CN100368519C - Aspergillus niger lipase and its preparation method - Google Patents
Aspergillus niger lipase and its preparation method Download PDFInfo
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- CN100368519C CN100368519C CNB200510035099XA CN200510035099A CN100368519C CN 100368519 C CN100368519 C CN 100368519C CN B200510035099X A CNB200510035099X A CN B200510035099XA CN 200510035099 A CN200510035099 A CN 200510035099A CN 100368519 C CN100368519 C CN 100368519C
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Abstract
The present invention belongs to the technical field of microorganism, which relates to the Aspergillus niger lipase, and the present invention also relates to a method for the preparing thereof. Wild-type bacterial strains S-8341 of Aspergillus niger are separated and screened out from nature, and superior production bacterial strains S-7749 can be obtained in a mode of mutation breeding through using industrial microorganism; the Aspergillus niger lipase can be prepared by the bacterial strains through enlarge cultivation and fermenting in a mode of low temperature and high oxygen. The Aspergillus niger lipase which is prepared has the enzymology characteristic that the optimum PH value of enzyme action is in a scope ranging from 6.5 to 8.0, and the enzyme action of which the PH value in a scope ranging from 5.8 to 12 is stable; the optimum temperature of the enzyme action is in a scope ranging from 25 DEG C to 35 DEG C, and the thermostable temperature is less than 40 DEG C; molecular weight of each enzyme is 29.2Kda, and an N-terminal amino acid sequence of the enzyme is ATADAAAFPDLHRAAKLSSA; the specificity of an enzyme action ester linkage position is lipase of a hydrolysis Sn-1 and Sn-3 position. The present invention has the advantages of simple preparation method, low cost high activity of the prepared Aspergillus niger lipase, wide range of the PH value which is acted and wide purpose.
Description
Invention field
The present invention relates to lipase and preparation method thereof, especially microbial lipase and preparation method thereof.
Background technology
In recent years the research to microbial lipase significantly increases, because microbial lipase has been widely adopted in industry such as foodstuffs industry, flour industry, washing industry, pharmaceutical industry, textile industry, fodder industry, biochemical industry industry and environment-protecting industrial, its application development space is quite wide.At present, the microbial lipase of having reported produces nearly 65 genus of bacterium both at home and abroad, wherein: bacterium, 28 genus; Filamentous fungus, 23 genus; Yeast, 10 genus; Actinomycetes, 4 genus.Filamentous fungus is that a kind of lipase of excellence is produced bacterial classification at the cell exocrine enzyme.The strongest bacterial strain of enzymatic productivity mainly concentrates on head mold (Rhizopas), aspergillus (Aspergillus), mould (Penicillium), Mucor (Mucor), must plant by genus such as mould (Phycomyces) at present.At the lipase of 4.5-5.5 report [Torossian K.and Bell A.W (1991) Biotechnol.Appl.Biochem.13,205-211] was arranged once by aspergillus niger (Aspergillus niger) preparation effect optimum pH field of activity.U.S. Pat 6534303 discloses a kind of method for preparing aspergillus niger lipase, and the effect optimum pH scope of obtained enzyme is at 2.0-3.0.These the two kinds of prepared aspergillus niger action of lipase of method optimum pH narrow range, and unstable under alkaline condition.
Summary of the invention
An object of the present invention is to provide the suitableeest a kind of action pH value wide ranges and can be in wide pH value scope stable aspergillus niger lipase and preparation method thereof.
Aspergillus niger lipase of the present invention adopts the single bacterial strain S-7749 preparation of aspergillus niger (Aspergillus niger), and bacterial strain S-7749 is obtained through selection by mutation by the single bacterial strain of original aspergillus niger (Aspergillus nigerS-8341) that filters out.Bacterial strain S-7749 preserves management committee common micro-organisms center (CGMCC) on March 23rd, 2005 at Chinese microorganism strain and has carried out preservation, the preservation centre address is in the Microbe Inst., Chinese Academy of Sciences of Zhongguancun, Beijing City, postcode is 100080, and deposit number is CGMCC No.1334.
