CN102586208B - 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 protein and encoding gene and the application of a kind of β of having-2-Acetamido-2-deoxy-D-glucose glycosides enzymic activity.
Background technology
Chitin has another name called chitin, chitosan, is β-Isosorbide-5-Nitrae glycosidic link polymer of 2-Acetamido-2-deoxy-D-glucose.Chitin is extensively present in lower plant, mushroom, algae, the 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 cellulosic second largest natural biological organic resource, a year biosynthesizing amount reaches 10,000,000,000 tons.
Microorganism passes through chitinase (chitinase to chitinous effective degraded, EC 3.2.1.14) and chitobiose enzyme be β-2-Acetamido-2-deoxy-D-glucose glycosides enzyme (β-N-acetylglucosaminidase, Nag, EC 3.2.1.52) etc. synergy is achieved.
Summary of the invention
The object of this invention is to provide protein and encoding gene and the application of a kind of β of having-2-Acetamido-2-deoxy-D-glucose glycosides enzymic activity.
Protein provided by the invention, available from Aeromonas veronii (Aeromonas veronii), called after NAG565 albumen is following (a) or (b) or (c):
(a) protein that in sequence table, sequence 1 forms from N-terminal the 23rd to 883 amino acids residues;
(b) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;
(c) by the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and there is the protein being derived by sequence 1 of β-2-Acetamido-2-deoxy-D-glucose glycosides enzyme function.
For make (a) or (b) in protein be convenient to purifying, the N-terminal of the protein that can form at the aminoacid sequence shown in sequence in sequence table 1 or C-terminal connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Protein in above-mentioned (c) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the protein in above-mentioned (c) can be by lacking the codon of one or several amino-acid residue in (a) or encoding gene (b), and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.
The gene of encoding said proteins also belongs to protection scope of the present invention.
Described gene can be following 1) or 2) or 3) or 4) DNA molecular:
1) in sequence table sequence 2 from the DNA molecular shown in the 67th to 2649 Nucleotide of 5 ' end the;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) the DNA sequence dna hybridization that limits and the coding DNA molecular with the albumen of β-2-Acetamido-2-deoxy-D-glucose glycosides enzymic activity;
4) with 1) or 2) DNA sequence dna that limits has the DNA molecular that 90% above homology and coding have the albumen of β-2-Acetamido-2-deoxy-D-glucose glycosides enzymic activity.
Described stringent condition is in the solution of 0.1 * SSPE (or 0.1 * SSC), 0.1%SDS, hybridizes and wash film under 65 ℃ of conditions.
The recombinant expression vector that contains described gene, expression cassette, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.
The recombinant expression vector that available existing expression vector establishment contains described gene.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.While using described gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can be used alone or are combined with other promotor; In addition, while using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions 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 described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.For the ease of identifying and screening, can process expression carrier used thereof, as add coding can produce the enzyme of colour-change or the gene of luminophor, there are the antibiotic marker thing of resistance or anti-chemical reagent marker gene etc.Also can not add any selected marker, directly according to phenotypic screen.
Described recombinant expression vector specifically can be the recombinant plasmid that the multiple clone site of described gene insertion vector pET28a (+) obtains.
Described recombinant bacterium specifically can be described recombinant expression vector is imported to the recombinant bacterium that intestinal bacteria obtain.Described intestinal bacteria are preferably e. coli bl21 (DE3).
The present invention also protects a kind of described method of protein of preparing, and comprises the steps:
(1) cultivate described recombinant bacterium and in culturing process, add IPTG to induce;
(2), by the culture system centrifugal collecting precipitation of completing steps (1), after ultrasonication, collect supernatant liquor and be the solution that contains described protein.
Described method specifically comprises the steps:
(1) described recombinant bacterium is cultured to OD
600=0.6, then adding IPTG and making its starting point concentration is 1mol/L, then 18 ℃ of shaking culture 12h;
(2) by the culture system centrifugal collecting precipitation of completing steps (1), after ultrasonication, collect supernatant liquor, be the solution that contains described protein.
