CN103865867A - Engineering bacteria based on extracellular Alpha-L-arabinofuranosidase and implementation method thereof - Google Patents

Abstract

The invention relates to an engineering bacteria based on extracellular Alpha-L-arabinofuranosidase and implementation method thereof, belonging to a field of bio-engineering technology. The arabinofuranosidase is prepared by cloning arabinofuranosidase gene from Streptomyces griseus JSD-1, linking the arabinofuranosidase gene with an expression vector to obtain a recombinant expression vector, further introducing the recombinant expression vector into E. coli and carrying out induced synthesis. The deficiencies of induced expression and low expression levels in prior art are overcome and a new method is provided for the industrial fermentative production of Alpha-L-Arab furan-glucosidasethe by applying gene engineering bacteria to the expression of the arabinofuranosidase prepared by the gene according to the invention.

Description

Based on engineering bacteria and its implementation of the outer α-l-arabfuranglycosidase of born of the same parents

Technical field

What the present invention relates to is gene and its implementation of a kind of technical field of biological genetic engineering, and specifically one can heterogenous expression born of the same parents colibacillus engineering and its implementation of α-l-arabfuranglycosidase (Sg-Abl) outward.

Background technology

Lignocellulose is natural high moleculer eompound the abundantest in vegitabilia, is the main dry-matter that plant produces by photosynthesis, mainly comprises Mierocrystalline cellulose, hemicellulose and xylogen.According to estimates, the lignocellulose gross dry weight that annual whole world green plants photosynthesis produces is 1,730 hundred million t, and contained total energy can reach 2 × 10 ¹⁸kJ, is equivalent to catabiotic 10 times of the annual whole world.The biological degradation of lignocellulose and unzipping are the processes of a high complexity, and it relates to the participation that numerous enzymes are.

Hemicellulose component in lignocellulose is the heterogeneous polymer being made up of several dissimilar monose, and these sugar are five-carbon sugar and hexose, comprises wood sugar, Ah 's sugar, seminose and semi-lactosi etc.Hemicellulose is mainly divided three classes: polyxylose class, poly-grape sweet dew carbohydrate and poly-semi-lactosi grape sweet dew carbohydrate, and wherein polyxylose class is to form main chain, the polysaccharide taking 4-oxygen methyl-glucopyranose aldehydic acid as side chain with Isosorbide-5-Nitrae-β-D-pyranose form wood sugar; Poly-grape sweet dew carbohydrate is to connect into main chain by D-glucopyranose base and pyranose form mannose group with Isosorbide-5-Nitrae-β type; Another semi-lactosi grape sweet dew carbohydrate of birdsing of the same feather flock together also has the form of D-galactopyranose base side chain to be connected on the some D-pyranose form mannose groups and D-glucopyranose base on this main chain with 1,6-α type.Hemicellulose accounts for 50% of total amount in lignum, and it is combined in the surface of cellulose micro-fibers, and interconnects, and these fibers have formed the interconnective network of hard cell.

The main degrading enzyme of hemicellulose has inscribe-β-1,4-zytase (Endo-β-1,4-xylanases) and β-1,4-xylosidase (β-1,4-xylosidase), in addition, the degraded of hemicellulose also needs the effect of the auxiliary enzymes such as xylan esterase, α-glucuronidase (α-glucuronidases), α-l-arabfuranglycosidase (α-L-arabinofuranosidase) and acetylase (Acetylesterase).As can be seen here, inscribe-β-Isosorbide-5-Nitrae-zytase plays important booster action in cellulosic enzyme liberating process.

At present, in many microorganisms, have been found that and clone and obtain α-l-arabfuranglycosidase.Compared with eukaryotic gene, prokaryotic gene is simple in structure, intronless, has the advantages such as easy clone, easily expression and kind are many.Ash slightly red streptomyces (Streptomyces griseus) is a kind of common soil bacteria (or actinomycetes).Ash slightly red streptomyces research emphasis is before how to transform its pathways metabolism to improve the especially fermentation yield of Streptomycin sulphate of microbiotic, to the also relative deficiency of development and utilization of other functional genes in its genome.

Compared with most of bacteriums, streptomycete has comparatively complicated growth, differentiation mechanism, and genome is also larger compared with other prokaryotic organism, can reach 8-9Mb.Generally, streptomycete has very strong decomposition to utilize ability to macromolecular polysaccharide material, as hemicellulose etc.Therefore, half-and-half cellulosic decomposition using mechanism of research streptomycete, finds brand-new arabinofuranosidase gene order and by transgenosis, goal gene is carried out to heterogenous expression, exploitation and production to novel hemicellulose zymin are significant.

Summary of the invention

The present invention is directed to prior art above shortcomings, propose a kind of engineering bacteria and its implementation based on the outer α-l-arabfuranglycosidase of born of the same parents.

