CN1683520A - A strain for producing composite enzyme and process for producing compoiste emzyme using said strain fermentation - Google Patents
A strain for producing composite enzyme and process for producing compoiste emzyme using said strain fermentation Download PDFInfo
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- CN1683520A CN1683520A CN 200510038488 CN200510038488A CN1683520A CN 1683520 A CN1683520 A CN 1683520A CN 200510038488 CN200510038488 CN 200510038488 CN 200510038488 A CN200510038488 A CN 200510038488A CN 1683520 A CN1683520 A CN 1683520A
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Abstract
The present invention belongs to the field of biological engineering technology. The strain of the present invention is Bacillus amyloliquefaciens SYB-001, and has the preservation number of CGMCC No. 1314 in Common microbe center. The strain may be used in preparing composite enzyme with relatively high activity in the fermenting culture medium with corn powder and barley powder as composite carbon source and soybean cake powder as nitrogen source. The present invention lays the foundation for directing breeding and ultimate industrial production.
Description
Technical field
The bacterial strain of prozyme and the method for producing prozyme with this strain fermentation are produced in one strain, the present invention relates to the bacterial strain of a kind of product prozyme (comprising beta-glucanase and proteolytic enzyme) and the method for fermentative production prozyme, belong to technical field of bioengineering.
Background technology
Abroad, beta-glucanase is applied to beer technology the earliest, and it can decompose the beta-glucan gel in barley and the Fructus Hordei Germinatus, reduce wort viscosity, improve filtration velocity, the wheat juice leaching yield of wheat juice, reduce gelatinous precipitate, improve the turbidity of beer, improve the quality of products.And the large-scale application of this technology of China is just inchoate in 20 end of the centurys, remarkable benefit.In recent years, development along with wheat class feed, to domestic animal, the poultry feed that contains barley, beta-glucan in the barley is the non-starch viscous polysaccharide of a class antinutritional factor, after adding beta-glucanase, not only can eliminate the influence of antinutritional factor, improve the nutrition balance of these feeds, and can obtain the good social benefit.Utilize microbial fermentation to produce prozyme and have many advantages such as raw materials cost is low, wide material sources, caused investigator's extensive interest.
Summary of the invention
The objective of the invention is to propose that the bacterial strain of prozyme is produced in a strain and with the method for this strain fermentation production prozyme, the present invention can prepare and has the active prozyme of high enzyme (comprising beta-glucanase and proteolytic enzyme).
Technical scheme of the present invention
The bacterial strain of the prozyme that comprises beta-glucanase and proteolytic enzyme is produced in one strain, this bacterial strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SYB-001, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.1314.
Utilize this strain fermentation production to comprise that the method for the prozyme of beta-glucanase and proteolytic enzyme is:
Inclined-plane and seed culture medium are in g/L: peptone 10.0, and extractum carnis 5, NaCl 5, and slant culture adds agar 20, pH7.0 again;
Fermention medium is in g/L: Semen Maydis powder 36-44, and barley meal 18-22, soybean cake powder 36-44, the mass ratio of control Semen Maydis powder and barley meal is 2: 1, Na
2HPO
48, (NH
4)
2SO
44, NH
4Cl 0.75, CaCl
21.0, pH7.0;
Culture condition is:
Shake flask fermentation, fermention medium is 25mL in the 250mL Erlenmeyer flask, temperature is 35～40 ℃ (best 37 ℃), the initial pH4.0 of substratum～10.0 (best 7.0), and rotating speed 150～200r/min (best 200 r/m), fermentation time is 60h;
Ferment tank, 5L fermentation cylinder for fermentation culture volume is 3L, and inoculum size is 8% (v/v), and temperature is 37 ℃, and air flow 3L/Lmin, mixing speed are 600r/min, fermentation time is 35-40h.
With Semen Maydis powder and barley meal with 2: 1 (mass ratio) composite during as culture medium carbon source fermentation to produce the vigor of beta-glucanase higher.
The vigor of fermentation product beta-glucanase is higher when being nitrogenous source with 4% soybean cake powder.
CGMCC No.1314 produces enzyme at the 5L fermentor tank, connects a ring CGMCC No.1314 bacterial classification from the inclined-plane and goes into seed culture medium (25mL/250mL Erlenmeyer flask), under 37 ℃, 200r/min, cultivate 12h after, the inoculum size with 8% (v/v) inserts in the 5L jar.Volume is 3L in the 5L jar, and air flow is 3L/Lmin, and mixing speed is 600r/mim, and leavening temperature is 37 ℃.Fermentation equipment is that the 5L of Shanghai Baoxing Biology Equipment Engineering Co., Ltd controls fermentor tank (Biotech-2001) automatically.Fermentation time is 35-40h, and beta-glucanase enzyme maximum alive is 123U/mL, and the highest enzyme of proteolytic enzyme is lived and is 3800U/mL.
