CN101225369B - Bacillus MP-2 with high-yield marine microorganism esterase and marine microorganism esterase generated thereby - Google Patents
Bacillus MP-2 with high-yield marine microorganism esterase and marine microorganism esterase generated thereby Download PDFInfo
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Abstract
The invention relates to bacillus MP-2 of high-yield marine microorganism esterase, which is characterized in that, the bacteria MP-2 is Bacillus sp.MP-2 of high-yield marine microorganism esterase which is filtered and separated from the silt on the Pohai floor, and is preserved in China Center for Type Culture Collection on Jun 9, 2007, with a preservation number CCTCC No: M207078; the high-yield marine microorganism esterase is widely used in the fields of medicine, chemical industry, food industry, energy industry, environmental protection and other fields.
Description
Technical field
The present invention relates to the marine microorganism field, particularly the genus bacillus MP-2 of a kind of high-yield marine microorganism esterase that screening and separating obtains from the bed mud of marine site, the Bohai Sea and the marine microorganism esterase of generation thereof.
Background technology
Microorganism esterase derives from natural microorganism, the Microbial resources that can produce esterase at occurring in nature have Lu Yuan biology and marine microorganism, the Lu Yuan microorganism is for example: the heat-stable extreme microorganism Thermus of the strain aquaticus that obtains from the Yellowstone hot spring, this strain culturing temperature is 80 ℃, people such as DongG from Thermus aquaticus outside the isolated born of the same parents alkaline phosphatase optimum temperuture be 75～80 ℃; Obtain from Thermotaga neaplitana separation and purification that the alkaline phosphatase optimum temperuture is 85 ℃ in the born of the same parents.Thermus S p.FD3041 is that domestic hot spring separates a strain that the obtains hot bacterium that dwells, and culture temperature is 70 ℃.Alkaline phosphatase FD2TAP in the born of the same parents that obtain, optimum temperuture is 70 ℃.Pyrococcus abyssi separates the heterotrophism archeobacteria obtain from the volcano, deep-sea, optimum growth temperature is 100 ℃, and the optimum temperuture that obtains alkaline phosphatase in the born of the same parents is 70 ℃ etc.Yet existing Lu Yuan microorganism esterase is often because of acidproof, alkaline-resisting or resistance toheat is poor, range of application is very limited, therefore seek microorganism esterase and produce the great attention that bacterial strain is subjected to countries in the world, become one of center of paying close attention in this field with special property.
Microbial resources in the ocean environment are very abundant, and because the uniqueness of ocean environment condition, comprise high salt, high pressure, low nutrition, low temperature etc., the many specificitys that marine microorganism is different from the Lu Sheng microorganism have been brought up, ocean environment microorganism and the enzyme that is produced thereof be also corresponding to have some special nature, and especially marine microorganism esterase has more special physiological properties and wide Application Areas.In addition, marine microorganism is difficult for cultivating, form is various and be easy to death etc. in preservation and culture transferring process, cause difficulty for the differentiation of their kinds, be unfavorable for going deep into of marine microorganism research and development, therefore as if the directed generation bacterium that separates various special property esterases from ocean environment, make up the strain library that an esterase produces bacterium, have great practicality.
Press for exploitation at present and reach quick, easy, accurately and reliably and the seed selection separation means and the method for good economy, obtain useful microorganism strains, for the development and use of marine microorganism resource lay the foundation so that can separate.
Meanwhile, need set up some classification authentication methods simple, convenient, easy handling analyzes marine microorganism, make people to a certain extent more science, more accurate, find the classification position of ocean isolate more quickly, for the development and use of marine microorganism resource lay the foundation.At present, macromole rRNA has become a molecular indexes, be widely used for the heredity of various microorganisms and the research of molecular difference, add that the DNA of a large amount of known microorganisms is determined and import international gene database, become the very useful reference system of microorganism identification classification, thereby can reach the purpose of identifying classification with it fast and effectively by mensuration and comparative analysis to unknown microbial DNA sequence.
Microorganism esterase is a kind of important industrial enzyme, relate to that ester is synthetic, lactone synthetic, catalyzed reactions such as transesterify, polypeptide are synthetic, the conversion of steric isomer and fractionation, esterase catalyzed do not need coenzyme and have the reaction conditions gentleness, method is easy, catalytic activity is high, selectivity is strong, product is easy to separation, be easy to advantage such as recovery.Therefore be widely used in fields such as foodstuffs industry, pharmaceutical industries, daily-use chemical industry industry, military affairs and biological protection.Especially the pyroreaction activity of thermophilic esterase, and to the strong resistance of organic solvent, stain remover and denaturing agent makes its potentiality that all are widely used at aspects such as food, medicine, process hides, oil production and waste treatment.
