CN109679935A

CN109679935A - Fast hydrolyzing xylan generates encoding gene and its application of the glycoside hydrolase of single xylose

Info

Publication number: CN109679935A
Application number: CN201910081708.7A
Authority: CN
Inventors: 楼慧强; 姜伟; 刘军权; 阿卜杜拉·巴西特; 苗挺; 宋丽娜; 温家琦; 李颖; 李季伦
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-01-28
Filing date: 2019-01-28
Publication date: 2019-04-26

Abstract

The present invention relates to genetic engineering fields, and in particular, to fast hydrolyzing xylan generates encoding gene and its application of the glycoside hydrolase of single xylose.The nucleotide sequence of the gene is as shown in SEQ ID NO:2.The nucleic acid as shown in SEQ ID NO:2 of offer of the invention can in yeast great expression, so as to largely obtain glycoside hydrolase Ttxy43, the glycoside hydrolase Ttxy43 has the activity of three kinds of xylobiase, inscribe beta-xylanase and α-l-arabfuranglycosidase enzymes simultaneously, furthermore, the glycoside hydrolase Ttxy43 enzyme of the invention birch xylan that can directly degrade generates single product xylose, and it combines and compares with commercialized inscribe beta-xylanase and xylobiase, xylose yield is higher, has more commercial application value.

Description

Fast hydrolyzing xylan generate the glycoside hydrolase of single xylose encoding gene and its Using

Technical field

The present invention relates to genetic engineering fields, and in particular, to a kind of fast hydrolyzing xylan generates the sugar of single xylose The encoding gene of glycosides hydrolase and its application.

Background technique

It is xylan that content is highest in hemicellulose, its complete hydrolysis needs a variety of enzymes to act synergistically jointly could be complete At wherein most important includes endo-xylanase (EC 3.2.1.8) and xylosidase (EC3.2.1.37).Inscribe xylan Enzyme can cut the main chain of xylan to generate the inhomogenous wood oligose of length, and wood oligose then can be by xylosidase into one The hydrolysis of step generates xylose, and xylose can be utilized by pichia stipitis or modified saccharomyces cerevisiae and produce second Alcohol.

Glycoside hydrolase Families 43 (GH43) (http://www.cazy.org) mainly contain xylobiase, α-L- Ah Primary furanoside enzyme, inscribe α-L- araban enzyme and 1,3- betagalactosidase activity are drawn, wherein having xylobiase Quantity it is more, and be often multifunctional enzyme, a variety of difference enzyme activity collaborations play a role, and have in terms of hemicellulose degradation extensive Application prospect.

Although multifunctional enzyme related with xylan hydrolysis it has been reported that still can effectively hydrolyzing xylan generate it is single The enzyme for playing xylobiase, beta-xylanase and α-l-arabfuranglycosidase while product xylose is not reported.

Summary of the invention

The present inventor has found there is the glucosides water of the amino acid sequence as shown in SEQ ID NO:2 under study for action Solution enzyme is provided simultaneously with the enzyme activity of xylobiase, beta-xylanase and α-l-arabfuranglycosidase, while can also be quick Hydrolyzed xylan generates single xylose, but the base of the glycoside hydrolase of amino acid sequence shown in existing coding SEQ ID NO:2 Because expression is lower, the glycoside hydrolase with the amino acid sequence as shown in SEQ ID NO:2 can not be largely obtained.

In order to largely obtain the glycoside hydrolase with the amino acid sequence as shown in SEQ ID NO:2, first party Face, the present invention provides the gene that a kind of coding fast hydrolyzing xylan generates the glycoside hydrolase of single xylose, the genes Nucleotide sequence as shown in SEQ ID NO:2.

Second aspect, the present invention provides a kind of recombinant vectors containing gene as described above.

The third aspect, the present invention provides a kind of recombinant bacterial strains containing recombinant vector as described above.

Fourth aspect, the present invention provides a kind of sides for preparing fast hydrolyzing xylan and generating the glycoside hydrolase of xylose Method, method includes the following steps: (1) cultivates recombinant bacterial strain as described above, induction coding fast hydrolyzing xylan generates single The expression of the gene of the glycoside hydrolase of one xylose；(2) glycoside hydrolase expressed by separating-purifying.

5th aspect, the present invention provides gene as described above, carrier as described above, recombinant bacteriums as described above Kind, the glycoside hydrolase of method as described above preparation, the fast hydrolyzing xylan as shown in SEQ ID NO:1 generate single wood The glycoside hydrolase of sugar or the glycoside hydrolase containing method as described above preparation or the fast hydrolyzing as shown in SEQ ID NO:1 The composition that xylan generates the glycoside hydrolase of single xylose is playing inscribe beta-xylanase and/or α-L- arabinofuranosidase Application in glycosidase activity.

6th aspect, the present invention provides gene as described above, carrier as described above, recombinant bacteriums as described above Kind, the glycoside hydrolase of method as described above preparation, the fast hydrolyzing xylan as shown in SEQ ID NO:1 generate single wood The glycoside hydrolase of sugar or the glycoside hydrolase containing method as described above preparation or the fast hydrolyzing as shown in SEQ ID NO:1 The composition that xylan generates the glycoside hydrolase of single xylose generates the application in single xylose in hydrolyzed xylan.