In fermented liquid, add different tensio-active agents and certain influence is arranged producing enzyme, in the basic medium with barley meal 2%, Semen Maydis powder 4% and soybean cake powder 4% and inorganic ion composition, different surfaces promoting agent SDS, the Tween-80, polyoxyethylene glycol or the defoamer polyethers that add 1% content respectively, shake-flask culture, the result shows, Tween-80, polyoxyethylene glycol or defoamer polyethers all can promote the generation of beta-glucanase, especially the effect with the defoamer polyethers is the most remarkable, has improved about 20% than control enzyme work.
Analytical procedure
The plastic centrifuge tube that will contain the 4mL fermented liquid places table model high speed centrifuge (TGL-16G), centrifugal 5min under 10000r/min, and it is standby to get supernatant.
Beta-glucan method of analyzing enzyme: 0.1mL dilution enzyme liquid adds the 0.4mL substrate, in 40 ℃ of reaction 10min, adds the 3ml precipitated liquid in each sample, and is centrifugal, surveys the absorption value A of clear liquid at the 590nm place, replaces enzyme liquid to do blank with 0.1mL distilled water equally.
Formula is calculated in enzyme work: enzyme (u/mL)=[(A alive
590+ 0.001238)/0.8903] * n
N-fermented liquid extension rate
The beta-glucanase definition: the 1ml fermented liquid is in 40 ℃, pH6.5, and per minute decomposition barley beta-glucan produces 1 μ mol reducing sugar and is defined as 1 enzyme unit alive.
The neutral protein method of analyzing enzyme: Folin-phenol method is measured.
Beneficial effect of the present invention: the prozyme that is provided produces bacterium CGMCC No.1314, can be that the fermented substrate fermentation produces the prozyme with high beta-glucanase enzymic activity with cheap raw materials such as barley meal, Semen Maydis powder, soybean cake powder, has reduced production cost; Shortened fermentation period greatly by improving rotating speed in the test of 5L jar.More than be found to be and instruct the design breeding and improve output by the metabolic regulation optimization of fermentation conditions and lay a good foundation, also for the production cost that reduces prozyme provide may, promoted the practical application process of prozyme in industrial production.
Description of drawings
The tensio-active agent that Fig. 1 is different produces enzyme to CGMCC No.1314 influence
The product enzyme process curve of Fig. 2 CGMCC No.1314 in the 5L fermentor tank.Enzyme unit alive is U/mL.The biological material specimens preservation
The bacterial strain of the prozyme that comprises beta-glucanase and proteolytic enzyme is produced in one strain, this bacterial strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SYB-001, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.1314, and preservation date is on February 2nd, 2005.
Embodiment
Following example has been explained some key point among the present invention in more detail, and foundation is provided for a better understanding of the present invention.
Embodiment 1
Choose several different carbon sources respectively, ferment under the situation that (as described in specification sheets) is identical in its zymotechnique, what CGMCC No.1314 produced beta-glucanase and proteolytic enzyme the results are shown in Table 1.
The different carbon sources of table 1 are produced the influence of beta-glucanase and proteolytic enzyme to CGMCC No.1314
| Carbon source | The relative enzyme of beta-glucanase % alive | The relative enzyme of proteolytic enzyme % alive |
| Barley meal+Semen Maydis powder | ?100 | ????100 |
| Barley meal | ??92 | ????56 |
| Semen Maydis powder | ??93 | ????123 |
| Glucose | ??83 | ????105 |
| Sucrose | ??68 | ????66 |
| Glycerine | ??22 | ????72 |
| Lactose | ??42 | ????81 |
| Starch | ??56 | ????299 |
The result shows with Semen Maydis powder and barley meal (mass ratio 2: 1) is composite when be carbon source, and the ability of product beta-glucanase is higher.
Embodiment 2
Composite with Semen Maydis powder and barley meal (mass ratio 2: 1) is carbon source, selects different nitrogenous sources respectively, under the identical situation of its zymotechnique (as described in specification sheets), ferments, and what CGMCC No.1314 produced beta-glucanase and proteolytic enzyme the results are shown in Table 2.
Table 2 different nitrogen sources is produced the influence of beta-glucanase and proteolytic enzyme to CGMCC No.1314
| Nitrogenous source | The relative enzyme work/% of beta-glucanase | The relative enzyme work/% of proteolytic enzyme |
| ????(NH 4) 2SO 4 | 42 | ???72 |
| ????NH 4Cl | 40 | ???35 |
| ????NH 4NO 3 | 50 | ???5 |
| Soybean cake powder | 100 | ???100 |
| Semen Maydis powder | 25 | ???80 |
| Peptone | 78 | ???11 |
| Extractum carnis | 75 | ???6 |
The result shows, 4% soybean cake powder is during as nitrogenous source, and enzyme is lived higher.
Embodiment 3
The different surfaces promoting agent is to producing the influence of enzyme
In the basic medium with barley meal 2%, Semen Maydis powder 4% and soybean cake powder 4% and inorganic ion composition, different surfaces promoting agent SDS, the Tween-80, polyoxyethylene glycol or the defoamer polyethers that add 1% content respectively, shake-flask culture, the result shows, Tween-80, polyoxyethylene glycol or defoamer polyethers all can promote the generation of beta-glucanase, and the effect with the defoamer polyethers is the most remarkable especially, has improved about 20% than control enzyme work.