In the world from the early 1990s till now, warm esterase, high-temperature alkaline esterase, the alkaline esterase of middle temperature etc. in developing in succession, but the zymologic property of esterase still can't satisfy the demand of modern industry.Therefore from the ocean, filter out marine microorganism esterase with unique zymologic property, a real much progress for the microorganism esterase exploitation, and will bring new vigor and vitality to zymin industry.From the seventies in last century, abroad just begin to screen, but do not see that relevant marine microorganism produces the report (M.Chandrasekaran, 1997) of esterase preparation and properties producing the esterase marine bacteria.The research of China marine microorganism esterase is started late, and people such as Yang Congfa is only arranged from seawater, and the halobiontic digestive tube of ooze is separated to alkaline esterase and produces bacterium (Yang Congfa, 2000).But, limited the ocean esterase in the further exploitation of preparation with application facet because there are problems such as seawater cultivation and esterase separation and purification difficulty in marine microorganism.
Summary of the invention
The objective of the invention is to overcome above-mentioned prior art exists not enough.Through the contriver be engaged in for a long time marine microorganism zymetology engineering development research and the screening bacterial strain fundamental research in, develop quick, easy, accurately and reliably and the seed selection separation means that combines of good economy, thereby the genus bacillus MP-2 that a plant height produces marine microorganism esterase and the marine microorganism esterase of generation thereof are provided.
The genus bacillus MP-2 of high-yield marine microorganism esterase provided by the invention, be the bacillus sp.MP-2 (being called for short bacterial strain MP-2) of the high-yield marine microorganism esterase that screening and separating obtains from the bottom silt of the Bohai Sea, be preserved in Chinese typical culture collection center on June 9th, 2007, this CCTCC No:M207078 of preserving number.
The morphological specificity of the genus bacillus MP-2 of high-yield marine microorganism esterase provided by the invention:
Morphological specificity
The bacterium colony smooth surface of bacterial strain MP-2, sorrel, translucent, neat in edge has tree root shape protuberance; Cell is shaft-like, and size is the μ m of (0.3-0.8) μ m * (2.0-3.0), the binary fission breeding, and with the peritrichous motion, it is positive that no pod membrane, bacterial strain are removed from office blue look dyeing, can observe oval-shaped gemma once in a while, life in the gemma, sporangium does not have obviously expands.
Cultural characteristic
Strict aerobic, moderate growth on nutrient agar plate, bacterium colony is flat, smooth, yellow-gray, no soluble pigment; If surface moisture can be enlivened expansion, the edge is irregular.It is thicker to grow on the agar glucose flat board, becomes gauffer.
Nucleotide sequence in specification sheets not the page or leaf
The phylogenetic tree based on 16S rDNA sequence (Figure 18) that bacterial strain MP-2 and the relevant genus kind of assembling from databases such as GenBank thereof make up
It is compared with the 16S rDNA sequence of the relevant bacterial strain of assembling from databases such as GenBank of bacillus.
Can find out obviously that from constructed phylogenetic tree bacterial strain MP-2 and an effective publication kind of this genus Bacillus licheniformis (B.licheniformis) form an independent branch, 16S rDNA sequence homology reaches 97%.A group is gathered into other several genus bacillus by this branch, wherein bacterial strain MP-2 and B.licheniformis, and the homology of B.subtilis is respectively 96.3%, 95.0%, and the relation of growing is nearer.The division bacteria scholar generally believes when 16S rRNA sequence homology and is higher than 97%, of the same race in can thinking to belong to, and being lower than 93%-95% then may be for belonging to outer member.And the morphological specificity of this bacterial strain and Bacillus licheniformis, physiological and biochemical property are not done the yin and yang attribute contrast respectively than big-difference and with intestinal bacteria and streptococcus aureus.Therefore, bacterial strain MP-2 is genus bacillus (Bacillus sp.MP-2).
Bacterial strain MP-2 screening is from the deep-sea bed mud; Can be in 4 ℃ of-50 ℃ of growths, 30 ℃ of optimum growth temperatures; And can grow in the 0%-9%NaCl substratum by this bacterial strain of salt tolerant experiment discovery, the suitableeest salt concn of growth is 4%.Therefore according to the definition of marine microorganism,, think that this bacterial strain is a marine bacteria in conjunction with source, low temperature adaptability, the salt tolerance of this bacterial strain.Compare with terrestrial bacterium, the general salt tolerant of marine bacteria, optimal salinity can be utilized the real marine bacteria of tolerance of salinity screening at 2%-5%.
