CN107267483B - A kind of zytase XynMF10 and its gene and application - Google Patents
A kind of zytase XynMF10 and its gene and application Download PDFInfo
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- CN107267483B CN107267483B CN201710584732.3A CN201710584732A CN107267483B CN 107267483 B CN107267483 B CN 107267483B CN 201710584732 A CN201710584732 A CN 201710584732A CN 107267483 B CN107267483 B CN 107267483B
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- xynmf10
- zytase
- enzyme
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Classifications
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- 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)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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Abstract
The present invention relates to genetic engineering fields, specifically provide a kind of zytase XynMF10 and its application, the zytase XynMF10, amino acid sequence encode the gene of above-mentioned zytase as shown in SEQ ID NO.1xynMF10, nucleotide sequence is as shown in SEQ ID NO.2, and the recombinant vector comprising the gene, recombinant bacterial strain, recombinase and application.Zytase XynMF10 optimal pH 6.0,50 DEG C of optimal reactive temperature.The enzyme has good salt tolerance and stability.Zytase XynMF10 has very high Rate activity, is 4376.9U/mg to the Rate activity of beech xylan under optimum condition.Zytase of the invention has the characteristics that neutrality, salt tolerant, high than living, can be applied to the preparation of wood oligose, the field of food industry such as steamed bun production, bread baking, marine product are handled.
Description
Technical field
The present invention relates to genetic engineering fields, in particular it relates to which a kind of mangrove fungi source is neutral, resistance to
Salt, height are than zytase XynMF10 living and its gene and the application in xylooligosaccharides production and steamed bun production.
Background technique
Xylan is the main constituents of hemicellulose, is the second carbon hydrate abundant after coming cellulose
Object accounts for about the one third of recycling organic carbon on the earth.The degradation of xylan needs the synergistic effect of a variety of enzymes, including,
Including inscribe-Isosorbide-5-Nitrae-β-D- zytase (endo-1,4- β-D-xylanase), α-L- arabinofuranosidase (α-L-
Arabinofuranosidase), α-D- glucuronidase (α-D-glucuronidase), β-D- xylosidase (β-
) and acetylesterase (acetylxylan esterases) etc. D-xylosidase.Wherein main is also most importantly interior
- Isosorbide-5-Nitrae-β-D- zytase is cut, it can cut β-Isosorbide-5-Nitrae-xylose connecting key from the internal random of xylan.Based on catalytic structure
Sequence similarity, zytase is classified into the 5th, 7,8,10,11 and 43 family of glycoside hydrolase.Wood obtained is poly- now
Most of carbohydrase belongs to the 10th and the 11st Liang Ge family.
Zytase distribution is relatively broad, has presence in the biology such as bacterium, algae, protozoan, plant.Microorganism
The zytase in source due to its in the fields such as food, feed, papermaking, the energy, weaving have it is huge application and by pass
Note.Anti-oxidant action can be eliminated or reduced such as in feedstuff industry to improve the utilization rate of feed；In the food industry may be used
To increase the volume of bread and improve mouthfeel, for the Production by Enzymes of xylo-oligosaccharide and for fruit clarification of juice；In papermaking and paper
It can promote the release of lignin in slurry industry to reduce the utilization of bleaching agent；It can be with auxiliary degumming etc. in weaving.
Mangrove is the beach shoal that the torrid zone, subtropical zone seashore and land have a common boundary, and with a large amount of shrubs, arbor etc. it is woody and
The tidal beach wetland biocoene that a small amount of liana, herbaceous plant are constituted.The ecosystem is a kind of land to the special of ocean transition
The ecosystem is flooded due to periodically being soaked by tidewater, and microbial resources are abundant and special, is not only had and is adapted to extra large land two places
The analyst of the organic matters such as some dry branches and fallen leaves of the growth characteristics of environment or mangrove natural ecosystems has and produces wood
The great potential of dextranase microorganism exploitation.And due to the particularity of the environment, the xylan that is obtained from the environmental microorganism
Enzyme also very likely has a different characteristic with other environmental sources.
The present invention has separated one plant of bacterial strain aspergillus that can produce neutral salt-tolerant xylanase from Mangrove In Guangxi Autonomous Region bed mud
Fungi obtains the encoding gene of the neutrality salt-tolerant xylanase using the method for molecular biology and genetic engineering, and to this
Gene is recombinantly expressed, and recombinase has high ratio (Rate activity to beech xylan is 4376.9U/mg) living, in neutrality
Under the conditions of have very high activity.The enzyme has good salt tolerance and stability, feelings existing for the NaCl within 1 M
Enzyme activity is promoted under condition, there remains 55% or more enzyme activity in the presence of 4.5 M NaCl.In addition, the enzyme is to wood
The hydrolysate of glycan is a series of wood oligoses of the xylobiose to six sugar of wood, therefore can be used for preparing oligomeric xylose.By the wood
Dextranase, which is applied to steamed bun production, can reduce the hardness of steamed bun, improve the mouthfeel of steamed bun.Therefore, zytase XynMF10 institute
It is the neutrality that has, salt tolerant, high than feature living, it can be applied to production, steamed bun preparation, bread baking and the marine products of xylo-oligosaccharide
The field of food industry such as the processing of product.
