CN105524896A - Method used for fermentation enzyme production of Serratia marcescens lipase and improving enzyme activity of Serratia marcescens lipase - Google Patents
Method used for fermentation enzyme production of Serratia marcescens lipase and improving enzyme activity of Serratia marcescens lipase Download PDFInfo
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- CN105524896A CN105524896A CN201510993398.8A CN201510993398A CN105524896A CN 105524896 A CN105524896 A CN 105524896A CN 201510993398 A CN201510993398 A CN 201510993398A CN 105524896 A CN105524896 A CN 105524896A
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- 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Abstract
The invention discloses a method used for fermentation enzyme production of Serratia marcescens lipase and improving the enzyme activity of the Serratia marcescens lipase. A derivant is added in a fed batch manner in the fermentation process to greatly improve the output of lipase derived from Serratia marcescens, and a multiple-metal ion combination technology is adopted to activate the lipase without changing the enzyme amount in order to improve the enzyme activity of the lipase, so low-cost and high-enzyme activity is provided for production application. The method promotes application of the lipase derived from Serratia marcescens strains in production, greatly reduces the production cost, and makes fermentation enzyme production and enzyme activity improvement be simple and efficient.
Description
Technical field
The present invention relates to a kind of serratia marcescens lipase fermentation to produce enzyme and improve enzyme method alive, belong to technical field of bioengineering.
Background technology
Bizio in 1823 find serratia marcescens (
serratiamarcesens) belong to serratia under intestinal bacilli section, can secrete multiple as extracellular enzymes such as lipase, chitinase, metalloprotease, thiol proteinases.At present, the mechanism of secretion of serratia marcescens lipase is studied clear, is the ABC-transpoter mechanism of gram negative bacterium.Because application potential is larger, the lipase gene of more bacterial strain obtains cloning and expressing, what be worth proposition is, the lipase of serratia marcescens Sr418000 is first by Japanese Scientists cloning and expressing, and enzyme is fixed on membrane reactor is used for resolving racemic trans-4-p-methoxy-phenyl glycidic acid methyl esters (MPGM) obtains (2R, 3S)-4-methoxyphenyl glycidic acid methyl esters.(-)-MPGM can as synthesis of chiral cis (+) Odizem.
The chemistry of Odizem is called cis-(+)-5-[(2-dimethylamino) ethyl]-2-(4-p-methoxy-phenyl)-3-ethanoyl-2,3-dihydro-1,5-benzothiazepines-4 (5H)-one hydrochloride.Day Honda limit pharmacy exploitation first by FDA certification in 1973, its effect suppresses calcium ion by vascular smooth muscle and myocardium membrane channel slow transit system, thus have strong vasodilation and myocardial cell's excitement-contraction uncoupling, clinically, being mainly used in treating dissimilar stenocardia, irregular pulse and local asphyxia type heart trouble and pneumonia in aged etc., is one of key agents of cardiovascular disorder.
From the later stage eighties in last century, American-European and Japan and other countries successively successful application chemoenzymatic synthesis Odizem, comprises day Honda limit company, Dutch DSM N. V. etc.But, live not high enough because the serratia marcescens lipase existed at present all exists enzyme on fermentation expression, or the problem that expression amount is lower, make enzyme liquid fractionation time and yield all not reach desirable.
Summary of the invention
The object of the invention is to solve above-mentioned technical problem, provide a kind of serratia marcescens lipase fermentation to produce enzyme and improve enzyme method alive.