Physio-biochemical characteristics
The physio-biochemical characteristics of bacterial strain MP-2 are listed in table 1,
Annotate :+, the bacterial strain of 90-100% is positive;-, the bacterial strain of 90-100% is negative; D, the bacterial strain of 11-89% is positive; V, character instability in a bacterial strain; ND, undetermined.
By the polyphase sort research that bacterial strain MP-2 morphological specificity, physiological and biochemical property and 16S rDNA sequence are carried out, find that this bacterial strain has the morphological feature of typical bacillus.16S rDNA sequence and this genus bacterial strain sequence homology are all very high, wherein with this genus in the sequence homology of Bacillus licheniformis reach 97%, do the yin and yang attribute contrast respectively in conjunction with the morphological specificity of Bacillus licheniformis, physiological and biochemical property and with intestinal bacteria and streptococcus aureus, bacterial strain MP-2 is decided to be genus bacillus (Bacillus sp.MP-2).
The fermentation culture of bacterial strain MP-2
The growth curve of bacterial strain MP-2
Bacterial strain MP-2 is carried out activation culture, substratum: glucose 1%, NaCl0.5%, extractum carnis 1%, peptone 1% every the 2h sampling, is measured the OD value of bacteria suspension at the 550nm place, and its OD curve (being growth curve) is as shown in Figure 2.
As shown in Figure 2, bacterial strain MP-2 is in lag phase by being inoculated into 4h, and physiological status of cells becomes recovery from decline, be about to growth, cell number does not increase: be in acceleration period from 4h to 6h, initial division of cell is carried out during this, and cell number begins to increase; From 6h to 12h is exponential phase of growth, and assimilation of cell is greater than dissimilation during this, and metabolism is vigorous, is in the state of positive growth, the cellular constituent balanced growth; Be in retardation from 12h to the 16h cell, state is constant and stop growing after the cell fission, is in stationary phase behind the 16h, assimilation and dissimilation kept in balance.
Bacterial strain MP-2 fermentation diagram
As shown in Figure 3, bacterial strain MP-2 is at 0～32h, and esterase activity does not almost improve, and behind 36h, esterase activity rises rapidly, reaches maximum value at 56h.
The influence that substratum is formed
Carbon source is produced the influence of enzyme to bacterial strain MP-2
Composition according to this chapter basic medium, replace glucose with corresponding carbon source, activate according to cultural method, every the time sampling, to glucose, sucrose, Semen Maydis powder, peanut meal, Zulkovsky starch, carbon sources such as cottonseed are single factor experiment Fig. 4 under different concentration.
By to carbon source screening, find the production that some agricultural byproducts can fine promotion esterase.Wherein with the peanut meal best results, peanut meal is the natural medium composition, contains the required multiple nutritional components of cell, is the comprehensive action of carbon source and other nutritive ingredient to producing the enzyme raising therefore, and cheap, suitable culture medium raw material as industrial fermentation.By comparison, when only containing glucose in the substratum, esterase output is lower.Show that according to document and data in the past glucose generally is the sugar of easy utilization in the carbon source.Therefore when being carbon source with glucose, may be because microorganism be very fast that carbon source is run out, and cell density increases rapidly and causes shaking dissolved oxygen deficiency in the bottle, thus cause the output of esterase to descend.Therefore when the industrial production esterase, under the prerequisite of oxygen supply condition, can consider that glucose and peanut meal are jointly as the carbon source in the microbiological culture media.By optimizing proportioning, thereby reach the carbon source of quick utilization and slowly utilize the optimum combination of carbon source, so promptly can make microorganism reach certain stand density rapidly, the oxygen supply that guarantees have competent carbon source can be used for esterase synthetic not only but also be unlikely to aggravate in the fermenting process is simultaneously born.
Nitrogenous source produces the influence of enzyme to bacterial strain MP-2
Replace extractum carnis in the basic medium under different concns, to do inorganic nitrogen-sourced, organic nitrogen source and compound nitrogen source comprises that peptone, soybean cake powder, yeast extract paste, extractum carnis, ammonium sulfate, urea etc. produce enzyme test to bacterial strain MP-2, the results are shown in Figure 5 with corresponding nitrogenous source.
Nitrogenous source is used to constitute somatic cells material (amino acid, protein, nucleic acid etc.) and nitrogenous metabolite as the main component in the substratum, and nitrogenous source commonly used is divided into organic nitrogen source and inorganic nitrogen-sourced.Shown in the nitrogenous source The selection result (Fig. 5): when adding organic nitrogen source in the substratum, esterase output obviously is better than only containing in the substratum inorganic nitrogen-sourced situation, wherein with the combined compound nitrogen source best results of soybean cake powder and ammonium sulfate and cheap, wide material sources and become desirable fermentation raw material, compare with organic nitrogen source, ammonium sulfate and urea are not suitable for independent nitrogenous source as substratum.