Embodiment 4
CGMCC No.1314 produces enzyme at the 5L fermentor tank, connects a ring CGMCC No.1314 bacterial classification from the inclined-plane and goes into seed culture medium (25mL/250mL Erlenmeyer flask), under 37 ℃, 200r/min, cultivate 12h after, the inoculum size with 8% (v/v) inserts in the 5L jar.Volume is 3L in the 5L jar, and air flow is 3L/ (Lmin), and mixing speed is 600r/mim, and leavening temperature is 37 ℃.Fermentation equipment is that the 5L of Shanghai Baoxing Biology Equipment Engineering Co., Ltd controls fermentor tank (Biotech-2001) automatically.Fermentation time is 35-40h, and beta-glucanase enzyme maximum alive is 123U/mL, and the highest enzyme of proteolytic enzyme is lived and is 3800U/mL.
Claims (2)
1. the bacterial strain of the prozyme that comprises beta-glucanase and proteolytic enzyme is produced in a strain, its classification called after bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SYB-001, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.1314.
2. method of utilizing the described strain fermentation production of claim 1 to comprise the prozyme of beta-glucanase and proteolytic enzyme is characterized in that:
Inclined-plane and seed culture medium are in g/L: peptone 10.0, and extractum carnis 5, NaCl 5, and slant culture adds agar 20 again, and pH 7.0;
Fermention medium is in g/L: Semen Maydis powder 36-44, and barley meal 18-22, soybean cake powder 36-44, the mass ratio of control Semen Maydis powder and barley meal is 2: 1, Na
2HPO
48, (NH
4)
2SO
44, NH
4Cl 0.75, CaCl
21.0, pH7.0;
Culture condition is:
Shake flask fermentation, fermention medium is 25mL in the 250mL Erlenmeyer flask, temperature is 35～40 ℃, the initial pH4.0 of substratum～10.0, rotating speed 150～200r/min, fermentation time are 60h;
Ferment tank, 5L fermentation cylinder for fermentation culture volume is 3L, and inoculum size is a volume ratio 8%, and temperature is 37 ℃, and air flow 3L/Lmin, mixing speed are 600r/min, fermentation time is 35-40h.
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| CNB2005100384888A CN1329504C (en) | 2005-03-17 | 2005-03-17 | A strain for producing composite enzyme and process for producing compoiste emzyme using said strain fermentation |
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|---|---|---|---|
| CNB2005100384888A CN1329504C (en) | 2005-03-17 | 2005-03-17 | A strain for producing composite enzyme and process for producing compoiste emzyme using said strain fermentation |
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| CN1329504C CN1329504C (en) | 2007-08-01 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280290B (en) * | 2008-05-13 | 2010-06-02 | 江南大学 | Genetic engineering bacteria for producing high-thermal stability recombinant beta-glucanase and construction thereof |
| CN102676483A (en) * | 2012-01-15 | 2012-09-19 | 河南科技大学 | Method for producing protease through one-bacterium multi-enzyme strain |
| CN102864091A (en) * | 2012-01-15 | 2013-01-09 | 河南科技大学 | One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof |
| CN104328097A (en) * | 2014-07-17 | 2015-02-04 | 河南科技大学 | Method for producing cellulase, mannase and glucoamylase from bacillus subtilis |
| CN104928262A (en) * | 2015-05-28 | 2015-09-23 | 南昌大学 | Preparation method of complex enzyme for extracting vegetable fat through aqueous enzymatic method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD282028A5 (en) * | 1989-02-16 | 1990-08-29 | Akad Wissenschaften Ddr | PROCESS FOR PREPARING THERMOSTABILES, HYBRIDEN BACILLUS BETA-1,3-1,4-GLUCANASE |
| CN1202241C (en) * | 2003-09-23 | 2005-05-18 | 中国科学院武汉病毒研究所 | Prepn process and application of amylolytic bacillus CH-2 strain |
-
2005
- 2005-03-17 CN CNB2005100384888A patent/CN1329504C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280290B (en) * | 2008-05-13 | 2010-06-02 | 江南大学 | Genetic engineering bacteria for producing high-thermal stability recombinant beta-glucanase and construction thereof |
| CN102676483A (en) * | 2012-01-15 | 2012-09-19 | 河南科技大学 | Method for producing protease through one-bacterium multi-enzyme strain |
| CN102864091A (en) * | 2012-01-15 | 2013-01-09 | 河南科技大学 | One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof |
| CN104328097A (en) * | 2014-07-17 | 2015-02-04 | 河南科技大学 | Method for producing cellulase, mannase and glucoamylase from bacillus subtilis |
| CN104928262A (en) * | 2015-05-28 | 2015-09-23 | 南昌大学 | Preparation method of complex enzyme for extracting vegetable fat through aqueous enzymatic method |
| CN104928262B (en) * | 2015-05-28 | 2018-10-19 | 南昌大学 | A kind of preparation method of aqueous enzymatic extraction vegetable fat complex enzyme |
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| Publication number | Publication date |
|---|---|
| CN1329504C (en) | 2007-08-01 |
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