Summary of the invention
The purpose of invention is to provide a kind of zytase XynMF10 and its gene and application.The present invention to be solved first
The technical issues of be overcome the deficiencies of the prior art and provide a kind of good properties, be suitable for applying in the food industry it is new
Zytase.The present invention obtains a kind of neutrality, salt tolerant, high than zytase XynMF10 living, has under neutral, high salt conditions
There is very high enzyme activity, and the enzyme has preferable resistance to some common metal ions and chemical reagent.The zytase
It can be by xylan hydrolysis at xylobiose to a series of wood oligoses such as wooden six sugar.In addition, the zytase is applied to steamed bun system
Make the hardness that can reduce steamed bun, the mouthfeel for improving steamed bun.These properties meet in food industry to be produced using the enzyme low cost
Oligomeric xylose, applied to steamed bun preparation and bread baking and marine product processing.
A kind of neutrality, salt tolerant, high-specific-activity xylanase XynMF10, amino acid sequence is as shown in SEQ ID NO. 1.
The present invention provides encode above-mentioned neutrality, salt tolerant, high-specific-activity xylanasexynMF10Gene, the gene nucleotide
Sequence is as shown in SEQ ID NO. 2.
The present invention also provides include above-mentioned neutrality, salt tolerant, high-specific-activity xylanasexynMF10The recombinant vector of gene,
PreferablypPICZ-α-C-xynMF10。
The present invention also provides include above-mentioned neutrality, salt tolerant, the high recombinant bacterium than zytase XynMF10 gene living
Strain, the preferably described bacterial strain are saccharomycete, filamentous fungi, bacillus and Escherichia coli.
The present invention also provides a kind of method for preparing neutrality, salt tolerant, high-specific-activity xylanase XynMF10, including it is following
Step:
1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；
2) recombinant bacterial strain, induction recombined xylanase expression are cultivated；And
3) it recycles and purifies expressed zytase XynMF10.
The present invention also provides the applications of above-mentioned neutrality, salt tolerant, high-specific-activity xylanase XynMF10.
Neutrality of the present invention, high derives from aspergillus fungi than zytase XynMF10 living at salt tolerantAspergillus
Sp. MF10, amino acid sequence is as shown in SEQ ID NO. 1:
MVSLSALLFACTTAIGVFAAPKPSEESSLIERSTPSSTGWHNGYYYSFWTDGGGDVTYTNGGGGSYSV
QWSNVGNFVGGKGWNPGSTRSISYSGNFNPSGNGYLAVYGWTQNPLIEYYIVESYGTYNPGSGGTYRGTVSSDGGT
YDIYTAVRYNAPSIEGTATFTQFWSVRQSKRTSGSVNTANHFQAWARLGMSLGTHNYQIVATEGYQSSGSASITVY。
Zytase XynMF10 of the invention contains 210 amino acid in total, and 19 amino acid are the signal peptide of prediction before N,
Theoretical molecular weight is 23.54 kDa, and theoretical isoelectric point is 6.31.
The optimal pH of zytase XynMF10 of the invention is 6.0, and is able to maintain that 60% between pH 5.0-8.0
Above remaining enzyme activity；And as pH<4 or pH>9, xylanase activity only has 20% remaining enzyme activity below.The wood is poly-
Carbohydrase between pH6.0-pH10.0 in have 70% or more remaining enzyme activity, this illustrate this enzyme have preferable pH stability.
The optimum temperature of zytase XynMF10 of the invention is 50 DEG C, when temperature is between 40-60 DEG C, also possesses 50%
Above remaining enzyme activity.Stablize at 50 DEG C, the activity of 74 % or more is still able to maintain after 60 min of processing.At 55 DEG C
Under it is unstable, half-life period is less than 10 min.
Zytase XynMF10 of the invention also has the tolerance and stability of good salt.In 0.25-4.5 M
The enzyme activity of 55% or more residue in the presence of NaCl.Handle 60 min under the concentration of 3 M and 4 M NaCl, residue 60% with
On enzyme activity.Show that the zytase has extraordinary tolerance and stability to salt.
Cr3+And Ni2+To the activity slightly facilitation of XynMF10；And Pb2+、Co2+、Mn2+、Cu2+Then have one to the enzyme
Fixed inhibiting effect (remaining enzyme activity 50-80%), Hg2+There is apparent inhibiting effect to the enzyme；Remaining metal ion K+、Na+ 、Mg2 +、Zn2+、 Li+ 、Fe3+ 、Ag+、Ca2+、Fe2+、Fe2+The action activity of the enzyme is influenced little.In the chemical reagent of 5mM,
EDTA, SDS, β-Mercaptoethanol have different degrees of inhibiting effect to the activity of the enzyme, wherein SDS and β-
Mercaptoethanol has stronger inhibiting effect to the activity of XynMF10.
XynMF10 is mainly a series of wood oligoses to the enzymolysis product of Corncob Xylan, including xylobiose, xylotriose,
Six sugar of Xylotetrose, the wooden pentasaccharides and wood.It digesting in the shorter time (within 30min), each product assay is substantially suitable, but with
The increase of enzymolysis time, xylobiose content have increased slightly, while without there is the apparent wooden monosaccharide.