Inorganic salt produce the influence of enzyme to bacterial strain MP-2
Corresponding composition in the replacement basic medium of common inorganic salt is done the product enzyme test of metal ion to bacterial strain MP-2, the results are shown in Figure 6.
Find out MgSO by Fig. 6
4And KH
2PO
4Product enzyme at 0.05% couple of bacterial strain MP-2 of concentration has bigger promoter action; CaCl
2Almost do not influence producing enzyme, but along with the increase of concentration is inhibited to producing enzyme; And FeSO
4, ZnSO
4, CuSO
4, PbCl
2Inhibited in inorganic salt to producing enzyme, ZnSO wherein
4, PbCl
2Inhibition particularly evident.Hence one can see that, and concentration is 0.05% MgSO
4And KH
2PO
4Two kinds of inorganic salt are to producing the enzyme best results.
Ester class, fatty acid ester, analog and tensio-active agent produce the influence of enzyme to bacterial strain MP-2
Ester class, fatty acid ester, analog and the tensio-active agent of different sorts and quantity added in the substratum (see Fig. 7 for details), measure esterase activity after the inoculation culture.
By Fig. 7 analysis as can be known, when peanut oil that adds 0.8 (v/v) and Tween-80 (polyethylene oxide dehydrating sorbitol monooleate), enzyme work increases relatively; When adding tributyrin, triolein, Tween-20 (polyethylene oxide Span 20),, when adding sweet oil, the obvious suppression effect is arranged to producing enzyme then to producing almost not influence of enzyme.This shows that the kind and the quantity of adding inductor have a significant effect to producing enzyme, bacterial strain enzyme that MP-2 produces may be inducible enzyme.
Culture condition
Initial pH produces the influence of enzyme to bacterial strain MP-2
Na with pH6.0～7.5
2HPO
4-citric acid, the damping fluid of pH8～8.5 glycine-NaOH is measured esterase activity as the initial pH of substratum after the inoculation culture, and enzyme is lived as Fig. 8 relatively.
As shown in Figure 8, be that 7.0 o'clock bacterial strain MP-2 product enzymes are best with the initial pH of substratum, enzyme activity reaches maximum value relatively.
Inoculum size is produced the influence of enzyme to bacterial strain MP-2
With 3.0%, 4.0%, 5.0%, 6.0%, 7.0% cultivates back its enzyme of survey with 8.0% inoculum size under identical condition lives, and the results are shown in Figure 9.
As shown in Figure 9, inoculum size is more remarkable to the influence of bacterial strain MP-2 product enzyme, and the inoculum size with 6.0% is the best.
Culture temperature and time are produced the influence of enzyme to bacterial strain MP-2
Respectively at 25 ℃, 30 ℃, 32 ℃, under 35 ℃ of four kinds of differing tempss, cultivated from the beginning and carried out the sampling and measuring enzyme every 4h and live, draw enzyme under four kinds of differing tempss and the different incubation time change curve of living, as shown in figure 10.
As shown in Figure 10, when temperature of reaction is higher (30 ℃), may be owing to accelerate enzymatic reaction, and cause whole fermentation period to shorten, but, thereby cause the stationary phase in the esterase fermentation production process shorter simultaneously because the raising of temperature causes the esterase ratio to be easier to inactivation or the interior nutrient of substratum consumes rapidly.When leavening temperature is low (25 ℃), may be because thalli growth to be slow, and the peak time of esterase production is delayed, whole fermentation period prolongs, and the output of lipase descends.Therefore, mistake is hanged down with the fermentative production of too high temperature for lipase and all is not suitable for.Therefore 30 ℃ is the best enzyme temperature of producing, and finds also in the experiment that fermentation time is very remarkable to the influence of producing enzyme, and between 40-48h, enzyme work reaches the highest.
It is peanut meal, soybean cake powder and inoculum size that the product enzyme is had the factor of remarkably influenced (P＜0.05).Wherein peanut meal presents negative effect to producing enzyme, and soybean cake powder and inoculum size present positive-effect, and the variation of other factors is not remarkable to the influence of producing enzyme.Drawn by each factorial effect, desire will improve enzyme activity, should improve soybean cake powder concentration, prolongs kind of an age, reduces peanut meal concentration.When peanut meal concentration 0.75～1.25%, soybean cake powder concentration is 2～3%, inoculum size concentration is between 5～7%, esterase activity is the highest.When excessive concentration or cross when low, all can cause esterase activity to descend.Bacterial strain MP-2 produces the top condition of esterase fermentation: peanut meal 0.9%; Soybean cake powder 2.29%; (NH4)
2SO
40.2%; KH
2PO
40.1%; K
2HPO
40.1%; MgSO
40.05%; Tween800.8%; Peanut oil 0.8%; PH is 7.0; 30 ℃ of leavening temperatures; Plant 12h in age; Inoculum size 5.98%.With this understanding, optimize the back enzyme activity and bring up to 318.2U/mL by 258.8U/mL.