The nucleic acid as shown in SEQ ID NO:2 of offer of the invention can in yeast great expression, so as to big Amount obtains glycoside hydrolase Ttxy43, and the present inventor has found that glycoside hydrolase Ttxy43 has simultaneously by experimental verification There is the activity of three kinds of xylobiase, inscribe beta-xylanase and α-l-arabfuranglycosidase enzymes.β-xylose of Ttxy43 Glycosides enzyme enzyme activity optimal reaction pH is 5.0-7..0, and optimal reactive temperature is 45-55 DEG C；Inscribe beta-xylanase enzyme activity optimal reaction PH is 6.0-9.0, and optimal reactive temperature is 50-65 DEG C；α-l-arabfuranglycosidase enzyme activity optimal reaction pH is 5.0- 8.0, optimal reactive temperature is 45-55 DEG C.In addition, xylobiase enzyme activity is resistant to the suppression of product xylose in Ttxy43 of the invention Production is used, and when xylose concentration reaches 0.2M, xylobiase relative activity is greater than 40%, and xylose does not influence inscribe β-wood Dextranase enzyme activity.Further, the Ttxy43 enzyme of the invention birch xylan that can directly degrade generates single product xylose, and It combines and compares with commercialized inscribe beta-xylanase and xylobiase, xylose yield is higher, has more business application valence Value.

Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.

Detailed description of the invention

The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:

Fig. 1 is the SDS-PAGE of Pichia anomala expression foreign protein Ttxy43 as a result, wherein M is protein Marker, and 1 is Foreign protein Ttxy43；

Fig. 2 be influence of the pH to three kinds of enzyme activity of Ttxy43, Fig. 2A: xylobiase, Fig. 2 B: inscribe beta-xylanase, Fig. 2 C: α-l-arabfuranglycosidase；

Fig. 3 be influence of the temperature to three kinds of enzyme activity of Ttxy43, Fig. 3 A: xylobiase, Fig. 3 B: inscribe β-xylan Enzyme, Fig. 3 C: α-l-arabfuranglycosidase；

Fig. 4 is influence of the xylose concentration to the enzyme activity of xylobiase in Ttxy43 and inscribe beta-xylanase；

Fig. 5 is standard items wood oligose HPLC result；

Fig. 6 is Ttxy43 and the HPLC of commercialized enzyme hydrolysis birch xylan is as a result, Fig. 6 A: birch xylan, Fig. 6 B: It is commercialized inscribe beta-xylanase results of hydrolysis, Fig. 6 C: commercialization inscribe xylobiase results of hydrolysis, Fig. 6 D:Ttxy43 water It solves result and commercialization inscribe beta-xylanase+xylobiase mixed enzyme degradation results compares.

Specific embodiment

Detailed description of the preferred embodiments below.It should be understood that described herein specific Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.

The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more New numberical range, these numberical ranges should be considered as specific open herein.

In a first aspect, the present invention provides a kind of base for encoding fast hydrolyzing xylan and generating the glycoside hydrolase of single xylose Cause, the nucleotide sequence of the gene is as shown in SEQ ID NO:2.

SEQ ID NO:2:

ATGGCTCCATTGATCACCAACATCTTCACCGCTGACCCATCTGCTCACGTTTTCGAGGGTAAGTTGTT CATCTACCCATCTCACGACAGAGAGACCGACATCAAGTTCAACGACGACGGTGACCAATACGACATGGTTGACTAC CACGTTTTCTCTACCGAGTCTTTGGACCCAGCTGCTCCAGTTACCGACCACGGTGTTGTTTTGAGAGCTGAGGACG TTCCATGGGTTTCTAAGCAATTGTGGGCTCCAGACGCTGCTTACAAGGACGGTAGATACTACTTGTACTTCCCAGC TAGAGACAAGCAAGGTGTTTTCAGAATCGGTGTTGCTGTTGGTGACAGACCAGAGGGTCCATTCACCCCAGACCCA GAGCCAATCAGAGACTCTTACTCTATCGACCCAGCTGTTTTCGTTGACGACGACGGTAGAGCTTACATGTACTTCG GTGGTTTGTGGGGTGGTCAATTGCAATGTTACCAAAAGGGTAACGGTATCTTCGACCCAGAGTGGTTGGGTCCAAG AGAGCCATCTGGTGAGGGTGTTAGAGCTTTGGGTCCAAGAGTTGCTAGATTGGCTGACGACATGAGACAATTCGCT TCTGAGGTTAAGGAGATCTCTATCTTGGCTCCAGAGACCGGTGAGCCAATCGCTGCTGACGACCACGACAGAAGAT TCTTCGAGGCTGCTTGGATGCACAAGTACGACGGTAAGTACTACTTCTCTTACTCTACCGGTGACACCCACTACTT GGTTTACGCTGTTGGTGACTCTCCATACGGTCCATTCACCTACGCTGGTAGAATCTTGGAGCCAGTTTTGGGTTGG ACCACCCACCACTCTATCGTTGAGTTCCACGGTAGATGGTGGTTGTTCCACCACGACTGTGAGTTGTCTGGTGGTG TTGACCACTTGAGATCTGTTAAGGTTAAGGAGATCTTCTACGACAAGGACGGTAAGATCGTTACCG AGAAGCCAGA GTAA

According to the present invention, the end 5' and/or the end 3' of nucleotide sequence can also be connected with the code sequence of label shown in table 1 Column.