Object of the present invention is achieved through the following technical solutions:
Serratia marcescens lipase fermentation produces enzyme and improves an enzyme method alive, comprises the steps:
S1, enzymatic production step;
Step is added in the combination of S2, metal ion;
Wherein, described S1 specifically comprises:
S11, seed culture medium and fermention medium preparation step:
A, seed culture medium: prepare sucrose, peptone, extractum carnis, NaCl, its weight part is: 1:3:2:1, dissolved by above-mentioned starting material tap water, regulate pH to be 7.5 with NaOH, tap water is settled to volume required, 121 DEG C of sterilizing 20min or more;
B, fermention medium: prepare sucrose, corn dextrin, peptone, corn starch, ammonium sulfate, K
2hPO
43H
2o, NaCl, MgSO
4, CaCl
2, FeSO
47H
2o is 5:10:15:10:4:6:2:0.5:0.1:0.01 by weight, is dissolved by above-mentioned starting material tap water, and regulate pH to be 7.5 with NaOH, tap water is settled to volume required, 121 DEG C of sterilizing 20min or more;
S12, seed culture step:
The serratia marcescens Db11 (deriving from East China University of Science) that freeze-drying pipe is preserved is inoculated on the seed culture medium solid plate of interpolation 2% agar, 30 DEG C of constant temperature culture 12h;
Select the single bacterium colony on flat board, get an articulating and enter to be equipped with in the triangular flask of described seed culture medium, be positioned in constant-temperature shaking incubator, 30 DEG C of constant-temperature shaking culture 12h or more, obtain seed liquor; The volume of seed culture medium is less than the half of triangular flask volume;
S13, fermentation culture step:
In the ratio of 5% (v/v), cultured for S12 seed liquor is accessed in described fermention medium, fermentation initial temperature is set as 32-35 DEG C, tank pressure is set as 0.05-0.07MPa, air velocity is set to 0.5-0.8vvm, by regulating mixing speed in fermenting process, the dissolved oxygen level maintaining fermented liquid is not less than 20%, and by stream acid liquid or alkali lye, maintaining fermented liquid pH is 7.5;
Enzyme step is produced in S14, induction:
After fermentation culture half the time, by miscarrying, the mode added adds enzyme inducer, then continues fermentation;
S15, fermentation liquor treatment step:
Cryogenic freezing whizzer is adopted to carry out centrifugal to the fermented liquid of S14, clear enzyme solution in collection;
Described S2 specifically comprises:
FeSO is added in the upper clear enzyme solution that S15 obtains
4, ZnCl
2, CaCl
2, make Fe
2+, Zn
2+, Ca
2+concentration be 50mol/L.
Wherein, in S14, described product enzyme inducer is tween-80 diluent, and after fermentation culture 8h, the mode added by stream adds the described tween-80 diluent of 5g/L, adds be as the criterion with 0.5-1h stream; Fermentation time is 16h;
Preferably, described tween-80 diluent is tween-80 and the distilled water dilution proportion according to 1:4.
Preferably, the detecting step detecting enzyme after described S2 alive comprises:
Getting the enzyme liquid that 100 μ L suitably dilute joins in the phosphate buffered saline buffer of 2.85mL, pH8.0, the DMSO solution of 50 μ L p-NP butyric esters is added after 30 DEG C of insulation 5min, concentration is 200mmol/L, quick mixing, be placed in spectrophotometer, constant temperature 30 DEG C detection, determined wavelength is 450nm, reads absorbance every 10s; The per minute enzyme amount generated needed for 1 μm of oL p-NP is defined as a Ge Meihuo unit (U),
Enzyme calculation formula alive:
Fermenting enzyme unit (U/L)=Δ A alive of enzyme
450* 1.823*10
4* enzyme liquid extension rate
Wherein: Δ A
450for the product average of every 10s light absorption value change.
When the enzyme of fermented liquid is lived as 10000-20000U/L, enzyme liquid measures after should diluting 5 times; When fermentation broth enzyme lives >20000U/L, measure again after 10 times should be diluted.
Preferably, in S12, the articulating being specially the single bacterium colony of making even on plate enters to be equipped with in the 500mL triangular flask of 100mL seed liquor substratum.
Preferably, in S13, fermentation initial temperature is set as 35 DEG C, and tank pressure is set as 0.05MPa, and air velocity is set to 0.8vvm.
Beneficial effect of the present invention is mainly reflected in: Serratia bacterial strain has the ability of yielding lipase, but under spontaneous fermentation state, yield of enzyme is lower, adopt tween-80 as inductor, and the mode adopting stream to add is added, greatly improves and produce enzyme efficiency.
Different ions affects enzyme by the physico-chemical property changing enzyme molecule and lives, and by optimizing and screening, adopts the mode of metal ion combination, adds, have stronger activation to enzyme in enzyme liquid after fermentation, can improve enzyme significantly and live.
The advantage of the method: 1, fermentation process is simple; 2, inductor is simple and easy to get, and inducing action is strong; 3, metal ion adds combination, and when without the need to other steps, activation is strong.