The zymologic property of marine microorganism esterase (abbreviation esterase)
The marine microorganism esterase optimum temperature
As shown in Figure 11, this esterase enzymic activity in the time of 60 ℃ is the highest, and relatively enzyme work remains between 80% between 50 ℃ to 70 ℃, be higher than 70 ℃ or when being lower than 50 ℃ then enzyme live on a declining curvely, can only maintain 25% 4 ℃ of relative enzyme work during with 100 ℃.Hence one can see that, and 50～70 ℃ of esterase optimum temperature scopes have shown high reactivity at 60 ℃, are optimum temperature.
The suitableeest action pH of esterase
The suitableeest action pH of so-called enzyme just is meant that enzyme reaction rate reaches maximum value, and is higher or lower than a certain pH value, and enzymatic reaction speed all can reduce.The optimal pH of various enzymes has nothing in common with each other, and different along with the difference of substrate sometimes.In 2.5～12 scopes, in the enzyme reaction system, add the Na that differs 1 pH of unit respectively
2HPO
4-citric acid (pH2.5～8), glycine-NaOH damping fluid (pH8～12) has been measured the activity of enzyme under different pH buffer systems.The result as shown in figure 12
Enzyme relative reactivity when pH is 10 is the highest as shown in Figure 12, the suitableeest action pH for esterase, but enzyme work can only maintain below 70% when pH2.5～9, think that major cause is because the potential of hydrogen variation that pH value causes changes to the ionization situation of the intermediate of enzyme-substrate or enzyme-substrate, makes zymoprotein irreversible denaturation occur.With regard to this enzyme, the pH variable effect of reaction medium combines to esterase and substrate p-nitrophenyl phosphoric acid ester, makes curve present down " V " type, and the pH that medium is described changes has very strong effect to this esterase enzyme work.
The thermostability of esterase
Esterase is dissolved in the damping fluid of NaOH-glycine of pH10, places under the differing temps (4～70 ℃), and 2h is handled in water bath with thermostatic control, every the 20min sampling, its remnant enzyme activity is measured in cooling rapidly, with not treated enzyme work is 100%, and all the other amount to into the percentage ratio of residual enzyme vigor, and the result is in Figure 13
As shown in Figure 13, after handling 2h through water bath with thermostatic control, esterase can keep more than 80% 4～40 ℃ of enzyme work, and 50～60 ℃ of enzymes are lived and still can be maintained more than 60%, show that this enzyme has good thermostability.
The pH stability of esterase
Esterase is dissolved in the damping fluid of different pH (2～12), places under 4 ℃, behind the insulation 48h, again enzyme liquid is adjusted back to optimal pH 10, and the enzyme activity that is incubated gained with optimal reaction pH10 down is 100%, and all the other amount to into the percentage ratio of residual enzyme vigor, and the result is in Figure 14.
As can be seen from Figure 14, esterase places under 4 ℃, behind the insulation 48h, can only keep below 30% in pH 2～9 enzyme work, and maintain more than 70% in the pH10 enzymic activity, shows in that this enzyme of pH10 is stable to belong to alkaline enzyme.
Metal ion is to the influence of esterase activity
Survey in the live body system at esterase and to add each metal ion species, and keep surveying and live that concentration of metal ions is 0.01mol/L in the reaction system, compare in 60 ℃, pH10 with damping fluid and measure esterase activity, the result is in Figure 15.
Co as shown in Figure 15
2+, Li
+Enzyme had activation, Ca
2+Vigor to esterase has no significant effect, and Cu
2+, Pb
2+, Ag
+, Mn
2+, Zn
2+, Fe
3+, Ba
2+, Mg
2+Activity to esterase has restraining effect.