Described method more specifically comprises the steps:
(1) described recombinant bacterium in liquid LB substratum (containing 100 μ g/mL kantlex) 37 ℃ of shaking culture to OD
600=0.6, then adding IPTG and making its starting point concentration is 1mol/L, then 18 ℃, 180 * g shaking culture 12h;
(2) by the centrifugal 5min collecting precipitation of culture system 10, the 000 * g of completing steps (1), after ultrasonication, collect supernatant liquor, be the solution that contains described protein.
The primer pair of described full length gene or its any fragment of increasing all belongs to protection scope of the present invention.Described primer pair specifically can be the primer pair that shown in the sequence 4 of DNA fragmentation shown in the sequence 3 of sequence table and sequence table, DNA fragmentation forms.
The present invention also protects described albumen as the application of β-2-Acetamido-2-deoxy-D-glucose glycosides enzyme.In described application, the substrate of described albumen can be pNP-GlcNAc, pNP-(GlcNAc)
2, pNP-(GlcNAc)
3, at least one in chitobiose, chitin trisaccharide, chitin tetrose, chitin pentasaccharides and chitin six sugar.
The present invention also protects the application of described albumen in preparing feed.
Protein provided by the invention is a kind of chitobiose enzyme, has higher specific activity, loose range of application and satisfactory stability, can be used as the cultivation that fodder additives is applied to hydrocoles, has major application prospect.
Accompanying drawing explanation
Fig. 1 is that the SDS-PAGE of CTP solution and NAG565 protein liquid analyzes, M:Marker; 1:CTP solution; 2:NAG565 protein liquid.
Fig. 2 is the optimal pH of NAG565 albumen.
Fig. 3 is the pH stability of NAG565 albumen.
Fig. 4 is the optimum temperuture of NAG565 albumen.
Fig. 5 is the thermostability of NAG565 albumen.
Fig. 6 is the protease resistant of NAG565 albumen.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.Experimental technique in following embodiment, if no special instructions, is ordinary method, all according to the related Sections in following laboratory manual or document or part, carry out, comprise: the 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 in embodiment is Bradford method.Test materials used in following embodiment, if no special instructions, is from routine biochemistry reagent shop and purchases available domestic analytical reagent.
Intestinal bacteria (Escherichia coli) DH5 α competent cell, intestinal bacteria (Escherichia coli) BL21 (DE3) competent cell are all purchased from Beijing Quan Shi King Company.Ligase enzyme is purchased from NEB company.Trypsin Trypsin), Chymetin (α-Chymotrypsin), Proteinase K (Proteinase K), Collagenase (Collagenase) are Sigma (USA) product.Stomach en-(Pepsin) is Amano Enzyme Inc. (Japan) product.IPTG is Promega company product.Anti-His antibody, sheep anti-mouse igg-HRP and Western Blot membrane closure liquid are all purchased from Beijing Tian Gen biochemical technology company limited.
Carrier pET28a (+): purchased from Invi trogen company.
P-nitrophenol (4-Nitrophenol or pNP): purchased from Sigma company, catalog number N7660.
PNP-GlcNAc (4-Nitrophenyl N-acetyl-α-D-glucosaminide): purchased from Sigma company, catalog number N8759.
PNP-(GlcNAc)
2(4-Nitrophenyl N, N '-Diacetylchitobiose): purchased from Sigma company, catalog number N6133.
PNP-(GlcNAc)
3(4-Nitrophenyl N, N ', N "-Triacetylchitotriose): purchased from Sigma company, catalog number N8638.
Aeromonas (Aeromonas sp.) B565, on December 03rd, 2010, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.4403.
Protein called after NAG565 albumen by shown in the sequence of sequence table 1, is comprised of 883 amino-acid residues, from the 1st to 22 of N-terminal, is signal peptide.By the encoding gene called after NAG565 gene of NAG565 albumen, its open reading frame is as shown in the sequence 2 of sequence table (2649bp).