Embodiment
The present invention uses the strain serratia marcescens provided by East China University of Science, and the yielding lipase that ferments, the method for being induced by inductor, and the mode adopting stream to add is added, and makes yield of enzyme larger; Meanwhile, by use metal ion combination add, activated lipase in conjunction with catalytic center, improve enzyme activity and the catalytic efficiency of lipase, achieve good effect.Preparation method of the present invention is illustrated below in conjunction with embodiment:
Serratia marcescens lipase fermentation of the present invention produces enzyme and improves enzyme method alive, comprises the steps:
S1, enzymatic production step;
Step is added in the combination of S2, metal ion;
Wherein, described S1 specifically comprises:
S11, seed culture medium and fermention medium preparation step:
Seed culture medium:
Sucrose 5g/L, peptone 15g/L, extractum carnis 10g/L, NaCl5g/L, above-mentioned starting material tap water dissolved, regulate pH to be 7.5 with NaOH, tap water is settled to volume required, and prepare 5 bottles of seed culture mediums, 121 DEG C of sterilizing 20min or more are stand-by;
Fermention medium:
Sucrose 5g/L, corn dextrin 10g/L, peptone 15g/L, corn starch 10g/L, ammonium sulfate 4g/L, K
2hPO
43H
2o6g/L, NaCl2g/L, MgSO
40.5g/L, CaCl
20.1g/L, FeSO
47H
2o0.01g/L, dissolves above-mentioned starting material tap water, and regulate pH to be 7.5 with NaOH, tap water is settled to volume required; Prepare 10L fermention medium, add water about 8.5L, puts into 15L fermentor tank, and according to fermenter sterilizing method, 121 DEG C of sterilizing 20min or more are stand-by, and the cumulative volume after sterilizing is about 9.5L.
S12, seed culture step:
Dull and stereotyped seed culture step: inclined-plane seed or-80 DEG C of freeze-drying pipe seeds preserved of getting 4 DEG C of Refrigerator stores, be inoculated on the seed culture medium solid plate of interpolation 2% agar, line, has inoculated rear inversion and has been incubated in 30-35 DEG C of constant incubator, cultivates 10-16 hour;
Shake-flask seed is cultivated: select the single bacterium colony on flat board, get an articulating and enter to be equipped with in the 500mL triangular flask of 100mL seed liquor substratum, be positioned in constant-temperature shaking incubator, 30-35 DEG C of constant-temperature shaking culture 12h or more.
S13, fermentation culture step:
Being total to 500ml shake-flask seed liquid by cultured 5 bottles accesses in described fermention medium in the ratio of 5% (v/v), fermentation initial temperature is set as 32-35 DEG C, tank pressure is set as 0.05-0.07MPa, air velocity is set to 0.5-0.8vvm, by regulating mixing speed in fermenting process, the dissolved oxygen level maintaining fermented liquid is not less than 20%, and by stream acid liquid or alkali lye, maintaining fermented liquid pH is 7.5.
Enzyme step is produced in S14, induction:
Weigh 50g tween-80, add distilled water 200g, stirring heating mixes, and put into 1L feed supplement bottle, 121 DEG C of sterilizing 20min, cool stand-by.
In fermentation culture after 8 hours, by peristaltic pump on tank and feed supplement connector, the feed supplement bottle of above-mentioned tween-80 diluent is equipped with in access, and flow feeding, feed rate is 5ml/min.
Ferment about 14-16 hour, and backlash sampling measures enzyme according to the method for following S16 and lives, and enzyme stops fermentation after living and reaching 20000U/L, puts tank process.
S15, fermentation liquor treatment step:
Adopt cryogenic freezing whizzer to carry out centrifugal to the fermented liquid of S14, clear enzyme solution in collection, 10L fermentation system, the enzyme liquid measure of acquisition is about 9-9.5L, saves backup.
S16, detection enzyme activating method:
Getting the enzyme liquid that 100 μ L suitably dilute joins in the phosphate buffered saline buffer of 2.85mL, pH8.0, the DMSO solution of 50 μ L p-NP butyric esters is added after 30 DEG C of insulation 5min, concentration is 200mmol/L, quick mixing, be placed in spectrophotometer, constant temperature 30 DEG C detection, determined wavelength is 450nm, reads absorbance every 10s; The per minute enzyme amount generated needed for 1 μm of oL p-NP is defined as a Ge Meihuo unit (U),
Enzyme calculation formula alive:
Fermenting enzyme unit (U/L)=Δ A alive of enzyme
450* 1.823*10
4* enzyme liquid extension rate
Wherein: Δ A
450for the product average of every 10s light absorption value change.
When the enzyme of fermented liquid is lived as 10000-20000U/L, enzyme liquid measures after should diluting 5 times; When fermentation broth enzyme lives >20000U/L, measure again after 10 times should be diluted.