The present invention also provides encode above-mentioned neutrality, salt tolerant, the high gene than zytase XynMF10 livingxynMF10,
The gene order is as shown in SEQ ID NO. 2:
ATGGTTTCGCTCTCTGCCCTCCTCTTCGCTTGCACCACTGCAATCGGTGTCTTCGCCGCCCCTAAACC
ATCTGAAGAGTCAAGCCTAATTGAGCGCTCCACGCCAAGCTCCACCGGCTGGCACAATGGCTACTATTACTCCTTC
TGGACCGACGGCGGCGGCGATGTGACCTACACCAACGGCGGCGGCGGATCGTATTCAGTGCAGTGGTCTAATGTTG
GAAACTTTGTCGGTGGAAAGGGTTGGAATCCTGGAAGTACACGGTCCATAAGCTACAGCGGAAACTTCAACCCCAG
CGGTAACGGCTACCTTGCCGTATACGGCTGGACCCAGAACCCTCTAATCGAGTACTACATTGTTGAATCATACGGC
ACCTACAACCCCGGCAGTGGGGGGACGTATCGCGGAACAGTGAGCTCTGATGGCGGGACATACGACATCTACACTG
CGGTTCGGTACAATGCGCCCTCGATTGAAGGGACGGCCACATTTACGCAGTTCTGGTCGGTGCGCCAGTCGAAGCG
TACTTCGGGGAGCGTCAATACTGCTAATCATTTCCAGGCGTGGGCGAGACTAGGCATGAGTCTGGGGACGCATAAT
TATCAGATTGTGGCCACGGAGGGGTATCAGAGTAGTGGGTCGGCTTCGATTACTGTTTACTAG。
The present invention has cloned this xylanase gene by the method separation of PCRxynMF10, DNA complete sequence analysis knot
Fruit shows zytase XynMF10 structural genexynMF10Overall length 663 bp, initiation codon ATG, terminator codon
For TAG, 50.8 % of GC content, the polypeptide (XynMF10) of 220 amino acid composition is encoded, 19 amino acid of N-terminal are pre-
The signal peptide sequence of survey.Comparison result in GenBank show it with fromAspergillus nidulans FGSC
The β of A4-Isosorbide-5-Nitrae-D- endo xylanase genes sequence consistency highest, Identity value are 83%, show that XynMF10 is one
A new zytase.
The present invention also provides include above-mentioned xylanase genexynMF10Recombinant vector, preferablypPICZ-α-C- xynMF10.By xylanase gene of the inventionxynMF10It is inserted between suitable restriction enzyme cleavage sites of the expression vector,
Make its nucleotide sequence is operable to be linked to the expression control sequence.As the most preferred embodiment of the invention,
Preferably by xylanase genexynMF10It is inserted on plasmid pPICZ- α-CEcoR IWith Xba IRestricted digestion position
Between point, recombinant expression plasmid is obtainedpPICZ-α-C-xynMF10。
The present invention also provides include above-mentioned xylanase genexynMF10Recombinant bacterial strain, the preferably described bacterial strain be ferment
Female bacterium, filamentous fungi, bacillus and Escherichia coli, preferably recombinant pichia yeast strain X33/xynMF10, will recombinantly express
Plasmid converts Pichia pastoris X33, obtains recombinant bacterial strainX33/XynMF10。
The present invention provides a kind of good properties, the new zytase that is suitable for applying in the food industry.The present invention
Provided zytase has very high activity and stability at neutrallty condition (pH5-8).The enzyme has very high than living
Property, therefore under the applicable cases of same vigor enzyme, the amount that can be used is few, it is possible to reduce the use cost of enzyme.The enzyme also has
Good salt tolerance and stability, can satisfy the application of enzyme in some hypersaline environments.In addition, hydrolysis of the enzyme to xylan
Product is a series of wood oligoses of the xylobiose to six sugar of wood, can be used for having using preparations such as agricultural wastes such as corncob, stalk
There is the xylo-oligosaccharide of prebiotic function.In addition, experiment shows that the zytase is applied to steamed bun production and can reduce the hard of steamed bun
Degree, the mouthfeel for improving steamed bun.Therefore neutrality of the invention, the high zytase than work, salt tolerant can be in oligomeric xylose preparations, steamed bun
The field of food such as production, the processing of bread baking and marine product have important application.
Detailed description of the invention
Fig. 1: the recombined xylanase XynMF10 expressed in Pichia pastoris X33 SDS-PAGE analysis.Wherein, M: low
Molecular weight protein matter Marker；1: thick enzyme of the resuspension precipitating of ammonium sulfate precipitation after ultrafiltration of dialysing；2:XynMF10-GH11 warp
Product after ni-sepharose purification；
Fig. 2: the optimal pH of recombined xylanase XynMF10.
Fig. 3: recombined xylanase XynMF10 pH stability.
Fig. 4: the optimum temperature of recombined xylanase XynMF10.
Fig. 5: the thermal stability of recombined xylanase XynMF10.
