CN105420218B - A kind of xyloside of low temperature/arabinofuranosidase glucosides bifunctional enzyme AX543 and its gene and application - Google Patents

A kind of xyloside of low temperature/arabinofuranosidase glucosides bifunctional enzyme AX543 and its gene and application Download PDF

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CN105420218B
CN105420218B CN201510973509.9A CN201510973509A CN105420218B CN 105420218 B CN105420218 B CN 105420218B CN 201510973509 A CN201510973509 A CN 201510973509A CN 105420218 B CN105420218 B CN 105420218B
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low temperature
xyloside
enzyme
bifunctional enzyme
arabinofuranosidase glucosides
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CN105420218A (en
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李中媛
张同存
张明慧
马文建
罗学刚
宋亚囝
王楠
何红鹏
周浩
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Tianjin University of Science and Technology
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
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    • 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)
    • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)

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Abstract

The present invention relates to genetic engineering fields, in particular it relates to a kind of low temperature xylosidase/arabinofuranosidase bifunctional enzyme AX543 and its gene.Its amino acid sequence has the property that optimal pH 6.0 as shown in SEQ ID NO.1, all has 70% or more relative activity within the scope of pH 5.0-8.0；It 25 DEG C of optimum temperature, maintains 5%, 20% and 80% or more relative activity respectively at 0 DEG C, 4 DEG C and 15 DEG C, there is good thermal stability, 50% or more enzymatic activity can be kept by handling 1h at 40 DEG C.AX543 has xylosidase and nofuranosidase activity simultaneously, and has the ability for hydrolyzing a variety of natural substrates.Bifunctional enzyme of the invention is a kind of new low temperature xyloside/arabinofuranosidase, and it is industrial to can be applied to the energy, feed and food etc., reduces energy consumption.

Description

A kind of xyloside of low temperature/arabinofuranosidase glucosides bifunctional enzyme AX543 and its gene And application
Technical field
The present invention relates to genetic engineering fields, in particular it relates to a kind of low temperature xyloside/arabinofuranosidase glucosides Bifunctional enzyme AX543 and its gene and application.
Background technique
Hemicellulose is the important composition ingredient in lignocellulosic, its structure and composition is more complicated, and main chain is It is polymerize by way of linear chain or branched chain by pentose and is formed, and its side chain has a variety of substituent groups, such as galactolipin, Arab Sugar etc..Xylan is the main ingredient for constituting hemicellulose and the most abundant hemicellulose resource.Although hemicellulose is very It is easy by acid to resolve into monomer, but acid hydrolyzation is easy to pollute the environment, uses bio-enzyme degradation hemicellulose can be with It effectively avoids pollution, realizes the energy " cleaning " this target.The a variety of enzymes of degradable needs of xylan, first by zytase It is randomly cut to the xylo-oligosaccharide molecule of different chain length, xylo-oligosaccharide molecule is and then acted on by xylobiase Non reducing end, it is degradable at xylose, and the effects of arabinofuranosidase, galactosidase and feruloyl esterase in The glycosidic bond of side chain releases more monosaccharide and oligosaccharides.Xylosidase and arabinofuranosidase can be applied to traditional The industry such as industrial technology, energy resources field, food industry, feed, papermaking.
In the industrial production continuously run, the application of cold-adapted enzyme can largely reduce the contaminated wind of strain Danger, production process carry out at depressed temperatures, or at room temperature, without heating and cooling, can reduce the cost savings energy；Production process is just In monitoring；In the food industry, inactivation is easy under medium temperature due to cold-adapted enzyme, is reaching optimum response effect using cold-adapted enzyme Afterwards, it only need to be kept for the short period enzyme can be made to inactivate in moderate temperature, the flavor of food will not be destroyed because of temperature height in this way.But Be presently found most xylosidase and arabinofuranosidase optimum temperature mainly between 40-70 DEG C, because This, obtains the low temperature xylosidase and arabinofuranosidase of good properties, and expresses it, purifying, property, structure feature and The research of application has great significance.
25 DEG C of xyloside/arabinofuranosidase optimum temperature of the invention, keeps respectively at 0 DEG C, 4 DEG C and 15 DEG C 5%, 20% and 80% or more relative activity, and there is good thermal stability, it can keep in 40 DEG C of processing 1h 50% or more enzymatic activity is a kind of new low temperature xyloside/arabinofuranosidase with application potential.
