CN100467586C - Aspergillus niger WZ001 capable of producing naringinase and cirmtimase simultaneously and its application - Google Patents
Aspergillus niger WZ001 capable of producing naringinase and cirmtimase simultaneously and its application Download PDFInfo
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- CN100467586C CN100467586C CNB200610051963XA CN200610051963A CN100467586C CN 100467586 C CN100467586 C CN 100467586C CN B200610051963X A CNB200610051963X A CN B200610051963XA CN 200610051963 A CN200610051963 A CN 200610051963A CN 100467586 C CN100467586 C CN 100467586C
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- 108010001078 naringinase Proteins 0.000 title claims abstract description 39
- 241000228245 Aspergillus niger Species 0.000 title claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 238000000855 fermentation Methods 0.000 claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 23
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 239000007787 solid Substances 0.000 claims abstract description 3
- 108090000790 Enzymes Proteins 0.000 claims description 54
- 102000004190 Enzymes Human genes 0.000 claims description 54
- 108010030923 hesperidinase Proteins 0.000 claims description 35
- 230000001954 sterilising effect Effects 0.000 claims description 13
- 238000004659 sterilization and disinfection Methods 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- 238000010563 solid-state fermentation Methods 0.000 claims description 9
- 244000068988 Glycine max Species 0.000 claims description 7
- 235000010469 Glycine max Nutrition 0.000 claims description 7
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 7
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 235000015099 wheat brans Nutrition 0.000 claims description 7
- 238000001816 cooling Methods 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 3
- 238000009776 industrial production Methods 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 238000011084 recovery Methods 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 239000002994 raw material Substances 0.000 description 8
- 231100000350 mutagenesis Toxicity 0.000 description 7
- 238000002703 mutagenesis Methods 0.000 description 7
- 238000001556 precipitation Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 5
- 108010047754 beta-Glucosidase Proteins 0.000 description 5
- 102000006995 beta-Glucosidase Human genes 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- FTVWIRXFELQLPI-ZDUSSCGKSA-N (S)-naringenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 FTVWIRXFELQLPI-ZDUSSCGKSA-N 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- AIONOLUJZLIMTK-AWEZNQCLSA-N hesperetin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 AIONOLUJZLIMTK-AWEZNQCLSA-N 0.000 description 4
- 229960001587 hesperetin Drugs 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- WGEYAGZBLYNDFV-UHFFFAOYSA-N naringenin Natural products C1(=O)C2=C(O)C=C(O)C=C2OC(C1)C1=CC=C(CC1)O WGEYAGZBLYNDFV-UHFFFAOYSA-N 0.000 description 4
- 229940117954 naringenin Drugs 0.000 description 4
- 235000007625 naringenin Nutrition 0.000 description 4
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 description 3
- 239000001606 7-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-5-hydroxy-2-(4-hydroxyphenyl)chroman-4-one Substances 0.000 description 3
- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 description 3
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 description 3
- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 description 3
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 description 3
- 229940025878 hesperidin Drugs 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 229930019673 naringin Natural products 0.000 description 3
- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 description 3
- 229940052490 naringin Drugs 0.000 description 3
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 235000003239 Guizotia abyssinica Nutrition 0.000 description 1
- 241000228150 Penicillium chrysogenum Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- HIVLDXAAFGCOFU-UHFFFAOYSA-N ammonium hydrosulfide Chemical compound [NH4+].[SH-] HIVLDXAAFGCOFU-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008495 β-glucosides Chemical class 0.000 description 1
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
Abstract
The present invention provides one kind of Aspergillus niger WZ001 capable of realizing industrial production of producing naringinase and cirmtimase simultaneously in high yield and its application. The Aspergillus niger WZ001 has the preservation number of CCTCC No.206047. It has high yield of naringinase and cirmtimase over 14000 U/g in liquid fermentation culture or over 6000 U/g in solid fermentation culture.