The present invention is achieved by the following technical solutions:

The present invention relates to a kind of engineering bacteria based on the outer α-l-arabfuranglycosidase of born of the same parents, can the heterogenous expression born of the same parents DH5 α intestinal bacteria of α-l-arabfuranglycosidase outward.

Described engineering bacteria builds and obtains in the following manner:

1) designing and synthesizing PCR primer, from slightly red streptomyces JSD-1(CGMCC NO.5706 of ash) genomic dna is that template is carried out pcr amplification;

Described PCR primer refers to the primer that contains Nde I and EcoR I restriction enzyme site, specifically comprises:

Forward primer Abl-Nde I-F:

5'-GGAATTCCATATGGCGACCGTGGACACGAACGCCTCGTAC-3'

Reverse primer Abl-EcoR I-R:

5'-GGAATTCTCAGCGCCGCAGCGTCAGCAGACC-3'

Described pcr amplification adopts PrimeSTAR GXL high-fidelity enzyme (TaKaRa) to increase, and pcr amplification condition is: 98 DEG C of denaturation 3min; 98 DEG C of distortion 10s, 68 DEG C are extended 1min; 30 rear 68 DEG C of whole 3min that extend of circulation.

2) build the plasmid with Sg-Abl gene: the pcr amplification product that step 1 is obtained is cut after glue reclaims and is connected to cloning vector, and imports DH5 α competent escherichia coli cell; Picking has the clone of corresponding resistant and identifies by bacterium colony PCR, until obtain after positive colony picking and shake bacterium and extract and obtain pET-41a plasmid.

Described cloning vector adopts pMD ^tM19-T Vector.

3) build the expression vector with Sg-Abl gene, concrete steps comprise:

3.1) after the cloned plasmids that contains Sg-Abl gene with Nde I and the processing of EcoR I double digestion, reclaim by agarose gel electrophoresis the fragment that contains outer α-l-arabfuranglycosidase (Sg-Abl) gene of β-Isosorbide-5-Nitrae-born of the same parents;

3.2) process pET-41a plasmid with Nde I and EcoR I restriction endonuclease, and reclaim carrier segments by agarose gel electrophoresis;

3.3) DNA fragmentation of twice recovery is mixed, the connection of spending the night under the effect of T4DNA Ligase, then imports DH5 α competent escherichia coli cell, and picking positive colony enlarged culturing are extracted its plasmid, obtains the expression vector that contains Sg-Abl gene.

4) expression vector is converted into intestinal bacteria: the expression vector plasmid that contains Sg-Abl gene obtaining in step 3 is proceeded in BL21 (DE3) competent cell.Screen acquisition positive colony by thering is the LB liquid nutrient medium of kalamycin resistance, contain the engineering bacteria of Sg-Abl gene.

Described β-1, the outer α-l-arabfuranglycosidase of 4-born of the same parents, its original amino acid is as SEQ ID NO.3,490 amino acid of encoding altogether, the maturation protein excising after front 35 amino acid signal peptide sequences is 455 amino acid, molecular weight is 49.9KD, as SEQ ID NO.4 (adding initial amino acid M totally 456 amino acid).

Described Sg-Abl gene, its parent nucleotide sequence is as shown in SEQ ID NO.1, its length is 1473 base pairs, 105 amino acid corresponding to Nucleotide after ripening albumen corresponding to excision coded signal peptide are 1368 Nucleotide, as SEQ IDNO.2 (adding initiator codon ATG totally 1371 Nucleotide).

Brief description of the drawings

Fig. 1 goal gene sequence signal peptide analysis figure.

Fig. 2 goal gene pcr amplification result electrophorogram.

The cloning vector restriction enzyme digestion and electrophoresis figure that Fig. 3 contains goal gene.

The expression vector restriction enzyme digestion and electrophoresis figure that Fig. 4 contains goal gene.

Fig. 5 recombinant bacterium inducible protein SDS-PAGE electrophorogram.

Embodiment

Below embodiments of the invention are elaborated, the present embodiment is implemented under taking technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.

Embodiment 1

Ash is the cultivation of red streptomyces (Streptomyces griseus) JSD-1 slightly

Ash slightly red streptomyces (Streptomyces griseus) separates the soil that rots from Pujiang town, Shanghai, and deposit number is CGMCC No.5706; This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center Institute of Microorganism, Academia Sinica (address: No. 3 100101, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on January 9th, 2012

This bacterium is inoculated in LB liquid nutrient medium, cultivates 60h for 32 DEG C, centrifugal collection thalline also extracts the genomic dna of bacterium.