Fig. 6: recombined xylanase XynMF10 salt tolerance.
Fig. 7: the salt-stable of recombined xylanase XynMF10.
Fig. 8: the product analysis of recombined xylanase XynMF10 degrading maize core xylan.Wherein, M: standard items include wood
The wooden pentasaccharides (X5) of monosaccharide (X1), xylobiose (X2), xylotriose (X3), Xylotetrose (X4)；1: hydrolyzing the product of 10min；2: hydrolysis
The product of 30min；3: hydrolyzing the product of 1h；4: hydrolyzing the product of 4h；5: hydrolyzing the product of 10h.
Fig. 9: recombined xylanase XynMF10 makes for steamed bun.
Specific embodiment
Test material and reagent
1, bacterial strain and carrier: I-T1 of Escherichia coli (Escherichia coli) Trans is purchased from Beijing TransGen public affairs
Department, carrier pPICZ- α-C and pMD19-T carrier are purchased from Invitrogen company, the U.S. and TaKaRa company, Japan respectively.
2, enzyme and other biochemical reagents: genome extraction kit is purchased from Beijing Tiangen company, and purifying and plasmid mention
Kit is taken to be purchased from OMEGA company, the U.S..Beech xylan, Yeast Nitrogen Base without Amino Acids
(YNB medium), biotin are purchased from Sigma Co., USA；Cut fastly (Fast digest) restriction enzyme (EcoR I、Xba I、Pme I), T4 DNA Ligase, bleomycin (Zeocin) be purchased from Thermo Scientific company, the U.S.；Sorbierite
It is purchased from AMRESCO company；The purchase such as LA Taq DNA polymerase, dNTP mixtures, 5000 DNA Marker of DL
In Japanese TaKaRa company.；Peptone (Tryptone), yeast extract (Yeast Extract) are the production of Britain OXOID company
Product, remaining reagent are that domestic analysis is pure.
3, culture medium:
(1) PDA fluid nutrient medium: 200 g of peeling potatoes is cut into small pieces, and appropriate distilled water is added and boils half small
When, with 8 layers of filtered through gauze, 20 g glucose are added, 1000mL are settled to, in 121 °C of 30 min of sterilizing.
(2) PDA solid medium: i.e. in PDA liquid medium be added agar powder make its final concentration of 2%, in 121 DEG C
Sterilize 30 min.
(3) LB culture medium: yeast powder 5.0, peptone 10.0, NaCl 10.0 are settled to 1000mL, adjust pH to 7.0.
(4) LB solid medium: i.e. in LB liquid medium be added agar powder make its final concentration of 2%, go out in 121 °C
30 min of bacterium.
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.
Embodiment 1: xylanase geneXynMF10Clone
The extraction of mangrove separation fungal genomic DNA: using the bacterial genomes extracts kit of Tiangeng company
(DP302) genomic DNA is extracted, concrete operations are carried out fully according to the specification of the kit.
The present invention has separated one plant of bacterial strain aspergillus that can produce neutral salt-tolerant xylanase from Mangrove In Guangxi Autonomous Region bed mud
Fungi.When we clone xylanase gene from the bacterium, to obtain xylanase gene segment, the 11st family wood is used
1) the degenerate primer X11-F and X11-R(of dextranase, which are shown in Table, carries out PCR amplification by template of genomic DNA.The PCR that will be expanded
Product carries out glue recycling, is attached with pMD18-T, converts E. coli competentEscherichia coli Top10 chooses
Positive recombinant is selected to be sequenced.Therefore the nucleotide sequence of the xylanase gene conserved region obtained according to sequencing designs upstream and downstream
Each three TAIL-PCR specific primers: design direction is the zone of ignorance direction for needing to expand, and the Position Design of sp2 is in sp1
Inside, sp3 is located at the inside of sp2.The distance between every two primer without strict regulations (for convenience of electrophoresis recognition result,
General 100 bp of spacing of sp2 and sp3), general 22~30 nt of primer length, annealing temperature is in 60 or so (tables 1).According to
Program in TAIL-PCR expands 5 ' end flanking sequences.
Primer needed for 1 zytase XynMF10 TAIL-PCR of table amplification and overall length amplification
The flanking sequence of known sequence is obtained by TAIL-PCR, amplification send handsome company to survey after obtaining product recycling
Sequence.The upstream and downstream flanking sequence of the segment is obtained by sequence assembly, complete sequence is total to long 1.1kb, and finding an overall length is 729bp
Open reading frame.But after the sequence is carried out bioinformatic analysis, predict to have among the full length sequence one section of size be
The intron sequences of 66bp.Therefore by this full length sequence, primer xynMF10-F and xynMF10-R is designed, is expanded from cDNA
Increase the xylanase gene xynMF10 for being free of intron sequences out.
Xylanase genexynMF10By 663 base compositions, 210 amino acid and a terminator codon, In are encoded
Comparison result in GenBank shows it and derives fromAspergillus nidulans FGSC A4(XP_661217) β-
The consistency highest of Isosorbide-5-Nitrae-D- endo xylanase genes sequence, highest consistency is 83%, and does not do functional study.xynMF10Coding albumen estimated molecular weight is 23.54kDa, isoelectric point 6.31.It is predicted, preceding 19 amino acid of XynMF10
For signal peptide sequence, centre is the catalyst structure domain of the 11st family's glycoside hydrolase.