Table 1

Label	Residue number	Amino acid sequence
			Poly-Arg	5-6 (usually 5)	RRRRR (SEQ ID NO:3)
Poly-His	2-10 (usually 6)	HHHHHH (SEQ ID NO:4)
			FLAG	8	DYKDDDDK (SEQ ID NO:5)
Strep-tagⅡ	8	WSHPQFEK (SEQ ID NO:6)
			c-myc	10	EQKLISEEDL (SEQ ID NO:7)

Nucleotide sequence provided by the invention can usually use polymerase chain reaction (PCR) amplification, recombination method or people Work synthetic method obtains.For example, those skilled in the art according to the present invention provided by nucleotide sequence, can be easy to To template and primer, amplification is carried out using PCR and obtains related sequence.

Once obtaining related nucleotide sequence, so that it may obtain related amino acid sequence with recombination method is large batch of.It is logical Often gained nucleotide sequence is cloned into carrier, then transgene engineering bacteria, then through conventional method after proliferation The isolated related nucleotide sequence of host cell.

In addition, also related nucleotide sequence can be synthesized with well known artificial chemistry synthetic method.

Second aspect, the present invention also provides a kind of recombinant vector, the recombinant vector contains gene as described above.

According to the present invention, various carriers known in the art, such as city can be selected in " carrier " used in the recombinant vector Various plasmids, clay, bacteriophage and retrovirus for selling etc., the preferred pPICZ α A plasmid of the present invention.Construction of recombinant vector can be adopted With can vector multiple cloning site have cleavage site various endonucleases (for pPICZ α A plasmid, EcoR I can be used With Kpn I etc.) it carries out digestion and obtains linear plasmid, it connect, is weighed with the genetic fragment using the cutting of identical nucleic acid restriction endonuclease Group plasmid.Present invention preferably employs EcoR I and Kpn I double digestion pPICZ α A and genetic fragment connected to it, linked enzymes Connection, building obtain recombinant vector pPICZ α A-Ttxy43.

The third aspect, the present invention provides a kind of recombinant bacterial strain, the recombinant bacterial strain contains gene as described above or such as The upper recombinant vector.

The recombinant vector can be converted, transduceed or is transfected into host cell (bacterium by the method for this field routine Strain) in, such as Calcium Chloride Method chemical conversion, electroporation.The host cell can be prokaryotic cell or eukaryocyte, it is excellent It is selected as bacillus (such as Escherichia coli (Escherichia coli) or bacillus subtilis (Bacillus subtilis)) or yeast Bacterium (such as Pichia pastoris (Pichia pastoris) or saccharomyces cerevisiae (Saccharomyces cerevisiae)), it is highly preferred that The host cell is Pichia pastoris (Pichia pastoris).

Fourth aspect, the present invention provides a kind of glycoside hydrolases for preparing fast hydrolyzing xylan and generating single xylose Method, this method comprises: (1) cultivates recombinant bacterial strain as described above, induction coding fast hydrolyzing xylan generates single xylose Glycoside hydrolase gene expression；(2) glycoside hydrolase expressed by separating-purifying.

According to the present invention, the condition of culture is conventional condition of culture, such as using BMGY fluid nutrient medium (for example, containing Have yeast extract 1%, peptone 2%, yeast basic nitrogen source 1.34%, 6.0 phosphate buffer of 100mM pH, glycerol 1%), Culture is to overnight at 25-30 DEG C；Then again in BMMY culture medium (for example, yeast extract 1%, peptone 2%, yeast base Plinth nitrogen source 1.34%, 6.0 phosphate buffer of 100mM pH, methanol 0.5%) in continue to cultivate, every 20-28h be added 0.8- The methanol of 1.2 volume % cultivates 100-150h, and it is Ttxy43 crude enzyme liquid that centrifugation, which obtains supernatant,.The crude enzyme liquid can also be adopted It is purified with method known in this field, thus the Ttxy43 enzyme purified.

SEQ ID NO:1 (glycoside hydrolase Ttxy43):

MAPLITNIFTADPSAHVFEGKLFIYPSHDRETDIKFNDDGDQYDMVDYHVFSTESLDPAAPVTDHGVVL RAEDVPWVSKQLWAPDAAYKDGRYYLYFPARDKQGVFRIGVAVGDRPEGPFTPDPEPIRDSYSIDPAVFVDDDGRAY MYFGGLWGGQLQCYQKGNGIFDPEWLGPREPSGEGVRALGPRVARLADDMRQFASEVKEISILAPETGEPIAADDHD RRFFEAAWMHKYDGKYYFSYSTGDTHYLVYAVGDSPYGPFTYAGRILEPVLGWTTHHSIVEFHGRWWLFHHDCELSG GVDHLRSVKVKEIFYDKDGKIVTEKPE

Derived according to the invention, it is further possible to be modified glycoside hydrolase Ttxy43 shown in SEQ ID NO:1 Protein." derivative protein " of the present invention refers to not to be had with the glycoside hydrolase Ttxy43 with above-mentioned amino acid sequence Difference on amino acid sequence, namely the difference on the modified forms of sequence is not influenced.Modification (do not change primary structure usually, Do not change amino acid sequence) form include: internal or external albumen chemical derivative form such as acetylation or carboxylated.It repairs Decorations further include glycosylation, in the synthesis and processing of albumen or carry out glycosylation modified in further processing step and produce such as those Raw albumen.This modification can carry out glycosylated enzyme (glycosylase of such as mammal is gone by the way that albumen to be exposed to Glycosylase) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphorus Sour threonine) sequence.It further include being modified to improve its anti-proteolytic properties or optimize the albumen of solubility property.