Bacterial strain S-7749 prepares through seed, fermentation culture, and fermented liquid under the enzymatic protective reagent effect, by spraying drying, obtains product through ultrafiltration.
Aspergillus niger used in the present invention is the food safety bacterial strain that health ministry and FDA announce, fermention medium wide material sources cost is low, and technology is simple, prepared aspergillus niger lipase activity height, and applicable pH value wide ranges, of many uses.
Description of drawings
Fig. 1 is the preparation process block diagram of aspergillus niger lipase
Fig. 2 be bacterial strain S-7749 the mutagenesis pedigree.
Fig. 3 is that the optimum pH of aspergillus niger lipase of the present invention effect reaches the stability to the pH value.
Fig. 4 is the optimum temperuture of aspergillus niger lipase of the present invention effect and to thermostability.
Embodiment
The screening of bacterial classification and mutagenesis:
Separation screening obtains the single bacterial strain of original aspergillus niger (Aspergillus nigerS-8341) from banyan soil, bacterial strain S-8341 does under the mutagenic compound condition at ultraviolet or ultraviolet and lithium chloride, through mutagenic obtained production bacterial strain S-7749 used in the present invention of 7 generations.Bacterial strain S-7749 is accredited as aspergillus niger (Aspergillus niger) through Microbe Inst., Chinese Academy of Sciences, and preserve management committee common micro-organisms center (CGMCC) on March 23rd, 2005 at Chinese Chinese microorganism strain and carried out preservation, the preservation centre address is in the Microbe Inst., Chinese Academy of Sciences of Zhongguancun, Beijing City, postcode is 100080, and deposit number is CGMCC No.1334.The seed preparation of bacterial classification:
Bacterial strain S-7749 expands cultured potato plug seed in the eggplant bottle of potato culture and cultivates under 25 ℃ of-27 ℃ of temperature, then, expands in the KShi bottle of potato culture again and cultivates, and incubation time is 80-120 hour.
Fermentation:
Comprise first class seed pot fermentation, the fermentation of secondary seed jar and ferment tank, the substratum that first class seed pot fermentation and secondary seed jar ferment: 2.6% soybean cake powder, 0.5% W-Gum, 0.5% SODIUMNITRATE, 0.05% Trisodium Citrate and 0.05% sal epsom, the medium pH value is 6.3-6.4.Ferment tank substratum: 6.0% soybean cake powder, 0.4% W-Gum, 0.2% SODIUMNITRATE, 0.4% potassium hydrogen phosphate, 0.2% lime carbonate, 0.02% sal epsom, 0.1% Trisodium Citrate, medium pH value 6.5-6.8.The first class seed pot leavening temperature is 25 ℃-27 ℃, air air flow 1: 1V/V in the fermenting process, culture cycle 16-18 hour.Secondary seed jar leavening temperature is 25 ℃-27 ℃, air air flow 1: 1V/V in the fermenting process, culture cycle 8-10 hour, the ferment tank temperature is 24 ℃-25 ℃, the air air flow is 1 in the process: 1.2-1: increase stage by stage in 1.6 scopes, keep tank pressure 0.1Mpa, fermentation period 42-44 hour.
Aftertreatment:
After the fermentation ends, filtering fermentating liquid, solid-liquid separation, filtrate ultrafiltration and concentration, molecular weight are held back＜30Kda, add this area protective material and activator commonly used, and spraying drying obtains finished product.
Enzyme activity determination:
With the tributyrin is substrate, and the fermenting enzyme that adopts this area method commonly used to measure fermented liquid is lived.Under 36 ℃ of temperature and pH9.4 condition, hydrolysis glycerine tri-n-butyl per minute generates the enzyme amount of 1 μ mol lipid acid, is a unit, represents with U/ml or U/g.
Below with reference to specific embodiment method of the present invention is described more specifically, but the present invention is not limited to specific embodiment.
