CN107988085A - A kind of microorganism Aspergillus aculeatus bacterial strain of high yield acid pectase and its application - Google Patents
A kind of microorganism Aspergillus aculeatus bacterial strain of high yield acid pectase and its application Download PDFInfo
- Publication number
- CN107988085A CN107988085A CN201711343442.6A CN201711343442A CN107988085A CN 107988085 A CN107988085 A CN 107988085A CN 201711343442 A CN201711343442 A CN 201711343442A CN 107988085 A CN107988085 A CN 107988085A
- Authority
- CN
- China
- Prior art keywords
- aspergillus aculeatus
- pectase
- acid pectase
- microorganism aspergillus
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000002253 acid Substances 0.000 title claims abstract description 34
- 244000005700 microbiome Species 0.000 title claims abstract description 29
- 241000228215 Aspergillus aculeatus Species 0.000 title claims abstract description 25
- 230000001580 bacterial effect Effects 0.000 title description 4
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 8
- 230000004151 fermentation Effects 0.000 claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 abstract description 36
- 102000004190 Enzymes Human genes 0.000 abstract description 35
- 230000000694 effects Effects 0.000 abstract description 17
- 241000894006 Bacteria Species 0.000 abstract description 8
- 239000007787 solid Substances 0.000 abstract description 5
- 230000035784 germination Effects 0.000 abstract description 3
- 238000009418 renovation Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 23
- 239000000243 solution Substances 0.000 description 17
- 239000001814 pectin Substances 0.000 description 13
- 229920001277 pectin Polymers 0.000 description 13
- 235000010987 pectin Nutrition 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- 238000002703 mutagenesis Methods 0.000 description 6
- 231100000350 mutagenesis Toxicity 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 235000019345 sodium thiosulphate Nutrition 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 3
- 229930091371 Fructose Natural products 0.000 description 3
- 239000005715 Fructose Substances 0.000 description 3
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 231100000225 lethality Toxicity 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 241000233866 Fungi Species 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
- 108090000604 Hydrolases Proteins 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108010059820 Polygalacturonase Proteins 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 108020004410 pectinesterase Proteins 0.000 description 2
- 230000036647 reaction Effects 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 230000007306 turnover Effects 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 241000165940 Houjia Species 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010029182 Pectin lyase Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000498 cooling water Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- -1 galactolipin Chemical compound 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000001724 microfibril Anatomy 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108010070456 protopectinase Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000012879 subculture medium Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01015—Polygalacturonase (3.2.1.15)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to microorganism renovation technique field, particular content is related to microorganism Aspergillus aculeatus mutant strain and its application of plant height production acid pectase.The deposit number of the mutant strain is CCTCCNO:M2017783, can be widely applied to fermenting and producing acid pectase.After the mutant strain solid shake flask fermentation 3.5d, its acid pectase enzyme activity reaches 938u/g, relatively goes out bacterium germination and improves 47.7%, achieves unexpected technique effect.
Description
Technical field
The invention belongs to microorganism renovation technique field, particular content is related to the microorganism Aspergillus aculeatus of plant height production acid pectase
Mutant strain and its application.
Technical background
Pectin is the polysaccharide chain that a kind of galacturonic acid by different esterification degrees is polymerized with α-Isosorbide-5-Nitrae glycosidic bond, normal band
The side chain being made of rhamnose, arabinose, galactolipin, xylose, trehalose etc., free carboxy moiety or all can with calcium,
Potassium, sodium ion, are particularly combined together with boride.Pectin is present in all higher plants, is deposited on blastema
Wall and cellular layer, the microfibril and some stretching, extension eggs of cellulose, hemicellulose, lignin in primary wall with different content
It is cross-linked with each other in vain, makes various cell tissue structures hard, so as to show intrinsic form.
