CN105039271A - Method for increasing yield of various enzyme preparations - Google Patents

Method for increasing yield of various enzyme preparations Download PDF

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Publication number
CN105039271A
CN105039271A CN201510358623.0A CN201510358623A CN105039271A CN 105039271 A CN105039271 A CN 105039271A CN 201510358623 A CN201510358623 A CN 201510358623A CN 105039271 A CN105039271 A CN 105039271A
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zymin
output
general formula
raising according
glycine
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马兴群
刘雨
宋琦
吕丽娟
陈霞
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SHANDONG SUNWIN BIOTECHOLOGY CO Ltd
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SHANDONG SUNWIN BIOTECHOLOGY CO Ltd
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    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

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Abstract

The invention relates to a method for increasing the yield of various enzyme preparations. The method is characterized in that a fermentation culture medium of fermentation production comprises one or more of compounds with the general formula (1) (shown in the description), and in the general formula (1), R refers to C1-C20 alkyl groups. According to the method, through adding the compounds with the general formula (I) into the culture medium, the problem of bacterium respiratory depression and the problem of feedback inhibition caused by overhigh intracellular fermentation product concentration are alleviated, the cell metabolism pathway is enabled to quickly progress towards the pathway of target enzyme production all the time, and finally, the yield of various enzyme preparations is obviously increased, and the energy consumption is reduced, so that the production cost is reduced, and the economic benefit is increased.

Description

A kind of method improving multiple zymin output
Technical field
The present invention relates to a kind of production method of zymin, particularly a kind of method improving multiple zymin output.
Background technology
Enzymes Industry occupies vital role in national economy, and its Application Areas, throughout light industry, food, chemical industry, medicine, health, agricultural and the energy, environment protection etc., produces huge Social and economic benef@.At present, zymin Application Areas constantly expands, and production technology level improves constantly, but still has gap compared with Foreign Advanced Lerel.Utilize the weak point of Production by Microorganism Fermentation zymin to be that enzyme secretion efficiency is not high at present, cause that fermentation production rate is low, production cost is higher.
The fermentative production of most industry zymin mainly utilizes aerobic bacteria to carry out aerobic fermentation, is that seed culture or bulk fermentation all need to carry out ventilation oxygen supply.Because in bacterial strain aerobic fermentation process, oxygen requirement is large, particularly ferment the middle and later periods, in short period of time, oxygen requirement is very high, and now cause fermented liquid thickness because thalline and meta-bolites are on the increase, permeability of cell membrane reduces, be unfavorable for the transmission of oxygen, conventional dependence increases ventilation or improves constantly the modes such as mixing speed can not satisfy the demand sometimes to improve dissolved oxygen.Be ventilation also or mixing speed all has the upper limit, can not infinitely improve, moreover opening degree is larger, the damage degree of fermentation equipment and energy consumption are also larger, and maintenance cost and cost are increased.In addition, air flow is too high or rotating speed is too fast, also easily causes physical abuse to cell.
Current, most industry zymin is extracellular enzyme, and production process is secreted into extracellular by cytolemma after producing in born of the same parents.Along with the carrying out of fermentation, especially the middle and later periods, fermented liquid viscosity increases, intraor extracellular osmotic pressure increases, and cell permeability reduces, and now intracellular enzyme is also in a large amount of generation stage, if the enzyme secreting outside in born of the same parents not in time, can cause too high due to Fermentation Substance Concentration in born of the same parents and feedback that is that cause suppresses, the resultant quantity of enzyme is reduced, output reduces.
In order to improve the output of zymin, those skilled in the art both domestic and external have carried out a large amount of research work, and provide a series of method.
Such as: Chinese patent application CNN101851614 discloses a kind of production technique improving zymin output, and this processing method passes into the pressurized air of rich oxygen content in process of production, to reach the object increasing zymin output.But aforesaid method needs oxygen generator, complex process, cost is higher.
Japanese Patent JPS55148090 discloses the method being improved zymin output by the method for externally-applied magnetic field, and the method needs extra magnetic force generator, and same process is complicated, and cost is higher.