As described above, it purifies for convenience, it can also be using the common label in this field to glucosides provided by the invention Hydrolase Ttxy43 is added modification, for example, can pass through the ammonia in glycoside hydrolase Ttxy43 provided by the invention Base end and/or carboxyl terminal connect (such as II and of Poly-Arg, Poly-His, FLAG, Strep-tag of label shown in the following table 1 At least one of c-myc) and obtain.The label will not influence the activity of glycoside hydrolase Ttxy43 of the invention, in reality In the application process of border, addition label can be chosen whether according to demand.

Above-mentioned glycoside hydrolase Ttxy43 can be obtained by artificial synthesized, can also first synthesize its encoding gene, then lead to Cross biological expression acquisition.

, according to the invention it is preferred to, on the basis of the total weight of the composition, the content of the glycoside hydrolase is 1- 10 weight %.

It according to the present invention, can also be containing well known to a person skilled in the art solvent (such as glycerol, carbohydrate in the composition With the protein protective agents such as protease inhibitors), agonist (such as iron ion Fe²⁺With manganese ion Mn²⁺) etc..

In the present invention, the method using the glycoside hydrolase degradation of xylan may include: by xylan sample and institute State glycoside hydrolase incubation.Relative to every gram of the xylan sample in terms of xylan, the dosage of the glycoside hydrolase can be with For 24-240U.The condition of the contact may include: that temperature is 45-55 DEG C, pH value 6-10.The time of the incubation can be with For 5-360min.In order to obtain more preferably degradation effect, it is preferable that the contact is in Fe²⁺And/or Mn²⁺In the presence of carry out. The xylan sample can be the xylan in various sources, such as zelkova schneideriana xylan, birch xylan, ramie xylan, corn Core xylan etc..

The present invention will be described in detail by way of examples below.

It is purchased from embodiment for Pichiapastoris expression strain pichia pastoris X-33 and expression vector pPICZ α A Invitrogen company.

Enzyme and other biochemical reagents: restriction enzyme EcoR I and Kpn I and T4 ligase is purchased from Takara Company, pNPX (to nitro-β-D- xylopyranose glucosides), pNPAF (to nitro-α-L-arabinose glycosides), birch xylan, business Change inscribe beta-xylanase and is purchased from Sigma company, commercialization xylobiase, standard items xylose, xylobiose, xylotriose, wood four Sugar, the wooden pentasaccharides and six sugar of wood are purchased from Megazyme company, other all (to purchase from common biochemical Reagent Company for domestic reagent It buys).

Culture medium:

Escherichia coli culture medium LC: yeast extract 0.5%, peptone 1%, NaCl 0.5% prepare solid medium 1.5% agar powder of Shi Tianjia；

Culture medium of pichia pastoris YPD: yeast extract 1%, peptone 2%, glucose 2%；

BMGY culture medium: yeast extract 1%, peptone 2%, yeast basic nitrogen source 1.34%, 6.0 phosphorus of 100mM pH Acid buffer, glycerol 1%；

Pichia pastoris inducing expression culture medium BMMY: yeast extract 1%, peptone 2%, yeast basic nitrogen source 6.0 phosphate buffer of 1.34%, 100mM pH, methanol 0.5%.

Embodiment 1

The present embodiment is used to illustrate the building of the recombinant bacterial strain containing Ttxy43 gene of the invention.

(1) acquisition of Ttxy43 gene

Synthesize nucleic acid sequence shown in SEQ ID NO:2.

Design specific primer Ttxy43-F/Ttxy43-R

Ttxy43-F:CGGAATTCATGGCTCCATTGATCACCAA (SEQ ID NO:8)

Ttxy43-R:GGGGTACCTTACTCTGGCTTCTCGGTA (SEQ ID NO:9)

Using nucleic acid sequence shown in SEQ ID NO:2 as template, Ttxy43-F/Ttxy43-R is that primer carries out PCR amplification, The response parameter of PCR are as follows: 95 DEG C of denaturation 5min, then 95 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extensions, 30 are followed Continue to extend 10min for 72 DEG C after ring, obtains Ttxy43 genetic fragment (SEQ ID NO:2).

(2) recombinant bacterial strain is constructed

Digestion Ttxy43 genetic fragment and pPICZ α A plasmid, recycling are distinguished using restriction enzyme EcoR I and Kpn I Segment is connected using T4 ligase, obtains recombinant plasmid (pPICZ α A-Ttxy43), then arrive obtained recombinant plasmid transformed In Pichia pastoris pichia pastoris X-33, restructuring yeast strains are obtained.