Described LB liquid nutrient medium component is: peptone 10g, yeast extract 5g, NaCl5g, deionized water 1L, pH6.8-7.2.

Embodiment 2

Ash is the clone of red streptomyces arabinofuranosidase (Sg-Abl) gene slightly

By to ash slightly red streptomyces arabinofuranosidase gene order carry out signal peptide sequence analysis (as Fig. 1), the primer that contains Nde I and EcoR I restriction enzyme site with the two ends design of the coding gene sequence of maturation protein is as follows:

Forward primer Abl-Nde I-F:

5'-GGAATTCCATATGGCGACCGTGGACACGAACGCCTCGTAC-3'

Reverse primer Abl-EcoR I-R:

5'-GGAATTCTCAGCGCCGCAGCGTCAGCAGACC-3'

Taking ash, slightly red streptomyces JSD-1 genomic dna is as template, and employing PrimeSTAR GXL high-fidelity enzyme (TaKaRa) increases.Pcr amplification condition is: 98 DEG C of denaturation 3min; 98 DEG C of distortion 10s, 68 DEG C are extended 1min; 30 rear 68 DEG C of whole 3min that extend of circulation.

Embodiment 3

The structure that contains arabinofuranosidase (Sg-Abl) gene clone carrier

Pcr amplification product is carried out to electrophoresis detection, and result shows to obtain fragment and is about 1.4Kb (seeing Fig. 2); Then, after PCR product being cut to glue and being reclaimed, add A by A-Tailing Kit (TaKaRa) and react and be connected to pMD ^tM19-T Vector (TaKaRa), imports DH5 α competent escherichia coli cell subsequently.

Picking has the clone of corresponding resistant, and by bacterium colony, PCR identifies, until obtain positive colony.Picking positive colony, shakes bacterium and extracts and serve the biological company limited of extra large Sani after its plasmid and check order.

Embodiment 4

The structure that contains arabinofuranosidase (Sg-Abl) expression vector

Choose the plasmid that sequence is correct, with reclaiming by agarose gel electrophoresis the fragment (as Fig. 3) that contains arabinofuranosidase (Sg-Abl) gene after Nde I and EcoR I double digestion; Equally, with Nde I and EcoR I restriction endonuclease processing pET-41a expression vector, and reclaim larger carrier segments by agarose gel electrophoresis.

The DNA fragmentation of twice recovery is mixed, and the connection of spending the night under the effect of T4DNA Ligase, then imports DH5 α competent escherichia coli cell.Picking positive colony, enlarged culturing is extracted its plasmid and is just obtained and contain arabinofuranosidase (Sg-Abl) expression vector.Detect (as Fig. 4) by agarose gel electrophoresis with Nde I and EcoR I double digestion, confirm that vector construction is errorless.

Embodiment 5

The structure that contains arabinofuranosidase (Sg-Abl) genetic expression bacterial strain

To contain arabinofuranosidase (Sg-Abl) expression vector plasmid proceeds in BL21 (DE3) competent cell.Screen and obtain positive colony by the LB solid medium that contains that microbiotic of card (25 μ g/mL).The positive colony obtaining is the expression strain for containing arabinofuranosidase (Sg-Abl) gene just.

Embodiment 6

The heterogenous expression of arabinofuranosidase (Sg-Abl)

Picking positive colony is also inoculated in the LB liquid nutrient medium that contains kantlex (concentration is 25 μ g/mL), 32 DEG C, 150rpm concussion cultivation 2-3h, in the time that OD600 reaches 0.5-0.6, add IPTG that IPTG concentration in fermented liquid is reached for 0.1mM, 28 DEG C, 140rpm continue to cultivate and carry out abduction delivering in 8 hours, and get respectively blank (before induction), 2,4,6,8 hours samples.Treat that abduction delivering completes, centrifugal collection thalline, with broken born of the same parents' damping fluid washing thalline once, use again after the resuspended thalline of 1 × PBS damping fluid of 1/10 fermentating liquid volume ultrasonic disruption to bacterium liquid under condition of ice bath to clarify, the centrifugal 10min of 13000rpm/min collects supernatant, and supernatant liquor is the crude enzyme liquid of arabinofuranosidase.

Embodiment 7

The SDS-PAGE of recombinant bacterium abduction delivering arabinofuranosidase crude enzyme liquid

Preparation 12%SDS-PAGE glue, crude enzyme liquid is mixed with 5 × SDS-PAGE Loading Buffer ratio, with boiling water bath 10min, each point sample hole loading 10 μ L, 120V electrophoresis 10min heightens voltage to 160V after band enters separation gel, electrophoresis 65-75min, until tetrabromophenol sulfonphthalein instruction band is run out of glue completely.Stop immediately electrophoresis, with the dyeing of spending the night of coomassie brilliant blue R_250 solution, then wash with destainer, until background colour sloughs completely, band high-visible (as Fig. 5).