Japanese Patent JPS527481 discloses a kind of method improving zymin output, the method adopts organic solvent to be auxiliary agent, although to a certain degree improve the output of zymin, the organic solvent added is bringing adverse influence to enzymic activity and follow-up separating step.
Summary of the invention
For the deficiencies in the prior art, the object of the invention is to the multiple zymin output of a kind of raising and economy method easily.
To achieve these goals, the method of the multiple zymin output of raising provided by the invention is fermentative production, fermention medium wherein, comprises carbon source, nitrogenous source, inorganic salt, characterized by further comprising one in general formula (1) compound or two or more;
R represents C 1~ C 20alkyl.
Preferred general formula (1) compound R is C1 ~ C12 alkyl.Preferred compound is trimethyl-glycine, lauryl betaine (N-dodecyl-N, N-N-methylsarcosine), octyl group trimethyl-glycine (N-octyl group-N, N-N-methylsarcosine), sec.-propyl trimethyl-glycine (N-sec.-propyl-DMG).
General formula (1) compound role in the medium, may have following mechanism:
1, as efficient methyl donor, participate in methylation reaction and the enzyme work of key enzyme in pathways metabolism can be improved, accelerate biomass growth rate and metabolite output.
2, can permeability of cell membrane be improved, promote nutritive substance and the exchange of oxygen inside and outside born of the same parents, and improve the respiratory chain system of microorganism, significantly improve the respiratory characteristic of thalline.
3, as osmoprotectant, intraor extracellular osmotic pressure can be regulated, more be conducive to intracellular product to the release outside born of the same parents, solve and suppress problem by the too high feedback caused of Fermentation Substance Concentration in born of the same parents.Reduce the respiration inhibition that high osmotic pressure causes, accelerate the transfer rate of oxygen at intraor extracellular, reduce energy consumption.
4, there is as tensio-active agent the effect of emulsifying agent, more accelerate the transmission speed of oxygen in the fermented liquid of thickness.
Optimum general formula (1) compound R is methyl, is trimethyl-glycine, can be selected from one in BETAINE anhydrous, a water trimethyl-glycine, hydrochloride trimethyl-glycine, phosphoric acid betaine or two or more.
Trimethyl-glycine, has another name called N, N, Betaine, is a kind of quaternary ammonium type water-soluble alkaloid.Extract and trimethyl-glycine of gaining the name from beet because of initial, sterling is white flaky crystals, pleasantly sweet, the easy moisture absorption, quickly dissolving in water, easy digested absorption, use safety, has no side effect.Except the above-mentioned possible mechanism of action, trimethyl-glycine also has obvious beneficial effect to the dispersity and biomass that improve bacterium ball in seed liquor.
Concentration 0.015 ~ the 7.0g/dl of general formula (1) compound in yeast fermentation substratum, is preferably 0.05 ~ 0.5g/dl.
The zymin that the inventive method is suitable for comprises: cellulase, zytase, saccharifying enzyme, proteolytic enzyme, amylase, lipase, beta-glucanase, mannonase polygalacturonase, alpha-galactosidase, glucose oxidase, phytase etc.
The inventive method is applicable to the zymophyte of following various zymin:
Bacterium class:
Intestinal bacteria, (withered grass, lichens, solution starch, short and small, how sticky class) bacillus etc.
Mycophyta:
1. yeast class: (finishing red, false silk) yeast etc.;
2. mould fungi: (Richter scale, long handle) wood is mould, (rice, black) aspergillus, (rope form, special) mould etc.
According to the difference of substrate, fermentation condition also changes to some extent.Fermentation is carried out under aerobic conditions, needs to stir or vibration.
The present invention, on prior art basis, improving zymin output by adding general formula (1) compound in the fermentation medium, dealing with problems one: because fermenting process weakens for hypoxgia and oxygen transmission the thalline respiration inhibition caused; Deal with problems two: solve intracellular product (zymin) and occur not freely or not in time to the secretion outside born of the same parents, and in the born of the same parents caused, the too high feedback caused of Fermentation Substance Concentration suppresses problem.Thus improving enzymic synthesis efficiency, final raising zymin productive rate also reduces energy consumption.