The method of conversion: taking 1mL Pichia pastoris bacterium solution to be inoculated into 50mL YPD fluid nutrient medium, 28 DEG C, 200rpm training It supports to OD600 to 1.3-1.5.After 4000g is centrifuged 5min under the conditions of 4 DEG C, outwells supernatant and collect Pichia pastoris thallus, then use 50mL Thallus is resuspended in the sterile water of pre-cooling.Thalline were collected by centrifugation under the same terms, then thallus is resuspended with the sterile water of 25mL pre-cooling.It is identical Under the conditions of thalline were collected by centrifugation, add 1mL pre-cooling 1M sorbierite, be resuspended thallus.Take 80 μ L bacteria suspensions to the 1.5mL of pre-cooling In centrifuge tube, the plasmid of 10 μ L conversion is added, is uniformly mixed, is transferred in 2mm electricity revolving cup, 5min is pre-chilled on ice.Electricity is turned The water wiped clean of cup outer wall, is put into electroporation, shocks by electricity under the conditions of 1500V.800 μ L pre-cooling is rapidly added after electric shock 1M sorbierite, resuspended bacterium solution are simultaneously transferred in 2mL centrifuge tube, and rejuvenation 1h is stood under the conditions of 28 DEG C, obtain restructuring yeast strains.

(3) verifying of recombinant bacterial strain

Restructuring yeast strains after conversion are coated on the YPD plate containing bleomycin (100 μ g/mL) resistance, 28 Culture to single colonie occurs in DEG C incubator.Picking single bacterium falls within sufficiently outstanding in the mixed liquor of 100 μ L 0.2M LiAcO and 1%SDS Floating, 70 DEG C of heating 5min are added 300 μ L dehydrated alcohols, are mixed well using vortex concussion instrument, and 12000rpm is centrifuged 5min, Supernatant is removed, 600 μ L70% ethyl alcohol is added, is mixed well using vortex concussion instrument, 12000rpm is centrifuged 2min, as far as possible will be upper It cleans and removes completely, add 20 μ L ddH₂Thallus is resuspended in O, and 12000rpm is centrifuged 30s, and supernatant is Pichia pastoris genomic DNA.

Using the Pichia pastoris genomic DNA of above-mentioned acquisition as template, 5 ' AOX of specific primer (GACTGGTTCCAATTGACAAGC, SEQ ID NO:10) and 3 ' AOX (GCAAATGGCATTCTGACATCC, SEQ ID NO: 11) PCR verifying, the response parameter of PCR are carried out for primer are as follows: 95 DEG C of denaturation 5min, then 95 DEG C of denaturation 30sec, 58 DEG C are annealed 30sec, 72 DEG C extend, and continue to extend 10min for 72 DEG C after 30 circulations.Obtain the restructuring yeast strains of correct expression Ttxy43.

Comparative example 1

This comparative example is used to illustrate the building of the recombinant bacterial strain containing existing Ttxy43 gene.

The building of restructuring yeast strains is carried out according to the method for embodiment 1, unlike, the nucleotides sequence of Ttxy43 gene Column are as shown in SEQ ID NO:8.

Test case 1

This test case is used to illustrate the inducing expression of Ttxy43.

1 recombinant yeast of embodiment 1 and comparative example of the fresh correct expression Ttxy43 grown on picking YPD plate Strain, and they is inoculated in respectively in BMGY culture medium, 28 DEG C, 200rpm overnight incubation, be then transferred to 1mL culture solution in In 50mL BMMY culture medium, every 1% methanol is added for 24 hours, 120h is cultivated, it is Ttxy43 crude enzyme liquid that centrifugation, which obtains supernatant,.

The Ttxy43 crude enzyme liquid is subjected to SDS-PAGE electrophoresis, as a result as shown in Figure 1, wherein M is protein Marker, The 1 foreign protein Ttxy43 encoded for gene provided by the invention；The results show that Ttxy43 provided by the invention can recombinated Great expression is carried out in yeast, and purity of protein is higher in supernatant, and the external source egg of the coding of gene shown in SEQ ID NO:12 The expression quantity of white Ttxy43 is extremely low (result is not shown), thus illustrates that gene coding provided by the invention can be improved destination protein Expression quantity.

Test case 2

This test case is for illustrating that Ttxy43 difference enzymatic properties are analyzed.

Each enzyme activity determination method of Ttxy43

(1) xylobiase: 10mM pNPX is prepared (to nitro-β-D- pyrans with the phosphate buffer of pH 6.0,100mM Xyloside), 100 μ L substrates (10mM pNPX) are added in 1.5mL centrifuge tube, add the crude enzyme liquid of 100 μ L, mix rapidly Uniformly, 10min is reacted under the conditions of 50 DEG C, and 100 μ L 1M Na are added₂CO₃Reaction is terminated, using buffer reaction system as blank pair According to measurement OD410.1U is defined as under optimum reaction conditions (pH 6.0, temperature 50 C), generates needed for 1 μm of ol pNP per minute Enzyme amount.

(2) inscribe beta-xylanase: with pH 7.0,100mM phosphate buffer prepare 1% birch xylan, 1.5mL from The enzyme solution that 100 μ L, 1% birch xylan adds 100 μ L is added in heart pipe, is uniformly mixed rapidly, is reacted under the conditions of 50 DEG C 10min is added 50 μ L 1M NaOH, 150 μ L DNS and terminates reaction, and boiling water bath 5min is immediately placed in cooled on ice, with buffer Reaction system is that blank control measures OD540.1U is defined as under optimum reaction conditions (pH 7.0, temperature 60 C), produces per minute Enzyme amount needed for raw 1 μm of ol reduced sugar.