Description
(1) technical field
The present invention relates to the black-koji mould WZ001 (Aspergillus niger WZ001) and the application thereof of a kind of high yield naringinase simultaneously and hesperidinase.
(2) background technology
Naringinase is by α-1, the enzyme system that 2-rhamnosidase and beta-glucosidase are formed, α-1, the 2-rhamnosidase can make the naringin hydrolysis obtain naringenin monoglycosides and rhamnosyl, beta-glucosidase further makes the hydrolysis of naringenin monoglycosides obtain naringenin and glucose, and naringinase can be produced by aspergillus niger, aspergillus oryzae, Penicillium notatum.
Hesperidinase is by α-1, the enzyme system that 6-rhamnosidase and beta-glucosidase are formed, α-1,6-rhamnosidase can make the Hesperidin hydrolysis obtain Hesperitin monoglycosides and rhamnosyl, and beta-glucosidase makes the hydrolysis of Hesperitin monoglycosides obtain Hesperitin and glucose again.
The Aspergillus niger strain that produces naringinase can produce hesperidinase usually simultaneously, but yield of enzyme is little, can't carry out suitability for industrialized production.The suitability for industrialized production of naringinase and hesperidinase early has report abroad, and domestic also have corresponding research, but is not applied to as yet produce, and major cause is to lack to be suitable for industrial high yield naringinase and hesperidinase bacterial strain, and fermentation condition is not optimized yet.
(3) summary of the invention
The present invention is for a kind of high yield naringinase simultaneously and hesperidinase being provided, and can realizing the black-koji mould WZ001 (Aspergillus niger WZ001) of suitability for industrialized production and use.
For reaching goal of the invention the technical solution used in the present invention be:
A kind of black-koji mould WZ001 (Aspergillus niger WZ001), be preserved in Chinese typical culture collection center, deposit number CCTCC No.206047, preservation date on May 15th, 2006, the classification called after black-koji mould WZ001 (Aspergillus niger WZ001) of proposal.
Described black-koji mould WZ001 colony characteristics is as follows: the speed of growth is very fast on the inclined-plane, under 30 ℃ of conditions, generally needs 3-5 days, and the bacterium colony initial stage fine hair shape that is white in color is diffusion type, and the surface is dry, and the bacterium colony later stage is born tan spore.In conidial head when children, is spherical in shape, has conidiophore, and wall is smooth, top capsule ball-type, the stigma bilayer, certainly the top capsule comprehensive give birth to, conidium ball-type or nearly ball-type, wall is coarse, is denseer thread behind the shake flask fermentation, incubation time is long, mycelium can self-dissolving.
Described black-koji mould WZ001 is obtained by following method mutagenesis screening:
From traditional fermented food such as distiller's yeast, bring out the higher aspergillus niger Aspergillus niger WZ1 for bacterial classification of enzyme activity and carry out ultraviolet mutagenesis and ethyl sulfate mutagenesis through primary dcreening operation, multiple screening, in naringin, the high-rise test tube of Hesperidin, cultivate again, get the transparent layer height big carry out shake flask fermentation, survey its naringinase and hesperidinase enzyme activity.The UV treatment condition is: at 20w ultraviolet lamp 40cm place, bacteria suspension is mutagenesis while stirring under magnetic stirring apparatus, and irradiation time is 16min, and lethality rate reaches 98.2%, positive mutation rate is 20%, selects a mutant strain Aspergillus niger WZ01 enzyme work and is significantly improved.Handle with ethyl sulfate with Aspergillus niger WZ01, actual conditions is again: wash with the spore of phosphoric acid buffer with the fresh inclined-plane of Aspergillus niger WZ01, make bacteria suspension, contain spore density 10
-6-10
-7Individual/ml, get DES solution and bacteria suspension equivalent (as 5:5) and join mixing in the sterile test tube, last concentration of treatment is 1%, at 30 ℃ of following oscillation treatment 30min, mutagenic treatment finishes the back and adds the physiological saline dilution, is applied in the plate, the incubator that plate is put into 30 ℃ is cultivated 72h, in the high-rise test tube of naringin, Hesperidin, cultivate again, get the transparent layer height big carry out shake flask fermentation, survey its naringinase and hesperidinase enzyme activity.Ultraviolet mutagenesis and DES mutagenesis enzyme activity have improved 74.2% and 99.4% respectively.Obtain a plant height at last and produce strains A spergillus niger WZ001.The enzyme that records hesperidinase wherein after the mutagenesis is lived and is that 14104.9U/g fermentation raw material, the enzyme of naringinase are lived and is the 14799.6U/g fermentation raw material.