Chemical reagent is to the influence of esterase
Add various chemical reagent in the esterase solution, and keep the concentration of reagent to be: 10mmol/L SDS (sodium lauryl sulphate), 1mol/LTris (Tutofusin tris), 10mmol/L halfcystine, 10mmol/L EDTA, 10% urea, 10%Tween-20,10%Tween-80, to leave standstill 30min for contrasting under normal temperature with damping fluid, in 60 ℃, pH10 mensuration esterase relative reactivity, the result is in Figure 16
As shown in Figure 16, SDS, EDTA, Tween-20 is remarkable to the inhibition effect of esterase.In addition, when adding the EDTA of 10mmol/L in the esterase solution, the activity inhibited to 4.91% of esterase can infer that thus metal ion may be contained in the active centre of this esterase.Metal chelator EDTA has restraining effect to heat resisting basic phosphatase.
The stability of esterase in organic solvent
Esterase is dissolved in organic solvent, reacts 4h at normal temperatures, and volatilization is after filtering removed organic solvent and surveyed its enzyme and live, and is contrast with the damping fluid of the pH10 of enzyme, and the result is in Figure 17.
As shown in Figure 17, the restraining effect of except dimethyl sulfoxide (DMSO) enzyme being lived in the common organic solvent significantly, other organic solvents are lived to enzyme does not almost have influence, wherein normal hexane is lived to enzyme influences minimum.The tolerance that this enzyme is good to having of common organic solvent is described.
Esterase is to the research of substrate selective
Esterase acted on have different carbon chain lengths, the triglyceride level of different saturation ratio and different branch degree.Get the aqueous solution or the emulsion mixing of certain enzyme liquid and the various esters of 1mmol/L and put into reactor, adopt sodium hydroxide volumetry and spectrophotometer method to measure esterase activity, the enzyme work that with p-NPP is substrate is 100%, and all the other amount to into the percentage ratio result of residual enzyme vigor in table 2.
Table 2 esterase is to the selectivity of substrate
Be that an important zymologic property of esterase finds that vigor descends the hydrolysis vigor of this esterase with the growth of fatty acid chain length in the substrate during to the hydrolysis of the various glyceryl ester of different carbonatomss to the selectivity of substrate.The relative enzyme of the glyceryl ester of short carbon chain such as glycerine triacetate, tributyrin and ethyl acetate is lived higher, be respectively 66.5%, 45.8% and 56.9%, but to the higher fatty acid fat of long carbochain as 18 not hydrolysis of carbon triolein, as everyone knows, sweet oil is to judge one of best substrate that the lipase enzyme is lived, this esterase is minimum to the sweet oil catalysis activity, does not show enzyme and lives, and illustrates that there are the otherness of substrate in lipase and esterase.
By studies show that of above-mentioned essential property to this esterase: 50～70 ℃ of optimum temperature scopes, shown high reactivity at 60 ℃, belong to thermostable enzyme; Enzymic activity when pH is 10 is the highest, is the suitableeest action pH of esterase, belongs to alkaline enzyme, and its pH value sphere of action is narrow; Esterase can keep more than 80% 4～40 ℃ of enzyme work after handling 2h through water bath with thermostatic control, and 50～60 ℃ of enzymes are lived and still can be maintained more than 60%, show that this enzyme has good thermostability; Esterase places under 4 ℃, behind the insulation 48h, can only keep below 30% in pH2～9 enzyme work, and maintain more than 70% in the pH10 enzymic activity, shows at this enzyme of pH10 more stable.
By metal ion, chemical reagent, organic solvent to esterase activity influence study, the result shows: Co
2+, Li
+Enzyme had activation, Ca
2+Vigor to esterase has no significant effect, and Cu
2+, Pb
2+, Ag
+, Mn
2+, Zn
2+, Fe
3+, Ba
2+, Mg
2+Activity to esterase has restraining effect; SDS, EDTA, nonionogenic tenside Tween-20 is remarkable to the inhibition effect of esterase; The restraining effect of except dimethyl sulfoxide (DMSO) enzyme being lived in the common organic solvent significantly, other organic solvents are lived to enzyme does not almost have influence, wherein normal hexane is lived to enzyme influences minimum.The tolerance that this enzyme is good to having of common organic solvent is described.
By studies show that the substrate selective of this esterase: the enzyme work that with p-NPP is substrate is 100%, the relative enzyme of the glyceryl ester of short carbon chain such as glycerine triacetate, tributyrin and ethyl acetate is lived higher, be respectively 66.5%, 45.8% and 56.9%, but to the higher fatty acid fat of long carbochain as 18 carbon trioleins and sweet oil show activity not.Hydrolysis vigor vigor decline with the growth of fatty acid chain length in the substrate of this esterase is described.
The genus bacillus MP-2 of high-yield marine microorganism esterase provided by the invention and generation marine microorganism esterase thereof are widely used in fields such as medical science, chemical industry, food, the energy and environmental protection.