The preparation of 2 recombined xylanase of embodiment.
To be introduced respectively in gene 5 ' and 3 ' endsEcoR I、With Xba IRestriction enzyme siteXynMF10-m-F andXynMF10-m-R is primer pair (see Table 1), using the cDNA of aspergillus fungi as template, carries out PCR amplification.PCR reaction ginseng
Number are as follows: 94 DEG C of 5 min of initial denaturation；94 DEG C of 30 sec of denaturation, 57 DEG C of 30 sec of annealing, 72 DEG C of 1 min of extension, 30 recycle, and 72
DEG C heat preservation 5 min.By expression vector pPICZ- α-C carry out double digestion (EcoR I、With Xba I), while xylan will be encoded
The gene of enzymexynMF10Double digestion (EcoR I、With Xba I), the genetic fragment and expression of the encoding mature zytase cut
Carrier pPICZ- α-C connection, and Escherichia coli Top10 is converted, acquisition contains xylanase genexynMF10Recombinant plasmid
pPICZ-α-C-xynMF10。
Recombinant expression plasmid pPIC-xynMF10 PmeI single endonuclease digestion is linearized, it is electroporated to Pichia pastoris Pichia
Pastoris X33 competent cell, coated plate is on the YPDS plate of the Zeocin containing 100ug/ml, and 30 DEG C are cultivated 2-3 days, screening
Recombinant pichia yeast strain.According to Pichia anomala expression handbook, the good recon of growth conditions is chosen into BMGY Liquid Culture
In base, 30 DEG C, after 200 r/min shaken cultivation, 48 h, by thallus centrifugation replacement to BMMY fluid nutrient medium, 30 DEG C, 200 r/
120 h of min shaken cultivation, and every 12h adds 0.5%(v/v) methanol progress inducing expression.
To purify the recombinant protein XynMF10 for having histidine mark, the bacterium solution of Fiber differentiation is centrifuged (12,000 first
× g, 4 DEG C of 15 min of centrifugation), supernatant is further concentrated using doughnut, and concentrate progress ammonium sulfate fractionation is sunk
It forms sediment, with McIlvaine buffer (the 0.2M Na of pH7.02HPO4/ 0.1M citric acid) thallus is suspended.Secondly,
The concentrate of suspension is packed into bag filter, is dialyzed overnight with 0.02 M Tris-HCl of pH7.6, and replaces a buffer, is removed
Remove ammonium sulfate.Finally, the enzyme solution dialysed is crossed nickel column, and use the Tris-HCl buffer of the imidazoles of 20-300mM containing final concentration
(20mM Tris-HCl, 500mM NaCl, 10% glycerol, pH7.6) carries out gradient elution.SDS-PAGE result (Fig. 1) shows weight
Group zytase is expressed in Pichia pastoris X33.After purifying, protein contains expressed zytase
Amount reaches 95% of total protein or more.
The property of 3 recombined xylanase XynMF10 of embodiment measures
1, the activity analysis of recombined xylanase
The method that the measurement of xylanase activity detects reduced sugar production quantity by dinitrosalicylic acid (DNS).To each
1% beech xylan of 0.9ml (6.0 McIlvaine buffer of pH preparation) is first added in reaction tube to keep the temperature in 50 DEG C of water-baths
The 100ul enzyme solution reaction 10min suitably diluted, and boiling water bath 5min is added, after being cooled to room temperature at OD540nm in 10min
Colorimetric estimation.Using xylose standard curve, absorbance is converted into restore glycogenetic molal quantity.Every group of reaction setting one is right
According to parallel with three.Under certain reaction condition, the enzyme amount of hydrolyzed xylan release 1umol xylose per minute is defined as 1 enzyme
Unit (U) living.
2, the measurement of the optimal pH of recombined xylanase XynMF10 and pH stability
The measurement of recombined xylanase XynMF10 optimal pH after purification is that enzyme solution is used to pH3.0-11.0 buffer respectively
The beech xylan substrate of configuration, carries out enzymatic reaction at 37 DEG C.Used buffer: pH3.0-7.0's
McIlvaine buffer (0.2M Na2HPO4/ 0.1M citric acid)；The 0.1M Tris-HCl buffer of pH 7.0-9.0, pH
9.0-11.0 0.1M Gly-NaoH buffer.For the pH stability of recombined xylanase XynMF10 after purification, be by
Enzyme solution is appropriate to dilute in the buffer of different pH at 37 DEG C after water-bath heat preservation 1h, and remaining enzyme activity is measured under optimum condition.
The result shows that: the optimal pH of XynMF10 is 6.0,60% or more (figure of residue maximum enzyme activity when between pH 5.0 to 8.0
2)；This zytase is very stable between pH 6.0-10.0, and remaining enzymatic activity exists after 60min is handled within the scope of this pH
70% or more, this illustrates that this enzyme has preferable pH stability (Fig. 3).
