CN102586208A - Protein with Beta-N-acetamido glucosaminidase activity as well as encoding gene and application thereof - Google Patents
Protein with Beta-N-acetamido glucosaminidase activity as well as encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a protein with Beta-N-acetamido glucosaminidase activity as well as an encoding gene and an application of the protein. According to the invention, the protein is as follows: (a) a protein formed by the 23th to the 883th amino acid residues from N terminal of a sequence 1 in a sequence table; (b) a protein formed by an amino acid sequence illustrated by the sequence 1 in the sequence table; or (c) a protein formed in the way that the amino acid sequence of the sequence 1 is substituted and/or deleted and/or added with one or more amino acid residues and having the function of the Beta-N-acetamido glucosaminidase. By using the protein disclosed by the invention, the pNP-G1cNAc can be high efficiently degraded so that the protein can be used for preparing feeds as a feed additive. The protein with the Beta-N-acetamido glucosaminidase activity, disclosed by the invention, has a broad application prospect.
Description
Technical field
The present invention relates to a kind of β of the having-active protein of N-N-Acetylhexosaminidase and encoding sox and application.
Background technology
Regitex FA has another name called chitin, chitosan, is β-1, the 4 glycosidic link polymer of N-acetylglucosamine.Regitex FA extensively is present in lower plant, mushroom, algae, shell of arthropods shrimp, crab and insect and cartilage, and the cell wallss of higher plant etc. are that occurring in nature is only second to the organic resource of cellulosic second largest natural biological, a year biosynthesizing amount reaches 10,000,000,000 tons.
Mikrobe passes through chitinase (chitinase to chitinous effective degraded; EC 3.2.1.14) and the chitobiose enzyme be β-N-N-Acetylhexosaminidase (β-N-acetylglucosaminidase; Nag, EC 3.2.1.52) etc. synergy is achieved.
Summary of the invention
The purpose of this invention is to provide a kind of β of the having-active protein of N-N-Acetylhexosaminidase and encoding sox and application.
Protein provided by the invention, available from Aeromonas veronii (Aeromonas veronii), called after NAG565 albumen is (a) or (b) or (c) as follows:
(a) protein that sequence 1 is formed from N-terminal the 23rd to 883 amino acids residue in the sequence table;
(b) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(c) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have β-N-N-Acetylhexosaminidase function by sequence 1 deutero-protein.
For make (a) or (b) in protein be convenient to purifying, label as shown in table 1 on proteinic N-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table or C-terminal connect.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | |
| FLAG | ||
| 8 | DYKDDDDK | |
| Strep-tag?II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (c) but in the protein synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Proteinic encoding sox in above-mentioned (c) can be through the codon with one or several amino-acid residue of disappearance in (a) or the encoding sox (b); And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The gene of encoding said proteins also belongs to protection scope of the present invention.
Said gene can be following 1) or 2) or 3) or 4) dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 67th to 2649 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization and the coding that limit have the active proteic dna molecular of β-N-N-Acetylhexosaminidase;
4) with 1) or 2) dna sequence dna that limits has 90% above homology and coding has the active proteic dna molecular of β-N-N-Acetylhexosaminidase.
Said stringent condition be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said gene all belong to protection scope of the present invention.
Available existing expression vector establishment contains the recombinant expression vector of said gene.Said expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor.When using said gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can use separately or be used in combination with other promotor; In addition; When using gene constructed recombinant expression vector of the present invention; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of identifying and screening, can process used expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change as adding to encode with resistance.Also can not add any selected marker, directly according to phenotypic screen.
Said recombinant expression vector specifically can be the recombinant plasmid that the MCS of said gene insertion vector pET28a (+) obtains.
Said reorganization bacterium specifically can be said recombinant expression vector is imported the reorganization bacterium that intestinal bacteria obtain.Said intestinal bacteria are preferably e. coli bl21 (DE3).
The present invention also protects the said method of protein of a kind of preparation, comprises the steps:
(1) cultivate said reorganization bacterium and in culturing process adding IPTG induce;
(2) with the culture system centrifugal collecting precipitation of completing steps (1), the collection supernatant is and contains said proteinic solution after the ultrasonication.
