CN1150316C - Chitinase produced by fumigacin - Google Patents
Chitinase produced by fumigacin Download PDFInfo
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Abstract
The present invention obtains Aspergillus fumigatus YJ-407 and separates and purifies a novel chitinase named chiM. The molecular weight of the chitinase is 46KDa, the isoelectric point has the pH value of 5.6, and the N-terminal amino acid sequence is A-S-S-G-Y-R-S-V-V-Y-F-V. The chitinase is stable at the range of pH4.0 to 7.0 and is most stable when the pH value is 5.0; the optimal reaction temperature of the chitinase is 60 DEG C; Fe<2+>, Zn<2+>, Mn<2+>, Ag<+>, Pb<2+> and Hg<2+> can strongly suppress enzyme activity. The hydrolyzing capability of the chitinase to 6-0-hydroxyl ethane chitin and chitosan (80% deacylated) is higher than that to chitin powder and colloidal chitin, and the chitinase can hydrolyze the chitin into chitobiose and chitotriose and has exoenzyme activity and endoenzyme activity. In addition, the chitinase has obvious activity of transglycosylation and is capable of transferring the chitobiose onto the chitotriose to form chitopentose. The property can be used for the enzymatic preparation of bioactive chitooligose.
Description
Chitin is that occurring in nature content is only second to cellulosic second largest carbon source, and in development and use Mierocrystalline cellulose, chitinous research, the mankind have made huge effort, though obtained some progress, but still fail really effectively to utilize.Chitin is not only the carbon source of enriching that can support utilization, and reaches in vivo and undertaking important function in the biological interphase interaction.Be cell wall constituent in fungi, constitute exoskeleton in invertebrates, these biologies itself all produce chitinase again, and enzyme is having important physiological significance aspect form generation and the nutrition intake again to chitinous degraded; Chitin also plays a very important role in the interaction of plant and pathogenic epiphyte and insect, as in the disease-resistant mechanism of plant, plant is being subjected to will synthesizing and accumulate antimicrobial plant alexin (phytoalexins) after infected by microbes or inducer (elicitor) are handled.Contained structural constituent chitin and chitosan (chitosan) are degraded into the oligosaccharides sheet by chitinase and have no progeny can bring out disease resistance response in various plants in cell walls of fungi and the exoskeleton of invertebrates.
Finding to take off the acetyl chitin oligo saccharide in recent years again has aspects such as antitumor, hypertension, mastitis-resisting, reducing cholesterol, the suppurative wound of treatment, sacroiliitis pain relieving obvious curative effects is all arranged.Therefore take off that the acetyl chitin oligo saccharide can be used as that the functional oligosaccharide product is used for that medicine is clinical, functional food, wound, pain relieving etc., therefore the research of taking off the acetyl chitin oligo saccharide also be subjected to the various countries scholar pay close attention to.Take off the acetyl chitin oligo saccharide by chitinase with the chitosan hydrolysis, the functional polymerization degree of taking off the acetyl chitin oligo saccharide is 2-10.Be that control enzyme reaction condition obtains the degraded product of suitable polymeric degree at present, produce the bioactive oligosaccharides segment of tool but be very difficult to control condition in actual applications.Multiple chitinase is Separation Research from different biologies.The mykose enzyme the form of fungi take place and nutrition intake aspect the important physical function is arranged, in addition, the mykose enzyme also can be applicable to biological control and contains chitinous insect and develop chitin.Separate from fungi in recent years and studied several chitinases, these enzymes mainly are isolating from parasitic fungi and insect pathomycete, and the aspergillus of some saprophytic property is degrade chitin very effectively also.Yet the structure and the character of the chitinase that aspergillus is produced are but known little about it.From several mykose enzymes of having reported, finding no changes the active enzyme of glycosyl, and only find also that in the research of the chitinase that all different biological species are originated a circumscribed chitinase that derives from bacterium has the glycosyl of commentaries on classics activity, not seeing has the endochitinase tool to change the active report of glycosyl.