Pectase (pectinase) is the general name for referring to decompose a variety of enzymes of pectin substance, is broadly divided into pectin water
Solve enzyme (pectin hydrolases), pectin lyase (pentinlyases), pectinesterase (pectin esterases) and
Protopectinase etc..According to the difference of effect optimal pH, pectase can be divided into acid pectase and alkaline pectase again.
Study at present and what application was more is acid pectase, acid pectase refers to interior polygalacturonase, can be random
The α in hydrolysis of pectin acid molecule-Isosorbide-5-Nitrae glycosidic bond then, it acts on optimal pH in slant acidity scope, is mainly used in food service industry
Fruit squeeze the juice and juice clarification, reduce grape wine viscosity, and feedstuff industry can be jointly used in other hydrolases.It is led
Animals and plants and microorganism, especially microorganism are derived from, because its speed of growth is fast, growth conditions is simple, metabolic process
The feature such as special, becomes the important sources of pectase, and wherein the pectase of originated from fungus is most of has height at acidic phs
Activity.Most of pectases commercially produced also are derived from fungi, especially from aspergillus niger and mould.Some pectin
Enzyme gene has been cloned, and is sequenced and is expressed.
At present using the having some limitations property of pectase of Natural strains production, mainly include that producing enzyme performance is relatively low, pectin
The heat resistance of enzyme is poor and action pH scope is smaller etc..Therefore, as pectase is using more and more extensive, there is an urgent need to right
Pectase production strain is improved accordingly, to improve the yield of pectase.
The content of the invention
The present invention is solution prior art problem, there is provided a plant height produces the microorganism Aspergillus aculeatus of acid pectase
(Aspergiulls aculeatus) bacterial strain and its application.Applicant with purchased from Germany Microbiological Culture Collection Center (DSMZ),
The microorganism Aspergillus aculeatus that preserving number is DSM 2334 are starting strain, and by the method for ultraviolet mutagenesis, screening obtains a plant mutant bacterial strain,
The yield of acid pectase can be increased substantially, may advantageously facilitate the extensive use of the enzyme.
One aspect of the present invention provides a kind of mutant strain microorganism Aspergillus aculeatus T18 (Aspergiulls aculeatus T18),
In the China typical culture collection center for being preserved in Wuhan, China Wuhan University on December 11st, 2017, deposit number is
CCTCC NO:M2017783。
One aspect of the present invention provides application of the microorganism Aspergillus aculeatus in acid pectase is produced.
Present invention also offers a kind of method for producing acid pectase, is using the microorganism Aspergillus aculeatus as fermentation strain.
Present invention also offers a kind of acid pectase, is to be fermented to obtain by the microorganism Aspergillus aculeatus.
The present invention, as starting strain, passes through ultraviolet mutagenesis using microorganism Aspergillus aculeatus (Aspergiulls aculeatus) DSM2344
Method screening obtains mutant strain microorganism Aspergillus aculeatus T18.After the mutant strain solid shake flask fermentation 3.5d, its acid pectase enzyme
Work reaches 938u/g, relatively goes out bacterium germination and improves 47.7%, achieves unexpected technique effect.Microorganism Aspergillus aculeatus provided by the invention
Mutant strain can be widely applied to the fermenting and producing of acid pectase, advantageously reduce the production cost of the enzyme, promote acid fruit
The popularization and application of glue enzyme.
Embodiment
The routine techniques and method that the present invention has used genetic engineering and biology field uses, such as
MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT
Described method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general bibliography
Provide definition well known by persons skilled in the art and method.But those skilled in the art can be described in the present invention
Technical solution on the basis of, using this area other conventional method, experimental program and reagents, and be not limited to of the invention specific
The restriction of embodiment.
With reference to embodiment, the present invention will be described in detail.
1 microorganism Aspergillus aculeatus DSM2344 solids shake flask fermentation of embodiment and Enzyme activity assay
Microorganism Aspergillus aculeatus (Aspergiulls aculeatus) DSM2344 is purchased from Germany Microbiological Culture Collection Center
(DSMZ), preserving number is DSM No.2334.The strains ferment produces acid pectase.
