CN107354165B - 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 present invention provides the applications that there is a kind of coding the special wood two of hydrolyzed xylan substrate high yield, the improvement gene of zytase of trisaccharide product its Recombinant organism to prepare xylo-oligosaccharide.86 asparagine mutations of zytase its N-terminal amino acid sequence after improvement are glutamine.Recombinase XYNGH10 zymologic property are as follows: optimal pH 5.5,65 DEG C of optimum temperature, enzyme activity 160U/ml, Xylose Content is greater than 14% in product when hydrolyzed xylan substrate；And construct mutant GH10-N86Q hydrolyzed xylan substrate when product in Xylose Content be below 2% hereinafter, and hydrolysate 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 enzyme activity is to improve 25.6% before 201U/ml is relatively transformed.There is its Recombinant organism of improvement gene of the invention good utilize 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

The invention belongs to genetic engineering and protein engineering field be related to it is a kind of from streptomycete (Streptomyces sp. L10608) with 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 technique

China is a large agricultural country, along with the production and processing of agricultural product, generates a large amount 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 hot spot for becoming the research of nearly more than ten years various countries.It is rich in agricultural wastes Containing cellulose and hemicellulose, wherein hemicellulose accounts 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.Since structure is complicated for xylan, Its thorough hydrolysis needs a series of synergistic effect of enzymes, and wherein the enzyme of most critical is zytase.Zytase category hydrolase Class has played irreplaceable function during carbon cycle, has important potential using value, has been successfully applied at present The fields such as food, feed, brewing, papermaking, 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 be by 2 ~ 7 xylose molecules with glucosides key connection at functional polymerization sugar, wherein wooden two, xylotriose For 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 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 beverage, Sugar-free low energy product in the food such as candy, cake, ice cream, dairy products and flavouring, pharmaceuticals industry, to supply obesity Disease, diabetes, hypertension, artery sclerosis, eurodonticus are edible.At present because there is biological enzyme environmental-friendly, product to be easy to control The advantages that system, good product specificity, is increasingly becoming the main method using agricultural wastes production xylo-oligosaccharide.And current enzyme process The specific components content such as wood two, xylotriose is big in product when producing the microbe-derived xylanase hydrolysis xylan used Mostly lower than 30% and containing monosaccharide and other non-functional ingredients such as a large amount of xyloses, arabinose, the later period need more complex technique and Great number at being purified originally, to improve function xylo-oligosaccharide purity (60% ~ 70%).The only xylan of only a few bacterial strain production When enzyme hydrolysis substrate, wood two, xylotriose ratio have been more than 50% in product.Therefore it is improved in the production of Enzymatic xylo-oligosaccharide The special functional component of xylobiose, xylotriose in catalytic process is eliminated and is reduced generation xylose, xylo-oligosaccharide is avoided further to change It is a critical issue urgently to be resolved for xylose.Significantly improve containing for specific function ingredient during Production by Enzymes xylo-oligosaccharide Amount will reduce Production by Enzymes cost, reduce energy consumption, sewage discharge etc. during later-period purification, reduce environmental pollution, realize high Effect produces the large-scale industrial production application of high added value xylo-oligosaccharide using low value agricultural wastes.

Summary of the invention

The first purpose of this invention obtains a kind of with hydrolyzed xylan substrate high yield special wooden two, 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), nucleotide sequences as shown in SEQ-1, Its amino acid sequence is as shown in SEQ-2.

The present invention obtains a kind of xylanase improved base with the special wood two of hydrolyzed xylan substrate high yield, trisaccharide product Cause.The zytase source (XYNGH10) and streptomycete as improvement basisStreptomyces sp. L10608(China is micro- The common micro- raw center of biological inoculum preservation administration committee, deposit number CGMCC No. 13271, address: Chaoyang District, Beijing City The institute 3 of North Star 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 86 asparagines of original zytase N-terminal amino acid sequence Sport glutamine.

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

The present invention also provides the recombination bacillus coli genetic engineerings containing original xylanase gene and the improvement gene Bacterial strain.The recombinase XYNGH10 zymologic property of Bacillus coli expression are as follows: optimal pH 5.5,65 DEG C of optimum temperature, enzyme activity 160U/ ML, Xylose Content is greater than 14% in product when hydrolyzed xylan substrate；And the mutant GH10-N86Q hydrolyzed xylan constructed When substrate in product Xylose Content be below 2% hereinafter, and hydrolysate to have the xylobiose of stronger beneficial function and xylotriose group It is divided into master.Mutant enzyme optimal pH 5.5,55 DEG C of optimum temperature, enzyme activity is to improve 25.6% before 201 U/mL are relatively transformed.