Embodiment
Be described in further details the present invention below by embodiment, these embodiments are only used for the present invention is described, do not limit the scope of the invention.
Embodiment 1
Seed culture medium (g/dl): corn steep liquor 3, glucose 3, microcrystalline cellulose 0.6, MgSO 47H 2o0.05, KH 2pO 40.3, (NH4) 2sO 40.2, NaNO 30.15, urea 0.1, pH is adjusted to 5.0, at 115 DEG C, heats and carries out sterilizing in 15 minutes.
Trichodermareesei CICC40358 is inoculated in 5L seeding tank, at 30 DEG C, shaking culture 40h.
The fermention medium (g/dl) of cellulase is: wheat bran 3, corn stalk powder 3, microcrystalline cellulose 0.6, MgSO 47H 2o0.05, KH 2pO 40.3, (NH4) 2sO 40.2, NaNO 30.15, urea 0.1, add respectively in substratum concentration shown in following table lauryl betaine, adjustment pH to 5.0.3L substratum is put into the automatic fermenter of 5L, at 121 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 30 DEG C.In culturing process, adopt liquid caustic soda and phosphoric acid to regulate the pH of substratum, make it maintain 4.0-5.5, fermentation culture terminates for 100 hours.Cellulase activity measures and adopts Filter paperlyase (FPA) mensuration alive.Enzyme is lived and is defined: the enzyme amount of decomposing substrate generation l μm of ol glucose with lml enzyme liquid 1min is defined as 1 Ge Meihuo unit.The results are shown in following table:
Table 1.
Embodiment 2
Seed culture medium (g/dl): Semen Maydis powder 5, corn steep liquor 5, soybean cake powder 3, pH4.5, at 115 DEG C, heats and carries out sterilizing in 20 minutes.
Black-koji mould CICC2108 is inoculated in the 5L seeding tank of liquid amount 3L, at 30 DEG C, cultivates 42h.
Saccharifying enzyme fermention medium (g/dl) is: Semen Maydis powder 14, corn steep liquor 6, soybean cake powder 5, (NH4) 2sO 40.2.The octyl group trimethyl-glycine of concentration shown in following table is added respectively, adjustment pH to 4.5 in substratum.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 30 DEG C.In culturing process, adopt liquefied ammonia to regulate the pH of substratum, make it maintain 4.4-4.6.Fermentation culture, after 160 hours, is fermented complete.The glucoamylase enzyme measured in substratum is lived.40 DEG C, under the condition of pH4.6,1ml liquid enzymes (1g solid enzyme powder) degraded Zulkovsky starch per hour produces 1mg glucose, is 1 enzyme activity unit, U/ml (U/g).Hypoiodite volumetric determination glucoamylase enzyme is lived.The results are shown in following table:
Table 2.
Embodiment 3
Seed culture medium (g/dl): glucose 3, corn steep liquor 3, yeast powder 0.5, NaCL1.5, K 2hPO 40.1, MgSO 47H 2o0.04, pH10.0, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
Bacillus licheniformis CICC20203 is inoculated in the 5L seeding tank of liquid amount 3L, at 34 DEG C, cultivates 42h.
Proteolytic enzyme fermention medium (g/dl) is: dregs of beans 6, sucrose 5, glucose 2, CaCl 20.07, Mg 2sO 40.04, KCl0.03.The sec.-propyl trimethyl-glycine of concentration shown in following table is added respectively, adjustment pH to 10.0 in substratum.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 150ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 34 ± 1 DEG C.In culturing process, adopt liquid caustic soda and hydrochloric acid to regulate the pH of substratum, make it maintain 9.5-10.0.Fermentation culture, after 78 hours, is fermented complete.Measure the proteinase activity in substratum.Enzyme is lived and is measured employing 3,5-dinitrosalicylic acid (DNS) colorimetry.1mL enzyme liquid 40 DEG C, under the condition of pH10.5, the enzyme amount that per minute caseinhydrolysate produces needed for 1 μ g tyrosine is defined as an enzyme activity unit.The results are shown in following table:
Table 3
Embodiment 4:
Seed culture medium (g/dl): glucose 5, peptone 1, yeast extract paste 1.5, KH 2pO40.2, MgSO 47H2O0.1, pH5.0, in 121 DEG C of sterilizings 20 minutes.