Microorganism Aspergillus aculeatus DSM2344 is inoculated into fresh PDA plate by applicant first, and (potato 200g/L, boils 20-
Filtering and removing slag after 30min；Glucose 2%；Agar powder 1.5%), 30 DEG C of culture 5d.
The sterile water elutions of 5ml are drawn, obtain spore liquid, are inoculated with 50ml liquid CSL- fructose seed culture medium (maltose
10%；Fructose 5%；Glucose 1%；Corn pulp 10%；Magnesium sulfate 0.05%；Sodium dihydrogen phosphate 0.1%；PH 5.8), 30 DEG C of trainings
Support 2d.After cultivating 2d, 2ml mycelium inoculation 5g solid Shake flask mediums are drawn, 30 DEG C of culture 3.5d, turn over song daily.Culture knot
Shu Houjia 40ml sterile waters, stirring 2h elutions, it is crude enzyme liquid that centrifugation, which obtains supernatant,.Crude enzyme liquid is subjected to acid pectase enzyme
Vitality test, the results show that acid pectase enzyme activity reaches 635u/g in the crude enzyme liquid, so as to illustrate microorganism Aspergillus aculeatus
DSM2344 can actually high yield acid pectase.
1st, acid pectase enzyme activity detects
(1) definition of acid pectase enzyme-activity unit
At 50 DEG C, under the conditions of pH is 3.5,1h is reacted, 1g enzyme powders or 1ml enzyme liquids decompose pectin and produce 1mg galacturonic acids
It is defined as a unit of activity (IU).
(2) enzyme activity determination method
(2.1) reagent and solution：
1% pectin amidin：Jelly powder (Sigma P9135) 1g is weighed, is dissolved in water, cooling is boiled, if any insoluble
Thing then needs to be filtered, and adjusts pH 3.5, is settled to 100ml with water, is stored for future use in refrigerator, usage time is no more than 3d；
Sodium carbonate liquor 1mol/L：Natrium carbonicum calcinatum 106g is weighed, is dissolved in water, is settled to 1L；
Iodine standard solution：0.1mol/L；
Sulfuric acid solution 2mol/L：Concentrated sulfuric acid 5.6ml is taken, is slowly added in suitable quantity of water, 100ml is settled to water after cooling；
Soluble starch indicator solution：10g/L；
0.1mol/L citric acid-sodium citrate buffer solutions (pH 3.5)：Weigh citric acid (C6H8O7·H2O)14.7098g
With trisodium citrate (C6H5Na3O7·2H2O) 8.823g, is dissolved with water and is settled to 1L；
0.05mol/L hypo solutions：By the 0.1mol/L thiosulfuric acid mother liquid of sodium 500ml of quantitative analysis with boiling
The cooling water dilution boiled, the cumulative volume of resulting solution is 1000ml.It after preparing, should be demarcated, obtain concentration correction system
Number f values；
(2.2) preparation of sample solution：
The accurate enzyme liquid 1ml that draws is diluted and determined with citric acid-sodium citrate buffer solution in the volumetric flask of certain volume
Hold.Enzyme liquid needs to be diluted to certain multiple, and enzyme liquid concentration should be controlled in consumption 0.05mol/L sodium thiosulfate standard solutions (A-
B difference) is in the range of 0.5~0.7ml.
(2.3) enzyme activity determination：
In blank tube, 10g/L pectin solutions 5ml and citric acid-sodium citrate buffer solution 5ml are separately added into, in sample
10g/L pectin solutions 5ml and citric acid-sodium citrate buffer solution 4ml is added in pipe, 5~10min is preheated in 50 DEG C of water-baths.
The enzyme liquid that 1ml has diluted is added into sample cell, shakes up timing at once, at this temperature accurate response 0.5h, is stood
Take out, boil 5min, terminate reaction, cooling.
