CN103667208B - A kind of low temperature inscribe β-2,6-levanase LevAGN25 and encoding gene thereof - Google Patents
A kind of low temperature inscribe β-2,6-levanase LevAGN25 and encoding gene thereof Download PDFInfo
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- CN103667208B CN103667208B CN201310711448.XA CN201310711448A CN103667208B CN 103667208 B CN103667208 B CN 103667208B CN 201310711448 A CN201310711448 A CN 201310711448A CN 103667208 B CN103667208 B CN 103667208B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01064—2,6-Beta-fructan 6-levanbiohydrolase (3.2.1.64)
Abstract
The present invention relates to a kind of levanase and encoding gene thereof.Levanase of the present invention has following character: optimal pH 6.0; Optimum temperuture 55 DEG C, the enzyme respectively at 10 DEG C and 20 DEG C with 13.5% and 32.6% is lived; At 37 DEG C, process 60min through 0.1M pH5.0 – 10.0 damping fluid, still can keep the activity of more than 70%; When 50 DEG C, the transformation period is greater than 60min; Inscribe Levan generates Nutriflora P.Levanase of the present invention can be applicable to food, aquatic feeds and medical industry.
Description
Technical field
The invention belongs to gene engineering technology field, particularly relate to a kind of low temperature inscribe β-2,6-levanase LevAGN25 and encoding gene thereof.
Background technology
Polylevulosan (Fructan) is the saccharan be formed by connecting by β-2,6 glycosidic link or β-2,1 glycosidic link by fructose.The Polylevulosan be formed by connecting by β-2,6 glycosidic link is called Levan, is mainly present in monocotyledons, as the exocellular polysaccharide of thimothy grass and bacterium, as plaque forms relevant bacterium Streptococcus salivarius and Actinomyces viscosus etc.; Levan can be used as the moiety of bacterial biof iotalm, helps bacterial adhesion, surely grows and resist microbiotic etc., causes to get rusty, pollute and the problem such as plaque.Inulin (Inulin) is a kind of Polylevulosan be formed by connecting by β-2,1 glycosidic link, is mainly present in dicotyledons, as in the root of the plants such as jerusalem artichoke, witloof, Garden Dahlia, burdock and yacon and stem.The Polylevulosan be simultaneously formed by connecting by β-2,1 and β-2,6 glycosidic link is called Graminan, is present in many higher plants, as (Han, Adv Appl Microbiol, 1990,35:171 – 194 in wheat, barley and oat; Rozen et al., FEMSMicrobiol Lett, 2001,195:205 – 210).
Glycoside hydrolase the 32nd family comprises endoinulase, exoinulinase, sucrase and inscribe β-2,6-levanase (Endolevanase) (Finn et al.Nucleic Acids Res, 2008,36:D281 – D288).Inscribe β-2,6-levanase, from Levan internal random hydrolysis β-2,6 glycosidic link, can be applicable in the industries such as feed, food and medical treatment.In feedstuff industry, inscribe β-2,6-levanase can assist cultivated animals hydrolyzing plant, as β-2, the 6 glucosides of bonding Polylevulosan in thimothy grass, promotes that animal is to the digestion of nutritive substance, improves efficiency of feed utilization; In food service industry, inscribe β-2,6-levanase can be hydrolyzed β-2,6 glucosides of bonding Polylevulosan and generate Nutriflora P, destroys the formation of bacterial biof iotalm and then reduce bacterial contamination and the reagent constituents etc. as mensuration Polylevulosan type; In medical industry, inscribe β-2,6-levanase can prevent bacterium formed microbial film, and then control plaque or bacterium to the adhesion of medicine equipment and pollution; In addition, inscribe β-2,6-levanase also can be used as additive application (U.S.Pat.No.6524827) in cleaning supplies.