Summary of the invention
The object of the present invention is to provide a kind of low temperature xylosidase/arabinofuranosidase of energy efficient application is difunctional Enzyme.
Another object of the present invention is to provide the above-mentioned low temperature xylosidase/arabinofuranosidase bifunctional enzyme base of coding Cause.
It is a further object of the present invention to provide the recombinant vectors comprising said gene.
It is a further object of the present invention to provide the recombinant bacterial strains comprising said gene.
The new low temperature xyloside of the present invention isolated one kind from ring-band shape Acremonium bacterium/arabinofuranosidase glucosides is double Functional enzyme AX543.
The present invention provides a kind of low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543, and amino acid sequence is such as Shown in SEQ ID NO.1.
SEQ ID NO.1:
MPPLITSIYTADPSAHVFNDKIYIYPSHDRETDIAFNDNGDQYDMADYHVFSTSDFKEVTDHGVVLKTE DVPWASKQLWAPDAAHKNGKYYLYFPARDKEGIFRIGVAVGDKPEGPFTADPEPIKGSYSIDPASFVDDDGQAYLYF GGLWGGQLQCYQKGDDTYDPEWQGPKEVSGEGVAAQGPRAAKLTDDMHQFESPAQELLILDPETKEPILGDDHARRF FEAAWMHKHNGKYYFSYSTGDTHFLCYAVGDSPMGPFTYGGKILEPVLGWTTHHSIVEYKGKTYLFFHDCELSKGVD HLRSVKAKEIFYDDQGRIITTKAD
Wherein, which encodes 324 amino acid and a terminator codon, does not include signal peptide, therefore, mature The theoretical molecular weight of bifunctional enzyme AX543 is 36.52kDa.
AX543 of the invention is a cold-adapted enzyme, and optimum temperature is 25 DEG C, while having good thermal stability, room temperature Under, activity is all had in faintly acid and neutral range.The bifunctional enzyme AX543 that the present invention clones, optimum pH It is 6.0, using pNPX as substrate, measures the enzyme activity that its xylosidase activity maintains 50% or more in the range of PH5.0~7.0 Property；Using pNPAf as substrate, the work that its nofuranosidase activity maintains 50% or more in the range of PH5~9 is measured Property；The enzyme activity that 1h latter two enzymatic activity all has 50% or more is handled at 40 DEG C.
The present invention provides encode above-mentioned low temperature bifunctional enzyme AX543.Specifically, the genome sequence of the gene such as SEQ Shown in ID NO.2.
SEQ ID NO.2:
ATGCCGCCCCTCATTACCTCCATCTACACAGCTGATCCCTCAGCCCACGTCTTCAATGACAAGATCTAC ATCTACCCGTCTCACGACCGCGAGACGGACATTGCCTTCAACGACAATGGCGACCAGTATGACATGGCCGACTACCA TGTCTTCTCCACGTCGGACTTCAAGGAGGTGACCGACCACGGCGTCGTGCTCAAGACAGAGGACGTGCCGTGGGCGA GCAAGCAGCTCTGGGCCCCCGACGCGGCGCACAAGAACGGAAAATACTACCTCTACTTCCCGGCACGAGACAAGGAA GGCATCTTTCGGATTGGCGTGGCCGTGGGTGACAAGCCCGAGGGCCCCTTCACCGCCGACCCAGAGCCCATCAAGGG CAGCTACTCCATCGACCCGGCCAGTTTCGTCGACGATGACGGCCAGGCCTACCTCTACTTTGGCGGTCTCTGGGGTG GCCAGCTCCAATGCTACCAAAAGGGCGACGACACGTACGACCCGGAGTGGCAAGGACCCAAGGAAGTCTCTGGCGAG GGCGTCGCAGCGCAGGGCCCTCGCGCCGCCAAGTTGACAGACGACATGCACCAATTCGAGTCGCCAGCCCAAGAGCT CCTCATTCTGGACCCAGAGACCAAGGAACCCATTCTCGGCGACGACCACGCGCGCCGCTTCTTTGAAGCCGCGTGGA TGCACAAGCACAATGGCAAGTACTACTTCTCCTACTCGACGGGCGACACCCACTTCCTCTGCTACGCCGTGGGCGAC TCACCCATGGGTCCGTTCACGTATGGCGGCAAGATCCTGGAGCCCGTGCTGGGCTGGACGACGCACCACTCGATTGT CGAGTACAAGGGCAAGACATATCTCTTCTTCCACGACTGTGAGCTGAGCAAGGGTGTGGACCACCTCAGGAGCGTCA AGGCCAAGGAGATCTTTTACGACGATCAGGGTAGGATCATCACGACAAAGGCAGAT
The present invention has cloned bifunctional enzyme AX543 by the separation of the method for PCR, DNA complete sequence analysis the result shows that, double function It can enzyme AX543 structural gene AX543 overall length 972bp.