Described black-koji mould WZ001 can be used for the fermentative preparation naringinase, also can be used for the fermentative preparation hesperidinase.
Described black-koji mould WZ001 can be used for preparing simultaneously naringinase, hesperidinase.Can adopt liquid state fermentation cultivation and solid state fermentation to cultivate dual mode prepares.
The liquid state fermentation cultural method is as follows: liquid state fermentation substratum quality content is: wheat bran 1～10%, and soybean cake powder 1～10%, bean dregs 5～15%, surplus is a water, the pH nature; Aspergillus niger kind song is inserted with volume ratio 2% inoculum size in described liquid state fermentation medium sterilization cooling back, cultivate 5～7 days under 30～37 ℃ of leavening temperatures, the rotating speed 150～200r/min condition after, filter the crude enzyme liquid of described naringinase, hesperidinase mixed enzyme.Described sterilization is at 0.1Mpa vapor pressure sterilization 20min.Described substratum is formed and is represented with weight/volume percent, contains this component of 1g in certain component concentration 1% expression 100mL substratum.
The solid state fermentation cultural method is as follows: the solid-state fermentation culture medium quality is composed as follows: 70～80 parts in wheat bran, 5～15 parts in bean dregs, 10～20 parts of soybean cake powder, the adding quality is a 1:1.0-1.5 water doubly with the ratio of above-mentioned solid mixt total mass, be sub-packed in the container, charging thickness is 2.5～3.0cm, aspergillus niger kind song is inserted with volume ratio 2% inoculum size in described solid-state fermentation culture medium sterilization cooling back, after cultivating 5～7 days under 32～37 ℃, culture is soaked 4～10h, filter the crude enzyme liquid of described naringinase, hesperidinase mixed enzyme.Described sterilization is 0.1Mpa vapor pressure sterilization 30min.
Enzyme is lived and defined: at certain condition, the 1min conversion of substrate obtains 1 μ g Hesperitin monoglycosides or the required enzyme amount of naringenin monoglycosides is 1 enzyme U of unit alive, and unit is μ g/min.
The effect of having a mind to of black-koji mould WZ001 of the present invention and application thereof is mainly reflected in: described strain enzyme-producing amount height, and when adopting liquid state fermentation to cultivate, naringinase and hesperidinase yield of enzyme all can reach more than the 14000U/g fermentation raw material; When adopting solid state fermentation to cultivate, naringinase and hesperidinase yield of enzyme all can reach more than the 6000U/g fermentation raw material.
(4) description of drawings
Black-koji mould WZ001 is preserved in Chinese typical culture collection center, deposit number CCTCCNo.206047, preservation date on May 15th, 2006.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
Black-koji mould WZ001 bacterial classification after 5 days, is got 30ml distilled water through the activation of Cha Shi substratum, makes spore suspension.
Liquid state fermentation substratum quality is formed: wheat bran 5%, soybean cake powder 4%, bean dregs 10%, surplus is a water, pH nature, 0.1Mpa vapor pressure sterilization 20min, the black-koji mould suspension is inserted with volume ratio 2% in every bottle of cooling back, cultivated 5-7 days under leavening temperature 30-37 ℃, rotating speed 150-200r/min condition, filter crude enzyme liquid, the enzyme that records hesperidinase is wherein lived and is that 14104.9U/g fermentation raw material, the enzyme of naringinase are lived and is the 14799.6U/g fermentation raw material.