Detailed description of the invention

Fig. 1xynGH10The mutational site schematic diagram of selection.

Fig. 2 Overlap extension PCR rite-directed mutagenesis splices sequence.M:marker；Road 1,2 is that parallel N86Q splices sequence

86 amino acids active force prediction of recombinase N-terminal before and after Fig. 3 site mutation.(A) preceding 86 asparagines are mutated to make It firmly predicts, 86 glutamine active force predictions after (B) mutation.

The optimal pH of Fig. 4 recombinase.

The optimum temperature of Fig. 5 recombinase.

Specific embodiment

The present invention is described further below in conjunction with specific example, but is not the limitation present invention.It is right with reference to the accompanying drawing The present invention further illustrates.Test method without specific conditions in embodiment, usually can routinely item, such as J. Pehanorm cloth Shandong Condition described in " the Molecular Cloning:A Laboratory guide third edition " that gram (Sambrook) etc. writes, or according to kit manufacturer Proposed condition carries out.Those skill in the art related can more fully understand and grasp the present invention by embodiment.

1. general reagent: Congo red；Kanamycins (U.S. AMRESCO)；Agarose (Spain Biowest)；Ammonia benzyl is green Mycin (U.S. AMRESCO)；Birch xylan (U.S. Sigma)；LB solid medium；The Congo red screening flat board of xylan-； NaCl eluent: 1 M；Oat xylan and Corncob Xylan (VETEC)；Xylose mark product (xylose, xylobiose, xylotriose, wood Tetrose) Chinese medicines group chemical reagent；LB solid medium: 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)；Plastic recovery kit (OMEGA, beauty State)；La Taq archaeal dna polymerase (with GC buffer)；Restriction enzymeEcoRI、XhoI；1kb DNA Ladder Marker (Japanese TAKARA)；La Taq archaeal dna polymerase (with GC buffer) (TAKARA, Japan)；200bp DNA Ladder (U.S. Biomega)；PCR amplification primer (the prosperous synthesis of Beijing AudioCodes)；The U.S. 200bp DNA ladder( Biomega)；1kbDNALadderMarker (Japanese TAKARA)；

3. bacterial strain: streptomycete (Streptomyces sp.) L10608；Escherichia coli DH5a and Transtetta (DE3) competent cell is purchased from Quan Shi King Company；

4. carrier: carrier T: pMD18-T is purchased from TAKARA company；PET-28a (+) is purchased from Merk company；

5. primer: carrier T sequencing: M13+ sequence: 5 '-GTTTTCCCAGTCACGAC-3 ', M13- sequence: 5 '- CAGGAAACAGCTATGAC-3'；

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

The building of 1 mutant of embodiment and prokaryotic expression

Obtain target genexynGH10Afterwards, it usesNcoRIWithXhoISimultaneously to expression vector pET28a and with restriction enzyme site 'sxynGH10Carry out double digestion, 37 DEG C of reaction 3h.Digestion passes through 1%(m/v later) agarose gel electrophoresis detect and cuts glue time Take-up toughness endxynGH10With linearisation pET28a.Thereafter with T4DNA ligase by recycling with cohesive endxynGH101:7 is placed in reaction system and connects in molar ratio with linear pET28a, 1h is reacted in 25 DEG C, to construct recombination matter Grain pET28a- xynGH10。

It has been connected to genexynGH10PET28a plasmid as mutation raw material.Using the side of Overlap extension PCR Method carries out Amino Acid-Induced Site-Directed Mutation with the rite-directed mutagenesis primer of designed the 86th amino acids of N-terminal.Obtain target genexynGH10-N86QAfterwards, it usesNcoRIWithXhoISimultaneously to expression vector pET28a and with restriction enzyme siteGH10-N86QIt carries out Double digestion, 37 DEG C of reaction 3h.Digestion gel extractionxynGH10-N86QWith linearisation pET28a.Thereafter with connection building recombination matter Grain pET28a-xynGH10-N86Q.By recombinant plasmid pET28a-xynGH10-N86QMutant nucleotide sequence imports competence BL21 (DE3), the verifying of mutant nucleotide sequence is then carried out.Position of the selected mutational site in complete sequence known in Fig. 1, N86 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 Two bar segments are extended to form 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 reaction pH is 5.5, and optimum temperature is 65 DEG C, and enzyme activity is measured as 160U/ through DNS method mL.And measuring Xylanase activity through DNS method into the mutation recombinase GH10-N86Q for crossing verifying is 201U/mL.To the weight of transformation For group zymoprotein, guarantee that its biological activity is to carry out the basis of practical application, and mutant GH10-N86Q is by 86 days Winter amide sports the active force that the non-covalent bond in protoenzyme protein molecular has been still kept after glutamine, on certain basis On still maintain space conformation.