The objective of the invention is to from fungus resource, seek the microorganism that tool changes the active endochitinase of glycosyl and produces this kind of enzyme.Concretely, provide a kind of generation to have inscribe, circumscribed and change the Aspergillus fumigatus Aspergillus fumigatus YJ-407 CGMCC NO.0386 of the active chitinase of glycosyl and utilize this bacterium to cultivate again on the fermention medium of cultivating, producing then chitinase on the potato dextrose culture-medium earlier, that the separation and purification of enzyme relates to is concentrated, saltout, DEAE column chromatography and polyacrylamide gel electrophoresis.A kind of chitinase that Aspergillus fumigatus Aspergillus fumigatus YJ-407 CGMCC NO.0386 produces, the molecular weight of this enzyme is 46KDa, iso-electric point is pH5.6, enzyme shows the typical absorption spectrum of minimum and maximum absorption respectively at 278nm wavelength and 252nm wavelength, the maximum fluorescence excitation wavelength is 342nm, zymoprotein-terminal amino acid sequence is: A-S-S-G-Y-R-S-V-V-Y-F-V, the optimal pH of enzyme reaction is 5.0, enzyme is stable in the scope of pH4.0-7.0, and the optimal reactive temperature of enzyme is 60 ℃; Enzyme is higher than chitin powder and tobacco brown spot pathogen the hydrolysis ability of 6-0-hydroxyl ethyl group chitin and chitosan, and above-mentioned chitosan is the chitin of 80% deacetylate.
The objective of the invention is to from fungus resource, seek the microorganism that tool changes the active endochitinase of glycosyl and produces this kind of enzyme.
We are separated to Aspergillus fumigatus and have the ability that produces chitinase from soil, wherein a strain Aspergillus fumigatus YJ-407 is best, this bacterium is preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", registering on the books is numbered CGMCCN0.0386.And by purifying in the fermented liquid of this bacterial strain a kind of new endochitinase chiM, this enzyme can be hydrolyzed to chitin chitobiose and chitin trisaccharide, in addition, also has the significant glycosyl activity of changeing, and chitobiose can be forwarded to and form the chitin pentasaccharides on the chitin trisaccharide.This character can be used for the enzymatic preparation of biological activity chitin oligo saccharide.The present invention provides possibility for exploitation biological activity chitin oligo saccharide is applied to clinical treatment.
Realize that concrete technical scheme of the present invention is:
1.Aspergillus the cultural method of fumigatus YJ-407
Aspergillus fumigatus YJ-407 is kept on the potato glucose agar medium inclined-plane.Composition part of potato glucose agar medium is: potato 200 gram adds 1 liter in water, boils after 10 minutes gauze and crosses the Lu, adds glucose 20 grams in the liquid of Lu, and agar 20 grams do not add agar in basal culture medium, can be used as potato liquid of glucose substratum.Be inoculated in after with 0.1%Tween-20 spore being collected in the 500ml triangular flask that 200ml potato dextrose culture-medium is housed that (inoculum size is 5 * 106 spores/ml), with the rotating speed of 200r/min, cultivated 24 hours for 35 ℃ on shaking table.Cultivate after-filtration and collect mycelium, and be: fine powder chitin 1%, peptone 0.05%, yeast extract paste 0.05% with the component that is suspended in chitin fermention medium in the 200ml chitinase fermention medium behind the aseptic water washing, glucose 0.1%, KH2PO4 0.07%, and K2HPO4 0.07%, MgSO47H200.05%, FeSO47H20 0.001%, ZnSO4 0.0001%, and pH 5.0, in 32 ℃ cultivate 96 hours after centrifugal collection fermented supernatant fluid be used for the purifying of enzyme.Per-cent in the above-mentioned substratum is to be the weight percent of matrix with water, and following method all together.
2.Aspergillus the preparation of the thick enzyme of fumigatus YJ-407 chitinase chiM
The purifying of enzyme carries out under 4 ℃.1 liter of fermented supernatant fluid concentrates with hollow fiber column ultrafilter, and the exclusion molecular weight of hollow fiber column ultrafilter can be in the 5000-20000 scope, and the general exclusion molecular weight that adopts is advisable about 10000.The ammonium sulfate precipitated protein that behind ultrafiltration and concentration, can add the 30%-70% saturation ratio, the protein precipitation of collection 30% ~ 60% saturation ratio also is dissolved in the deionized water, and dialysis is thick enzyme sample in 50mM Tris-HCl damping fluid (pH8.0) then.
3.Aspergillus the DEAE-52 column purification of fumigatus YJ-407 chitinase chiM
Thick enzyme is with DEAE-52 post (3 * 30cm) purifying of 50mM Tris-HCl (pH8.0) pre-equilibration.With the 600ml gradient is that 0.15 ~ 0.5M NaCl makes linear elution (flow velocity is 15ml/h), collects to contain the active part of chitinase chiM, concentrates the back 50mM Tris-HCl (pH8.0) damping fluid is dialysed.