The preparation of embodiment 1, NAG565 albumen
One, the structure of recombinant plasmid NAG565-pET28a (+)
1, extract the genomic dna of Aeromonas B565.
2, take the genomic dna of step 1 is template, with the primer pair that F1 and R1 form, carries out pcr amplification, obtains pcr amplification product.
F1 (sequence 3 of sequence table): 5 '-CCC
gAATTCgCCGATGCCGCCAAGCAGGC-3 ';
R1 (sequence 4 of sequence table): 5 '-TAT
gCGGCCGCgACGCGGCTGGTGCGCT-3 '.
3, with the pcr amplification product of restriction enzyme EcoRI and NotI double digestion step 2, reclaim enzyme and cut product.
4, with restriction enzyme EcoRI and NotI double digestion carrier pET28a (+), reclaim carrier framework (about 5300bp).
5, the carrier framework of the enzyme of step 3 being cut to product and step 4 is connected, and obtains recombinant plasmid NAG565-pET28a (+).According to sequencing result, recombinant plasmid NAG565-pET28a (+) is carried out to structrual description as follows: the sequence 2 of having inserted sequence table between the EcoRI of carrier pET28a (+) and NotI restriction enzyme site is from the DNA fragmentation shown in the 67th to 2649 Nucleotide of 5 ' end.
Two, the structure of recombinant bacterium and contrast bacterium
Recombinant plasmid NAG565-pET28a (+) heat shock is transformed to e. coli bl21 (DE3) competent cell, obtain recombinant bacterium.Carrier pET28a (+) heat shock is transformed to e. coli bl21 (DE3) competent cell, obtain contrasting bacterium.
Three, the preparation of NAG565 albumen and reference protein
1, recombinant bacterium step 2 being built 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 10mL liquid LB substratum (containing 100 μ g/mL kantlex) to 37 ℃ of about 3h (OD of shaking culture
600=0.6) (making its starting point concentration is 1mol/L, to add IPTG; IPTG is inductor), 18 ℃, 180 * g shaking culture 12h (from adding IPTG to start timing).
3, the centrifugal 5min of nutrient solution 10,000 * g step 2 being obtained, collects respectively culture supernatant and cell precipitation.
4, the Tris-HCl damping fluid of pH8.0,20mM for cell precipitation step 3 being obtained is resuspended, ultrasonication, and then the centrifugal 10min of 10,000 * g, collects supernatant, is the solution (being called for short CTP solution) containing total protein of cell.
The contrast bacterium that step 2 is built replaces recombinant bacterium to carry out the operation of step 1 to 4, obtains the solution (abbreviation contrast solution) containing total protein of cell.
Four, the enzyme activity determination of CTP solution and contrast solution (pNP-GlcNAc method)
Enzyme (U) unit definition alive: per minute decomposition pNP-GlcNAc discharges the required enzyme amount of 1 μ mol p-nitrophenol (pNP) and is defined as Yi Gemeihuo unit (1U).In unit volume, the enzyme work of solution to be measured, divided by the protein content of solution to be measured in unit volume, is the ratio vigor of albumen.
Enzyme activity determination method: pNP-GlcNAc is dissolved in 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 and shaken up in test tube, and 37 ℃ of incubation 5min, then add 2mL 0.5M NaOH aqueous solution termination reaction, surveys OD value at 405nm place.Control tube: add successively 10 μ L solution to be measured in test tube, 240 μ L damping fluids, the 2mL 0.5M NaOH aqueous solution and 250 μ L substrate solutions, survey OD value at 405nm place.Using p-nitrophenol as standard substance production standard curve, typical curve function is: y=(0.169x-0.0153) * 100 * N/5; Y: enzyme is lived (U/mL), x:OD value, 100: by the conversion alive of the enzyme in 10 μ L solution to be measured, be the enzymic activity of 1mL, N: extension rate.