Embodiment 2:
After 5 days, inoculation one ring is cultivated 36h to black-koji mould WZ001 bacterial classification to liquid seed culture medium (surplus is a water for wheat bran 4%, soybean cake powder 3%, bean dregs 8%), obtain seed liquor through the activation of Cha Shi substratum.
Solid-state fermentation culture medium is wheat bran 100g, bean dregs 10g, soybean cake powder 20g, the water that adds 100ml, be sub-packed in the triangular flask of 500ml, charging thickness is 2.5-3.0cm, 0.1Mpa vapor pressure sterilization 30min, the aspergillus niger seed liquor is inserted with volume ratio 2% in every bottle of cooling back, cultivated 5-7 days down in 32 ℃, culture is soaked 4h, filter crude enzyme liquid, the enzyme that records hesperidinase is wherein lived and is that 6378.5U/g fermentation raw material, the enzyme of naringinase are lived and is the 6601.7U/g fermentation raw material.
Embodiment 3:
Press in the embodiment 2 method gained crude enzyme liquids toward 100mL, add 200mL ethanol, leave standstill 20min, centrifugal, oven dry gets naringinase and hesperidinase mixed enzyme powder.The hesperidinase rate of recovery is 75.2%, and the naringinase rate of recovery is 68.7%.
Embodiment 4:
Press in the embodiment 1 method gained crude enzyme liquid toward 100mL, add 150mL ethanol, centrifugal gained precipitation goes out with 100mL is water-soluble, and in the gained mixed enzyme solution, the hesperidinase rate of recovery is 71.2%, and the naringinase rate of recovery is 61.7%.
Embodiment 5:
Press in the embodiment 1 method gained crude enzyme liquid toward 100mL, add 220mL ethanol, centrifugal gained precipitation goes out with 100mL is water-soluble, and in the gained mixed enzyme solution, the hesperidinase rate of recovery is 81.6%, and the naringinase rate of recovery is 80.3%.
Embodiment 6:
Press in the embodiment 1 method gained crude enzyme liquid toward 100mL, add 270mL ethanol, centrifugal gained precipitation goes out with 100mL is water-soluble, and in the gained mixed enzyme solution, the hesperidinase rate of recovery is 83.0%, and the naringinase rate of recovery is 88.9%.
Embodiment 7:
Press in the embodiment 1 method gained crude enzyme liquid toward 100mL, add 300mL ethanol, centrifugal gained precipitation 100mL distilled water stripping, in the gained mixed enzyme solution, the hesperidinase rate of recovery is 81.3%, the naringinase rate of recovery is 85.7%.
Embodiment 8:
Press in the embodiment 1 method gained crude enzyme liquid toward 100mL, add 350mL ethanol, centrifugal gained precipitation 100mL distilled water stripping, in the gained mixed enzyme solution, the hesperidinase rate of recovery is 80.7%, the naringinase rate of recovery is 84.3%.
Embodiment 9:
Press in the embodiment 1 method gained crude enzyme liquid toward 100mL, add 270ml ethanol, centrifugal must the precipitation, oven dry gets the mixed enzyme powder, and wherein the hesperidinase enzyme is lived and is 48150U/g, and total yield is 68.1%, and the naringinase enzyme is lived and is 39596.5U/g, and total yield is 66.4%.
Embodiment 10:
Utilize twice sulphur ammonium precipitation, Hiprep
TM16/10Phenyl hydrophobic chromatography, Hiprep
TMQ ion-exchange, Superdex
TMMethods such as G75 gel-filtration are carried out separation and purification with hesperidinase and naringinase, get electrophoresis level α-rhamnosidase and beta-glucosidase, α-1, and 6-rhamnoside enzyme purification multiple is 38.5 times, and enzyme work is recovered as 15.0%, and molecular weight is about 66kDa; α-1, the purifying multiple of 2-rhamnosidase are doubly 57.3 times, and enzyme work is recovered as 9.4%, and the molecular weight size is about 104kDa; Beta-glucoside enzyme purification multiple is doubly 41.2 times, and enzyme work is recovered as 15.4%, and molecular weight is about 97kDa.
