CN107354165A - The xylanase improved gene and its engineering bacteria of a kind of high yield specific product prepare the application of xylo-oligosaccharide - Google Patents

Abstract

The invention provides a kind of encode to prepare the application of xylo-oligosaccharide with the special wood two of hydrolyzed xylan substrate high yield, the improvement gene of zytase of trisaccharide product its Recombinant organism.The asparagine mutation of 86 of zytase its N-terminal amino acid sequence after improvement is glutamine.Recombinase XYNGH10 zymologic properties are：Optimal pH 5.5,65 DEG C, enzyme activity 160U/ml of optimum temperature, Xylose Content is more than 14% in product during its hydrolyzed xylan substrate；And build mutant GH10 N86Q hydrolyzed xylan substrates when product in Xylose Content be below less than 2%, and hydrolysate is to have based on the xylobiose of stronger beneficial function and xylotriose component.Mutant enzyme optimal pH is 5.5, and optimum temperature is 55 DEG C, and its enzyme activity is to improve 25.6% before 201U/ml is relatively transformed.There is its Recombinant organism of the improvement gene of the present invention good utilization to be rich in hemicellulose agricultural wastes Production by Enzymes xylo-oligosaccharide industrial production application prospect, can be widely applied to the fields such as food, medical industry.

Description

The xylanase improved gene and its engineering bacteria of a kind of high yield specific product prepare low The application of xylan

Technical field

It is related to one kind the invention belongs to genetic engineering and protein engineering field and derives from streptomycete (Streptomyces Sp. L10608) have hydrolyzed xylan substrate high yield specificity wood two, the xylanase improved gene of trisaccharide product and its Engineered strain prepares the application of xylo-oligosaccharide.

Background technology

China is a large agricultural country, along with the production and processing of agricultural product, produces substantial amounts of agricultural wastes every year.This A little agricultural wastes resource regeneration rates are extremely low, are largely wasted.Devoting Major Efforts To Developing using these resources can alleviate environment, Resource and crisis in food, therefore agricultural wastes recycle the focus for turning into the research of nearly more than ten years various countries.It is rich in agricultural wastes Containing cellulose and hemicellulose, wherein hemicellulose account for 20%-30%, and annual newly-generated hemicellulose is known as 3 × 10¹⁰Ton It is more.Xylan is the more complicated hemicellulose of structure, is second-biggest-in-the-world renewable resource.Because xylan is complicated, Its thorough hydrolysis needs a series of synergy of enzymes, and the enzyme of wherein most critical is zytase.Zytase category hydrolase Class, irreplaceable function is played during carbon cycle, there is important potential using value, be successfully applied at present Food, feed, brewage, papermaking, the field such as medicine and the energy.In numerous applications, produced with xylan compared with high added value Function oligosaccharide is effective value-added route of the agricultural wastes regeneration rich in hemicellulosic materials, is had huge potential Economic benefit and environment protection significance.

Xylo-oligosaccharide is the feature polymerization sugar connected into by 2~7 xylose molecules with glycosidic bond, wherein wood two, wood three Sugar is principle active component.Its added value is high, market prospects are good, and 70% xylo-oligosaccharide syrupy product price is up to 200,000 yuan of people People's coin/t, and content is then up to 630,000 yuans/t up to 95% solid powder price.Xylo-oligosaccharide can be widely applied to drink Sugar-free low energy product in the food such as material, candy, cake, ice cream, dairy products and flavouring, pharmaceuticals industry, to supply fertilizer Fat disease, diabetes, hypertension, artery sclerosis, eurodonticus eat.At present because there is biological enzyme environment-friendly, product to be easy to The advantages that control, good product specificity, it is increasingly becoming the main method using agricultural wastes production xylo-oligosaccharide.And current enzyme The specific component content such as wood two, xylotriose in product during the microbe-derived xylanase hydrolysis xylan that method production uses Mostly more complicated work is needed less than 30% and containing monose and other non-functional compositions such as a large amount of xyloses, arabinose, later stage Skill is into purified originally with great number, to improve function xylo-oligosaccharide purity (60%-70%).Only only a few bacterial strain produces During xylanase hydrolysis substrate, wood two, xylotriose ratio have exceeded 50% in product.Therefore the production of Enzymatic xylo-oligosaccharide Xylobiose, the special functional component of xylotriose in middle raising catalytic process, generation xylose is eliminated and reduced, avoids xylo-oligosaccharide from entering one It is a key issue urgently to be resolved hurrily that step, which is changed into xylose,.Significantly improve during Production by Enzymes xylo-oligosaccharide specific function into Point content will reduce Production by Enzymes cost, reduce energy consumption during later-period purification, sewage discharge etc., reduce environmental pollution, Realize and efficiently utilize the large-scale industrial production application of low value agricultural wastes production high added value xylo-oligosaccharide.