Said method specifically comprises the steps:
(1) said reorganization bacterium is cultured to OD
600=0.6, adding IPTG then and making its starting point concentration is 1mol/L, then 18 ℃ of shaking culture 12h;
(2) with the culture system centrifugal collecting precipitation of completing steps (1), collect supernatant after the ultrasonication, be and contain said proteinic solution.
Said method more specifically comprises the steps:
(1) said reorganization bacterium in liquid LB substratum (containing 100 μ g/mL kantlex) 37 ℃ of shaking culture to OD
600=0.6, adding IPTG then and making its starting point concentration is 1mol/L, 18 ℃ then, 180 * g shaking culture 12h;
(2) with the centrifugal 5min collecting precipitation of culture system 10,000 * g of completing steps (1), collect supernatant after the ultrasonication, be and contain said proteinic solution.
Increase said full length gene or its any segmental primer to all belonging to protection scope of the present invention.The primer that said primer is formed dna fragmentation shown in the sequence 4 of dna fragmentation shown in the sequence 3 that specifically can be sequence table and sequence table is right.
The present invention also protects the application of said albumen as β-N-N-Acetylhexosaminidase.In the said application, said proteic substrate can be pNP-GlcNAc, pNP-(GlcNAc)
2, pNP-(GlcNAc)
3, at least a in chitobiose, chitin trisaccharide, chitin tetrose, chitin pentasaccharides and chitin six sugar.
The present invention also protects the application of said albumen in the preparation feed.
Protein provided by the invention is a kind of chitobiose enzyme, has height ratio vigor, loose range of application and satisfactory stability property, can be used as the breed that fodder additives is applied to hydrocoles, has the major application prospect.
Description of drawings
Fig. 1 is that the SDS-PAGE of CTP solution and NAG565 protein liquid analyzes M:Marker; 1:CTP solution; The 2:NAG565 protein liquid.
Fig. 2 is the proteic ph optimum of NAG565.
Fig. 3 is the proteic pH stability of NAG565.
Fig. 4 is the proteic optimum temperuture of NAG565.
Fig. 5 is the proteic thermostability of NAG565.
Fig. 6 is the proteic protease resistant of NAG565.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Experimental technique among the following embodiment like no specified otherwise, is ordinary method; All carry out according to related Sections in following laboratory manual or the document or part; Comprise: people such as Sambrook, Molecular Cloning, ALaboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); With Curren t Protocols inMolecularBiology (people such as Ausubel compiles, 1994).The method that detects protein concentration among the embodiment is the Bradford method.Used test materials among the following embodiment like no specified otherwise, is and buys the homemade AR that obtains from routine biochemistry reagent shop.
Intestinal bacteria (Escherichia coli) DH5 α competent cell, intestinal bacteria (Escherichia coli) BL21 (DE3) competent cell are all available from the full formula in Beijing King Company.Ligase enzyme is available from NEB company.Trypsin Trypsin), (α-Chymotrypsin), Proteinase K (Proteinase K), Collagenase (Collagenase) are Sigma (USA) product to Chymetin.Stomach en-(Pepsin) is Amano Enzyme Inc. (Japan) product.IPTG is the Promega Company products.Anti-His antibody, sheep anti-mouse igg-HRP and Western Blot membrane closure liquid are all available from sky, Beijing root biochemical technology ltd.
Carrier pET28a (+): available from Invi trogen company.
P-nitrophenol (4-Nitrophenol or pNP): available from Sigma company, catalog number N7660.
PNP-GlcNAc (4-Nitrophenyl N-acetyl-α-D-glucosaminide): available from Sigma company, catalog number N8759.
PNP-(GlcNAc)
2(4-Nitrophenyl N, N '-Diacetylchitobiose): available from Sigma company, catalog number N6133.
PNP-(GlcNAc)
3(4-Nitrophenyl N, N ', N "-Triacetylchitotriose): available from Sigma company, catalog number N8638.
Aeromonas (Aeromonas sp.) B565; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 03rd, 2010 and (be called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.4403.
With the protein called after NAG565 albumen shown in the sequence 1 of sequence table, form by 883 amino-acid residues, be signal peptide from the 1st to 22 of N-terminal.With the proteic encoding sox called after of NAG565 NAG565 gene, its ORFs is shown in the sequence 2 of sequence table (2649bp).