4.Aspergillus the polyacrylamide gel electrophoresis purifying of fumigatus YJ-407 chitinase chiM
1. Tou Xi enzyme (2mg) is with preparation type polyacrylamide gel electrophoresis (100 * 120 * 30mm, 7.5%gel, pH8.9) purifying, the mobility Rf of chitinase chiM on running gel is 0.43, chitinase chiM band is downcut from glue, and (pH5.0) comes out the enzyme wash-out with the 50mM sodium-acetate buffer, and elutriant is dialysed in deionized water, at last, the freeze-drying of enzyme liquid is stored in-20 ℃.
The final purification that carries out enzyme with preparation type high pressure liquid chromatography also can obtain identical result.
5. enzyme activity determination method
Add 100 μ l chitinase liquid in the reaction system that contains 0.4ml 0.5% tobacco brown spot pathogen and 0.5ml 100mM sodium-acetate buffer (pH5.0), 60 ℃ were reacted 15 minutes, and boiled 5 minutes termination reactions in boiling water baths.Reducing sugar content in the assaying reaction mixture.The enzyme work of chitinase is defined as per minute catalysis under these conditions and produces that to be equivalent to the required enzyme amount of 1 μ mol N-acetylglucosamine be 1 unit.
6. protein determination method
The mensuration of protein concentration is pressed the Lowry method and is measured.
7. electrophoresis
5% and 12% polyacrylamide gel electrophoresis is undertaken by the method for Davis, and the SDS-PAGE electrophoresis is undertaken by the method for Laemmli, and the IEF-SDS-2-D electrophoresis is undertaken by the method for Hames etc.
8. other analytical procedure
The n terminal amino acid sequence is measured with 477A Protein Sequencer (ABI), and amino acid composition is analyzed with Beckman 121MB Amino Acid Analyzer.Enzyme reaction product silica gel thin-layer chromatography analysis, developping agent are propyl carbinol: acetic acid: water (2: 1: 1).
Embodiment
To be kept on the potato glucose agar medium inclined-plane with 0.1%Tween-20 and to be inoculated in after Aspergillus fumigatus YJ-407 spore is collected that (inoculum size is 5 * 10 in the 500ml triangular flask that 200ml potato dextrose culture-medium is housed
6Spore/ml), on shaking table,, cultivated 24 hours for 35 ℃ with the rotating speed of 200r/min.Cultivate after-filtration and collect mycelium, and being suspended in behind the aseptic water washing in the 200ml chitinase fermention medium, in 32 ℃ cultivate 96 hours after centrifugal collection fermented supernatant fluid be used for the purifying of enzyme.
The purifying of enzyme carries out under 4 ℃.1 liter of fermented supernatant fluid is concentrated with hollow fiber column ultrafilter, and the exclusion molecular weight of hollow fiber column ultrafilter is 6000.Add ammonium sulfate precipitated protein behind ultrafiltration and concentration, the protein precipitation of collection 30% ~ 60% saturation ratio also is dissolved in the deionized water, and dialysis is thick enzyme sample in 50mM Tris-HCl damping fluid (pH8.0) then.
Thick enzyme is with DEAE-52 post (3 * 30cm) purifying of 50mM Tris-HCl (pH8.0) pre-equilibration.With the 600ml gradient is that 0.15 ~ 0.5M NaCl makes linear elution (flow velocity is 15ml/h), collects to contain the active part of chitinase chiM, concentrates the back 50mM Tris-HCl (pH8.0) damping fluid is dialysed.
The enzyme (2mg) of dialysis preparation type polyacrylamide gel electrophoresis (100 * 120 * 30mm, 7.5%gel, pH8.9) purifying, the mobility Rf of chitinase chiM on running gel is 0.43, chitinase chiM band is downcut from glue, and (pH5.0) comes out the enzyme wash-out with the 50mM sodium-acetate buffer, and elutriant is dialysed in deionized water, at last, the freeze-drying of enzyme liquid is stored in-20 ℃.The final purification that carries out enzyme with preparation type high pressure liquid chromatography also can obtain identical result.
By above-mentioned steps, purifying chitinase chiM from Aspergillus fumigatus YJ-407 obtains the pure enzyme of PAGE and IEF-SDS-2D electrophoresis homogeneous.