Claims (8)
1. a black-koji mould WZ001 (Aspergillus niger WZ001) is preserved in Chinese typical culture collection center, deposit number CCTCC No.206047, preservation date on May 15th, 2006.
2. the application of black-koji mould WZ001 as claimed in claim 1 in the fermentative preparation naringinase.
3. the application of black-koji mould WZ001 as claimed in claim 1 in the fermentative preparation hesperidinase.
4. the application of black-koji mould WZ001 as claimed in claim 1 in preparation naringinase, hesperidinase mixed enzyme.
5. the application of black-koji mould WZ001 as claimed in claim 4 in preparation naringinase, hesperidinase mixed enzyme, it is characterized in that described mixed enzyme preparation method is as follows: liquid state fermentation substratum quality content is: wheat bran 1～10%, soybean cake powder 1～10%, bean dregs 5～15%, surplus is a water, the pH nature; Aspergillus niger kind song is inserted with volume ratio 2% inoculum size in described liquid state fermentation medium sterilization cooling back, cultivate 5～7 days under 30～37 ℃ of leavening temperatures, the rotating speed 150～200r/min condition after, filter the crude enzyme liquid of described naringinase, hesperidinase mixed enzyme.
6. the application of black-koji mould WZ001 as claimed in claim 5 in preparation naringinase, hesperidinase mixed enzyme is characterized in that described liquid state fermentation medium sterilization is the 20min that sterilizes at the 0.1Mpa vapor pressure.
7. black-koji mould WZ001 as claimed in claim 4 is at the preparation naringinase, application in the hesperidinase mixed enzyme, it is characterized in that described mixed enzyme preparation method is as follows: the solid-state fermentation culture medium quality is composed as follows: 70～80 parts in wheat bran, 5～15 parts in bean dregs, 10～20 parts of soybean cake powder, adding quality is above-mentioned solid mixt total mass 1.0-1.5 water doubly, be sub-packed in the container, charging thickness is 2.5～3.0cm, aspergillus niger kind song is inserted with 2% volume ratio inoculum size in described solid-state fermentation culture medium sterilization cooling back, after cultivating 5～7 days under 32～37 ℃, culture is soaked 4～10h, filter described naringinase, the crude enzyme liquid of hesperidinase mixed enzyme.
8. the application of black-koji mould WZ001 as claimed in claim 7 in preparation naringinase, hesperidinase mixed enzyme is characterized in that described solid-state fermentation culture medium sterilization is 0.1Mpa vapor pressure sterilization 30min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200610051963XA CN100467586C (en) | 2006-06-14 | 2006-06-14 | Aspergillus niger WZ001 capable of producing naringinase and cirmtimase simultaneously and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200610051963XA CN100467586C (en) | 2006-06-14 | 2006-06-14 | Aspergillus niger WZ001 capable of producing naringinase and cirmtimase simultaneously and its application |
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| Publication Number | Publication Date |
|---|---|
| CN101089175A CN101089175A (en) | 2007-12-19 |
| CN100467586C true CN100467586C (en) | 2009-03-11 |
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Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732491A (en) * | 2012-07-10 | 2012-10-17 | 福州大学 | Culture medium and method for producing naringinase by fermenting citrus peel powder and beam dregs by aspergillus niger |
| CN103740610B (en) * | 2013-12-17 | 2015-08-19 | 河北农业大学 | Streptococcus AUH-JLD109 and application thereof in naringenin biosynthesis |
| CN106148446A (en) * | 2015-04-22 | 2016-11-23 | 中国科学院大连化学物理研究所 | A kind of method preparing hesperetin by enzymatic hydrolysis neohesperidin or Hesperidin |
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