The content of the invention

First purpose of the present invention, which is that acquisition is a kind of, has the special wood two of hydrolyzed xylan substrate high yield, trisaccharide product Xylanase improved gene.

Second object of the present invention is to obtain the engineering strain for expressing above-mentioned xylanase improved gene.

Third object of the present invention is to prepare xylo-oligosaccharide to engineered strain to carry out Preliminary Applications.

Present invention obtains a kind of xylanase improved gene (xynGH10-N86Q), its nucleotide sequences as shown in SEQ-1, Its amino acid sequence is as shown in SEQ-2.

The present invention obtains a kind of with the special wood two of hydrolyzed xylan substrate high yield, the xylanase improved base of trisaccharide product Cause.Zytase (XYNGH10) source and streptomycete Streptomyces sp.10608 (the micro- lifes of China as improvement basis The common micro- raw center of thing culture presevation administration committee, deposit number CGMCC No.13271, address：Chaoyang District, Beijing City north The institute 3 of occasion West Road 1, preservation date on November 14th, 2016).The zytase optimal pH 5.5,65 DEG C of optimum temperature.Change Zytase after good is compared with original zytase, by the asparagine of 86 of original zytase N-terminal amino acid sequence Sport glutamine.

The invention provides a kind of plasmid of the nucleotide coding sequence containing above-mentioned xylanase improved gene.The plasmid Can be protokaryon or eucaryon plasmid.Xylanase improved gene coded sequence containing the present invention is cloned by conventional method Multiple cloning sites to carrier can form transfer vector plasmid.The plasmid of the present invention can be prokaryotic expression plasmid, such as large intestine Bacillus expression plasmid PET21a, PET28a, PET30a etc..The plasmid can also be eukaryon expression plasmid such as pPIC9K, pPIC9 Deng.

Present invention also offers the recombination bacillus coli genetic engineering containing original xylanase gene and the improvement gene Bacterial strain.The recombinase XYNGH10 zymologic properties of Bacillus coli expression are：Optimal pH 5.5,65 DEG C of optimum temperature, enzyme activity 160U/ Ml, Xylose Content is more than 14% in product during its hydrolyzed xylan substrate；And the mutant GH10-N86Q hydrolyzed xylans built Xylose Content is below less than 2% in product during substrate, and hydrolysate is to have the xylobiose of stronger beneficial function and xylotriose Based on component.Mutant enzyme optimal pH 5.5,55 DEG C of optimum temperature, its enzyme activity are to improve 25.6% before 201U/ml is relatively transformed.

Brief description of the drawings

The mutational site schematic diagram of Fig. 1 xynGH10 selections.

Fig. 2 Overlap extension PCRs rite-directed mutagenesis splices sequence.M：marker；Splice sequence for parallel N86Q in road 1,2

The amino acids active force of recombinase N-terminal 86 is predicted before and after Fig. 3 site mutations.(A) 86 asparagines are made before being mutated Firmly predict, 86 glutamine active force predictions after (B) mutation.

The optimal pH of Fig. 4 recombinases.

The optimum temperature of Fig. 5 recombinases.

Embodiment

The present invention is described further below in conjunction with instantiation, but is not the limitation present invention.It is right below in conjunction with the accompanying drawings The present invention further illustrates.The experimental method of unreceipted actual conditions in embodiment, generally can routinely bar, such as J. Pehanorm cloth Shandong Gram (Sambrook) etc. writes《The Molecular Cloning:A Laboratory guide third edition》Described in condition, or according to kit manufacturer Proposed condition is carried out.Those skill in the art related can more fully understand and grasp the present invention by embodiment.