Aspergillus niger CICC40616 is inoculated in the 5L seeding tank of liquid amount 3L, at 30 DEG C, shaking culture 42h.
The fermention medium (g/dl) of zytase is: corn cob meal 3, glucose 0.5, peptone 1, MgSO 47H 2o0.1, KH 2pO 40.5, urea 0.3, adds water trimethyl-glycine and an empgen BB of concentration shown in following table respectively in substratum, adjustment pH to 5.0.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 30 DEG C.In culturing process, adopt ammoniacal liquor and hydrochloric acid to regulate the pH of substratum, make it maintain 5.0-5.5, fermentation culture terminates for 120 hours.Xylanase activity measures and adopts DNS method.40 DEG C, under the condition of pH5.2, the 1mL liquid enzymes per minute hydrolyzed xylan enzyme amount generated required for 1 μm of ol wood sugar is defined as 1 Ge Meihuo unit (U).The results are shown in following table:
Table 4.
Embodiment 5
Seed culture medium (g/dl): glucose 3, corn steep liquor 3, (NH4) 2sO 40.5, L-glutamic acid 1, KH 2pO40.5, pH6.0, in 115 DEG C of sterilizings 20 minutes.
Bacillus licheniformis CICC10181 is inoculated in the 5L seeding tank of liquid amount 3L, at 37 DEG C, shaking culture 16h.
Diastatic fermention medium (g/dl) is: white dextrin 12, soybean cake powder 2, (NH 4) 2sO 40.5, L-glutamic acid 1, KH 2pO 40.8, add octyl group trimethyl-glycine and the sec.-propyl trimethyl-glycine of concentration shown in following table in substratum respectively, adjustment pH to 6.0.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 150ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 37 ± 1 DEG C.In culturing process, adopt liquid caustic soda and hydrochloric acid to regulate the pH of substratum, make it maintain 6.0-6.5, fermentation culture terminates for 108 hours.Amylase activity measures and adopts YoungJ.Yoo improved method.At 40 DEG C, in 5min, the enzyme amount of hydrolysis 1mg starch (0.5% starch) is a unit of activity.The results are shown in following table:
Table 5
Embodiment 6
Seed culture medium (g/dl): corn steep liquor 3, glucose 3, microcrystalline cellulose 0.6, MgSO 47H 2o0.05, KH 2pO 40.3, (NH4) 2sO 40.2, NaNO 30.15, urea 0.1, pH is adjusted to 5.0, at 115 DEG C, heats and carries out sterilizing in 15 minutes.
Trichodermareesei CICC40358 is inoculated in 5L seeding tank, at 30 DEG C, shaking culture 40h.
The fermention medium (g/dl) of cellulase is: wheat bran 3, corn stalk powder 3, microcrystalline cellulose 0.6, MgSO 47H 2o0.05, KH 2pO 40.3, (NH4) 2sO 40.2, NaNO 30.15, urea 0.1, adds the hydrochloride trimethyl-glycine of concentration shown in following table respectively in substratum, adjustment pH to 5.0.3L substratum is put into the automatic fermenter of 5L, at 121 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 30 DEG C.In culturing process, adopt liquid caustic soda and phosphoric acid to regulate the pH of substratum, make it maintain 4.0-5.5, fermentation culture terminates for 100 hours.Cellulase activity measures and adopts Filter paperlyase (FPA) mensuration alive.Enzyme is lived and is defined: the enzyme amount of decomposing substrate generation l μm of ol glucose with lml enzyme liquid 1min is defined as 1 Ge Meihuo unit.The results are shown in following table:
Table 6
Embodiment 7
Seed culture medium (g/dl): corn steep liquor 3, glucose 3, microcrystalline cellulose 0.6, MgSO 47H 2o0.05, KH 2pO 40.3, (NH4) 2sO 40.2, NaNO 30.15, urea 0.1, pH is adjusted to 5.0, at 115 DEG C, heats and carries out sterilizing in 15 minutes.