2 recombination mutation enzyme hydrolysis substrate of embodiment generates the research of specific product property

Building mutant enzyme first hydrolyzes substrate system.It configures 1mL reaction system: being 20mg/mL's including 500 μ L concentration Make water-insoluble Corncob Xylan, Corncob Xylan (VETEC), oat xylan (Biotopped) etc. by oneself or not laboratory Same substrate, the enzyme solution of 10U complement to 1mL with NaAc_HAc buffer solution (pH=5.5).By configured 1mL reaction system In 55 DEG C of water-bath heating in water bath for reaction 12h, 0.22 μm of water phase film injection sample injection bottle is medium to be detected.The detector bar of hydrolysate Part are as follows: amino acid analysis column, mobile phase are acetonitrile: water 70:30, flow velocity 0.8mL/min, show poor folding by 30 DEG C of column oven temperature 30 DEG C of photodetector temperature, 20 μ L of sample volume.

It is soluble in water that xylose, xylobiose, xylotriose, Xylotetrose standard items are drawn respectively, are successively diluted to concentration are as follows: The standard solution mark of 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL make quasi- working curve, with 0.45 μm filtering with microporous membrane is to get xylose, xylobiose, xylotriose, Xylotetrose standard liquid series.Take 20 μ L standard series molten respectively Sample introduction is analyzed for liquid, and using xylose, xylobiose, xylotriose, Xylotetrose concentration as abscissa, peak area is ordinate, draws xylose, wood Disaccharides, xylotriose, Xylotetrose peak area-concentration standard curve.

As shown in table 1, it selects using the xylan not of the same race such as beech, oat and birch as substrate, verifying recombination mutation enzyme Hydrolysis properties and its industrial circle application a possibility that.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 hydrolysate 2.49%, and the content of the wooden xylo-oligosaccharides such as three and Xylotetrose also increases significantly, respectively 56.53% and 15.97%.With GH10-N86Q still maintains higher xylo-oligosaccharide yield when oat xylan is substrate, produces after original XYNGH10 hydrolysis substrate Xylose Content is 14.20% in object, and the accounting of xylose is remarkably decreased only in improved GH10-N86Q hydrolysate 1.45%, and the content of the wooden xylo-oligosaccharides such as three and Xylotetrose also increases significantly, respectively 52.76% and 24.22%.With When birch xylan is hydrolysis substrate, the Xylose Content in hydrolysate is equally aobvious from 21.23% in original XYNGH10 product Write 1.01% fallen in GH10-N86Q, the content of the wooden xylo-oligosaccharides such as three and Xylotetrose also significantly improve as 61.89% and 11.42%。

The case where different xylans generate product comprehensive analysis: original recombinase is hydrolyzed according to mutant enzyme GH10-N86Q The hydrolysis feature of xynGH10 be in product based on xylose and xylobiose, have very strong hydrolysing activity to xylotriose substrate, lead It causes to generate a large amount of wooden monosaccharide in product.And improved mutant GH10-N86Q be applied to hydrolyzed xylan when, maintain Good hydrolysis properties, predominantly xylo-oligosaccharide and Xylose Content is below 2% hereinafter, also maintaining simultaneously higher in product Catalytic activity, activity are the 125.6% of xynGH10.The mutant has the good agriculture waste for utilizing and being rich in hemicellulose Object Production by Enzymes xylo-oligosaccharide industrial production application prospect.

The space structure discovery of 86 zymoprotein mutant of N-terminal, 86 amino acids main compositions two are analyzed by software Hydrogen bond ASN86:N-GLU87:OE1, LYS89:N-ASN86:O can see ASN86 from spatial position and Multiple Sequence Alignment - 2 binding sites of substrate and enzyme are in LYS89, it is clear that 86 amide group and p- 2 substrate of hydroxyl group and enzyme knot The spatial stability building of coincidence point plays the role of important building (Fig. 3 (A)).And mutant GH10-N86Q not only improves water Solution product composition also improves enzyme activity simultaneously, this may be apart from catalytic site due to the position amino acid compared with into and numerous Amino acid has mutual left and right, and after asparagine is substituted by glutamine, the effect of amide group is retained, and is changing While enzyme-to-substrate combination, the stabilization (Fig. 3 (B)) of entire enzyme molecule space conformation is maintained.