(3) α-l-arabfuranglycosidase: preparing 10mM pNPAF with pH 7.0,100mM phosphate buffer, 100 μ L10mM pNPAF are added in 1.5mL centrifuge tube, add the enzyme solution of 100 μ L, are uniformly mixed rapidly, it is anti-under the conditions of 50 DEG C 10min is answered, 100 μ L 1M Na are added₂CO₃Reaction is terminated, measures OD410 by blank control of buffer reaction system.1U definition For (pH 7.0, temperature 50 C) under optimum reaction conditions, enzyme amount needed for generating 1 μm of ol pNP per minute.

1, tri- kinds of enzyme activity optimal pH measurements of Ttxy43

Influence using the phosphate buffer measurement pH of different pH to tri- kinds of enzyme activity of Ttxy43, as a result as seen in figs. 2a-2c, Show: the optimal reaction pH of xylobiase, inscribe beta-xylanase and α-l-arabfuranglycosidase is 7.0.

2, tri- kinds of enzyme activity optimum temperature measurements of Ttxy43

Every 10 DEG C of measurement tri- kinds of enzyme activity of Ttxy43 under the conditions of 20-70 DEG C of temperature, as a result as shown in figs. 3 a-3 c, as a result Show: xylobiase optimal reactive temperature is 50 DEG C, and inscribe beta-xylanase optimal reactive temperature is 60 DEG C, and α-L- is Arabic Furanoside enzyme optimal reactive temperature is 50 DEG C.

3, different metal ions influence tri- kinds of enzyme activity of Ttxy43

Under optimum reaction conditions, certain density metal ion is added in the reaction system, tri- kinds of Ttxy43 of measurement is not Same enzyme activity, as a result as shown in table 2 below, Fe²⁺And Mn²⁺There is facilitation to each enzyme activity of Ttxy43.

Table 2

	Concentration	Inscribe beta-xylanase	Xylobiase	α-l-arabfuranglycosidase
					control		100±4.3	100±4.2	100±4.6
Mg²⁺	5mM	74.3±6.5	64.6±4.5	100.3±5.3
					Ca²⁺	5mM	140.1±3.7	102.0±2.4	95.5±5.2
Co²⁺	5mM	74.6±1.2	50.4±1.2	103.8±6.3
					Fe²⁺	5mM	141.6±3.1	121.4±3.1	114.3±4.0
Mn²⁺	5mM	160.7±7.0	118.2±6.8	125.2±3.1
					Ni²⁺	5mM	32.5±3.6	93.3±4.6	83.2±4.1
Zn²⁺	5mM	55.8±1.5	10.0±1.1	10.0±2.2
					Cu²⁺	5mM	30.6±3.3	12.0±3.1	22.0±3.1
EDTA	5mM	18.9±3.1	20.6±3.7	26.6±2.6
					Tween 20	1%	79.1±2.2	58.0±2.8	60.0±3.6
SDS	1%	31.8±2.2	7.0±2.9	10.0±1.2

4, various concentration xylose influences xylobiase and inscribe beta-xylanase enzyme activity

Standard items xylose concentration measures it to xylobiase and inscribe β-xylan every 100mM between 0-1000mM The influence of enzyme enzyme activity, as a result as shown in Figure 4, the results showed that with the raising of standard items xylose concentration, the enzyme activity of xylobiase It is gradually reduced, when concentration is 1000mM, enzyme activity is close to zero, and xylose does not influence the enzyme activity of inscribe beta-xylanase.

Test case 3

The present embodiment is for illustrating that Ttxy43 hydrolyzes birch xylan interpretation of result.

Using 1% weight birch xylan as substrate, it is utilized respectively Ttxy43, commercialization inscribe beta-xylanase, commercialization Xylobiase, commercialization inscribe beta-xylanase and xylobiase mixed enzyme (pH 7.0, temperature under optimum reaction conditions 50 DEG C) it is hydrolyzed, it is centrifuged after hydrolysis, supernatant utilizes Shimadzu LC-20A HPLC system, selects organic acid column ROA-Organic Acid H+ (8%) (Phenomenex) carries out analytical standard product xylose, xylobiose, xylotriose, Xylotetrose, the wooden pentasaccharides and wood six The generation situation of sugar, as a result as shown in Figure 6 (Fig. 6 A: birch xylan, Fig. 6 B: commercialization inscribe beta-xylanase results of hydrolysis, Fig. 6 C: commercialization inscribe xylobiase results of hydrolysis, Fig. 6 D:Ttxy43 results of hydrolysis and commercialization inscribe beta-xylanase/ The comparison of xylobiase mixed enzyme degradation results), the results showed that Ttxy43 is compared with commercialized enzyme, and degradation birch xylan is raw It is single xylose at product.Standard items wood oligose HPLC result is as shown in Figure 5.

The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this A little simple variants all belong to the scope of protection of the present invention.

It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can No further explanation will be given for the combination of energy.

In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally The thought of invention, it should also be regarded as the disclosure of the present invention.