Albumen theoretical molecular weight is 36.52kDa, the amino acid sequence that will bifunctional enzyme Gene A X543 sequence and derive Carry out BLAST comparison in GenBank, the gene with from Acremonium chrysogenum ATCC11550 Xylosidase/arabinosidase-like protein Amino acid sequence identity is 86%.Illustrate that AX543 is a kind of new Bifunctional enzyme.
The present invention also provides the recombinant vectors comprising above-mentioned bifunctional enzyme Gene A X543, are named as pPIC-AX543.It will Bifunctional enzyme gene of the invention is inserted between suitable restriction enzyme cleavage sites of the expression vector, and grasp its nucleotide sequence can That makees is linked to the expression control sequence.As the most preferred embodiment of the invention, preferably by wood of the invention Glycosidase genes are inserted between EcoR I and Not the I restriction enzyme site on plasmid pPIC9, make the nucleotide sequence position In AOX1 promoter downstream and regulated and controled by it, obtain expression of recombinant yeast plasmid pPIC9-AX543.
The present invention also provides the recombinant bacterial strain comprising above-mentioned low temperature bifunctional enzyme AX543, the preferably described bacterial strain is large intestine Bacillus, saccharomycete, preferably recombinant bacterial strain GS115/AX543.
The present invention also provides a kind of methods for preparing low temperature bifunctional enzyme AX543, comprising the following steps:
1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；
2) recombinant bacterial strain is cultivated, recombination double functions expression of enzymes is induced；
3) it recycles and purifies expressed bifunctional enzyme AX543.
Wherein, the preferably described host cell be Pichia pastoris, beer yeast cells or many types of inferior yeast cells, preferably Expression of recombinant yeast plasmid is converted into Pichia pastoris (Pichia pastoris) GS115, obtains recombinant bacterial strain GS115/ AX543。
Detailed description of the invention
The ni-sepharose purification of Fig. 1 recombination double functions enzyme.
The optimal pH of Fig. 2 recombination double functions enzyme.
The pH stability of Fig. 3 recombination double functions enzyme.
The optimum temperature of Fig. 4 recombination double functions enzyme.
The thermal stability of Fig. 5 recombination double functions enzyme.
The metal ion stability of Fig. 6 recombination double functions enzyme.
Specific embodiment
Test material and reagent
1, bacterial strain and carrier: the present invention is isolated one from from the ring-band shape Acremonium bacterium 54-7 of pedotheque The new low temperature bifunctional enzyme AX543 of kind.Yeast expression vector pPIC9 and bacterial strain GS115 is purchased from Invitrogen company.
2, enzyme and other biochemical reagents: restriction endonuclease is purchased from TaKaRa company, and ligase is purchased from Invitrogen company.Purchase It is other all (to be commercially available from common biochemical Reagent Company) for domestic reagent from Sigma company.
3, culture medium:
(1) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
(2) BMGY culture medium: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% is sweet Oily (V/V).
(3) BMMY culture medium: glycerol is replaced divided by 0.5% methanol, remaining composition is identical as BMGY.
(4) NB culture medium: 0.3% yeast powder, 0.5% peptone, 0.6 glucose, 1%Nacl.
Illustrate: not making the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning: A Laboratory Guide " specific method listed in book of (third edition) J. Pehanorm Brooker one carries out, or according to kit and product description It carries out.