The contrast solution respectively 4 of 3 of the step 3 culture supernatant that obtain, the step 3 CTP solution that obtain and step 3 being obtained makes to carry out enzyme activity determination.The damping fluid adopting in enzyme activity determination is Sodium phosphate dibasic-citrate buffer solution of pH7.0,20mM.The enzyme of contrast solution is lived as 0U/ml, and the enzyme of CTP solution is lived as 1562U/ml (being 1243U/mg than vigor).Result shows, NAG565 albumen is mainly at intestinal bacteria intracellular expression.
Five, the purifying of NAG565 albumen
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 Ni-NTA resin, and purchased from Shanghai Sheng Gong bio-engineering corporation (catalog number BSP033), dress post amount is 1mL.
Each damping fluid following (pH is 7.6) in elution process:
NTA-0: containing 20mmol/L Tris-HCl, 0.5mol/L NaCl, 10g/100ml glycerine, other is water;
NTA-20: containing 20mmol/L Tris-HCl, 20mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is water;
NTA-40: containing 20mmol/L Tris-HCl, 40mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is water;
NTA-60:20mmol/L Tris-HCl, 60mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is water;
NTA-80:20mmol/L Tris-HCl, 80mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is water;
NTA-100:20mmol/L Tris-HCl, 100mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is water;
NTA-200:20mmol/L Tris-HCl, 200mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is water;
NTA-300:20mmol/L Tris-HCl, 300mmol/L imidazoles, 0.5mol/L NaCl, 10g/100ml glycerine, other is water.
Elution process (at every turn add 5mL, flow out pillar completely then add again 5mL):
(1) pillar watering balance 20mL;
(2) with NTA-0 balance 20mL, loading 5mLCTP solution;
(3) with NTA-0 wash-out 25mL (foreigh protein removing), then use successively NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, each 5mL wash-out of NTA-300, and collect the solution (NAG565 protein liquid) after post of crossing of NTA40-80 wash-out.
NAG565 protein liquid prepared by CTP solution prepared by step 4 and step 5 carries out SDS-PAGE electrophoresis, sees Fig. 1, and the albumen after purifying shows the single band of 95kDa, conforms to expection molecular weight.
Six, the enzyme activity determination of NAG565 albumen
The NAG565 protein liquid that step 5 is obtained is as solution to be measured, other Complete Synchronization rapid four.The enzyme activity of NAG565 protein liquid is 2257U/mL, than vigor, is 7328U/mg.
The zymologic property of embodiment 2, NAG565 albumen
The NAG565 protein liquid that the step 5 of embodiment 1 is obtained, as solution to be measured, carries out between following each zymologic property successively:
One, the optimal pH of NAG565 albumen
Enzyme activity determination method is with the step 4 of embodiment 1.
In enzyme activity determination, adopt respectively the sodium acetate buffer of following damping fluid: pH3.0,0.1M, 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, glycine-NaOH damping fluid of pH10.0,0.1M, glycine-NaOH damping fluid of pH11.0,0.1M.
NAG565 albumen enzyme in the environment of pH7.0 is lived the highest, than vigor, is 7301U/mg.Take this higher specific activity is 100%, calculates the relative enzyme of NAG565 albumen in other pH environment and lives, and sees Fig. 2.NAG565 albumen, in the environment of pH8.0, can keep more than 80% relative enzyme to live.
Two, the pH stability of NAG565 albumen
Enzyme activity determination method:
PNP-GlcNAc is dissolved in 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, then add 250 μ L substrate solutions to mix and 37 ℃ of incubation 5min, then add 2mL 0.5M NaOH aqueous solution termination reaction, at 405nm place, survey OD value.
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, then add successively the 2mL 0.5M NaOH aqueous solution and 250 μ L substrate solutions, in 405nm place survey OD value.
NAG565 albumen stability in the environment of pH7.0 is the highest, and 37 ℃ of incubations ratio vigor after 12 hours is 6350U/mg.Take this higher specific activity 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 pH scope, keeps more than 70% relative enzyme to live.