1. general reagent：It is Congo red；Kanamycins (U.S. AMRESCO)；Agarose (Spain Biowest)；Ammonia benzyl is blue or green Mycin (U.S. AMRESCO)；Birch xylan (U.S. Sigma)；LB solid mediums；Xylan-Congo red screening flat board； NaCl eluents：1M；Oat xylan and Corncob Xylan (VETEC)；Xylose mark product (xylose, xylobiose, xylotriose, wood Tetrose) Chinese medicines group chemical reagent；LB solid mediums：Tryptone 1% (m/v), yeast extract 0.5% (m/v), NaCl1% (m/v), agar powder 1.5% (m/v)；Other reagents are that domestic analysis is pure.

2. biochemical reagents：Bacterial genomes extracts kit (U.S. OMEGA)；Glue reclaim kit (OMEGA, the U.S.)； La Taq archaeal dna polymerases (with GC buffer)；Restriction enzyme EcoRI, XhoI；1kb DNA Ladder Marker (Japanese TAKARA)；La Taq archaeal dna polymerases (with GC buffer) (TAKARA, Japan)；200bp DNA ladder (U.S. Biomega)；Pcr amplification primer thing (the prosperous synthesis of Beijing AudioCodes)；200bp DNA ladder (U.S. Biomega)； 1kbDNALadderMarker (Japanese TAKARA)；

3. bacterial strain：Streptomycete (Streptomyces sp.) 10608；Escherichia coli DH5a and Transtetta (DE3) senses Quan Shi King Companies are purchased from by state cell

4. carrier：Carrier T：PMD18-T is purchased from TAKARA companies；PET-28a (+) is purchased from Merk companies

5. primer：Carrier T is sequenced：M13+ sequences：5 '-GTTTTCCCAGTCACGAC-3 ', M13- sequence：5’- CAGGAAACAGCTATGAC-3’；

Rite-directed mutagenesis primer：xynGH10N86QF：5 '-ACCGCCGAGGGCGAGATGAAG-3 ', xynGH10N86QR： 5’-CTTCATCTCGCCCTCGGCGGT-3’

Embodiment 1：The structure and prokaryotic expression of mutant

After obtaining target gene xynGH10, with NcoR I and Xho I simultaneously to expression vector pET28a and with digestion position The xynGH10 of point carries out double digestion, 37 DEG C of reaction 3h.Digestion is detected by 1% (m/v) agarose gel electrophoresis later and cuts glue XynGH10 and linearisation pET28a of the recovery with cohesive end.Thereafter recovery is carried into cohesive end with T4DNA ligases XynGH10 and linear pET28a be placed in reaction system connect at 1: 7 in molar ratio, 1h is reacted in 25 DEG C, so as to build restructuring Plasmid pET28a-xynGH10.

It has been connected to raw material of the gene xynGH10 PET28a plasmids as mutation.Using the side of Overlap extension PCR Method carries out Amino Acid-Induced Site-Directed Mutation with the rite-directed mutagenesis primer of the designed amino acids of N-terminal the 86th.Obtain target gene After xynGH10-N86Q, with NcoR I and Xho I simultaneously to expression vector pET28a and the GH10-N86Q with restriction enzyme site Carry out double digestion, 37 DEG C of reaction 3h.Digestion gel extraction xynGH10-N86Q and linearisation pET28a.Thereafter with connection structure weight Group plasmid pET28a-xynGH10-N86Q.Recombinant plasmid pET28a-xynGH10-N86Q mutant nucleotide sequences are imported into competence BL21 (DE3) checking of mutant nucleotide sequence, is then carried out.By understanding position of the selected mutational site in complete sequence, N86 in Fig. 1 Position in the sequence is more forward, is not in the catalytic domain of enzyme.

Using Overlap extension PCR method mutant fragments when, a complete genetic fragment is from using mutational site as midpoint Extend to form two bar segments to both sides.As shown in Fig. 2 road 1,2 is the complete genome sequence for the about 1600bp length spliced.