Above-mentioned each 5ml of blank tube and sample cell reaction solution is taken, is put into iodine flask, it is accurate to add 1mol/L sodium carbonate liquors
1ml, 0.1mol/L iodine solution 5ml, shake up, and 20min is placed in dark place.
Take out, add 2mol/L sulfuric acid solution 2ml, be titrated to 0.05mol/L hypo solutions light yellow, add shallow lake
Powder indicator solution 3 drips, and it is terminal to continue to be titrated to blueness and just disappear, record blank tube, sample cell reaction solution consumption thiosulfuric acid
The volume of sodium standard solution.
Enzyme activity calculation formula：
X=(A-B) × c × 0.51 × 194.14 × n × 10/ (5 × 1 × 0.5)=(A-B) × c × n × 396.05
In formula：The enzyme activity of X-- sample, U/ml；
The volume of A-- blank solution titration consumption sodium thiosulfate standard solution, ml；
The volume of B-- sample solution titration consumption sodium thiosulfate standard solution, ml；
The concentration of c-- sodium thiosulfate standard solution, mol/L；
Free galacturonic acid of the 0.51-- 1mol sodium thiosulfate equivalent to 0.51mol；
MM quality of 194.14-- galacturonic acid, mg；
N-- enzyme liquid extension rate；
10-- reaction solution cumulative volume, ml；
The volume extracted reaction solution during 5-- titration, ml；
The volume of dilution enzyme liquid, ml are added during 1-- reaction；
0.5-- reaction time, h；
Enzyme activity (U/g) Y=8X, 8 add the conversion multiple of 40ml water elutions for 5g solid materials.
2 mutagenesis screening of embodiment
Applicant, as starting strain, further passes through purple using microorganism Aspergillus aculeatus (Aspergiulls aculeatus) DSM2344
The mutant strain of the method screening acid pectase output increased of outer mutagenesis.
Determine lethality：Above-mentioned microorganism Aspergillus aculeatus strain DSM 2344 is inoculated in PDA plate, 30 DEG C of culture 5d.Treat bacterium colony table
When face produces a large amount of spores, the sterile water elutions of 5ml are drawn, spore liquid is obtained, is resuspended after centrifugation with sterile water, uses blood counting chamber
Count.A 90mm culture dish is taken, (concentration is about 1 × 10 to the spore suspension that addition 5ml has diluted7A/mL), add rotor simultaneously
Stirring makes spore liquid be in uniform state on magnetic stirring apparatus.In aseptic superclean bench, the ultraviolet lamp for being 9w with power
Irradiated in the top of vertical range 20cm, irradiate 30s, 60s, 90s, 120s, 150s, 180s respectively, take the spore liquid after irradiation
Dilution 10,100,1000 times, take 100ul be coated with PDA plate, 30 DEG C culture 2-3d after count, using non-irradiated spore liquid as pair
According to calculating lethality.When wherein irradiating 150s, lethality 98%, chooses the irradiation time and carries out follow-up Mutagenesis experiments.
Mutagenesis screening：A 90mm culture dish is taken, (concentration is 1 × 10 to the spore suspension that addition 5ml has diluted7), add
Rotor and on magnetic stirring apparatus stirring spore liquid is in uniform state.It is 9w's with power in aseptic superclean bench
Ultraviolet lamp is irradiated in the top of vertical range 20cm, dilutes 1000 times after irradiating 150s, takes 100ul to be coated with PDA plate, 30 DEG C of trainings
Support 2-3d.
100 pieces of PDA plates are coated with altogether, and after 30 DEG C are cultivated 2-3d, each tablet grows 10-20 bacterium colony, first passes through observation
Colonial morphology, filters out the mutant bacteria totally 51 that significant changes occur for colonial morphology, is inoculated into PDA plate, 30 DEG C of cultures respectively
5d；Each mutant bacteria bacterium colony is eluted with 5ml sterile waters, obtains spore liquid；50ml liquid CSL- fructose kinds are seeded to respectively
Sub- culture medium, 30 DEG C of culture 2d；Then 2ml mycelium are drawn respectively is seeded to 5g solids Shake flask medium (wheat bran 2g；Wooden fibre
Tie up element 2g；Gum arabic powder 1g), 30 DEG C of culture 3.5d, turn over song daily；After culture plus 40ml sterile waters, stirring 2h are washed
De-, it is crude enzyme liquid that centrifugation, which obtains supernatant,.