Three, the optimum temperuture of NAG565 albumen
In enzyme activity determination except heated culture temperature, the step 4 of the other the same as in Example 1.In enzyme activity determination, adopt respectively following heated culture temperature: 0 ℃, 20 ℃, 30 ℃, 37 ℃, 50 ℃, 60 ℃ and 70 ℃.In enzyme activity determination, adopt Sodium phosphate dibasic-citrate buffer solution of pH7.0,0.1M.
NAG565 albumen enzyme in the environment of 37 ℃ is lived the highest, than vigor, is 7321U/mg.Take this higher specific activity is 100%, calculates the relative enzyme of NAG565 albumen in other temperature environment and lives, and sees Fig. 4.NAG565 albumen, in the buffering of 20 ℃, can keep the enzyme of 40% left and right to live.
Four, the thermostability of NAG565 albumen
Enzyme activity determination method:
PNP-GlcNAc is dissolved in damping fluid, and making its final concentration is 1mM, is substrate solution.
Developmental tube: in test tube by 10 μ L solution differing temps to be measured (0 ℃, 20 ℃, 30 ℃, 37 ℃, 50 ℃, 60 ℃ or 70 ℃) incubation 30min, then add 240 μ L damping fluids and 250 μ L substrate solutions, mix, 37 ℃ of incubation 5min, then add 2mL 0.5M NaOH aqueous solution termination reaction, at 405nm place, survey OD value.
Control tube: by 10 μ L solution differing temps to be measured incubation 30min, then add successively 240 μ L damping fluids, the 2mL 0.5M NaOH aqueous solution and 250 μ L substrate solutions in test tube, survey OD value at 405nm place.
In enzyme activity determination, adopt Sodium phosphate dibasic-citrate buffer solution of pH7.0,0.1M.
NAG565 albumen stability in the environment of 30 ℃ is the highest, and the ratio vigor after 30 ℃ of incubation 30min is 6187U/mg.Take this higher specific activity is 100%, calculates NAG565 albumen relative enzyme after 30 ℃ of incubation 30min in other temperature environment and lives, and sees Fig. 5.NAG565 albumen loses activity above completely at 70 ℃, and between 30-70 ℃, along with the rising for the treatment of temp, enzyme is lived and reduced gradually, and 30 ℃ of following processing are on not impact of enzymic activity.
The kinetic constant of embodiment 3, NAG565 albumen
The NAG565 albumen obtaining 37 ℃ of step 5 of measuring embodiment 1, to the initial velocity of reaction of the substrate of different concns (1,2,5,10,50,100,500,1000 μ mol/L) (enzyme live the step 4 of detection method with embodiment 1), is drawn and is drawn the K of this enzyme to 3 kinds of substrates through Lineweaver-Burk
mand V
maxvalue.Substrate is respectively pNP-GlcNAc, pNP-(GlcNAc)
2and pNP-(GlcNAc)
3.
The results are shown in Table 2.In 3 kinds of substrates, the K of pNP-GlcNAc
mvalue is minimum, V
maxvalue is maximum.
The kinetic constant of table 2NAG565 albumen to various substrates
The substrate specificity of embodiment 4, NAG565 albumen
One, the degraded of NAG565 albumen to chitin oligo saccharide
1, by substrate (chitin oligo saccharide), the concentration with 4mM is dissolved in the phosphoric acid buffer of pH7.0,10mM, is substrate solution.Chitin oligo saccharide adopts respectively following several: chitobiose, chitin trisaccharide, chitin tetrose, chitin pentasaccharides or chitin six sugar, and all purchased from TRC company.
2, get the chitin oligo saccharide substrate solution of 0.1mL step 1, add NAG565 albumen prepared by the step 5 of 5 μ g embodiment 1, be initial system (reducing sugar that contains 0 μ g/mL in initial system); By 37 ℃ of enzymolysis 6h of initial system, obtain termination system; Using the phosphoric acid buffer of 0.1mLpH7.0,10mM as the negative control of chitin oligo saccharide substrate solution.