The enzyme with low temperature active can be applicable to low temperature habitat and machining at low temperature process, as the food-processing under low temperature, medicine equipment cleaning and the feeding enzyme field of aquatic products.The course of processing under low temperature can prevent microbiological contamination, nutritive loss and Food Quality from reducing, transfer the process of middle temperature or high temperature process to effect (Zhou et al. that machining at low temperature process also can play reduction energy consumption, J Biosci Bioeng, 2012,113:568 – 574).
Summary of the invention
Object of the present invention the object of this invention is to provide a kind of low temperature inscribe β-2,6-levanase LevAGN25.
Another object of the present invention is to provide the gene of above-mentioned levanase of encoding.
Another object of the present invention is to provide the carrier comprising said gene.
Another object of the present invention is to provide the bacterial strain comprising said gene.
Another object of the present invention is to provide the host cell comprising said gene.
Levanase of the present invention is inscribe β-2,6-levanase LevAGN25, Sphingobacterium (Sphingobacterium sp.) can be derived from, its aminoacid sequence as shown in SEQ ID NO.1 or by have the aminoacid sequence shown in SEQ ID NO:1 modified, replace, lack or add one or several amino acid obtain.Wherein, described modification comprise amidation, phosphorylation, methylate, acetylize, ubiquitination, glycosylation or carbonylation.
Inscribe β-2,6-levanase LevAGN25 of the present invention is altogether containing 501 amino acid, and theoretical molecular is 56.4kDa.The optimum pH of this enzyme is 6.0, maintains the enzymic activity of more than 50% in the scope of pH5.0 – 7.0; Through the damping fluid process 1h of pH5.0 – 10.0, this enzyme enzyme residue alive reaches more than 70%; This enzyme optimum temperuture is 55 DEG C, and the enzyme respectively 10 DEG C and 20 DEG C with 13.5% and 32.6% is lived, and the enzyme at 37 DEG C with about 75% is lived; When 50 DEG C, the transformation period of this enzyme is greater than 60min, and when 60 DEG C, the transformation period is about 10min; At pH6.0 and 55 DEG C, this enzyme is to 0.5%(w/v) specific activity of Levan is 219.5 ± 4.2Umg
-1, and inulin, sucrose, Zulkovsky starch, raffinose (Raffinose), stachyose (Stachyose), birch xylan (Birchwood xylan), beech wood glycan (Beechwood xylan), 4-O-methyl-d-glucurono-d-xylan, araboxylan (Wheat flour arabinoxylan), p-nitrophenyl-β-d-xylopyranoside and p-nitrophenyl-α-l-arabinofuranoside are all lived without enzyme; Inscribe Levan generates Nutriflora P.
The invention provides the above-mentioned inscribe β-2 of coding, the gene levAGN25 of 6-levanase LevAGN25, this gene order as shown in SEQ ID NO.2 or modified by the nucleotide sequence shown in SEQ ID NO.2, replace, lack or add one or several base obtain.
The present invention passes through the method separating clone of PCR the encoding gene levAGN25 of inscribe β-2,6-levanase LevAGN25, its total length 1506bp, and initiation codon is ATG, and termination codon is TAA.Through BLAST comparison, in this inscribe β-2,6-levanase LevAGN25 and GenBank, the glycoside hydrolase the 32nd family protein (WP_019540151) in Proteiniphilumacetatigenes source has the highest Amino acid sequence identity, is 66.1%.The protein-active that this Proteiniphilum acetatigenes originates also is not studied, just range glycoside hydrolase the 32nd family protein by sequence similarity, and glycoside hydrolase the 32nd family comprises endoinulase, exoinulinase, sucrase and inscribe β-2,6-levanase, so the activity not deriving LevAGN25 by this Proteiniphilum acetatigenes derived Protein.Above BLAST comparison result illustrates that this inscribe β-2,6-levanase LevAGN25 is a kind of new levanase.
Present invention also offers the recombinant vectors comprising above-mentioned inscribe β-2,6-levanase gene levAGN25, be preferably pEasy-E1-levAGN25.Inscribe β-2,6-levanase gene of the present invention is inserted in expression vector, its nucleotide sequence is connected with expression regulation sequence.As the most preferred embodiment of the present invention, inscribe β-2,6-levanase gene of the present invention is connected by T-A mode with expression vector pEasy-E1, obtains expression of recombinant e. coli plasmid pEasy-E1-levAGN25.