Trichodermareesei CICC40358 is inoculated in 5L seeding tank, at 30 DEG C, shaking culture 40h.
The fermention medium (g/dl) of cellulase is: wheat bran 3, corn stalk powder 3, microcrystalline cellulose 0.6, MgSO 47H 2o0.05, KH 2pO 40.3, (NH4) 2sO 40.2, NaNO 30.15, urea 0.1, adjustment pH to 5.0.3L substratum is put into the automatic fermenter of 5L, at 121 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 30 DEG C.In culturing process, adopt liquid caustic soda and phosphoric acid to regulate the pH of substratum, make it maintain 4.0-5.5.Respectively in fermentation culture 0, after 3,8,14,18 hours, add the hydrochloride trimethyl-glycine of 0.15g/dl, fermentation culture, after 100 hours, is fermented complete.Cellulase activity measures and adopts Filter paperlyase (FPA) mensuration alive.Enzyme is lived and is defined: the enzyme amount of decomposing substrate generation l μm of ol glucose with lml enzyme liquid 1min is defined as 1 Ge Meihuo unit.The results are shown in following table:
Table 7
Embodiment 8
Seed culture medium (g/dl): Semen Maydis powder 5, corn steep liquor 5, soybean cake powder 3, pH4.5, at 115 DEG C, heats and carries out sterilizing in 20 minutes.
Black-koji mould CICC2108 is inoculated in the 5L seeding tank of liquid amount 3L, at 30 DEG C, cultivates 42h.
Saccharifying enzyme fermention medium (g/dl) is: Semen Maydis powder 14, corn steep liquor 6, soybean cake powder 5, (NH4) 2sO 40.2.The phosphoric acid betaine of concentration shown in following table is added respectively, adjustment pH to 4.5 in substratum.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 30 DEG C.In culturing process, adopt liquefied ammonia to regulate the pH of substratum, make it maintain 4.4-4.6.Fermentation culture, after 160 hours, is fermented complete.The glucoamylase enzyme measured in substratum is lived.40 DEG C, under the condition of pH4.6,1ml liquid enzymes (1g solid enzyme powder) degraded Zulkovsky starch per hour produces 1mg glucose, is 1 enzyme activity unit, U/ml (U/g).Hypoiodite volumetric determination glucoamylase enzyme is lived.The results are shown in following table:
Table 8
Embodiment 9
Seed culture medium (g/dl): glucose 3, corn steep liquor 3, yeast powder 0.5, NaCL1.5, K 2hPO 40.1, MgSO 47H 2o0.04, pH10.0, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
Bacillus licheniformis CICC20203 is inoculated in the 5L seeding tank of liquid amount 3L, at 34 DEG C, cultivates 42h.
Proteolytic enzyme fermention medium (g/dl) is: dregs of beans 6, sucrose 5, glucose 2, CaCl 20.07, Mg 2sO 40.04, KCl0.03.The BETAINE anhydrous of concentration shown in following table is added respectively, adjustment pH to 10.0 in substratum.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 150ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 34 ± 1 DEG C.In culturing process, adopt liquid caustic soda and hydrochloric acid to regulate the pH of substratum, make it maintain 9.5-10.0.Fermentation culture, after 78 hours, is fermented complete.Measure the proteinase activity in substratum.Enzyme is lived and is measured employing 3,5-dinitrosalicylic acid (DNS) colorimetry.1mL enzyme liquid 40 DEG C, under the condition of pH10.5, the enzyme amount that per minute caseinhydrolysate produces needed for 1 μ g tyrosine is defined as an enzyme activity unit.The results are shown in following table:
Table 9
Embodiment 10
Seed culture medium (g/dl): glucose 5, peptone 1, yeast extract paste 1.5, KH 2pO40.2, MgSO 47H2O0.1, pH5.0, in 121 DEG C of sterilizings 20 minutes.