3, the optimum temperature and thermal stability determination of recombined xylanase XynMF10
The measurement of the optimum temperature of enzyme: in the buffer of pH 6.0, enzymatic reaction is carried out at 20-70 DEG C.The heat of enzyme
Stability Determination: the enzyme solution of same enzyme amount is placed in 45 DEG C, 50 DEG C and 55 DEG C, after handling 0-60min, in pH 6.0 and 50
Enzymatic reaction is carried out at DEG C, using untreated enzyme solution as control.Enzyme reaction optimum temperature measurement result (Fig. 4) shows it most
Thermophilic degree is 50 DEG C.When temperature is between 40-60 DEG C, also possess 50% or more remaining enzyme activity, and when temperature is higher than 60 DEG C,
Enzyme activity then sharply drops to 10% remaining enzyme activity.The thermal stability of enzyme is experiments have shown that (Fig. 5), recombinase is at 50 DEG C or less
When stability it is very good.30 min are kept the temperature at 55 DEG C, remaining enzymatic activity is 11.3%, 60 DEG C of 15 min enzyme activity almost all of processing
It loses.
4, recombined xylanase XynMF10V maxWithK mMeasurement
With the xylan substrate of different final concentrations (1,1.5,2,4,6,8 and 10 mg/mL), enzyme is measured under optimum condition
Activity calculates corresponding reaction speed, acquires K using the double counting backward techniques of Michaelis-Menten equationmValue and Vmax.The result shows that: the enzymeV max
For 9615.38 ± 0.09 μm of ol min–1mg–1,K mFor 5.38 ± 0.22 mg mL–1。
5, influence measurement of the different metal ions chemical reagent to XynMF10 enzyme activity
Influence for research different metal ions and chemical reagent to recombined xylanase XynMF10 enzyme activity, it is anti-in enzymatic
The metal ion and chemical reagent of final concentration of 5mM are separately added into answering, and using the reaction tube without any processing as control,
Remaining enzyme activity is measured under optimum condition.Using to metal ion and chemical reagent include: NaCl, KCl, CaCl2 、
CoCl2、NiSO4、CuSO4、MgSO4、FeSO4、FeCl3、MnSO4、ZnSO4、Pb(CH3COO)2、AgCl、HgCl2、LiCl、
CrCl2、EDTA、β-mercaptoethanol、SDS。
The metal ion of 5 mM is added in enzymatic reaction system, studies its influence to enzymatic activity.At 50 DEG C and pH
Under the conditions of 6.0, enzymatic activity is measured by substrate of beech xylan.The result shows that: Mn2+Have to the activity of rXynMF13-GH10 bright
Aobvious facilitation, enzymatic activity have reached 1.49 times；And Fe2+、 Pb2+、Cu2+ then have certain inhibiting effect (surplus the enzyme
Remaining enzyme activity 50-80%), Hg2+There is strong inhibiting effect to the enzyme；Remaining metal ion Na+ 、K+ 、Fe3+ 、Zn2+、 Li+ 、
Mg2+ 、Ca2+ 、Ni2+ 、Co2+(table 2) in a slight decrease to the action activity of the enzyme.
In the chemical reagent of 5 mM, EDTA has facilitation to the activity of rXynMF13-GH10, is added to the enzyme of EDTA
Activity has reached 1.12 times, and β-Mercaptoethanol and SDS then almost have the work of complete inhibition to the activity of the enzyme
With.
2 metal ion of table and chemical reagent are on the active influence of recombined xylanase XynMF10
6, activity and stable measurement of the various concentration NaCl to recombined xylanase XynMF10
Activity influence of the NaCl to recombined xylanase XynMF10: various concentration is added in enzymatic reaction system
The NaCl of (0.25-4.5M) studies its influence to enzymatic activity.Under the conditions of 50 DEG C and 6.0 pH, using beech xylan the bottom of as
Object measures enzymatic activity.Influence of the NaCl to the stability of recombined xylanase XynMF10: enzyme solution is respectively placed in containing 3M and 4
In the buffer of the pH 9.0 of M, 1 h is handled at 37 DEG C, then enzymatic reaction is carried out at pH 6.0 and 50 DEG C, not locate
The enzyme solution of reason is as control.
The result shows that: containing when the NaCl of concentration, which is significantly swashed within 1mol/L in enzymatic reaction system
Effect living, and in the NaCl containing 0.75mol/L, the maximum activity of the enzyme is detected, is 122.1%；When NaCl concentration is greater than
After 1mol/L, remaining enzyme activity slowly declines, but as the NaCl in reaction system containing 4.5mol/L, enzyme activity is also protected
There is 50% or more initial (Fig. 6), shows that the enzyme has good salt tolerance.37 DEG C of guarantors in the NaCl solution of 3 mol/L
Temperature, within preceding 20min enzyme activity increase, have obvious activation, later with the increase enzyme activity of soaking time gradually under
Drop；Simultaneously in the NaCl solution of 4 mol/L after 37 DEG C of heat preservation 1h, there are 50% or more remaining enzyme activities (Fig. 7), show this
Enzyme has extraordinary salt-stable.