Described S2 specifically comprises: in the upper clear enzyme solution that S15 obtains, add FeSO
4, ZnCl
2, CaCl
2, make Fe
2+, Zn
2+, Ca
2+concentration be 50mol/L, adopt detection method described in S16 to detect enzyme and live, enzyme can be improved and to live about 30%-40%.Different ions adds, and can change the physico-chemical property of enzyme, thus has different impacts, description of test, Fe to enzyme is alive
2+, Zn
2+, Ca
2+add to enzyme live have larger promoter action, select this three kinds of materials, SO
4 2-, Cl
-add on enzyme live without impact, ionic concn and combination reason illustrate in comparative example.
Below provide relevant comparative's example better to show the selection of preferred parameter in the present invention:
Comparative example 1: adopt different inductors, fermented after 8 hours, and the mode adopting stream to add is added, and the enzyme recorded is lived and contrasted:
Table 1: different inductors is on the impact of producing enzyme
| Inductor (5g/L) | Fermenting enzyme lives (U/L) |
| Blank | 6346±347 |
| TritonX-100 | 4448±101 |
| Soybean oil | 8472±714 |
| Glycerine | 6653±255 |
| Peanut oil | 9542±586 |
| Oleic acid | 6288±671 |
| Tween-80 | 19264±421 |
As shown in Table 1: TritonX-100 has restraining effect to enzymatic production, soybean oil and peanut oil have promoter action slightly to product enzyme, and oleic acid and glycerine are on product enzyme almost without impact, and tween-80 is the strongest to the promoter action of producing enzyme, improves more than 3 times.
Comparative example 2: adopt different metal ions to add respectively, detects the impact of living on enzyme:
Table 2: the impact that different metal ions enzyme is lived
| Metal ion adds (50mM) | Enzyme lives (U/L) |
| Blank | 22296±234 |
| Fe 2+ | 24129±196 |
| Fe 3+ | 22385±338 |
| Al 3+ | 19010±512 |
| Mn 2+ | 18861±429 |
| Zn 2+ | 23574±101 |
| Co 2+ | 18851±422 |
| Cu 2+ | 14155±250 |
| Ca 2+ | 28252±205 |
As shown in Table 2: Ca
2+enzyme is lived and has larger promoter action, Fe
2+and Zn
2+enzyme is lived and has slight promoter action, but also obviously, Fe
3+enzyme is lived almost without impact, and Al
3+, Co
2+, Cu
2+, Mn
2+to the enzyme restraining effect had in various degree alive.
Table 3: have the impact that the metal ion combination of promoter action is lived on enzyme
| Metal ion adds (50mM) | Enzyme lives (U/L) |
| Blank | 25147±223 |
| Ca + | 30482±341 |
| Fe 2++Ca + | 31241±222 |
| Zn 2++Ca + | 31885±394 |
| Fe 2++Zn 2+ | 29954±204 |
| Fe 2++Zn 2++Ca + | 34890±524 |
As shown in Table 3: superposed by metal ion and contrast, Fe
2++ Zn
2++ Ca
+the raising action effect mixed enzyme is alive is best, can improve enzyme and live 30%-40%.
Relative to the method in comparative example, method of the present invention improves a lot in raising ferment tank enzyme is lived, and by the inductor of uniqueness, yield of enzyme is increased substantially; Utilize metal ion on the impact of lipase, the mode reasonably adopting many kinds of metal ions combination to add, adds the activation to deriving from Serratia lipase, improves enzyme and lives.
The present invention still has multiple concrete embodiment, and all employings are equal to replacement or equivalent transformation and all technical schemes of being formed, all drop within the scope of protection of present invention.