Recombinase XYNGH10 optimal reactions pH is 5.5, and optimum temperature is 65 DEG C, and enzyme activity is determined as 160U/ through DNS methods ml.And it is 201U/ml that the mutation recombinase GH10-N86Q for entering checking determines Xylanase activity through DNS methods.To the weight of transformation For group zymoprotein, it is to carry out the basis of practical application to ensure its biological activity, and mutant GH10-N86Q is by 86 Asparagine mutation is has remained on the active force of the non-covalent bond in protoenzyme protein molecular after glutamine, in certain base Space conformation is still maintained on plinth.

Embodiment 2：Recombination mutation enzyme hydrolysis substrate generates specific product property research

Mutant enzyme hydrolysis substrate system is built first.Configure 1ml reaction systems：It is 20mg/ml's including 500 μ L concentration Laboratory self-control water-insoluble Corncob Xylan, Corncob Xylan (VETEC), oat xylan (Biotopped) etc. are no Same substrate, 10U enzyme liquid, 1ml is complemented to NaAc_HAc buffer solution (pH=5.5).The 1ml reactants that will have been configured 55 DEG C of water-bath heating in water bath for reaction 12h are lain in, 0.22 μm of aqueous phase film injection sample injection bottle is medium to be detected.The detection of hydrolysate Condition is：Amino acid analysis post, mobile phase are acetonitrile: water 70: 30, flow velocity 0.8ml/min, 30 DEG C of column oven temperature, show difference 30 DEG C of refraction detector temperature, the μ l of sample size 20.

Absorption xylose, xylobiose, xylotriose, Xylotetrose standard items are soluble in water respectively, and being diluted to concentration successively is： 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml standard liquid mark make quasi- working curve, with 0.45 μm filtering with microporous membrane, produces xylose, xylobiose, xylotriose, Xylotetrose standard liquid series.20 μ l standard series are taken respectively Solution sample introduction is analyzed, and using xylose, xylobiose, xylotriose, Xylotetrose concentration as abscissa, peak area is ordinate, drafting xylose, Xylobiose, xylotriose, peak area-concentration standard curve of Xylotetrose.

As shown in table 1, select using the xylan not of the same race such as beech, oat and birch as substrate, checking recombination mutation enzyme Hydrolysis properties and its possibility in industrial circle application.When using beech xylan as substrate, original XYNGH10 hydrolyzes substrate Xylose Content is 20.66% in product afterwards, and the accounting of xylose is remarkably decreased only in improved GH10-N86Q hydrolysates 2.49%, and the content of wood three and the xylo-oligosaccharide such as Xylotetrose also increases significantly, respectively 56.53% and 15.97%. After GH10-N86Q remains in that higher xylo-oligosaccharide yield, original XYNGH10 hydrolyze substrate during using oat xylan as substrate Xylose Content is 14.20% in product, and the accounting of xylose is remarkably decreased only in improved GH10-N86Q hydrolysates 1.45%, and the content of wood three and the xylo-oligosaccharide such as Xylotetrose also increases significantly, respectively 52.76% and 24.22%. When using birch xylan as hydrolysis substrate, the Xylose Content in hydrolysate is equally from original XYNGH10 products 21.23% is remarkably decreased as 1.01% in GH10-N86Q, the content of the wooden xylo-oligosaccharide such as three and Xylotetrose also significantly improve for 61.89% and 11.42%.

The information summary analysis of different xylan generation products is hydrolyzed according to mutant enzyme GH10-N86Q：Original recombinase XynGH10 hydrolysis feature is that it has very strong hydrolysing activity to xylotriose substrate, leads in product based on xylose and xylobiose Cause to generate substantial amounts of wooden monose in product.And improved mutant GH10-N86Q is maintained when being applied to hydrolyzed xylan Good hydrolysis properties, predominantly xylo-oligosaccharide and Xylose Content is below less than 2%, while also maintain higher in product Catalytic activity, its activity are the 125.6% of xynGH10.There is the mutant agricultural of the good utilization rich in hemicellulose to give up Gurry Production by Enzymes xylo-oligosaccharide industrial production application prospect.