Description of drawings
Fig. 1 is the electromicroscopic photograph (3000 *) of bacterial strain MP-2
Fig. 2 is the growth curve of bacterial strain MP-2
Fig. 3 is the fermentation diagram of bacterial strain MP-2
Fig. 4 is a carbon source to the influence of esterase output (diagram: 1. glucose 2. sucrose 3. Semen Maydis powder 4. peanut meals 5. Zulkovsky starches 6. cottonseed meals)
Fig. 5 is a nitrogenous source to the influence of esterase output (diagram: 1. peptone 2. soybean cake powder+ammonium sulfate 3. yeast extract pastes 4. extractum carniss 5. soybean cake powder 6. ammonium sulfate 7. urea)
Fig. 6 is inorganic salt to the influence of esterase output (diagram: 1. blank 2. calcium chloride 3. sal epsom 4. potassium primary phosphates 5. ferric sulfate 6. copper sulfate 7. bariumchlorides 8. zinc sulfate 9. lead chlorides)
Fig. 7 is ester class, lipid acid, analog and tensio-active agent to the influence of esterase output (diagram: 1. blank 2. peanut oil 3. Semen Maydis oils 4. soya-bean oil 5. Trisun Oil R 80s 6. sweet oil 7. tween-80s 8. tween 20s 9. tributyrins 10. trioleins)
Fig. 8 is the influence of initial p H to esterase output
Fig. 9 is the influence of inoculum size to esterase output
Figure 10 is the influence of temperature to esterase output
Figure 11 is the influence of temperature to esterase activity
Figure 12 is the influence of PH to esterase activity
Figure 13 is the thermostability of esterase
Figure 14 is the PH stability of esterase
Figure 15 is the influence of metal ion to esterase activity
Figure 16 is the influence of chemical reagent to esterase activity
Figure 17 is the stability of esterase in the machine solvent
The phylogenetic tree that Figure 18 makes up for bacterial strain MP-2 and the relevant genus kind of assembling from databases such as GenBank thereof based on 16S rDNA sequence
Embodiment
The present invention further specifies the present invention with the following example, but protection scope of the present invention is not limited to the following example
Primary election
Marine site, Bohai Sea bed mud 1 gram is added in the 250mL triangular flask that the 20mL sterilized water is housed (in put tens of granulated glass spherees), vibration evenly back fills in the 250mL triangular flask of 25mL enrichment medium with the adding of transfer pipet suction 5mL suspension, under 30 ℃, the 200r/min shake flask fermentation is cultivated.Screening bacterium enrichment 48h, mould enrichment 96h pipettes the 5mL nutrient solution again and continues to cultivate in another fills the triangular flask of fresh culture.So repeat 3 times, carry out the flat board dilution then and separate.
The composition of substratum: glucose 1.0; Extractum carnis 1.0; Peptone 1.0; NaCl0.5; Transfer pH to 7.0.The composition of primary dcreening operation plate culture medium: primary dcreening operation bacteria culture medium+glycerine triacetate 1.0 (V/V) (all after the biofilter degerming, adding)+agar 2.0.
The nutrient solution of enrichment is coated on the primary dcreening operation flat board, puts into constant incubator, 30 ℃ of cultivations, the observations that the 24h of being separated by is regular.The operation of employing coating method, the bacterium colony that will have the color changeable transparent circle is selected, and bacterium is chosen to the bacterium slant medium and preserves,
Shake flask fermentation is cultivated
The bacterial strain of primary dcreening operation inserted be equipped with in the triangular flask that 25mL sieves substratum again, under 30 ℃, 200r/min carries out the liquid shaking bottle fermentation culture.Microbial culture 36h, mould is cultivated 72h.Under 4 ℃, the fermented liquid after the centrifugal cultivation of 15000r/min gets supernatant liquor and is crude enzyme liquid.Fermention medium is a glucose 1.0; Extractum carnis 1.0; Peptone 1.0; NaCl0.5; Transfer pH to 7.0.
The detection culture medium flat plate that purpurum bromocresolis is housed is injected in above-mentioned fermentation culture punching, in 30 ℃ of constant incubators, react, with p-Npp (p-nitrophenyl cetylate) is that substrate is surveyed work, screens the bacterial strain MP-2 that a plant height produces marine microorganism esterase, and enzyme work reaches 218.6u/ml.Through being accredited as bacillus sp.MP-2.
By fermentation culture in the triangular flask shaking table, 30 ℃ of leavening temperatures are planted 12h in age with bacterial strain MP-2, inoculum size 5.98%,
Fermention medium: peanut meal 0.9%; Soybean cake powder 2.29%; (NH4)
2SO
40.2%; KH
2PO
40.1%; K
2HPO
40.1%; MgSO
40.05%; Tween-800.8%; Peanut oil 0.8%; PH is 7.0.