Example 1
Bacterial classification: Aspergillus niger strain S-7749
The preparation of the seed of bacterial classification: bacterial strain S-7749 expands the potato plug seed in the eggplant bottle to and to cultivate under 27 ℃ of temperature, expands in the KShi bottle again and cultivates, and per step incubation time was all 100 hours.
Fermentation:
1) first class seed pot fermentation
Substratum (%):
Soybean cake powder 2.6
W-Gum 0.5
NaNo
3 0.5
Trisodium Citrate 0.08
MgSo
4 0.04
Inoculation method: spore inoculating method
Culture condition:
Temperature: 26 ℃
Air flow: 1: 1V/V
PH： 6.3-6.4
Culture cycle: 17 hours
2) secondary seed jar fermentation
Substratum (%): ferment with the one-level seeding tank
Inoculation method: pressure reduction culture transferring method
Culture condition
Temperature: 26 ℃
Air flow: 1: 1V/V
PH value: 6.3-6.4
Culture cycle: 9 hours
3) zymotechnique:
Substratum (%):
Soybean cake powder 6.0
W-Gum 0.4
NaNo
3 0.2
K
2HPO
4 0.4
CaCo
3 0.2
MgSo
4 0.02
Trisodium Citrate 0.1
PH 6.5-6.8
Seed culture transferring amount 15
Inoculation method: pressure reduction culture transferring method
Culture condition:
PH control: 6.8-7.8
Temperature: 24 ℃
Fermentation period: 43 hours
Air flow: fermentation beginning air air flow is for being controlled to be 1: 1.2V/V, fermenting increased to 1 after 16 hours: 1.4V/V, fermenting increased to 1 after 32 hours: 1.6V/V.
Keep tank pressure 0.1MPa in the fermenting process.
With 40 order filter cloth Plate Filtrations, filtrate is through 5m with fermented liq for fermentation ends
3And 1m
3After the bag filter, use the hollow fiber ultrafiltration membrane ultrafiltration of 20,000 molecular weight again, molecular weight is held back＜30Kda, and the ultrafiltration dope adds enzyme stabilizers and activator, through the press spray drying, gets finished product, moisture content＜6%.
The mensuration enzyme is lived: the enzyme of liquid is lived and is 3478U/ml after the interior fermentation ends of mensuration fermentor tank.
Example 2
Similar with example 1, difference is: temperature is 26 ℃ in the seed preparation process of bacterial classification, and incubation time was all 120 hours in eggplant bottle and the KShi bottle; Leavening temperature is 27 ℃ in the first class seed pot, and culture cycle is 18 hours; Leavening temperature is 27 ℃ in the secondary seed jar, and culture cycle is 10 hours, and the fermentation cylinder for fermentation temperature is 25 ℃, and fermentation period is 44 hours; Fermentation beginning air air flow is for being controlled to be 1: 1.2V/V, fermenting increased to 1 after 18 hours: 1.4V/V, fermenting increased to 1 after 35 hours: 1.6V/V.
The enzyme of liquid is lived and is 3216U/ml after the interior fermentation ends of mensuration fermentor tank.
Example 3
Similar with example 1, difference is: temperature is 25 ℃ in the seed preparation process of bacterial classification, and incubation time was all 80 hours in eggplant bottle and the KShi bottle; Leavening temperature is 25 ℃ in the first class seed pot, and culture cycle is 16 hours; Leavening temperature is 25 ℃ in the secondary seed jar, and culture cycle is 8 hours, and the fermentation cylinder for fermentation temperature is 25 ℃, and fermentation period is 42 hours; Fermentation beginning air air flow is for being controlled to be 1: 1.2V/V, fermenting increased to 1 after 15 hours: 1.4V/V, fermenting increased to 1 after 30 hours: 1.6V/V.。
The enzyme of liquid is lived and is 3016U/ml after the interior fermentation ends of mensuration fermentor tank.
Example 4
Being substrate with the tributyrin detects the influence of the aspergillus niger lipase activity that the pH value makes example 1 with method conventional in the field, show that institute's zymogenesis optimal pH is at 6.5-8.0, not only keep higher enzyme activity in the alkalescence zone, and also maintain suitable enzyme activity at acidic region, has wide pH sphere of action, its pH stable range is at 5.8-12.0, and is of many uses.Detected result is seen Fig. 3.