Aspergillus niger CICC40616 is inoculated in the 5L seeding tank of liquid amount 3L, at 30 DEG C, shaking culture 42h.
The fermention medium (g/dl) of zytase is: corn cob meal 3, glucose 0.5, peptone 1, MgSO 47H 2o0.1, KH 2pO 40.5, urea 0.3, adds a water trimethyl-glycine of concentration shown in following table respectively in substratum, adjustment pH to 5.0.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 30 DEG C.In culturing process, adopt ammoniacal liquor and hydrochloric acid to regulate the pH of substratum, make it maintain 5.0-5.5, fermentation culture terminates for 120 hours.Xylanase activity measures and adopts DNS method.40 DEG C, under the condition of pH5.2, the 1mL liquid enzymes per minute hydrolyzed xylan enzyme amount generated required for 1 μm of ol wood sugar is defined as 1 Ge Meihuo unit (U).The results are shown in following table:.
Table 10
Embodiment 11
Seed culture medium (g/dl): glucose 3, corn steep liquor 3, (NH4) 2sO 40.5, L-glutamic acid 1, KH 2pO40.5, pH6.0, in 115 DEG C of sterilizings 20 minutes.
Bacillus licheniformis CICC10181 is inoculated in the 5L seeding tank of liquid amount 3L, at 37 DEG C, shaking culture 16h.
Diastatic fermention medium (g/dl) is: white dextrin 12, soybean cake powder 2, (NH 4) 2sO 40.5, L-glutamic acid 1, KH 2pO 40.8, add the phosphoric acid betaine of concentration shown in following table in substratum respectively, adjustment pH to 6.0.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 150ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 37 ± 1 DEG C.In culturing process, adopt liquid caustic soda and hydrochloric acid to regulate the pH of substratum, make it maintain 6.0-6.5, fermentation culture terminates for 108 hours.Amylase activity measures and adopts YoungJ.Yoo improved method.At 40 DEG C, in 5min, the enzyme amount of hydrolysis 1mg starch (0.5% starch) is a unit of activity.The results are shown in following table:
Table 11
Embodiment 12
Seed culture medium (g/dl): glucose 3, corn steep liquor 3, (NH4) 2sO 40.2, MgSO 40.05, KH 2pO40.01, pH6.5, in 115 DEG C of sterilizings 20 minutes.
Candidiasis CICC1973 is inoculated in the 5L seeding tank of liquid amount 3L, at 28 DEG C, shaking culture 42h.
Diastatic fermention medium (g/dl) is: soya-bean oil 4, dregs of beans 4, (NH 4) 2sO 40.1, MgSO 40.05, KH 2pO 40.1, K 2hPO 40.2.The hydrochloride trimethyl-glycine of concentration shown in following table is added respectively, adjustment pH to 6.0-6.5 in substratum.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 28 DEG C.In culturing process, adopt liquid caustic soda and hydrochloric acid to regulate the pH of substratum, make it maintain 6.0-6.5, fermentation culture terminates for 70 hours.Adopt sweet oil emulsion hydrolytic titration method (abbreviation volumetry), enzyme is lived definition: 40 DEG C, per minute produces the enzyme amount needed for 1 μm of ol lipid acid under pH8, is 1 lipase international unit (IU).The results are shown in following table:
Table 12
Embodiment 13
Seed culture medium (g/dl): glucose 2, corn steep liquor 3, peptone 1, MgSO 40.1, (NH4) 2sO 40.2, K 2hPO41.2, pH, 7.0,0.1MPa, 115 DEG C of sterilizings 20 minutes.
Paenibacillus polymyxa CICC20185 is inoculated in the 5L seeding tank of liquid amount 3L, at 35 DEG C, shaking culture 24h.