By carrying out acid pectase enzyme activity detection to the crude enzyme liquid of above-mentioned acquisition, applicant finally filters out one plant of acid
The property highest mutant strain of pectin production of enzyme, is named as microorganism Aspergillus aculeatus T18 (Aspergiulls aculeatus T18), the bacterium
The enzyme activity of acid pectase reaches 938u/g in the crude enzyme liquid that strain fermentation obtains, and relatively goes out bacterium germination and improves 47.7%, achieves expectation
Less than technique effect.
Applicant is on December 11st, 2017 by above-mentioned mutant strain microorganism Aspergillus aculeatus T18 (Aspergiulls
Aculeatus T18) it is preserved in the China typical culture collection center of Wuhan, China Wuhan University, deposit number CCTCC
NO:M2017783。
Claims (4)
1. a kind of microorganism Aspergillus aculeatus, it is characterised in that the deposit number of the microorganism Aspergillus aculeatus is CCTCC NO:M2017783.
2. application of the microorganism Aspergillus aculeatus in acid pectase is produced described in claim 1.
A kind of 3. method for producing acid pectase, it is characterised in that the method is bent with the spine spore described in claim 1
It is mould to be used as fermentation strain to carry out fermenting and producing acid pectase.
4. a kind of acid pectase, it is characterised in that the acid pectase is made as the microorganism Aspergillus aculeatus described in claim 1
Fermentation acquisition is carried out for fermentation strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711343442.6A CN107988085B (en) | 2017-12-14 | 2017-12-14 | A kind of microorganism Aspergillus aculeatus bacterial strain of high yield acid pectase and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711343442.6A CN107988085B (en) | 2017-12-14 | 2017-12-14 | A kind of microorganism Aspergillus aculeatus bacterial strain of high yield acid pectase and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107988085A true CN107988085A (en) | 2018-05-04 |
| CN107988085B CN107988085B (en) | 2019-11-08 |
Family
ID=62037921
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711343442.6A Active CN107988085B (en) | 2017-12-14 | 2017-12-14 | A kind of microorganism Aspergillus aculeatus bacterial strain of high yield acid pectase and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107988085B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961852A (en) * | 2019-12-12 | 2021-06-15 | 青岛蔚蓝生物集团有限公司 | Promoter and application thereof in aspergillus aculeatus gene self-cloning expression |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1139457A (en) * | 1993-12-01 | 1997-01-01 | 诺沃诺尔迪斯克生物技术有限公司 | Aspergillus expression system |
| CN1273273A (en) * | 1999-04-01 | 2000-11-15 | 刘海森 | Process for preparing pectinase by solid fermentation of coom aspergillus mutant K58 |
| CN104059857A (en) * | 2014-06-30 | 2014-09-24 | 广西大学 | Aspergillus and application of aspergillus in fructosyltransferase preparing |
| CN105838693A (en) * | 2016-05-16 | 2016-08-10 | 北京科为博生物科技有限公司 | High-temperature-resistant acidic pectinase PAGL8 and gene and application thereof |
-
2017
- 2017-12-14 CN CN201711343442.6A patent/CN107988085B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1139457A (en) * | 1993-12-01 | 1997-01-01 | 诺沃诺尔迪斯克生物技术有限公司 | Aspergillus expression system |
| CN1273273A (en) * | 1999-04-01 | 2000-11-15 | 刘海森 | Process for preparing pectinase by solid fermentation of coom aspergillus mutant K58 |
| CN104059857A (en) * | 2014-06-30 | 2014-09-24 | 广西大学 | Aspergillus and application of aspergillus in fructosyltransferase preparing |