The clone of 1 ring-band shape Acremonium bacterium xylosidase encoding gene ax543 of embodiment
Extract the ring-band shape Acremonium bacterium 54-7 genomic DNA for deriving from pedotheque:
Letter has been synthesized according to conserved region (SKQLWAPD and CWTTHHSIVE) sequence design of the 43rd xylosidase gene And primer XylF, XylR.Touch-down PCR amplification is carried out by template of ring-band shape Acremonium bacterium 54-7 total DNA.PCR is anti- Answer parameter are as follows: 94 DEG C of denaturation 5min；Then 94 DEG C of denaturation 30sec, 65-55 DEG C of annealing 45sec (0.5 degree of each cycle down), 72 DEG C extend 40sec 10 circulations, 94 DEG C of denaturation 30s, 55 DEG C of annealing 45sec, 72 DEG C of extension 1min, 30 recycle after 72 DEG C protect Warm 10min.An about 622bp segment is obtained, will be connected and be sequenced with PMD-19T carrier after segment recycling.It is obtained according to sequencing Nucleotide sequence, design each three TAIL-PCR specific primers of upstream and downstream: design direction be needs expand it is unknown Region direction, for the Position Design of sp2 in the inside of sp1, sp3 is located at the inside of sp2.The distance between every two primer is without tight Lattice regulation, the general 22~30nt of primer length, annealing temperature is at 60~65 DEG C.And they are respectively designated as usp1, usp2, Usp3 (upstream specific primer), dsp1, dsp2, dsp3 (downstream specific primer) are shown in Table 1.
1. xylosidase Xyl543-1TAIL-PCR specific primer of table
The flanking sequence of known sequence is obtained by TAIL-PCR, amplification obtains product recycling Hou Songjinwei intelligence company Sequencing.AX543 xylosidase gene overall length 972bp after splicing encodes 324 amino acid and a terminator codon.The gene The theoretical molecular weight of encoded maturation protein is 36.52kDa.
The preparation of 2 recombination double functions enzyme Xyl/Abf543-1 of embodiment
Expression vector pPIC9 is subjected to double digestion (EcoR I+Not I), while the gene ax543 that xylosidase will be encoded Double digestion (EcoR I+Not I), the genetic fragment for cutting out encoding mature xylosidase is connect with expression vector pPIC9, is contained There is the recombinant plasmid pPIC-ax543 conversion Pichia pastoris GS115 of xylosidase gene ax543, obtains recombinant pichia yeast strain GS115/ax543。
The GS115 bacterial strain containing recombinant plasmid is taken, is inoculated in 100mL BMGY culture solution, 30 DEG C of 250rpm shaken cultivations After 48h, thalline were collected by centrifugation.Then it is resuspended in 50mL BMMY culture medium, 30 DEG C of 250rpm shaken cultivations.After inducing 48h, from The heart collects supernatant.Measure the vigor of xylosidase.After ni-sepharose purification, SDS-PAGE the result shows that, recombination double functions enzyme exists It is expressed in Pichia pastoris.As shown, swimming lane 1 is after purification as a result, swimming lane 2 is unpurified result.Measure it Xylosidase is than living for 15.076U/mg；Arabinofuranosidase is than living for 2.083U/mg.
The activity analysis of 3 bifunctional enzyme of embodiment
The specific method is as follows: the pNPX/pNPAf substrate of 250 μ L 2mM and 150 μ L citrate-phosphate disodium hydrogen buffers It mixes, the suitably diluted enzyme solution of 100 μ L is added, in 25 DEG C of reaction 10min, 1.5mL, the Na of 1M is added2CO3Reaction is terminated, is used Spectrophotometric determination OD405Value, to calculate the amount of product p-nitrophenol.1 xylosidase activity unit (U) is defined as Under the conditions of given respective reaction, p-nitrophenyl β-D- xyloside (pNPX) substrate is decomposed per minute and generates 1 μm of ol to nitro Enzyme amount required for phenol (pNP).1 α-l-arabfuranglycosidase active unit (U) is defined as in given reaction condition Under, enzyme amount needed for substrate pNPAf generates 1 μm of ol p-nitrophenol is decomposed per minute.
The property of 4 bifunctional enzyme AX543 of embodiment measures
1, the measuring method of the optimal pH of recombination double functions enzyme AX543 and pH stability is as follows:
The recombination xylosidase that embodiment 2 purifies is subjected to enzymatic reaction at different pH to measure its optimal pH.With PNPX/pNPAf is substrate, final concentration of 2mM, takes 250 μ L substrates and the 150 corresponding buffers of μ L are added.Buffer solution delays Rush gradient difference: 100mMcitrate-Na2HPO4(pH 3.0-8.0), 100mM glycine-NaOH (pH 9.0-12.0).Substrate 5min is first pre-chilled under 25 DEG C of reaction condition with the mixed solution of buffer, the suitably diluted enzyme solution of 100 μ L is added, mixes simultaneously 1.5ml Na is added in accurate response 10min2CO3Reaction is terminated, measures OD after being cooled to room temperature405Value.As a result (Fig. 1) shows weight Optimal pH of group enzyme when using pNPX as substrate is 6.0, there is 30% or more relative activity between PH4.0~7.5.With Optimal pH when pNPAf is substrate is also 6.0, there is 50% or more relative activity between PH5.0~9.Recombinase in 37 DEG C in the buffer of various different PHs, 1h is handled then at the buffer of PH6.0 and measures relative surplus under conditions of 20 DEG C Enzyme activity, with the PH stability of studying enzyme.As a result (Fig. 2) shows to measure its xylosidase activity all very stable between PH5~7, Relative surplus enzyme activity is 50% or more.Nofuranosidase activity is also comparatively steady in the range of PH5.5~7.5 It is fixed.