SEQUENCE LISTING

<110>China Agricultural University

<120>fast hydrolyzing xylan generates encoding gene and its application of the glycoside hydrolase of single xylose

<130> I55672NYD

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 327

<212> PRT

<213>glycoside hydrolase Ttxy43

<400> 1

Met Ala Pro Leu Ile Thr Asn Ile Phe Thr Ala Asp Pro Ser Ala His

1 5 10 15

Val Phe Glu Gly Lys Leu Phe Ile Tyr Pro Ser His Asp Arg Glu Thr

20 25 30

Asp Ile Lys Phe Asn Asp Asp Gly Asp Gln Tyr Asp Met Val Asp Tyr

35 40 45

His Val Phe Ser Thr Glu Ser Leu Asp Pro Ala Ala Pro Val Thr Asp

50 55 60

His Gly Val Val Leu Arg Ala Glu Asp Val Pro Trp Val Ser Lys Gln

65 70 75 80

Leu Trp Ala Pro Asp Ala Ala Tyr Lys Asp Gly Arg Tyr Tyr Leu Tyr

85 90 95

Phe Pro Ala Arg Asp Lys Gln Gly Val Phe Arg Ile Gly Val Ala Val

100 105 110

Gly Asp Arg Pro Glu Gly Pro Phe Thr Pro Asp Pro Glu Pro Ile Arg

115 120 125

Asp Ser Tyr Ser Ile Asp Pro Ala Val Phe Val Asp Asp Asp Gly Arg

130 135 140

Ala Tyr Met Tyr Phe Gly Gly Leu Trp Gly Gly Gln Leu Gln Cys Tyr

145 150 155 160

Gln Lys Gly Asn Gly Ile Phe Asp Pro Glu Trp Leu Gly Pro Arg Glu

165 170 175

Pro Ser Gly Glu Gly Val Arg Ala Leu Gly Pro Arg Val Ala Arg Leu

180 185 190

Ala Asp Asp Met Arg Gln Phe Ala Ser Glu Val Lys Glu Ile Ser Ile

195 200 205

Leu Ala Pro Glu Thr Gly Glu Pro Ile Ala Ala Asp Asp His Asp Arg

210 215 220

Arg Phe Phe Glu Ala Ala Trp Met His Lys Tyr Asp Gly Lys Tyr Tyr

225 230 235 240

Phe Ser Tyr Ser Thr Gly Asp Thr His Tyr Leu Val Tyr Ala Val Gly

245 250 255

Asp Ser Pro Tyr Gly Pro Phe Thr Tyr Ala Gly Arg Ile Leu Glu Pro

260 265 270

Val Leu Gly Trp Thr Thr His His Ser Ile Val Glu Phe His Gly Arg

275 280 285

Trp Trp Leu Phe His His Asp Cys Glu Leu Ser Gly Gly Val Asp His

290 295 300

Leu Arg Ser Val Lys Val Lys Glu Ile Phe Tyr Asp Lys Asp Gly Lys

305 310 315 320

Ile Val Thr Glu Lys Pro Glu

325

<210> 2

<211> 984

<212> DNA

<213>glycoside hydrolase Ttxy43 encoding gene

<400> 2

atggctccat tgatcaccaa catcttcacc gctgacccat ctgctcacgt tttcgagggt 60

aagttgttca tctacccatc tcacgacaga gagaccgaca tcaagttcaa cgacgacggt 120

gaccaatacg acatggttga ctaccacgtt ttctctaccg agtctttgga cccagctgct 180

ccagttaccg accacggtgt tgttttgaga gctgaggacg ttccatgggt ttctaagcaa 240

ttgtgggctc cagacgctgc ttacaaggac ggtagatact acttgtactt cccagctaga 300

gacaagcaag gtgttttcag aatcggtgtt gctgttggtg acagaccaga gggtccattc 360

accccagacc cagagccaat cagagactct tactctatcg acccagctgt tttcgttgac 420

gacgacggta gagcttacat gtacttcggt ggtttgtggg gtggtcaatt gcaatgttac 480

caaaagggta acggtatctt cgacccagag tggttgggtc caagagagcc atctggtgag 540

ggtgttagag ctttgggtcc aagagttgct agattggctg acgacatgag acaattcgct 600

tctgaggtta aggagatctc tatcttggct ccagagaccg gtgagccaat cgctgctgac 660

gaccacgaca gaagattctt cgaggctgct tggatgcaca agtacgacgg taagtactac 720

ttctcttact ctaccggtga cacccactac ttggtttacg ctgttggtga ctctccatac 780

ggtccattca cctacgctgg tagaatcttg gagccagttt tgggttggac cacccaccac 840

tctatcgttg agttccacgg tagatggtgg ttgttccacc acgactgtga gttgtctggt 900

ggtgttgacc acttgagatc tgttaaggtt aaggagatct tctacgacaa ggacggtaag 960

atcgttaccg agaagccaga gtaa 984

<210> 3

<211> 5

<212> PRT

<213>Poly-Arg label

<400> 3

Arg Arg Arg Arg Arg

1 5

<210> 4

<211> 6

<212> PRT

<213>Poly-His label

<400> 4

His His His His His His

1 5

<210> 5

<211> 8

<212> PRT

<213>FLAG label

<400> 5

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 6

<211> 8

<212> PRT

<213>II label of Strep-tag

<400> 6

Trp Ser His Pro Gln Phe Glu Lys

1 5

<210> 7

<211> 10

<212> PRT

<213>c-myc label

<400> 7

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 8

<211> 28

<212> DNA

<213>Ttxy43-F primer

<400> 8

cggaattcat ggctccattg atcaccaa 28

<210> 9

<211> 27

<212> DNA

<213>Ttxy43-R primer

<400> 9

ggggtacctt actctggctt ctcggta 27

<210> 10

<211> 21

<212> DNA

<213>primer 5'AOX

<400> 10

gactggttcc aattgacaag c 21

<210> 11

<211> 21

<212> DNA

<213>primer 3'AOX

<400> 11

gcaaatggca ttctgacatc c 21

<210> 12

<211> 984

<212> DNA