One, the structure of recombinant plasmid NAG565-pET28a (+)
1, extracts the genomic dna of Aeromonas B565.
2, the genomic dna with step 1 is a template, and the primer of forming with F1 and R1 obtains pcr amplification product to carrying out pcr amplification.
F1 (sequence 3 of sequence table): 5 '-CCC
GAATTCGCCGATGCCGCCAAGCAGGC-3 ';
R1 (sequence 4 of sequence table): 5 '-TAT
GCGGCCGCGACGCGGCTGGTGCGCT-3 '.
3,, reclaim enzyme and cut product with the pcr amplification product of restriction enzyme EcoRI and NotI double digestion step 2.
4,, reclaim carrier framework (about 5300bp) with restriction enzyme EcoRI and NotI double digestion carrier pET28a (+).
5, the carrier framework of the enzyme of step 3 being cut product and step 4 is connected, and obtains recombinant plasmid NAG565-pET28a (+).According to sequencing result, it is following that recombinant plasmid NAG565-pET28a (+) is carried out structrual description: the sequence 2 of between the EcoRI of carrier pET28a (+) and NotI restriction enzyme site, having inserted sequence table is from the dna fragmentation shown in 5 ' terminal the 67th to 2649 Nucleotide.
Two, the structure of reorganization bacterium and contrast bacterium
With recombinant plasmid NAG565-pET28a (+) heat shock transformed into escherichia coli BL21 (DE3) competent cell, obtain the bacterium of recombinating.With carrier pET28a (+) heat shock transformed into escherichia coli BL21 (DE3) competent cell, obtain contrasting bacterium.
Three, the preparation of NAG565 albumen and reference protein
1, the reorganization bacterium that step 2 is made up is inoculated in 3mL liquid LB substratum (containing 100 μ g/mL kantlex), and 37 ℃ of shaking culture are spent the night, and obtain seed liquor.
2,100 μ L seed liquor are forwarded in the 10mL liquid LB substratum (containing 100 μ g/mL kantlex) 37 ℃ of about 3h (OD of shaking culture
600=0.6) (making its starting point concentration is 1mol/L, to add IPTG; IPTG is an inductor), 18 ℃, 180 * g shaking culture 12h (picking up counting) from adding IPTG.
3, the centrifugal 5min of nutrient solution 10,000 * g that step 2 is obtained collects culture supernatant and cell precipitation respectively.
4, the cell precipitation that step 3 is obtained is resuspended with the Tris-HCl damping fluid of pH8.0,20mM, ultrasonication, and the centrifugal 10min of 10,000 * g collects supernatant then, is the solution (being called for short CTP solution) that contains total protein of cell.
The contrast bacterium that step 2 is made up replaces the reorganization bacterium to carry out the operation of step 1 to 4, obtains to contain the solution (abbreviation contrast solution) of total protein of cell.
Four, the enzyme activity determination of CTP solution and contrast solution (pNP-GlcNAc method)
Enzyme (U) unit definition alive: PM decomposition pNP-GlcNAc discharges the required enzyme amount of 1 μ mol p-nitrophenol (pNP) and is defined as enzyme unit (1U) alive.The enzyme work of solution to be measured is the proteic vigor that compares divided by the protein content of solution to be measured in the unit volume in the unit volume.
The enzyme activity determination method: pNP-GlcNAc is dissolved in the damping fluid, and making its final concentration is 1mM, is substrate solution.Developmental tube: 10 μ L solution to be measured, 240 μ L damping fluids and 250 μ L substrate solutions are mixed in test tube and shake up, and 37 ℃ of incubation 5min add 2mL 0.5M NaOH aqueous solution termination reaction then, survey the OD value at the 405nm place.Control tube: in test tube, add 10 μ L solution to be measured successively, 240 μ L damping fluids, the 2mL 0.5M NaOH aqueous solution and 250 μ L substrate solutions are surveyed the OD value at the 405nm place.As standard substance production standard curve, the typical curve function is with p-nitrophenol: y=(0.169x-0.0153) * 100 * N/5; Y: enzyme is lived (U/mL), x:OD value, 100: the conversion alive of the enzyme in the 10 μ L solution to be measured is the enzymic activity of 1mL, N: extension rate.