Present invention also offers the recombinant bacterial strain comprising above-mentioned inscribe β-2,6-levanase gene levAGN25, preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus, is preferably recombinant bacterial strain BL21 (DE3)/levAGN25.
The method that the present invention prepares inscribe β-2,6-levanase LevAGN25 is carried out according to the following steps:
1) with above-mentioned recombinant vectors transformed host cell, recombinant bacterial strain is obtained;
2) cultivate recombinant bacterial strain, induction recombined endo β-2,6-levanase LevAGN25 expresses;
3) inscribe β-2, the 6-levanase LevAGN25 also expressed by purifying is reclaimed.
Wherein, preferred described host cell is Bacillus coli cells, preferably by expression of recombinant e. coli plasmid transformation escherichia coli cell BL21(DE3), obtain recombinant bacterial strain BL21 (DE3)/levAGN25.
The invention provides new inscribe β-2, a 6-levanase gene, inscribe β-2, the 6-levanase optimal pH 6.0 of its coding; Optimum temperuture 55 DEG C, the enzyme respectively at 10 DEG C and 20 DEG C with 13.5% and 32.6% is lived; At 37 DEG C, process 60min through 0.1M pH5.0 – 10.0 damping fluid, still can keep the activity of more than 70%; When 50 DEG C, the transformation period is greater than 60min; Inscribe Levan generates Nutriflora P.Inscribe β-2,6-levanase of the present invention can be applicable to food, aquatic feeds and medical industry.
Accompanying drawing explanation
Fig. 1 analyzes at the SDS-PAGE of recombined endo β-2, the 6-levanase LevAGN25 of expression in escherichia coli, wherein, and M: protein Marker; S: unpurified LevAGN25 crude enzyme liquid; 0,60,80,100: be respectively 0,60,80,100mM imidazoles wash-out is affine in Nickel-NTA Agarose recombined endo β-2,6-levanase LevAGN25;
Fig. 2 is that the pH of recombined endo β-2, the 6-levanase LevAGN25 of purifying is active;
Fig. 3 is the pH stability of recombined endo β-2, the 6-levanase LevAGN25 of purifying;
Fig. 4 is the thermal activities of recombined endo β-2, the 6-levanase LevAGN25 of purifying;
Fig. 5 is the thermostability of recombined endo β-2, the 6-levanase LevAGN25 of purifying;
Fig. 6 is the product analysis that recombined endo β-2, the 6-levanase LevAGN25 of purifying is hydrolyzed Levan under different time, wherein, and M: kestose, GF3, GF4; 10min is processed) at CK:Levan and inactivation LevAGN25(100 DEG C.
Embodiment
Test materials and reagent
1, bacterial strain and carrier: Sphingobacterium (Sphingobacterium sp.), with bibliographical information bacterial classification character, as China General Microbiological culture presevation administrative center bacterial strain CGMCC1.6855; Intestinal bacteria Escherichia coli BL21(DE3) and expression vector pEasy-E1 be purchased from Beijing Quanshijin Biotechnology Co., Ltd.
2, enzyme and other biochemical reagents: archaeal dna polymerase and dNTP are purchased from TaKaRa company; Inulin (Inulin), birch xylan (Birchwood xylan), beech wood glycan (Beechwood xylan), 4-O-methyl-d-glucurono-d-xylan, p-nitrophenol(pNP), p-nitrophenyl-β-d-xylopyranoside and p-nitrophenyl-α-l-arabinofuranoside available from Sigma; Araboxylan (Wheat flour arabinoxylan) is purchased from Megazyme company; Levan(derives from Zymomonas mobilis) purchased from Advanced Technology & Industrial company; Other is all domestic reagent, all can buy from common biochemical Reagent Company and obtain.