4 recombined xylanase XynMF10 of embodiment prepares oligomeric xylose for Corncob Xylan hydrolysis
Sample handling processes are as follows: using 1% Corncob Xylan of the most stable pH configuration of excessive pure enzyme solution and recombinase
Substrate (ratio 2:1) mixes, and is placed on water-bath in the most stable of temperature of recombinase, respectively at 10 min, 30 min,
It is sampled under 1h, 4h, 10h, boiling water bath 5min inactivates enzyme solution, and 13000 r/min are centrifuged 5 min after being cooled to room temperature, and takes same volume
Long-pending sample point sample is on silica gel plate.
Silica gel plate preparation it is as follows: using distilled water configure 0.5% CMC solution, 100 mL, suitably heat and after completely dissolution,
Bubble is removed using ultrasonic wave (about 15 min)；30g thin layer silica white is added in the solution, it sufficiently will mixing using mortar
Object grinding is uniform.
The formula of solvent is n-butanol: glacial acetic acid: water (5:3:2, v/v/v)；Develop the color agent prescription: 2 mL of aniline, hexichol
0 mL of amine 2 g, φ=85% phosphatase 11, is dissolved in 200 mL acetone.First solvent is fitted into chromatography cylinder, covers chromatography cylinder cap
Son makes the gas of organic solvent in solvent full of entire cylinder body, by the thin of first-class sample and standard items (the wooden monosaccharide-wood pentasaccharides)
Laminate is put into cylinder, and to be deployed dose of forward position reaches and take out at whole plate about 2/3rds, after drying in oven (80 DEG C), takes out cooling
Colour developing is observed after about 20 min of drying in oven using spray bottle to color developing agent is uniformly sprayed after lamellae to room temperature.
XynMF10 carries out enzymolysis product analysis result as shown in figure 8, its hydrolysate is mainly one to Corncob Xylan
Serial wood oligose, including xylobiose, xylotriose, six sugar of Xylotetrose, the wooden pentasaccharides and wood.Digest in shorter time (30min with
It is interior), each product assay is substantially suitable, but with the increase of enzymolysis time, xylobiose content is had increased slightly, while bright without occurring
The aobvious wooden monosaccharide occurs.So the enzyme, which compares, produces oligomeric xylose suitable for using agricultural wastes such as corncobs.
5 recombined xylanase XynMF10 of embodiment is for making steamed bun
In the food industry, zytase is as a kind of common additive for making different food.Wherein, In
During making steamed bun, it is properly added the zytase of a certain range amount concentration, its quality and mouthfeel can be changed.The present invention couple
Application of the recombinase XynMF10 in steamed bun manufacturing process tentatively probe into, formula (the 100g flour, dry ferment of steamed bun
0.8g, salt 0.8g, sugar 10g and water 48g).
Preparation step is specific as follows: first with warm water the xylan of Different adding amount is arranged in activated yeast when preparing dough
Enzyme (0.25U/g, 0.50U/g, 0.75U/g), it is a small amount of several times to be added, and enzyme solution is replaced as a control group with clear water, it will be whole
Material mixes, and rubbing dough covers preservative film and be placed in 1.5 h of provocation under 36 °C and certain damp condition to uniformly and circle is rubbed with the hands,
Exhaust and 20 min of secondary provocation are taken out, the good dough of provocation is finally entered into whole 20 min of pot, takes out cooling.
It weighs (W) to steamed bun, measures steamed bun volume (V) using millet displacement method, and calculate specific volume (P=V/W)；Texture instrument
Parameter is set as TPA mode: Test Spee 1mm/sec, Trigger Type Auto, Tare Mode Auto, Trigger
Force 5g, Distance 10.0 mm, 10.00 sec of Time.An equal amount of steamed bun core (20mm or so) is cut, is measured
Hardness, elasticity and chewiness.
The result shows that the steamed bun specific volume of addition recombinase XynMF10 increases, it is more soft, and the number of the stomata in steamed bun
Measure more (Fig. 9) than being not added with enzyme.The steamed bun core hardness and chewiness parameter for adding recombinase XynMF10 are decreased obviously, In
Best with the enzyme amount effect of 0.50 U/g in three kinds of additive amount concentration, hardness and chewiness have dropped 436 and 242 respectively, that is, divide
30% and 24% are not reduced, and in the addition of the enzyme amount of 0.75 U/g, there is (table 3) respectively again in hardness and chewiness.
This also has been reported that in some documents, thus it is speculated that its possible cause is that recombined xylanase excessive in dough makes it in fermentation process
Middle stickiness increases, and causes dough internal structure inflexible, is unfavorable for swollen hair.In addition, the elasticity and hardness in texture parameter are in bright
The addition of aobvious negative correlation, recombinase XynMF10 increases the elasticity of steamed bun, improves the quality of steamed bun.