The contrast solution that CTP solution that 4 of the culture supernatant that respectively 3 of step 3 is obtained, step 3 obtains and step 3 obtain makes to carry out enzyme activity determination.The damping fluid that adopts in the enzyme activity determination is Sodium phosphate, dibasic-citrate buffer solution of pH7.0,20mM.The enzyme of contrast solution is lived and is that it is 1562U/ml (is 1243U/mg than vigor) that 0U/ml, the enzyme of CTP solution live.The result shows that NAG565 albumen is mainly expressed in Escherichia coli cell.
Five, the proteic purifying of NAG565
The CTP solution that step 4 is obtained carries out His tag fusion protein affinitive layer purification.
The weighting material of His tag fusion protein affinity chromatography is the Ni-NTA resin, gives birth to worker bio-engineering corporation (catalog number BSP033) available from Shanghai, and dress post amount is 1mL.
Each damping fluid in the elution process is (pH is 7.6) as follows:
NTA-0: contain 20mmol/L Tris-HCl, 0.5mol/L NaCl, 10g/100ml glycerine, other is a water;
NTA-20: contain 20mmol/L Tris-HCl, the 20mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is a water;
NTA-40: contain 20mmol/L Tris-HCl, the 40mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is a water;
NTA-60:20mmol/L Tris-HCl, the 60mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is a water;
NTA-80:20mmol/L Tris-HCl, the 80mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is a water;
NTA-100:20mmol/L Tris-HCl, the 100mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is a water;
NTA-200:20mmol/L Tris-HCl, the 200mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is a water;
NTA-300:20mmol/L Tris-HCl, the 300mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is a water.
Elution process (at every turn add 5mL, flow out pillar fully then add 5mL again):
(1) pillar watering balance 20mL;
(2) with NTA-0 balance 20mL, last appearance 5mLCTP solution;
(3) with NTA-0 wash-out 25mL (foreigh protein removing), use NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, each 5mL wash-out of NTA-300 then successively, and solution (NAG565 protein liquid) behind the post excessively of collection NTA40-80 wash-out.
The CTP solution of step 4 preparation and the NAG565 protein liquid of step 5 preparation are carried out the SDS-PAGE electrophoresis, see Fig. 1, the albumen behind the purifying shows the single band of 95kDa, conforms to the expection molecular weight.
Six, the proteic enzyme activity determination of NAG565
The NAG565 protein liquid that step 5 is obtained is as solution to be measured, other complete same step 4.The enzyme activity of NAG565 protein liquid is 2257U/mL, is 7328U/mg than vigor.
The NAG565 protein liquid that the step 5 of embodiment 1 is obtained carries out between following each zymologic property as solution to be measured successively:
One, the proteic ph optimum of NAG565
The enzyme activity determination method is with the step 4 of embodiment 1.
Adopt the sodium acetate buffer of following damping fluid: pH3.0,0.1M in the enzyme activity determination respectively; The sodium acetate buffer of pH4.0,0.1M; Sodium phosphate, dibasic-citrate buffer solution of Sodium phosphate, dibasic-citrate buffer solution of pH5.0,0.1M, pH6.0,0.1M; Sodium phosphate, dibasic-citrate buffer solution of pH7.0,0.1M, the Tris-HCl damping fluid of pH8.0,0.1M, the Tris-HCl damping fluid of pH9.0,0.1M; The glycocoll of pH10.0,0.1M-NaOH damping fluid, the glycocoll of pH11.0,0.1M-NaOH damping fluid.
NAG565 albumen enzyme in the environment of pH7.0 is lived the highest, is 7301U/mg than vigor.With this height ratio vigor is 100%, calculates the relative enzyme of NAG565 albumen in other pH environment and lives, and sees Fig. 2.NAG565 albumen can keep the relative enzyme more than 80% to live in the environment of pH8.0.
Two, the proteic pH stability of NAG565
The enzyme activity determination method:
PNP-GlcNAc is dissolved in the damping fluid, and making its final concentration is 1mM, is substrate solution.