3, substratum:
LB substratum: Peptone10g, Yeast extract5g, NaCl10g, adding distil water is to 1000ml, pH nature (being about 7).Solid medium adds 2.0%(w/v on this basis) agar.
Illustrate: in following examples, do not make the experimental methods of molecular biology illustrated, all carry out with reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book, or carry out according to test kit and product description.
The clone of embodiment 1: inscribe β-2,6-levanase gene levAGN25
Extract Sphingobacterium genomic dna: by the bacterium liquid centrifuging and taking thalline of liquid culture 2d, add 1mL N,O-Diacetylmuramidase, 37 DEG C of process 60min, then add lysate, lysate consists of: 50mM Tris, 20mM EDTA, Nacl500mM, 2%SDS(w/v), pH8.0,70 DEG C of water-bath cracking 60min, every 10min mixing once, the centrifugal 5min of 10000rpm at 4 DEG C.Get supernatant extrct foreigh protein removing in phenol/chloroform, then get supernatant and add equal-volume Virahol, after room temperature leaves standstill 5min, the centrifugal 10min of 10000rpm at 4 DEG C.Abandon supernatant, precipitate with 70% washing with alcohol twice, vacuum-drying, adds appropriate TE and dissolves, be placed in-20 DEG C for subsequent use.
According to the exoinulinase gene Z2-5 in patent (patent No.: ZL201210062425.6), design hot asymmetric interlaced PCR(and be called for short TAIL-PCR) upstream specific primer 4, and by their respectively called after usp1(base sequence as shown in SEQ ID No.3), usp2(base sequence is as shown in SEQ ID No.5), usp3(base sequence is as shown in SEQ ID No.6), usp4(base sequence is as shown in SEQ ID No.7), see table 1, table 1 is the cloning and expression primer of inscribe β-2,6-levanase gene levAGN25.Specific primer design direction is the zone of ignorance direction needing amplification, and the Position Design of usp2 is in the inner side of usp1, and the Position Design of usp4 is in the inner side of usp3.With Sphingobacterium genomic dna for template, the flanking sequence of known sequence is obtained by TAIL-PCR, TAIL-PCR reaction parameter reference literature (Zhou JP et al., Appl Biochem Biotech2010,160:1277 – 1292), Auele Specific Primer annealing temperature is 50 – 60 DEG C.Amplified production send Beijing Liuhe Huada Genomics Technology Co., Ltd's Guangzhou Branch order-checking.Sequencing result splices mutually with known sequence fragment, obtains the outside upstream sequence of full genome of the exoinulinase gene Z2-5 in patent (patent No.: ZL201210062425.6).This upstream sequence obtains this patent inscribe β-2,6-levanase gene levAGN25 after reading frame analysis, and this gene order, as shown in SEQ ID NO.2, does not comprise the base in any gene Z2-5.
Embodiment 2: the preparation of recombined endo β-2,6-levanase LevAGN25
With levAGN25F(base sequence as shown in SEQ ID No.7) and levAGN25R(base sequence as shown in SEQ ID No.8) for primer pair, see table 1, table 1 is inscribe β-2, the cloning and expression primer of 6-levanase gene levAGN25, Sphingobacterium genomic dna is template, carries out pcr amplification.PCR reaction parameter is: 94 DEG C of sex change 5min; Then 94 DEG C of sex change 30sec, 50 DEG C of annealing 30sec, 72 DEG C extend 1min30sec, 30 rear 72 DEG C of insulation 10min of circulation.PCR result obtains inscribe β-2,6-levanase gene levAGN25, and introduces outstanding A base at this gene 3 ' end.Inscribe β-2,6-levanase gene levAGN25 is connected by T-A mode with expression vector pEasy-E1, obtains the recombinant expression plasmid pEasy-E1-levAGN25 containing levAGN25.By pEasy-E1-levAGN25 transformation of E. coli BL21(DE3), obtain recombinant escherichia coli strain BL21 (DE3)/levAGN25.