Claims (6)
1. serratia marcescens lipase fermentation produces enzyme and improves enzyme method alive, it is characterized in that, comprises the steps:
S1, enzymatic production step;
Step is added in the combination of S2, metal ion;
Wherein, described S1 specifically comprises:
S11, seed culture medium and fermention medium preparation step:
A, seed culture medium: prepare sucrose, peptone, extractum carnis, NaCl, its weight part is: 1:3:2:1, dissolved by above-mentioned starting material tap water, regulate pH to be 7.5 with NaOH, tap water is settled to volume required, 121 DEG C of sterilizing 20min or more;
B, fermention medium: prepare sucrose, corn dextrin, peptone, corn starch, ammonium sulfate, K
2hPO
43H
2o, NaCl, MgSO
4, CaCl
2, FeSO
47H
2o is 5:10:15:10:4:6:2:0.5:0.1:0.01 by weight, is dissolved by above-mentioned starting material tap water, and regulate pH to be 7.5 with NaOH, tap water is settled to volume required, 121 DEG C of sterilizing 20min or more;
S12, seed culture step:
The serratia marcescens Db11 (deriving from East China University of Science) that freeze-drying pipe is preserved is inoculated on the seed culture medium solid plate of interpolation 2% agar, 30 DEG C of constant temperature culture 12h;
Select the single bacterium colony on flat board, get an articulating and enter to be equipped with in the triangular flask of described seed culture medium, be positioned in constant-temperature shaking incubator, 30 DEG C of constant-temperature shaking culture 12h or more, obtain seed liquor; The volume of seed culture medium is less than the half of triangular flask volume;
S13, fermentation culture step:
In the ratio of 5% (v/v), cultured for S12 seed liquor is accessed in described fermention medium, fermentation initial temperature is set as 32-35 DEG C, tank pressure is set as 0.05-0.07MPa, air velocity is set to 0.5-0.8vvm, by regulating mixing speed in fermenting process, the dissolved oxygen level maintaining fermented liquid is not less than 20%, and by stream acid liquid or alkali lye, maintaining fermented liquid pH is 7.5;
Enzyme step is produced in S14, induction:
After fermentation culture half the time, by miscarrying, the mode added adds enzyme inducer, then continues fermentation;
S15, fermentation liquor treatment step:
Cryogenic freezing whizzer is adopted to carry out centrifugal to the fermented liquid of S14, clear enzyme solution in collection;
Described S2 specifically comprises:
FeSO is added in the upper clear enzyme solution that S15 obtains
4, ZnCl
2, CaCl
2, make Fe
2+, Zn
2+, Ca
2+concentration be 50mol/L.
2. serratia marcescens lipase fermentation according to claim 1 produces enzyme and improves enzyme method alive, it is characterized in that: in S14, described product enzyme inducer is tween-80 diluent, after fermentation culture 8h, the mode added by stream adds the described tween-80 diluent of 5g/L, adds be as the criterion with 0.5-1h stream; Fermentation time is 16h.
3. serratia marcescens lipase fermentation according to claim 2 produces enzyme and improves the method that enzyme lives, and it is characterized in that: described tween-80 diluent is tween-80 and the distilled water dilution proportion according to 1:4.
4. serratia marcescens lipase fermentation according to claim 1 produces enzyme and improves enzyme method alive, it is characterized in that: the detecting step detecting enzyme after described S2 alive comprises:
Getting the enzyme liquid that 100 μ L suitably dilute joins in the phosphate buffered saline buffer of 2.85mL, pH8.0, the DMSO solution of 50 μ L p-NP butyric esters is added after 30 DEG C of insulation 5min, concentration is 200mmol/L, quick mixing, be placed in spectrophotometer, constant temperature 30 DEG C detection, determined wavelength is 450nm, reads absorbance every 10s; The per minute enzyme amount generated needed for 1 μm of oL p-NP is defined as a Ge Meihuo unit (U),
Enzyme calculation formula alive:
Fermenting enzyme unit (U/L)=Δ A alive of enzyme
450* 1.823*10
4* enzyme liquid extension rate
Wherein: Δ A
450for the product average of every 10s light absorption value change.
When the enzyme of fermented liquid is lived as 10000-20000U/L, enzyme liquid measures after should diluting 5 times; When fermentation broth enzyme lives >20000U/L, measure again after 10 times should be diluted.
5. serratia marcescens lipase fermentation according to claim 1 produces enzyme and improves enzyme method alive, and it is characterized in that: in S12, the articulating being specially the single bacterium colony of making even on plate enters to be equipped with in the 500mL triangular flask of 100mL seed liquor substratum.
6. serratia marcescens lipase fermentation according to claim 1 produces enzyme and improves enzyme method alive, it is characterized in that: in S13, and fermentation initial temperature is set as 35 DEG C, and tank pressure is set as 0.05MPa, and air velocity is set to 0.8vvm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106222152A (en) * | 2016-08-09 | 2016-12-14 | 苏州开元民生科技股份有限公司 | A kind of fermentation process producing () gamma-lactams enzyme recombination bacillus coli |
| CN109975280A (en) * | 2019-03-15 | 2019-07-05 | 广州聚佰生物科技有限公司 | A kind of pesticide residue detection method and card of recognizable false positive |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222152A (en) * | 2016-08-09 | 2016-12-14 | 苏州开元民生科技股份有限公司 | A kind of fermentation process producing () gamma-lactams enzyme recombination bacillus coli |
| CN109975280A (en) * | 2019-03-15 | 2019-07-05 | 广州聚佰生物科技有限公司 | A kind of pesticide residue detection method and card of recognizable false positive |
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