Developmental tube: 10 μ L solution to be measured and 240 μ L damping fluids are mixed in test tube and 37 ℃ of incubations 12 hours; Adding 250 μ L substrate solutions then mixes and 37 ℃ of incubation 5min; Add 2mL 0.5M NaOH aqueous solution termination reaction then, survey the OD value at the 405nm place.
Control tube: 10 μ L solution to be measured and 240 μ L damping fluids are mixed in test tube and 37 ℃ of incubations 12 hours, add the 2mL 0.5M NaOH aqueous solution and 250 μ L substrate solutions then successively, survey OD value at 405nm place.
NAG565 albumen stability in the environment of pH7.0 is the highest, and the ratio vigor of 37 ℃ of incubations after 12 hours is 6350U/mg.With this height ratio vigor is 100%, calculates NAG565 albumen 37 ℃ of incubations relative enzyme after 12 hours in other pH environment and lives, and sees Fig. 3.NAG565 albumen is all very stable between 5.0-9.0 in the pH scope, keeps the relative enzyme more than 70% to live.
Three, the proteic optimum temperuture of NAG565
Except heated culture temperature, other is with the step 4 of embodiment 1 in the enzyme activity determination.Adopt following heated culture temperature in the enzyme activity determination respectively: 0 ℃, 20 ℃, 30 ℃, 37 ℃, 50 ℃, 60 ℃ and 70 ℃.Adopt Sodium phosphate, dibasic-citrate buffer solution of pH7.0,0.1M in the enzyme activity determination.
NAG565 albumen enzyme in 37 ℃ environment is lived the highest, is 7321U/mg than vigor.With this height ratio vigor is 100%, calculates the relative enzyme of NAG565 albumen in other temperature environment and lives, and sees Fig. 4.NAG565 albumen can keep the enzyme about 40% to live in 20 ℃ buffering.
Four, the proteic thermostability of NAG565
The enzyme activity determination method:
PNP-GlcNAc is dissolved in the damping fluid, and making its final concentration is 1mM, is substrate solution.
Developmental tube: in test tube with 10 μ L solution differing temps to be measured (0 ℃, 20 ℃, 30 ℃, 37 ℃, 50 ℃, 60 ℃ or 70 ℃) incubation 30min; Add 240 μ L damping fluids and 250 μ L substrate solutions then; Mixing; 37 ℃ of incubation 5min add 2mL 0.5M NaOH aqueous solution termination reaction then, survey the OD value at the 405nm place.
Control tube: in test tube,, add 240 μ L damping fluids, the 2mL 0.5M NaOH aqueous solution and 250 μ L substrate solutions then successively, survey the OD value at the 405nm place with 10 μ L solution differing temps to be measured incubation 30min.
Adopt Sodium phosphate, dibasic-citrate buffer solution of pH7.0,0.1M in the enzyme activity determination.
NAG565 albumen stability in 30 ℃ environment is the highest, and the ratio vigor behind 30 ℃ of incubation 30min is 6187U/mg.With this height ratio vigor is 100%, calculates NAG565 albumen relative enzyme behind 30 ℃ of incubation 30min in other temperature environment and lives, and sees Fig. 5.NAG565 albumen is losing activity more than 70 ℃ fully, and between 30-70 ℃, along with the rising of treatment temp, enzyme is lived and reduced gradually, and the processing below 30 ℃ is to not influence of enzymic activity.
The NAG565 albumen that obtains 37 ℃ of step 5 of measuring embodiment 1 draws the K of this enzyme to 3 kinds of substrates to the initial velocity of reaction (the enzyme biopsy method of surveying is with the step 4 of embodiment 1) of the substrate of different concns (1,2,5,10,50,100,500,1000 μ mol/L) through the Lineweaver-Burk drawing
mAnd V
MaxValue.Substrate is respectively pNP-GlcNAc, pNP-(GlcNAc)
2And pNP-(GlcNAc)
3
The result sees table 2.In 3 kinds of substrates, the K of pNP-GlcNAc
mValue is minimum, V
MaxValue is maximum.
Table 2NAG565 albumen is to the kinetic constant of various substrates
One, NAG565 albumen is to the degraded of chitin oligo saccharide
1, the concentration of substrate (chitin oligo saccharide) with 4mM is dissolved in the phosphoric acid buffer of pH7.0,10mM, is substrate solution.Below chitin oligo saccharide adopts respectively several kinds: chitobiose, chitin trisaccharide, chitin tetrose, chitin pentasaccharides or chitin six sugar, all available from TRC company.