The cloning and expression primer of table 1 inscribe β-2,6-levanase gene levAGN25
Get recombinant escherichia coli strain BL21 (the DE3)/levAGN25 containing recombinant plasmid pEasy-E1-levAGN25, the inoculum size with 0.1% is inoculated in LB(containing 100 μ g/mL Amp) in nutrient solution, 37 DEG C of quick oscillation 16h.Then this bacterium liquid activated is inoculated into fresh LB(containing 100 μ g/mL Amp with 1% inoculum size) in nutrient solution, quick oscillation is cultivated about 2 – 3h(OD600 and is reached 0.6 – 1.0) after, the IPTG adding final concentration 0.7mM induces, and is about a 20h or 26 DEG C shaking culture is about 8h in 20 DEG C of continuation shaking culture.The centrifugal 5min of 12000rpm, collects thalline.After appropriate pH7.0Tris-HCl damping fluid suspension thalline, ultrasonic disruption thalline under low temperature water-bath.With first enzyme liquid concentrated in upper eye lid after the centrifugal 10min of 13,000rpm, draw supernatant and use the imidazoles of Nickel-NTA Agarose and 0 – 100mM affine and wash-out target protein respectively.SDS-PAGE the results are shown in Figure 1, Fig. 1 and analyzes at the SDS-PAGE of recombined endo β-2, the 6-levanase LevAGN25 of expression in escherichia coli, wherein, and M: protein Marker; S: unpurified LevAGN25 crude enzyme liquid; 0,60,80,100: be respectively 0,60,80,100mM imidazoles wash-out is affine in Nickel-NTA Agarose recombined endo β-2,6-levanase LevAGN25.As shown in Figure 1, recombined endo β-2,6-levanase LevAGN25 obtains expression in intestinal bacteria, and after the imidazoles wash-out of 100mM, product is single band.
Embodiment 3: the property testing of recombined endo β-2, the 6-levanase LevAGN25 of purifying
1, the activation analysis of recombined endo β-2, the 6-levanase LevAGN25 of purifying:
The activity determination method of recombined endo β-2, the 6-levanase LevAGN25 of embodiment 2 purifying adopts 3,5-dinitrosalicylic acid (DNS) method: be dissolved in by substrate in 0.1M damping fluid, make its final concentration be 0.5%(w/v); Reaction system suitably dilutes enzyme liquid, 500 μ L substrates containing 100 μ L; Substrate, at the reaction temperatures after preheating 5min, reacts 10min again after adding enzyme liquid, then adds 1.9mL DNS termination reaction, boiling water boiling 5min, measure OD value after being cooled to room temperature under 540nm wavelength; 1 Ge Meihuo unit (U) is defined as the raw enzyme amount needed for 1 μm of ol reducing sugar (in fructose) of per minute bottom exploded produce under given conditions.PNP method is adopted to the mensuration of substrate p-nitrophenyl-β-d-xylopyranoside and p-nitrophenyl-α-l-arabinofuranoside: be dissolved in by substrate in 0.1M damping fluid, make its final concentration be 2mM; Reaction system contains the appropriate enzyme liquid of 100 μ L, the 2mM substrate of 500 μ L; Substrate, at the reaction temperatures after preheating 5min, adds enzyme liquid and reacts 10min again, then add 1.9mL1MNa
2cO
3termination reaction, measures the pNP discharged under 405nm wavelength after being cooled to room temperature; 1 Ge Meihuo unit (U) is defined as the raw enzyme amount needed for 1 μm of ol pNP of per minute bottom exploded produce.
