43 and k317d 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.
The activated bacterial suspension was then 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), adding IPTG with the final concentration of 0.1mM for induction, and continuing shaking culture 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. The crude enzyme solution concentrated in the above cells was centrifuged at 12,000rpm for 1After 0min, sucking the supernatant and respectively carrying out affinity and elution on the target protein by using Nickel-NTAAgarose and 0-500 mM imidazole.
SDS-PAGE results (FIG. 1) show that the wild enzyme HJ14GH43 and the mutant enzyme K317D 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 K317D
The activity of the purified wild enzyme HJ14GH43 and mutant enzyme K317D was determined by the pNP method: 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 K317D 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 K317D were determined by reacting for 10min with pNPX as a substrate.
The results show that: the wild enzyme HJ14GH43 and the mutant enzyme K317D are not stable in NaCl, and after the wild enzyme HJ14GH43 and the mutant enzyme K317D are treated by 3.0-30.0% (w/v) NaCl for 60min, 20-44% of the activity of the wild enzyme HJ14GH43 and 29-54% of the activity of the mutant enzyme K317D are remained (figure 2).
2) Stability of purified wild enzyme HJ14GH43 and mutant enzyme K317D 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 K317D were determined by reacting for 10min with pNPX as a substrate.
The results show that: the stability of the wild enzyme HJ14GH43 and the mutant enzyme K317D in KCl is different, after the wild enzyme HJ14GH43 and the mutant enzyme K317H D are treated by KCl with the concentration of 3.0-30.0% (w/v) for 60min, the activity of the wild enzyme HJ14G H43 is reduced to 28% from 114%, and the activity of the mutant enzyme K317D is 106% at most and 68% at least (figure 3).
3) Purified wild enzyme HJ14GH43 and mutant enzyme K317D 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 enzymatic properties of the purified HJ14GH43 and the mutant enzyme K317D were determined by reacting for 10min with pNPX as a substrate.
The results show that: the wild enzyme HJ14GH43 and the mutant enzyme K317D are in Na2SO4Has similar stability in the presence of Na in an amount of 3.0-30.0% (w/v)2SO4After 60min of treatment, the enzyme activity of the wild enzyme H J14GH43 is basically reduced, 47-86% of the enzyme activity remains, and the mutant enzyme K317D is treated with 5.0-25.0% (w/v) of Na2SO4After 60min of treatment, 34-78% of activity remained (FIG. 4).
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> university of Yunnan Master
<120> potassium chloride-resistant xylosidase mutant K317D and application thereof
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<212>DNA
<213> mutant (k317d)
<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
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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 aagcgccaga tatcgaagaa 960
aaggtttttg caccaaccta tcatacagtc gatgaattta aagaatcaac tctaaataga 1020
cactttcaaa cattaagaat tccgtttacc gatcagattg gttcgttaac ggagaaacct 1080
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cttcctgctg attttcacta ttttcgctat gaggaaacag acgaataa 1608
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<210>4
<211>1608
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<213> wild enzyme gene (hJ14GH43)
<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 (6)

1. The potassium chloride-resistant xylosidase mutant K317D is characterized in that the mutant K317D is obtained by mutating 317 th lysine of a xylosidase sequence AQM74402 to aspartic acid, wherein the sequence of the xylosidase sequence AQM74402 is shown as SEQ ID NO. 3.
2. The gene K317d encoding the mutant K317D of claim 1, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2.
3. A recombinant vector comprising the coding gene of claim 2.
4. A recombinant bacterium comprising the coding gene according to claim 2.
5. The method for preparing the xylosidase mutant K317D according to claim 1, comprising the following steps:
1) the gene K317d of the synthetic mutant K317D;
2) connecting the sequence synthesized in the step 1) with an expression vector pEasy-E1 to obtain an expression vector containing k317 d;
3) transforming the expression vector containing K317d obtained in the step 2) into escherichia coli BL21(D E3) to obtain a recombinant strain expressing K317D;
4) culturing the recombinant strain, and inducing expression of a xylosidase mutant K317D;
5) the expressed xylosidase mutant K317D was recovered and purified.
6. The mutant K317D of claim 1 for use in agriculture and sewage treatment.
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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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