The fermention medium (g/dl) of polygalacturonase is: pectin 1, peptone 2, KH 2pO40.3, K 2hPO4,0.6, MgSO40.1, NaCl1.The phosphoric acid betaine of concentration shown in following table is added respectively, adjustment pH to 9.0 in substratum.3L substratum is put into the automatic fermenter of 5L, at 115 DEG C, heat and carry out sterilizing in 20 minutes.
In each fermentor tank, access the seed that 300ml obtains by above-mentioned cultivation, blowing air is cultivated, and temperature is 35 DEG C.In culturing process, adopt liquid caustic soda and hydrochloric acid to regulate the pH of substratum, make it maintain 8.5-9.0, fermentation culture terminates for 36 hours.Enzyme activity determination adopts DNS spectrophotometer method.Enzyme activity unit is defined as: under pH value is 10.0,50 DEG C of conditions, and it is 1 enzyme activity unit that per minute catalysis hydrolyzed pectin generates enzyme amount needed for 1 μ g galacturonic acid.The results are shown in following table:
Table 13
By adding general formula (1) compound in the medium, alleviate on the one hand due to fermenting process for hypoxgia, not in time and oxygen transmission weaken the thalline respiration inhibition caused; On the other hand owing to helping cell in time by a large amount of enzyme secretion of producing in born of the same parents to outside born of the same parents, reduce and suppress problem by the too high feedback caused of Fermentation Substance Concentration in born of the same parents, cellular metabolic pathways is carried out fast towards the approach of production object enzyme all the time, finally significantly improve various zymin output and reduce energy consumption (and can see that general formula (1) compound or should add before fermentation early stage in time from embodiment 7), thus reduction production cost, increase economic efficiency.
Should be understood that; the above is only the preferred embodiment of the present invention, for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (11)

1. improve the method for multiple zymin output, the method is fermentative production, it is characterized in that the fermention medium of described fermentative production contains one in general formula (1) compound or two or more;
R represents C 1~ C 20alkyl.
2. the method for the multiple zymin output of raising according to claim 1, is characterized in that described general formula (1) compound R is C 1~ C 12alkyl.
3. the method for the multiple zymin output of raising according to claim 1 and 2, is characterized in that described general formula (1) compound is trimethyl-glycine, lauryl betaine, octyl group trimethyl-glycine, sec.-propyl trimethyl-glycine.
4. the method for the multiple zymin output of raising according to claim 3, is characterized in that described general formula (1) compound is BETAINE anhydrous, a water trimethyl-glycine, hydrochloride trimethyl-glycine, phosphoric acid betaine.
5. the method for the multiple zymin output of raising according to claim 1, is characterized in that the concentration of described general formula (1) compound in described fermention medium is 0.015 ~ 7.0g/dl.
6. the method for the multiple zymin output of raising according to claim 5, is characterized in that the concentration of described general formula (1) compound in described fermention medium is 0.05 ~ 0.5g/dl.
7. the method for the multiple zymin output of raising according to claim 1, is characterized in that described zymin comprises cellulase, zytase, saccharifying enzyme, proteolytic enzyme, amylase, lipase, beta-glucanase, mannonase polygalacturonase, alpha-galactosidase, glucose oxidase, phytase.
8. the method for the multiple zymin output of raising according to claim 1,2,4,5,6 any one, is characterized in that the zymophyte producing above-mentioned zymin comprises intestinal bacteria, bacillus, yeast, mould, the mould of wood.
9. the method for the multiple zymin output of raising according to claim 8, is characterized in that described bacillus is the one in Bacillus subtillis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus pumilus, aerobacillus polymyxa Donker.
10. the method for the multiple zymin output of raising according to claim 8, is characterized in that described yeast is pichia spp or candiyeast; Described wood is mould is that Trichodermareesei or long handle wood are mould; Described aspergillus is aspergillus oryzae or aspergillus niger; Described mould is penicillium funiculosum or Penicllium notatum.
The method of the multiple zymin output of 11. raising according to claim 1, is characterized in that fermentation is carried out under aerobic conditions, needs to stir or vibration.
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