2, the active and pH Stability Determination of the pH of recombined endo β-2, the 6-levanase LevAGN25 of purifying:
The optimal pH of enzyme measures: inscribe β-2,6-levanase LevAGN25 is carried out enzymatic reaction at 37 DEG C and in the damping fluid of 0.1MpH4.0 – 9.0.The pH Stability Determination of enzyme: the damping fluid enzyme liquid of purifying being placed in 0.1M pH4.0 – 11.0, processes 60min at 37 DEG C, then carries out enzymatic reaction at pH6.0 and 37 DEG C, with untreated enzyme liquid in contrast.Damping fluid is: 0.1M McIlvainebuffer(pH4.0 – 8.0) and 0.1M glycine-NaOH(pH9.0 – 11.0).Take Levan as substrate, reaction 10min, measures the zymologic property of the LevAGN25 of purifying.Result is that the pH of recombined endo β-2, the 6-levanase LevAGN25 of purifying is active see Fig. 2 and Fig. 3, Fig. 2; Fig. 3 is the pH stability of recombined endo β-2, the 6-levanase LevAGN25 of purifying, Fig. 2 and Fig. 3 shows: the optimal pH of LevAGN25 is 6.0, maintains the enzymic activity of more than 50% in the scope of pH5.0 – 7.0; Through the damping fluid process 60min of pH5.0 – 10.0, the activity of more than 70% still can be kept.
3, the thermal activities of recombined endo β-2, the 6-levanase LevAGN25 of purifying and thermal stability determination:
The optimum temperuture of enzyme measures: in the damping fluid of pH6.0, at 10 – 70 DEG C, carry out enzymatic reaction.The thermal stability determination of enzyme: the enzyme liquid of same enzyme amount is placed in 37 DEG C, 50 DEG C and 60 DEG C, after processing 0 – 60min, carries out enzymatic reaction, with untreated enzyme liquid in contrast at pH6.0 and 37 DEG C.Take Levan as substrate, reaction 10min, measures the zymologic property of the LevAGN25 of purifying.Result is the thermal activities of recombined endo β-2, the 6-levanase LevAGN25 of purifying see Fig. 4 and Fig. 5, Fig. 4;
Fig. 5 is the thermostability of recombined endo β-2, the 6-levanase LevAGN25 of purifying, Fig. 4 and Fig. 5 shows: the optimum temperuture of LevAGN25 is 55 DEG C, and the enzyme respectively 10 DEG C and 20 DEG C with 13.5% and 32.6% is lived, and the enzyme at 37 DEG C with about 75% is lived; 37 DEG C and 50 DEG C time LevAGN25 transformation period be greater than 60min, when 60 DEG C, the transformation period of this enzyme is about 10min.
4, the Determination of Kinetic Parameters of recombined endo β-2, the 6-levanase LevAGN25 of purifying:
The kinetic parameter first order reaction timing of enzyme: at pH6.0 and 55 DEG C, with 0.3%(w/v) Levan be substrate, termination reaction measure enzymic activity in 1 – 10min of enzymatic reaction successively, calculate the ratio in enzymic activity and reaction times, if this ratio keeps stable within a certain period of time, then this time is the first order reaction time.With 0.05 – 1.0%(w/v) Levan be substrate, under pH6.0,55 DEG C and first order reaction time, measure K according to Lineweaver-Burk method
m, V
maxand k
cat.After measured, under 55 DEG C and pH6.0 condition, LevAGN25 is to the K of Levan
m, V
maxand k
catbe respectively 8.3mgmL
-1, 400.0 μm of ol min
-1mg
-1and 389.2s
-1.
5, different metal ion and chemical reagent are on the impact of the restructuring LevAGN25 vigor of purifying:
In enzymatic reaction system, add metal ion and the chemical reagent of 1mM or 10mM, study its impact on enzymic activity.Under 37 DEG C and pH6.0 condition, be that substrate measures enzymic activity with Levan.Result is see table 2, and table 2 is that metal ion and chemical reagent are to the impact of recombined endo β-2, the 6-levanase LevAGN25 vigor of purifying.
Table 2 metal ion and chemical reagent are to the impact of recombined endo β-2, the 6-levanase LevAGN25 vigor of purifying
Table 2 shows, HgCl
2and SDS suppresses LevAGN25 completely; PbAC, FeSO of 10mM
4and FeCl
3the restraining effect certain to LevAGN25 tool, residual enzyme 74.6 – 79.8% alive; And the β-Mercaptoethanol of 10mM has obvious promoter action to LevAGN25, the enzyme improving LevAGN25 is lived about 0.8 times; All the other metal ions and the impact of chemical reagent on LevAGN25 less.