Have following feature from the chitinase chiM of Aspergillus fumigatus YJ-407 purifying: pure enzyme molecular weight is 46KDa, and iso-electric point is pH5.6.Enzyme shows the typical absorption spectrum of minimum and maximum absorption respectively at 278nm wavelength and 252nm wavelength, and the maximum fluorescence excitation wavelength is 342nm.Zymoprotein-terminal amino acid sequence is: A-S-S-G-Y-R-S-V-V-Y-F-V.The optimal pH of enzyme is 5.0, and enzyme is stable in the scope of pH4.0-7.0, wherein pH be 5.0 o'clock the most stable.The optimal reactive temperature of enzyme is 60 ℃; Enzyme is dissolved in the 50mM acetate buffer solution (pH5.0), and it is alive that enzyme is measured in insulation after 30 minutes under differing temps, and the result shows that enzyme is stable when being lower than 45 ℃, enzyme complete deactivation when temperature is 65 ℃.Fe
2+, Zn
2+, Mn
2+, Ag
+, pb
2+, Hg
2+The strongly inhibited enzyme is lived, and simultaneously, EDTA lives to enzyme does not have influence.This enzyme is higher than chitin powder and tobacco brown spot pathogen the hydrolysis ability of the chitosan of 6-O-hydroxyl ethyl group chitin and 80% deacetylation, and the difference of these substrate abilities of prompting enzymic hydrolysis is the difference of substrate solubleness.This enzyme does not act on Mierocrystalline cellulose and CMC Mierocrystalline cellulose, and therefore, this enzyme is the chitinase of special hydrolysis linear pattern poly N-acetylglucosamine.The Km value of enzymic hydrolysis tobacco brown spot pathogen is 0.9mg/ml.Carboxyl and tryptophan residue are that enzyme is alive necessary, and sulfydryl and histidine residues are less to the contribution of enzyme active center.
Add tobacco brown spot pathogen and chitinase liquid in the reaction system that contains 0.5ml 100mM sodium-acetate buffer (pH5.0), 60 ℃ were reacted 15 minutes, and boiled 5 minutes termination reactions in boiling water baths.With its product of silica gel thin sheet chromatographic analysis, the result shows that this enzyme can become the tobacco brown spot pathogen hydrolysis chitobiose, chitin trisaccharide and N-acetylglucosamine, and wherein chitobiose is a main degradation products.
In the reaction system that contains 0.5ml 100mM sodium-acetate buffer (pH5.0), add chitin trisaccharide and chitinase liquid, after 60 ℃ of incubated overnight, can produce chitobiose and N-acetylglucosamine.
Add chitin six sugar and chitinase liquid in the reaction system that contains 0.5ml 100mM sodium-acetate buffer (pH5.0), 60 ℃ of reactions can produce the product from chitobiose to the chitin pentasaccharides in 1 hour.
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Claims (13)
1, a kind of generation has inscribe, circumscribed and change the Aspergillus fumigatus Aspergillus fumigatus YJ-407 CGMCC NO.0386 of the active chitinase of glycosyl.
2, a kind of method from Aspergillus fumigatus Aspergillus fumigatus separation and purification chitinase, it is characterized in that present method employed be Aspergillus fumigatus Aspergillus fumigatusYJ-407 CGMCC NO.0386, this bacterium is cultivated on the fermention medium of cultivating earlier, producing then chitinase on the potato dextrose culture-medium again, and the separation and purification of enzyme comprises the steps: successively to concentrate, saltouts, DEAE column chromatography and polyacrylamide gel electrophoresis.
3, method according to claim 2, being formulated as follows of potato dextrose culture-medium wherein: potato 200 grams add one liter in water, boil filtered through gauze after 10 minutes, add glucose 20 grams, agar 20 grams in filtrate, final volume is 1000ml, sterilizes 20 minutes in 8 pounds after the packing; Produce in the chitinase fermention medium with water is that the components in weight percent of matrix is: fine powder chitin 1%, peptone 0.05%, yeast extract paste 0.05%, glucose 0.1%, KH
2PO
40.07%, K
2HPO
40.07%, MgSO
47H
20 0.05%, FeSO
47H
2O 0.001%, ZnSO
40.0001%, pH5.0.
4, method according to claim 2, wherein the concentrated of chitinase is to adopt hollow fiber column ultrafilter to concentrate, the exclusion molecular weight of hollow fiber column ultrafilter is in the 5000-20000 scope.
5, method according to claim 4, the exclusion molecular weight 10000 of selection hollow fiber column ultrafilter.
6, method according to claim 2, wherein saltouing of enzyme liquid is the method that adopts the ammonium sulfate precipitation zymoprotein, the concentration of ammonium sulfate is in the scope of 30% to 70% saturation ratio.