3, according to the reducing sugar content in DNS method mensuration product, (adopt glucose as standard substance production standard curve, typical curve equation is as follows: Y=2521.1* Δ OD+20.41; The increasing amount of reducing sugar in termination system is compared in 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), using that this characterizes NAG565 albumen as the ability of β-2-Acetamido-2-deoxy-D-glucose glycosides enzyme liberating 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, 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 determine that NAG565 albumen has certain Degradation to chitin oligo saccharide.
Two, the identification of NAG565 albumen to other substrate
The same step 1 of method, difference is only 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)、
Carboxymethyl cellulose;
Chitosan powder.
NAG565 albumen does not all have degradation capability to above-mentioned substrate.
Result shows, NAG565 albumen has specificity to β-2-Acetamido-2-deoxy-D-glucose glycosidic bond.
The resistance of the NAG565 albumen that the step 5 of detection embodiment 1 obtains to different metal ion or chemical reagent.Enzyme detection method alive is referring to the step 4 of embodiment 1, and difference is only to add different metal ions or chemical reagent (K in reaction system
+, 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), with the reaction system that do not add metal ion and chemical reagent in contrast.In enzyme activity determination, adopt Sodium phosphate dibasic-citrate buffer solution of pH7.0,0.1M.
The results are shown in Table 3.Na
+, Mn
2+enzyme work to NAG565 albumen has promoter action.Co
2+, Cr
3+, Cu
2+deng the enzyme work to NAG565 albumen, there is restraining effect in various degree, Ag
+and Hg
2+almost completely suppress the enzyme of NAG565 albumen and live, and K
+, Ca
2+, Mg
2+the impact of living Deng the enzyme on NAG565 albumen is very little, negligible.NAG565 albumen has certain resistivity to radiolucent table surface-active agent-SDS.
Table 3 metal ion and the impact of relevant chemical reagent on NAG565 albumen
| Chemical reagent | Residual enzyme (%) alive | Metal 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 become respectively to the solution of 1mg/mL with following solution preparation: with 0.1M glycine-HCl of pH2.0, stomach en-is made into the solution of 1mg/mL; With the 0.1M Tris-HCl of pH7.0, respectively trypsinase and Chymetin are made into the solution of 1mg/mL; With the 0.1M Tris-HCl of pH7.5, Proteinase K and Collagenase are made into respectively to the solution of 1mg/mL.The NAG565 albumen that the step 5 of embodiment 1 is obtained and proteolytic enzyme are processed after 30min or 60min by 10: 1 (mass ratio) in this protein enzyme solution, by the residual enzyme of standard method Check processing enzyme (referring to the step 4 of embodiment 1) alive.With the reaction system that do not add proteolytic enzyme (CK) in contrast.
The results are shown in Figure 6.With after different protease treatment 60min, the residual enzyme work of NAG565 albumen 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 multiple protein enzyme.
Claims (1)
1. the application of the protein with β-2-Acetamido-2-deoxy-D-glucose glycosides enzymic activity in preparing feed; The protein of the described β of having-2-Acetamido-2-deoxy-D-glucose glycosides enzymic activity is the protein that in sequence table, sequence 1 forms from N-terminal the 23rd to 883 amino acids residues.
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| 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 |
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| Title |
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| 张宇婷.来源于维氏气单胞菌B565的两个几丁质降解基因的高效表达、性质研究及水产养殖应用.《中国优秀硕士学位论文全文数据库 农业科技辑》.2012,(第10期),D052-20. |
| 来源于维氏气单胞菌B565的两个几丁质降解基因的高效表达、性质研究及水产养殖应用;张宇婷;《中国优秀硕士学位论文全文数据库 农业科技辑》;20121015(第10期);全文 * |
| 登录号.CP002607 REGION:2996521-2999184.《NCBI,Genbank》.2011, * |
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