<213>wild-type glycoside hydrolase encoding gene

<400> 12

atggcgcccc tcatcaccaa catcttcacg gccgacccgt cggcccacgt cttcgagggc 60

aagctcttca tatacccgtc gcacgatcgc gagacggaca tcaagttcaa cgacgacggc 120

gaccagtacg acatggtcga ctaccacgta ttcagcaccg agtcgctgga cccggccgcc 180

cccgtgaccg accacggcgt cgtgctccgg gccgaagacg tcccctgggt gtccaagcag 240

ctctgggccc ccgacgccgc ctacaaggac ggcaggtact acctctactt ccccgcccgc 300

gacaagcagg gcgtcttccg catcggcgtc gccgtcggcg accgccccga gggccccttc 360

acccccgacc cggagcccat ccgggacagc tacagcatcg acccggccgt cttcgtcgac 420

gacgacggcc gggcctacat gtactttggc gggctctggg gcggccagct gcagtgctac 480

cagaagggca acggcatctt cgaccccgag tggctggggc ccagggagcc ctcgggcgag 540

ggcgtccggg cgctggggcc gcgcgtcgcc cggctggcgg acgacatgcg ccagttcgcc 600

agcgaggtga aggagatttc gatcctggcg cccgagacgg gcgagccgat cgcggccgac 660

gaccacgacc gccgcttctt cgaggccgcc tggatgcaca agtacgacgg caagtactac 720

ttcagctact ccaccggcga cacccactac ctcgtctacg ccgtcggcga cagcccctac 780

gggcccttca cctacgccgg ccgcatcctc gagcccgtcc tcggctggac cacgcaccac 840

tccatcgtcg agttccacgg ccgctggtgg ctcttccacc acgactgcga gctcagcggc 900

ggagtcgacc acctgcgctc cgtcaaggtc aaggagatct tctacgacaa ggacggcaag 960

attgtcactg aaaagcccga atag 984

Claims

1. the gene that a kind of coding fast hydrolyzing xylan generates the glycoside hydrolase of single xylose, which is characterized in that the base The nucleotide sequence of cause is as shown in SEQ ID NO:2.

2. a kind of recombinant vector, which is characterized in that the recombinant vector contains gene described in claim 1.

3. a kind of recombinant bacterial strain, which is characterized in that the recombinant bacterial strain contains gene described in claim 1 or wants containing having the right Recombinant vector described in asking 2.

4. recombinant bacterial strain according to claim 3, wherein the bacterial strain is bacillus or saccharomycete.

5. recombinant bacterial strain according to claim 3 or 4, wherein the bacterial strain is Escherichia coli (Escherichia Coli), bacillus subtilis (Bacillus subtilis), Pichia pastoris (Pichia pastoris) or saccharomyces cerevisiae (Saccharomyces cerevisiae)。

6. a kind of method for preparing fast hydrolyzing xylan and generating the glycoside hydrolase of single xylose, which is characterized in that this method The following steps are included:

(1) recombinant bacterial strain described in any one of claim 3-4 is cultivated, induction coding fast hydrolyzing xylan generates single The expression of the gene of the glycoside hydrolase of xylose；

(2) glycoside hydrolase expressed by separating-purifying.

7. gene described in claim 1, carrier as claimed in claim 2, recombinant strain as claimed in claim 3, right are wanted Glycoside hydrolase, the fast hydrolyzing xylan as shown in SEQ ID NO:1 of the preparation of method described in asking 6 generate single xylose Glycoside hydrolase or the glycoside hydrolase containing method of claim 6 preparation or the quick water as shown in SEQ ID NO:1 The composition that solution xylan generates the glycoside hydrolase of single xylose is playing inscribe beta-xylanase and/or α-L- Arab furan The application muttered in glycosidase activity.

8. gene described in claim 1, carrier as claimed in claim 2, recombinant strain as claimed in claim 3, right are wanted Glycoside hydrolase, the fast hydrolyzing xylan as shown in SEQ ID NO:1 of the preparation of method described in asking 6 generate single xylose Glycoside hydrolase or the glycoside hydrolase containing method of claim 6 preparation or the quick water as shown in SEQ ID NO:1 The composition for the glycoside hydrolase that solution xylan generates single xylose generates the application in single xylose in hydrolyzed xylan.

9. application according to claim 7 or 8, wherein on the basis of the total weight of the composition, the glycoside hydrolysis The content of enzyme is 1-10 weight %.

10. application according to claim 7 or 8, wherein also contain Fe in the composition²⁺And/or Mn²⁺。