| CN105838693A (en) * | 2016-05-16 | 2016-08-10 | 北京科为博生物科技有限公司 | High-temperature-resistant acidic pectinase PAGL8 and gene and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 何海燕 等: "产果胶酶棘孢曲霉的筛选鉴定及微波诱变育种", 《中国饲料》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961852A (en) * | 2019-12-12 | 2021-06-15 | 青岛蔚蓝生物集团有限公司 | Promoter and application thereof in aspergillus aculeatus gene self-cloning expression |
| CN112961852B (en) * | 2019-12-12 | 2023-11-21 | 青岛蔚蓝生物集团有限公司 | Promoter and application thereof in aspergillus aculeatus gene self-cloning expression |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107988085B (en) | 2019-11-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Omojasola et al. | Cellulase production by Trichoderma longi, Aspergillus niger and Saccharomyces cerevisae cultured on waste materials from orange | |
| CN110684673B (en) | L-malic acid high-yield strain and application thereof | |
| CN104893985B (en) | A kind of microorganism Aspergillus aculeatus bacterial strain and its application | |
| CN104498365A (en) | Bacterial strain capable of producing chitin deacetylase and application of bacterial strain in production of chitin deacetylase through fermentation | |
| CN107022541A (en) | Aspergillus niger immobilization method | |
| CN108359631A (en) | A kind of production method of trichoderma chlamydospore | |
| CN107488600A (en) | One plant height produces the aspergillus niger of resistance to oxidation low temperature glucose oxidase | |
| CN107488615B (en) | Pseudomonas capable of producing lipase at high yield and fermentation enzyme production method thereof | |
| CN102127515B (en) | Screening and application of L-proline high-producing Brevundimonas sp. (JNPP-1) | |
| CN107988085B (en) | A kind of microorganism Aspergillus aculeatus bacterial strain of high yield acid pectase and its application | |
| CN114292757A (en) | Culture medium for purification culture of eurotium cristatum conidia and preparation method and application thereof | |
| CN106520575B (en) | One plant height produces Aspergillus niger strain and its application of catalase | |
| CN105420143A (en) | Acetobacter orientalis and method for producing astragalus polysaccharide through same | |
| CN106282145B (en) | A kind of liquid state fermentation method of adenylate deaminase | |
| CN105802881B (en) | Method for improving biomass and acid yield of lactic acid bacteria | |
| CN102851328A (en) | Method for preparing citric acid through fermenting corn sugar solution by immobilized Aspergillus niger | |
| CN107603884A (en) | One plant height produces the trichoderma reesei mutant strain of neutral cellulase | |
| CN107446904B (en) | Lipase, and production method and application thereof | |
| CN107988086B (en) | A kind of bacterial strain of high yield rhamnosan galacturonic hydrolase | |
| CN110791436B (en) | Aspergillus niger strain capable of producing pectinase at high yield and application thereof | |
| CN105647812A (en) | Schizophyllum commune strain and application thereof in production of Pu'er tea | |
| CN110885760B (en) | Liquid fermentation method for producing pectinase | |
| CN108004153A (en) | The Li's Trichoderma strains of one plant height production pectin lyase and its application | |
| CN103352007A (en) | Method for improving fermentation-production citric acid yield through use of two-stage dissolved oxygen control technology | |
| CN114107105A (en) | A kind of fermentation medium containing fruit pomace enzymolysis liquid and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191025 Address after: Miao road Laoshan District 266061 Shandong city of Qingdao province Shandong No. 29 high building 12A07 Applicant after: Qingdao Weilan Biology Group Co., Ltd. Applicant after: Weifang KDN Biotechnology Co., Ltd. Address before: Miao road Laoshan District 266061 Shandong city of Qingdao province Shandong No. 29 high building 12A07 Applicant before: Qingdao Weilan Biology Group Co., Ltd. |
|
| TA01 | Transfer of patent application right |