The product composition ratio of recombinase when table 1 hydrolyzes different xylan substrates

。

3 recombination mutation enzyme zymologic property research of embodiment

Recombinase GH10-N86Q is diluted with certain proportion with optimal pH buffer, then respectively in 40 ~ 90 °C of difference At a temperature of, 10min is reacted, mutant xylanases vigor is measured, with maximum value for 100%.Simultaneously with optimal pH buffer by enzyme Liquid is diluted with certain proportion, the reaction of ice-water bath termination immediately after treatment of different temperature 30min is chosen, compared with untreated enzyme Compared with the opposite enzyme activity of calculating measures temperature stability when mutant enzyme hydrolysis.

As shown in figure 4, the optimal pH of recombinase xynGH10 and mutant enzyme GH10-N86Q are 5.5, this may be by It is not related to influencing the critical sites of enzyme pH characteristic and its space structure variation in the enzyme-to-substrate binding site of mutation, therefore does not lead Entire protein is caused to generate particularly evident pH characteristic changing.

The optimum temperature of GH-N86Q is 55 DEG C in the measurement of optimum temperature as shown in Figure 5, and the unmutated body of xynGH10 Optimum temperature be then 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, being 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, recombined xylanase catalysis reaction in working concentration be 1.5 μ g/mL.Using LineWeaver-Bur graphing method, make The recombined xylanase is calculated according to Michaelis-Menten equation in double (1 V-1 []) curves reciprocal of enzyme activity and concentration of substrate Kinetic parameter (Michaelis constantKm, catalytic constantkcatAnd catalytic efficiencykcat/Km).

As shown in table 2, the recombinase before mechanics parameter and mutation are urged from enzyme compares result: GH10-N86Q It does not significantly change the affinity of substrate after transformation, and its reaction constantKmIt is not apparent from increase compared to xynGH10, is shown 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 zytase with hydrolyzed xylan substrate high yield specificity wood two, trisaccharide product, which is characterized in that the wood The amino acid sequence of dextranase is as shown in SEQ-2.

2. zytase as described in claim 1, which is characterized in that the nucleotide coding sequence of the zytase such as SEQ-1 It is shown.

3. a kind of plasmid of the nucleotide coding sequence containing zytase described in claims 1 or 2.

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

5. plasmid as claimed in claim 4, which is characterized in that the plasmid is colibacillus expression plasmid.

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

7. the preparation method of zytase as described in claim 1, which is characterized in that using XYNGH10 amino acid sequence as template, By its amino acid coding N-terminal, the 86th sports glutamine；The amino acid sequence of XYNGH10 are as follows:

MGIQALPRAAVRQKLRTPLPALAAGVLGLTAALVPPTNADAAESTLGAAAAQSGRYFGVAIASGKLGDSTY TSIANREFNSVTAENEMKIDATEPNRGQFNFSSADRVYNRAVQNGKQVRGHTLAWHSQQPGWMQSLSGSSLRQAMI DHINGVMNHYKGKIAQWDVVNEAFADGSSGARRDSNLQRTGNDWTEVAFRTARAADPSAKLCYNDYNVENWNWAKT QAMYNMVKDFKSRGVPIGCVGFQSHFNSGSPYDSNFRTTLQNFAALGVDVAVTELDIQGASSSTYAAVVNDCLAVS RCLGVTVWGVRDSDSWRASDTPLLFNNDGSKKAAYSAVLNALNGGTTTPPPTGDGGQIKGVASGRCLDVPNASTTD GAGVQLYDCHSNSNQQWAVTDSGEIRVYGNKCLDAAGTGNGASVQIYSCWGGDNQKWRLNSDGSIVGVQSGRCLDA AGSGNGARIQLYACSGGSNQRWTRT*；

The XYNGH10 derives from streptomyceteStreptomyces sp. L10608, deposit number CGMCC No. 13271.

8. zytase as claimed in claim 1 or 2, or the plasmid as described in claim 3,4 or 5, or such as claim 6 The engineered strain is in food processing or the application of Medicines and Health Product field preparation function xylo-oligosaccharide.