2, the optimum temperature of bifunctional enzyme and thermal stability determination method are as follows:
The optimum temperature of bifunctional enzyme is measured as in citrate-phosphate disodium hydrogen buffer (pH6.0) buffer solution system And enzymatic reaction is carried out under different temperatures.Thermal stability determination handles different time for recombinase at different temperatures, then 25 Enzyme assay is carried out at DEG C.Enzyme reaction optimum temperature measurement result (Fig. 3) shows that two kinds of enzymatic activity optimum temperatures are 25 DEG C. The thermal stability of enzyme incubates 60min at 40 DEG C, is able to maintain experiments have shown that (Fig. 4), AX543 have good thermal stability 50% or more enzyme activity.
3, the K of recombinasemValues determination method is as follows:
It is respectively substrate with the pNPX of various concentration and pNPAf, it is slow in citrate-phosphate disodium hydrogen buffer (pH6.0) In fliud flushing system, enzymatic activity is measured at 25 DEG C, calculates separately out the K at 4 DEG C, 15 DEG C and 25 DEG CmValue and Vmax.Experiment knot It is as shown in the table for fruit.(table 2)
Km, Vmax value measurement under table 2.AX543 different temperatures
4, influence measurement of the different metal ions chemical reagent to AX543 enzyme activity is as follows:
Different metal ion and chemical reagent are added in enzymatic reaction system, studies its influence to enzymatic activity, respectively The kind final concentration of 5mmol/L of substance.Enzymatic activity is measured under the conditions of 20 DEG C, Ph6.0.The result shows that using pNPX and pNPAf the bottom of as Object, most of ions have a degree of inhibiting effect to its enzyme activity when concentration is 5mmol.Wherein Fe2+, Zn2+, Cu2 +Its vigor of energy strong inhibition, and Mn2+、Ca2+Enzyme activity is remarkably reinforced.
5, the substrate specificity of recombinase
The enzyme is active to pNPX, pNPAf outer, also has certain hydrolysis to beech xylan and birch xylan (table 3).
The analysis of 3. bifunctional enzyme AX543 substrate specificity of table

Claims (8)

1. a kind of low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543, which is characterized in that its amino acid sequence such as SEQ Shown in ID NO.1.
2. a kind of low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543 encoding gene, which is characterized in that the gene Encode low temperature xyloside described in claim 1/arabinofuranosidase glucosides bifunctional enzyme AX543.
3. low temperature xyloside according to claim 2/arabinofuranosidase glucosides bifunctional enzyme AX543 encoding gene, special Sign is that nucleotide sequence is as shown in SEQ ID NO.2.
4. being carried comprising low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543 encoding gene recombination described in claim 2 Body.
5. being carried comprising low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543 encoding gene recombination described in claim 2 Body pPIC-AX543, wherein be inserted into the low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543 encoding gene Between EcoR I and Not I restriction enzyme site on plasmid pPIC9, keep the low temperature xyloside/arabinofuranosidase glucosides double Functional enzyme AX543 encoding gene is located at the downstream of AOX1 promoter and is regulated and controled by it, obtains recombinant vector pPIC-AX543.
6. including low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543 encoding gene recombinant bacterium described in claim 2 Strain.
7. a kind of prepare the xyloside of low temperature described in claim 1/arabinofuranosidase glucosides bifunctional enzyme AX543 method, spy Sign is, the described method comprises the following steps:
1) host cell is converted with the recombinant vector of claim 4, obtains recombinant bacterial strain；
2) recombinant bacterial strain, induction recombination low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543 encoding gene table are cultivated It reaches；
3) it recycles and purifies expressed low temperature xyloside/arabinofuranosidase glucosides bifunctional enzyme AX543.
8. the xyloside of low temperature described in claim 1/arabinofuranosidase glucosides bifunctional enzyme AX543 is in the energy, feed or food work Application in industry.
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