2, get the chitin oligo saccharide substrate solution of 0.1mL step 1, add the NAG565 albumen of the step 5 preparation of 5 μ g embodiment 1, be initial system (reducing sugar that contains 0 μ g/mL in the initial system); With 37 ℃ of enzymolysis 6h of initial system, obtain the termination system; With the phosphoric acid buffer of 0.1mLpH7.0,10mM negative control as the chitin oligo saccharide substrate solution.
3, (adopt glucose as standard substance production standard curve, the typical curve equation is following: Y=2521.1* Δ OD+20.41 according to the reducing sugar content in the DNS method mensuration product; The increasing amount of reducing sugar in the termination system is compared in the Y representative with initial system; Unit is μ g/mL; Δ OD is the OD value that the OD value of termination system deducts initial system), characterize the ability of NAG565 albumen with this as β-N-N-Acetylhexosaminidase degraded chitin oligo saccharide.The reducing sugar increasing amount of negative control group is 0 μ g/mL; The reducing sugar increasing amount of chitobiose group is 1505.3 μ g/mL; The reducing sugar increasing amount of chitin trisaccharide group is 907.8 μ g/mL; The reducing sugar increasing amount of chitin tetrose group is 796.9 μ g/mL, and the reducing sugar increasing amount of chitin pentasaccharides group is 708.7 μ g/mL, and the reducing sugar increasing amount of chitin six sugar groups is 403.6 μ g/mL.Can confirm that NAG565 albumen has certain Degradation to chitin oligo saccharide.
Two, NAG565 albumen is to the identification of other substrate
The same step 1 of method, difference only are to replace chitin oligo saccharide with following substrate:
4-Methylumbelliferyl-N-Acetyl-α-D-Glucosaminide(Sigma)、
4-Methylumbelliferyl-α-D-Glucoside(Sigma)、
4-Methylumbelliferyl-α-D-Galactoside(Sigma)、
4-Methylumbelliferyl-α-D-Xyloside(Sigma)、
4-Methylumbel-Liferyl-α-D-Cellobiopyranoside(Sigma)、
Glycol-chi?tosan(Sigma)、
CMC 99.5;
The chitosan powder.
NAG565 albumen does not all have degradation capability to above-mentioned substrate.
The result shows that NAG565 albumen has specificity to β-N-acetylglucosamine glycosidic bond.
The NAG565 albumen that the step 5 of detection embodiment 1 obtains is to the resistance of different metal ion or chemical reagent.Enzyme biopsy survey method is referring to the step 4 of embodiment 1, and difference only is in reaction system, to add different metallic ion or chemical reagent (K
+, Na
+, Ca
2+, Li
+, Co
2+, Cr
3+, Ni
2+, Cu
2+, Mg
2+, Fe
2+, Mn
2+, Zn
2+, Ag
+, Hg
2+, EDTA, SDS or beta-mercaptoethanol; Final concentration is 5mM), not add metals ion and chemical reagents system as contrast.Adopt Sodium phosphate, dibasic-citrate buffer solution of pH7.0,0.1M in the enzyme activity determination.
The result sees table 3.Na
+, Mn
2+Proteic enzyme work has promoter action to NAG565.Co
2+, Cr
3+, Cu
2+Deng proteic enzyme work has restraining effect in various degree to NAG565, Ag
+And Hg
2+Almost completely suppress the proteic enzyme of NAG565 and live, and K
+, Ca
2+, Mg
2+Influence Deng the proteic enzyme of NAG565 is lived is very little, can ignore.NAG565 albumen has certain resistivity to radiolucent table surface-active agent-SDS.