Found by the space structure of 86 zymoprotein mutant of software analysis N-terminal, 86 amino acids main compositions two Hydrogen bond ASN86：N-GLU87：OE1, LYS89：N-ASN86：O, it can be seen from locus and Multiple Sequence Alignment ASN86 and LYS89 are in -2 binding sites of substrate and enzyme, it is clear that 86 acid amides group and p- 2 substrate of oh group with The spatial stability structure of enzyme binding site serves important structure effect (Fig. 3 (A)).And mutant GH10-N86Q not only improves Hydrolysate composition also improves enzyme activity simultaneously, and this is probably because the position amino acid relatively enters apart from catalytic site, with crowd More amino acid has mutual left and right, and after asparagine is substituted by glutamine, the effect of its amide group is retained, While changing enzyme-to-substrate combination, the stabilization (Fig. 3 (B)) of whole enzyme molecule space conformation is maintained.

The product proportion of composing of recombinase when table 1 hydrolyzes different xylan substrates

Embodiment 3：Recombination mutation enzyme zymologic property research

Recombinase GH10-N86Q is diluted with certain proportion with optimal pH buffer solution, then respectively at 40~90 DEG C not Under synthermal, 10rnin is reacted, mutant xylanases vigor is determined, using maximum as 100%.Simultaneously with optimal pH buffer solution Enzyme liquid is diluted with certain proportion, ice-water bath terminating reaction immediately is chosen after treatment of different temperature 30min, with undressed enzyme Compare, calculate relative enzyme activity, determine temperature stability during mutant enzyme hydrolysis.

As shown in figure 4, recombinase xynGH10 and mutant enzyme GH10-N86Q optimal pH are 5.5, this be probably by It is not related to the critical sites for influenceing enzyme pH characteristics and its space structure change in the enzyme-to-substrate binding site of mutation, therefore does not lead Whole protein is caused to produce particularly evident pH characteristic changings.

GH-N86Q optimum temperature is 55 DEG C in the measure of optimum temperature as shown in Figure 5, and the unmutated bodies of xynGH10 Optimum temperature then be 60 DEG C.Unmutated body keeps the ability of enzyme activity preferable, system of the recombinase after mutation in high temperature Stability inferior is poor, it may be possible to due to destroying the space structure of enzyme after mutation.

The activity of the recombined xylanase is detected using beech xylan as substrate.In addition, examined according to xylanase activity Standard conditions are surveyed, carry out the work of the recombined xylanase with the beech glycan of final concentration of 1,2,3,4,5,6 and 8mg/ml respectively Property detection, the working concentration in recombined xylanase catalytic reaction is 1.5 μ g/ml.Using LineWeaver-Bur graphing methods, make Double (1/V-1/ [S]) curves reciprocal of enzyme activity and concentration of substrate, according to Michaelis-Menten equation, are calculated the dynamic of the recombined xylanase Mechanics parameter (Michaelis constant Km, catalytic constant kcat and catalytic efficiency kcat/Km).

As shown in table 2, mechanics parameter is urged from enzyme and carries out comparing result with the recombinase before mutation：GH10-N86Q Its affinity to substrate does not significantly change after transformation, and its reaction constant Km is not apparent from increasing compared to xynGH10, shows Its affinity is preferable, higher to the catalytic efficiency of substrate, illustrates that the mutant has good advantage in transformation, can further answer For industrial production.

The enzyme kinetic analysis of recombinase when table 2 is using beech xylan as substrate

Claims (8)

1. a kind of have hydrolyzed xylan substrate high yield specificity wood two, the xylanase improved gene of trisaccharide product, its feature It is, the amino acid sequence of the xylanase improved gene is as shown in SEQ-2.

2. xylanase improved gene as claimed in claim 1, it is characterised in that the nucleotides of the xylanase improved gene Coded sequence is as shown in SEQ-1.

A kind of 3. plasmid of the nucleotide coding sequence containing xylanase improved gene described in claims 1 or 2.

4. plasmid as claimed in claim 3, it is characterised in that the plasmid is protokaryon or eukaryon expression plasmid.

5. plasmid as claimed in claim 4, it is characterised in that the plasmid is colibacillus expression plasmid.

A kind of 6. Recombinant organism strain containing expression plasmid described in claim 5.

7. the preparation method of gene as claimed in claim 1, it is characterised in that using XYNGH10 amino acid sequences as template, by it Amino acid coding N-terminal the 86th sports glutamine.

8. right 1,2,3,4,5,6,7, in food processing, Medicines and Health Product field prepares the application of function xylo-oligosaccharide.