Influence of 3 recombined xylanase of table to steamed bun texture parameter
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
SEQUENCE LISTING
<110>University of Fuzhou
<120>a kind of zytase XynMF10 and its gene and application
<130> 14
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 220
<212> PRT
<213>amino acid sequence
<400> 1
Met Val Ser Leu Ser Ala Leu Leu Phe Ala Cys Thr Thr Ala Ile Gly
1 5 10 15
Val Phe Ala Ala Pro Lys Pro Ser Glu Glu Ser Ser Leu Ile Glu Arg
20 25 30
Ser Thr Pro Ser Ser Thr Gly Trp His Asn Gly Tyr Tyr Tyr Ser Phe
35 40 45
Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn Gly Gly Gly Gly
50 55 60
Ser Tyr Ser Val Gln Trp Ser Asn Val Gly Asn Phe Val Gly Gly Lys
65 70 75 80
Gly Trp Asn Pro Gly Ser Thr Arg Ser Ile Ser Tyr Ser Gly Asn Phe
85 90 95
Asn Pro Ser Gly Asn Gly Tyr Leu Ala Val Tyr Gly Trp Thr Gln Asn
100 105 110
Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly Thr Tyr Asn Pro
115 120 125
Gly Ser Gly Gly Thr Tyr Arg Gly Thr Val Ser Ser Asp Gly Gly Thr
130 135 140
Tyr Asp Ile Tyr Thr Ala Val Arg Tyr Asn Ala Pro Ser Ile Glu Gly
145 150 155 160
Thr Ala Thr Phe Thr Gln Phe Trp Ser Val Arg Gln Ser Lys Arg Thr
165 170 175
Ser Gly Ser Val Asn Thr Ala Asn His Phe Gln Ala Trp Ala Arg Leu
180 185 190
Gly Met Ser Leu Gly Thr His Asn Tyr Gln Ile Val Ala Thr Glu Gly
195 200 205
Tyr Gln Ser Ser Gly Ser Ala Ser Ile Thr Val Tyr
210 215 220
<210> 2
<211> 663
<212> DNA
<213>gene order
<400> 2
atggtttcgc tctctgccct cctcttcgct tgcaccactg caatcggtgt cttcgccgcc 60
cctaaaccat ctgaagagtc aagcctaatt gagcgctcca cgccaagctc caccggctgg 120
cacaatggct actattactc cttctggacc gacggcggcg gcgatgtgac ctacaccaac 180
ggcggcggcg gatcgtattc agtgcagtgg tctaatgttg gaaactttgt cggtggaaag 240
ggttggaatc ctggaagtac acggtccata agctacagcg gaaacttcaa ccccagcggt 300
aacggctacc ttgccgtata cggctggacc cagaaccctc taatcgagta ctacattgtt 360
gaatcatacg gcacctacaa ccccggcagt ggggggacgt atcgcggaac agtgagctct 420
gatggcggga catacgacat ctacactgcg gttcggtaca atgcgccctc gattgaaggg 480
acggccacat ttacgcagtt ctggtcggtg cgccagtcga agcgtacttc ggggagcgtc 540
aatactgcta atcatttcca ggcgtgggcg agactaggca tgagtctggg gacgcataat 600
tatcagattg tggccacgga ggggtatcag agtagtgggt cggcttcgat tactgtttac 660
tag 663
<210> 3
<211> 26
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (15)..(15)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (23)..(23)
<223> n is a, c, g, or t
<400> 3
aactgctacc tgkcnItntay ggntgg 26
<210> 4
<211> 26
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (19)..(19)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (24)..(24)
<223> n is a, c, g, or t
<400> 4
ccgcacggac cagtaytgnk Iraangt 26
<210> 5
<211> 36
<212> DNA
<213>artificial sequence
<400> 5
ctaatcgagt actacattgt tgaatcatac ggcacc 36
<210> 6
<211> 25
<212> DNA
<213>artificial sequence
<400> 6
ggacgtatcg cggaacagtg agctc 25
<210> 7
<211> 29
<212> DNA
<213>artificial sequence
<400> 7
gacatctaca ctgcggttcg gtacaatgc 29
<210> 8
<211> 29
<212> DNA
<213>artificial sequence
<400> 8
gcattgtacc gaaccgcagt gtagatgtc 29
<210> 9
<211> 25
<212> DNA
<213>artificial sequence
<400> 9
gagctcactg ttccgcgata cgtcc 25
<210> 10
<211> 36
<212> DNA
<213>artificial sequence
<400> 10
ggtgccgtat gattcaacaa tgtagtactc gattag 36
<210> 11
<211> 27
<212> DNA
<213>artificial sequence
<400> 11
atggtttcgc tctctgccct cctcttc 27
<210> 12
<211> 31
<212> DNA
<213>artificial sequence
<400> 12
gtaaacagta atcgaagccg acccactact c 31
<210> 13
<211> 36
<212> DNA
<213>artificial sequence
<400> 13
cgcgaattca gcccctaaac catctgaaga gtcaag 36
<210> 14
<211> 37
<212> DNA
<213>artificial sequence
<400> 14
ccgtctagaa agtaaacagt aatcgaagcc gacccac 37
Claims (1)
1. a kind of neutrality, salt tolerant, the high application than zytase XynMF10 living in xylooligosaccharides production and steamed bun production,
The amino acid sequence of the zytase XynMF10 is as shown in SEQ ID NO. 1；Encode the wood of the zytase XynMF10
Xylanase genexynMF10Nucleotide sequence as shown in SEQ ID NO. 2.
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