7, method according to claim 6 is used the scope of ammonium sulfate concentrations as the 30%-60% saturation ratio.
8, method according to claim 2, wherein adopt DEAE-52 ion-exchange-resin process separation and purification chitinase, its condition is: with 50mM pH8.0 Tris-HCl damping fluid pre-equilibration DEAE-52 post, adopt gradient to make linear elution for 0.15-0.5M NaCl, the 50mM Tris-HCl damping fluid that this NaCl solution is dissolved in pH8.0 obtains.
9, method according to claim 2, wherein polyacrylamide gel electrophoresis is the method that adopts 7.5% polyacrylamide gel electrophoresis.
10, a kind of chitinase that produces by the described Aspergillus fumigatus Aspergillus of claim 1 fumigatus YJ-407CGMCC NO.0386, the molecular weight that it is characterized in that this enzyme is 46KDa, iso-electric point is pH5.6, enzyme shows the typical absorption spectrum of minimum and maximum absorption respectively at 278nm wavelength and 252nm wavelength, the maximum fluorescence excitation wavelength is 342nm, zymoprotein-terminal amino acid sequence is: A-S-S-G-Y-R-S-V-V-Y-F-V, the optimal pH of enzyme reaction is 5.0, enzyme is stable in the scope of pH4.0-7.0, and the optimal reactive temperature of enzyme is 60 ℃; Enzyme is higher than chitin powder and tobacco brown spot pathogen the hydrolysis ability of 6-O-hydroxyl ethyl group chitin and chitosan, and above-mentioned chitosan is the chitin of 80% deacetylate.
11, a kind of chitinase according to claim 10, this enzyme can be hydrolyzed to chitin chitobiose, chitin trisaccharide and N-acetylglucosamine, and wherein chitobiose is a main degradation products, and this enzyme has inscribe, circumscribed activity.
12, a kind of chitinase according to claim 10, this enzyme can forward chitobiose to and form the chitin pentasaccharides on the chitin trisaccharide, have the activity of changeing glycosyl.
13, a kind of chitinase according to claim 10 is in the application in the acetyl chitin oligo saccharide of taking off of preparation biologically active.
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| CNB991054156A CN1150316C (en) | 1999-04-06 | 1999-04-06 | Chitinase produced by fumigacin |
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| CNB991054156A CN1150316C (en) | 1999-04-06 | 1999-04-06 | Chitinase produced by fumigacin |
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| CN100526335C (en) * | 2006-05-10 | 2009-08-12 | 国家海洋局第三海洋研究所 | Chitin oligose preparing process |
| CN101759783B (en) * | 2008-12-24 | 2012-05-30 | 中国科学院微生物研究所 | Polypeptide for localizing proteins on cell membranes and application thereof |
| CN101759784B (en) * | 2008-12-24 | 2012-05-30 | 中国科学院微生物研究所 | Polypeptide for localizing proteins on cell walls and application thereof |
| CN101759782B (en) * | 2008-12-24 | 2012-05-30 | 中国科学院微生物研究所 | Polypeptide for localizing proteins on cell membranes and/or cell walls and application thereof |
| CN101518247B (en) * | 2009-04-13 | 2012-06-06 | 东北农业大学 | Preparation method of novel herbicide sugar adjuvant |
| CN101613685B (en) * | 2009-05-20 | 2011-04-13 | 广东省昆虫研究所 | Combined chitinase for deactivating varroa destructor and application thereof |
| US20120329135A1 (en) | 2011-06-23 | 2012-12-27 | Agrinos AS | Process for Making Chitin Derivatives |
| CN102286414B (en) * | 2011-09-02 | 2012-12-05 | 海南大学 | Chitin-degrading bacterial strain and method for preparing chitooligosaccharide by utilizing same |
| CN104054819B (en) * | 2014-07-03 | 2016-08-24 | 浙江大学 | Induction fruit resistance controls the method for disease and preparation used |
| CN104388496B (en) * | 2014-12-18 | 2017-12-22 | 南京工业大学 | A kind of method of enzymic degradation chitin production N acetylglucosamines |
| TWI670372B (en) * | 2017-06-27 | 2019-09-01 | 淡江大學 | Medium composition for providing fermentation of α-glucosidase inhibitor by Bacillus licheniformis |
| CN115851675A (en) * | 2022-08-04 | 2023-03-28 | 广西科学院 | N-terminal truncated mutant of chitinase and application thereof |
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