6, recombined endo β-2, the 6-levanase LevAGN25 of purifying is to the degraded of substrate:
At pH6.0 and 55 DEG C, LevAGN25 is to 0.5%(w/v) specific activity of Levan is 219.5 ± 4.2U mg
-1, and inulin, sucrose, Zulkovsky starch, raffinose (Raffinose), stachyose (Stachyose), birch xylan (Birchwood xylan), beech wood glycan (Beechwood xylan), 4-O-methyl-d-glucurono-d-xylan, araboxylan (Wheat flour arabinoxylan), p-nitrophenyl-β-d-xylopyranoside and p-nitrophenyl-α-l-arabinofuranoside are all lived without enzyme.This result shows that LevAGN25 is Levan or β-2,6-Polylevulosan selective degradation enzyme.
7, recombined endo β-2, the 6-levanase LevAGN25 of purifying is hydrolyzed the product analysis of Levan:
Product analysis reaction system is containing 10mL0.5%(w/v) Levan, 2mL suitably dilutes enzyme liquid (altogether 12U enzyme liquid).At pH6.0 and 37 DEG C, termination reaction analyze hydrolysate in the 5min – 12h of enzymatic reaction successively.Product analysis adopts thin layer chromatography (using the High Performance Thin Layer Chromatography silica-gel plate G type of Qingdao Marine Chemical Co., Ltd.), and chromatographic step is as follows:
(1) prepare developping agent (Glacial acetic acid 20mL, distilled water 20mL, propyl carbinol 40mL, mixing), get and pour separation chamber in right amount, leave standstill about 30min;
(2) silica-gel plate is placed in 110 DEG C of baking ovens and activates 30min, line after cooling, point sample (each 0.5 μ L, dries up, concurrent 3 times);
(3) one end silica-gel plate of point sample is put into separation chamber down, point of sample does not submerge developping agent;
(4) to be deployed dose to apart from silica-gel plate along 1.5cm time, take out silica-gel plate, dry up, then launch once;
(5), after second time expansion terminates, silica-gel plate directly immerses appropriate developer (1g pentanoic is dissolved in 50mL acetone, adds the phosphoric acid of 1mL aniline and 5mL85% after dissolving, mixing, matching while using);
(6), after a few second, take out silica-gel plate immediately and be positioned over 10 – 15min in 90 DEG C of baking ovens, making spot development.
Result is hydrolyzed the product analysis of Levan see recombined endo β-2, the 6-levanase LevAGN25 that Fig. 6, Fig. 6 are purifying under different time, wherein, and M: kestose, GF3, GF4; 10min is processed at CK:Levan and inactivation LevAGN25(100 DEG C), Fig. 6 shows: under different time, and the product that LevAGN25 is hydrolyzed Levan is Nutriflora P, shows that LevAGN25 is endo-type β-2,6-levanase.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1. a levanase, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:1.
2. the gene of coding levanase according to claim 1.
3. gene as claimed in claim 2, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.2.
4. comprise the recombinant vectors of gene described in Claims 2 or 3.
5. the host cell containing carrier described in claim 4.
6. comprise the bacterial strain of gene described in Claims 2 or 3, described bacterial strain is recombinant bacterial strain.
7. bacterial strain according to claim 6, is characterized in that, described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus.
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| low temperature and defoliation affect fructan-metabolizing enzymes in different regions of the rhizophores of vernonia herbacea.;portes mt et al.;《j plant physiol.》;20081009;第165卷(第15期);1572-1581 * |
| purification and characterization of the enzymes of fructan biosynthesis in tubers of helianthus tuberosus colombia;koops aj et al.;《plant physiol.》;19960430;第110卷(第4期);1167-1175 * |
| sphingobacterium siyangense sp.nov., isolated from farm soil;rui liu et al.;《international journal of systematic and evolutionary microbiology》;20080630;第58卷(第6期);摘要 * |
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