Described 12%SDS-PAGE is made up of concentrated glue and separation gel, and its component is respectively:

Separation gel: distilled water 1.6mL, 30% acrylamide soln 2.0mL, 1.5M Tris-HCl (pH8.8) 1.3mL, 10% ammonium persulphate (APS) 0.05mL, 10%SDS solution 0.05mL, TEMED0.002mL;

Concentrated glue: distilled water 0.68mL, 30% acrylamide soln 0.17mL, 1.0M Tris-HCl (pH6.8) 0.13mL, 10% ammonium persulphate (APS) 0.01mL, 10%SDS solution 0.01mL, TEMED0.001mL;

Described 5 × SDS-PAGE Loading Buffer component is: 1M Tris-HCl (pH6.8) 1.25mL, and SDS0.5g, tetrabromophenol sulfonphthalein 25mg, glycerine 2.5mL, deionized water is settled to 5mL.After aliquot packing (500 μ L) and room temperature preservation, before using, every aliquot adds beta-mercaptoethanol 25 μ L.

Described coomassie brilliant blue R_250 solution component is: coomassie brilliant blue R_250 1g, Virahol 250mL, Glacial acetic acid 100mL, deionized water 650mL.

Described destainer component is: Glacial acetic acid 100mL, dehydrated alcohol 50mL, deionized water 850mL.

Claims (5)

1. the engineering bacteria based on the outer α-l-arabfuranglycosidase of born of the same parents, is characterized in that, this project bacterium is the DH5 α intestinal bacteria of the outer α-l-arabfuranglycosidase of heterogenous expression born of the same parents;

The outer α-l-arabfuranglycosidase of described born of the same parents is specially the outer α-l-arabfuranglycosidase of β-Isosorbide-5-Nitrae-born of the same parents, and its aminoacid sequence is as shown in SEQ ID NO.4;

Described β-1, the outer α-l-arabfuranglycosidase of 4-born of the same parents has Sg-Abl gene, its nucleotide sequence is as shown in SEQ ID NO.2, its length is 1473 base pairs, 490 amino acid of encoding altogether, the maturation protein excising after front 35 amino acid signal peptide sequences adds initial amino acid M totally 456 amino acid, and molecular weight is 49.9KD.

2. the implementation method of engineering bacteria according to claim 1, is characterized in that, comprises the following steps:

1) designing and synthesizing PCR primer, from slightly red streptomyces JSD-1(CGMCC NO.5706 of ash) genomic dna is that template is carried out pcr amplification;

Forward primer Abl-Nde I-F:

5'-GGAATTCCATATGGCGACCGTGGACACGAACGCCTCGTAC-3'

Reverse primer Abl-EcoR I-R:

5'-GGAATTCTCAGCGCCGCAGCGTCAGCAGACC-3'

2) build the plasmid with Sg-Abl gene: the pcr amplification product that step 1 is obtained is cut after glue reclaims and is connected to cloning vector, and imports DH5 α competent escherichia coli cell; Picking has the clone of corresponding resistant and identifies by bacterium colony PCR, until obtain after positive colony picking and shake bacterium and extract and obtain pET-41a plasmid.

3) build the expression vector with Sg-Abl gene;

4) expression vector is converted into intestinal bacteria: the expression vector plasmid that contains Sg-Abl gene obtaining in step 3 is proceeded in BL21 competent cell.Screen acquisition positive colony by thering is the LB liquid nutrient medium of kalamycin resistance, contain the engineering bacteria of Sg-Abl gene.

3. method according to claim 2, is characterized in that, described pcr amplification adopts PrimeSTAR GXL high-fidelity enzyme to increase, and pcr amplification condition is: 98 DEG C of denaturation 3min; 98 DEG C of distortion 10s, 68 DEG C are extended 1min; 30 rear 68 DEG C of whole 3min that extend of circulation.

4. method according to claim 2, is characterized in that, described step 3 specifically comprises:

3.1) after the cloned plasmids that contains Sg-Abl gene with Nde I and the processing of EcoR I double digestion, reclaim by agarose gel electrophoresis the fragment that contains Sg-Abl gene;

3.2) process pET-41a plasmid with Nde I and EcoR I restriction endonuclease, and reclaim carrier segments by agarose gel electrophoresis;

3.3) DNA fragmentation of twice recovery is mixed, the connection of spending the night under the effect of T4DNA Ligase, then imports DH5 α competent escherichia coli cell, and picking positive colony enlarged culturing are extracted its plasmid, obtains the expression vector that contains Sg-Abl gene.

5. method according to claim 2, is characterized in that, described cloning vector adopts pMD ^tM19-T Vector.

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