Table 3 metals ion and relevant chemical reagent are to the proteic influence of NAG565
| Chemical reagent | Residual enzyme (%) alive | Metals ion or chemical reagent | Residual enzyme (%) alive |
| Na + | 108.52 | Mn 2+ | 108.51 |
| K + | 95.20 | Zn 2+ | 82.65 |
| Ca 2+ | 97.66 | Pb 2+ | 83.83 |
| Li + | 67.71 | Fe 3+ | 86.00 |
| Co 2+ | 43.40 | Ag + | 18.33 |
| Cr 3+ | 64.19 | |
0 |
| Ni + | 86.91 | SDS | 64.71 |
| Cu 2+ | 77.79 | beta-Met | 74.01 |
| Mg 2+ | 91.94 | EDTA | 89.11 |
Proteolytic enzyme is mixed with the solution of 1mg/mL respectively with following solution: the solution that stomach en-is made into 1mg/mL with 0.1M glycocoll-HCl of pH2.0; Respectively trypsinase and Chymetin are made into the solution of 1mg/mL with the 0.1M Tris-HCl of pH7.0; Proteinase K and Collagenase are made into the solution of 1mg/mL respectively with the 0.1M Tris-HCl of pH7.5.NAG565 albumen that the step 5 of embodiment 1 is obtained and proteolytic enzyme are handled 30min or 60min by 10: 1 (mass ratio) in this protein enzyme solution after, the residual enzyme that detects treat enzyme by standard method live (referring to the step 4 of embodiment 1).With the reaction system that do not add proteolytic enzyme as contrast (CK).
The result sees Fig. 6.Behind different protease treatment 60min; The proteic residual enzyme work of NAG565 is respectively 68.9% (stomach en-); 76.9% (trypsinase), 89.1% (Chymetin), 84.9% (Proteinase K); 78.9% (Collagenase) this shows that NAG565 albumen all has stronger resistance to the multiple protein enzyme.
Claims (10)
1. protein, for (a) as follows or (b) or (c):
(a) protein that sequence 1 is formed from N-terminal the 23rd to 883 amino acids residue in the sequence table;
(b) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(c) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have β-N-N-Acetylhexosaminidase function by sequence 1 deutero-protein.
2. coding claim 1 said proteic gene.
3. gene as claimed in claim 2 is characterized in that: it is for following 1) or 2) or 3) or 4) dna molecular:
1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 67th to 2649 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization and the coding that limit have the active proteic dna molecular of β-N-N-Acetylhexosaminidase;
4) with 1) or 2) dna sequence dna that limits has 90% above homology and coding has the active proteic dna molecular of β-N-N-Acetylhexosaminidase.
4. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said genes.
5. recombinant expression vector as claimed in claim 4 is characterized in that: the recombinant plasmid that said recombinant expression vector obtains for the MCS with claim 2 or 3 said gene insertion vector pET28a (+).
6. reorganization bacterium as claimed in claim 4 is characterized in that: said reorganization bacterium is for importing the reorganization bacterium that intestinal bacteria obtain with claim 4 or 5 said recombinant expression vectors.
7. one kind prepares the said method of protein of claim 1, comprises the steps:
(1) cultivate claim 6 said reorganization bacterium and in culturing process adding IPTG induce;
(2) with the culture system centrifugal collecting precipitation of completing steps (1), the collection supernatant is and contains the said proteinic solution of claim 1 after the ultrasonication.
8. amplification claim 2 or 3 said full length genes or its any segmental primer are right.
9. the said albumen of claim 1 is as the application of β-N-N-Acetylhexosaminidase.
10. the application of the said albumen of claim 1 in the preparation feed.
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| CN105400807A (en) * | 2015-12-23 | 2016-03-16 | 太原理工大学 | Gypsy moth beta-N-acetylglucosamine glycosidase gene based on gene silencing technology |
| CN105400807B (en) * | 2015-12-23 | 2019-02-12 | 太原理工大学 | Gypsymoth β-N-Acetyl-D-glucosamine glycosidase genes based on gene silent technology |
| CN105483102A (en) * | 2016-01-14 | 2016-04-13 | 云南师范大学 | Beta-N-acetylglucosamine enzyme hJ5nag resisting product inhibition and preparation method thereof |
| CN105483102B (en) * | 2016-01-14 | 2018-09-25 | 云南师范大学 | The β of resistance to Product inhibiton-N-acetylglucosaminidase HJ5nag and preparation method thereof |
| CN108070628A (en) * | 2016-11-14 | 2018-05-25 | 中国科学院微生物研究所 | A kind of method for producing Glucosamine |
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| CN102586208B (en) | 2014-03-05 |
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