Enzyme activity is up to 318.2u/ml after the fermentation culture.
Sequence table
＜110〉Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
＜120〉a kind of genus bacillus MP-2 of high-yield marine microorganism esterase and the marine microorganism esterase of generation thereof
<160>1
<210>1
<211>1417
<212>DNA
＜213〉genus bacillus (Bacillus sp.MP-2)
<400>1
5’-tgcgtgtcga?gcggactgat?gggagcttgg?tccctgatgt?tgtcggcggt?cggttgagta?60
acacgtgtat?aacctgcctg?gaagactggt?ataactccgg?gaaaccggcg?ctaataccgg 120
atgcttgatt?gaaccgcatg?gttcaattat?aaaaggtggc?ttttagctac?cacttacaga 180
tggacccgcg?gcgcattatc?tagctgctca?gttcggctca?ccaaggcgac?gatgcgtatc 240
cgacctgaga?gggtgatcag?tctcaatgtc?attgacacct?tccccagact?cctacgggag 300
gcagcactta?ggaatcttcc?gcaatggatg?aaagtctgac?ggagcaacgc?cgcgtgagtg 360
atgaaggttt?tcggatcgta?caactctgtt?gttaaggaag?aacatgtacc?gttcgaatat 420
ctgggtacct?tgacggtacc?taatctcaaa?gcctgggcta?actacgtgcc?agcagccgcg 480
gtaatacgta?ggtggtcaag?cgttgtcccg?gaattattgg?gcgtaaagcg?cgcgcaggcg 540
gtttcttaag?tctgatgtga?aagcccccgg?ctcaacccgg?ggagggtcat?tggaaactgg 600
ggaacttgag?tgcagaagag?gagagtggaa?ttccacgtgt?agcggtgaaa?tgcgtagaga 660
tgtggaggaa?caccagtggc?gaaggcgact?ctctggtctg?taactgacgc?tgaggcgcga 720
aagcgtgggg?agcgaacagg?attagatacc?ctggtagtcc?acgccgtaaa?cgatgagtgc 780
taagtgttag?agggtttccg?ccctttagtg?ctgcagcaaa?cgcattaagc?actccgcctg 840
gggagtacgg?tcgcaagact?gaaactcaaa?ggaattgacg?ggggcccgca?caagcggtgg 900
agcatgtggt?ttaattcgaa?gcaacgcgaa?gaaccttacc?aggtcttgac?atcctctgac 960
aaccctagag?atagggcttc?cccttcgggg?gcagagtgac?aggtggtgca?tggttgtcgt 1020
cagctcgtgt?cgtgagatgt?tgggttaagt?cccgcaacga?gcgcaaccct?tgatcttagt 1080
tgccagcatt?cagttgggca?ctctaaggtg?actgccggtg?acaaaccgga?ggaaggtggg 1140
gatgacgtca?aatcatcatg?ccccttatga?cctgggctac?acacgtgcta?caatgggcag 1200
acaaagggca?gcgaagccgc?gaggctaagc?caatcccaca?aatctgttct?cagttcggat 1260
cgcagtctgc?aactcgactg?cgtgaagctg?gaatcgctag?taatcgcgga?tcagcatgcc 1320
gcggtgaata?cgttcccggg?ccttgtacac?accgcccgtc?acaccacgag?agtttgtaac 1380
acccgaagtc?ggtgaggtaa?cctttgagcc?agccgct 1417
Claims (2)
1. the genus bacillus MP-2 of a high-yield marine microorganism esterase (Bacillus sp.MP-2), it is characterized in that this genus bacillus MP-2 is the genus bacillus of the high-yield marine microorganism esterase that screening and separating obtains from the bottom silt of the Bohai Sea, be preserved in Chinese typical culture collection center on June 9th, 2007, preserving number is CCTCC No:M207078.
2. the high-yield marine microorganism esterase of the genus bacillus MP-2 of the high-yield marine microorganism esterase of a claim 1.
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| CN102286441B (en) * | 2011-07-24 | 2012-12-12 | 国家海洋局第二海洋研究所 | Low-temperature esterase and coding gene and use thereof |
| CN102660460A (en) * | 2012-05-10 | 2012-09-12 | 同济大学 | Method for screening non-photosynthetic high-efficiency carbon immobilization microorganism under aerobic conditions |
| CN104140959B (en) * | 2014-07-14 | 2017-05-24 | 中国科学院南海海洋研究所 | Novel esterase as well as coding gene and application of esterase |
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