Example 5
The optimum temperuture and the thermostability of the aspergillus niger lipase effect that to be substrate with the tributyrin make with method test example 1 conventional in the field detect and show that the optimum temperature of prepared enzyme is 25 ℃-35 ℃; Surpass 40 ℃, enzyme activity begins obvious decline, and is lower than 20 ℃, still has good enzyme activity power, and the inclined to one side cold-adapted enzyme of temperature was poor slightly to thermostability during this enzyme belonged to.Detected result is referring to Fig. 4.
Example 6
With the aspergillus niger lipase that obtains in the example 1 after reduction is handled, carry out SDS-GAPE. together with standard protein, with the logarithm (ordinate zou) of each standard protein molecular weight and mobility (X-coordinate) the mutually typical curve that draws, the relative mobility of calculating this lipase is 0.509, and the molecular weight of being obtained this alkaline lipase by typical curve again is 29.2Kda.
Example 7
The N terminal amino acid sequence of measuring the aspergillus niger lipase that example 1 makes with the determined amino acid sequence instrument is: ATADAAAFPDLHRAAKLSSA.
Example 8
The aspergillus niger lipase effect ester bond location specific that adopts special-purpose ester bond location specific determinator mensuration example 1 to make in the biological production department of the Chinese Academy of Sciences of the Hiroshima University of Japan laboratory is a kind of hydrolysis Sn-1 and Sn-3 position lipase, is suitable for the food enzyme.
Claims (2)
1. an aspergillus niger (Aspergillus niger) bacterial strain S-7749 CGMCC No.1334.
2. Aspergillus niger strain as claimed in claim 1 is used to prepare lipase.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101518646B (en) * | 2009-04-02 | 2012-10-03 | 深圳市绿微康生物工程有限公司 | Lipase-containing composition |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8338133B2 (en) * | 2007-01-30 | 2012-12-25 | Mitsubishi-Kagaku Foods Corporation | DNA encoding glyceroglycolipid lipase |
| CN103125766B (en) * | 2011-12-05 | 2014-09-17 | 深圳市绿微康生物工程有限公司 | Duck feed containing lipase and preparation method of lipase |
| CN103125765B (en) * | 2011-12-05 | 2014-11-12 | 深圳市绿微康生物工程有限公司 | Chicken feed containing lipase and preparation method of lipase |
| CN103184159B (en) * | 2011-12-27 | 2016-09-21 | 丰益(上海)生物技术研发中心有限公司 | Closely branch acremonium bacterial strain and specific lipase thereof |
| CN106135648B (en) * | 2015-04-17 | 2020-01-14 | 深圳市汇尚科科技有限公司 | Lipase composition and application thereof |
| CN104762278A (en) * | 2015-04-23 | 2015-07-08 | 焦作健康元生物制品有限公司 | Method for fermenting lipase |
| CN105112303B (en) * | 2015-09-06 | 2018-10-16 | 江南大学 | A kind of Aspergillus niger strain of production wine complex enzyme |
| CN110938554B (en) * | 2019-11-27 | 2022-10-28 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain capable of stably producing lipase at high yield |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6534303B2 (en) * | 2001-03-13 | 2003-03-18 | Council Of Scientific & Industrial Research | Process for the preparation of acidic lipase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6534303B2 (en) * | 2001-03-13 | 2003-03-18 | Council Of Scientific & Industrial Research | Process for the preparation of acidic lipase |
Non-Patent Citations (1)
| Title |
|---|
| Purification and characterization of an acid-resistanttriacylglycerol lipase from Aspergillus niger. Torossian K. and Bell A.W.Biotechnol.Appl.Biochem.,Vol.13 No.2. 1991 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101518646B (en) * | 2009-04-02 | 2012-10-03 | 深圳市绿微康生物工程有限公司 | Lipase-containing composition |
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