CN101724592A - Bacillus subtilis and application thereof in biocatalytic production of L-lactic acid - Google Patents

Bacillus subtilis and application thereof in biocatalytic production of L-lactic acid Download PDF

Info

Publication number
CN101724592A
CN101724592A CN200910264303A CN200910264303A CN101724592A CN 101724592 A CN101724592 A CN 101724592A CN 200910264303 A CN200910264303 A CN 200910264303A CN 200910264303 A CN200910264303 A CN 200910264303A CN 101724592 A CN101724592 A CN 101724592A
Authority
CN
China
Prior art keywords
lactic acid
subtilis
cgmcc
application
lactonitrile
Prior art date
Application number
CN200910264303A
Other languages
Chinese (zh)
Other versions
CN101724592B (en
Inventor
杨寿海
姜志忠
孔国平
芮忠南
刘善和
Original Assignee
南京第一农药集团有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京第一农药集团有限公司 filed Critical 南京第一农药集团有限公司
Priority to CN2009102643033A priority Critical patent/CN101724592B/en
Publication of CN101724592A publication Critical patent/CN101724592A/en
Application granted granted Critical
Publication of CN101724592B publication Critical patent/CN101724592B/en

Links

Abstract

The invention belongs to the biochemical industry field and discloses a Bacillus subtilis and an application thereof in biocatalytic production of L-lactic acid. The method in biocatalytic production of L-lactic acid by using the Bacillus subtilis (the preservation number is CGMCC No.3242) comprises the steps as follows: taking immobilized cells or flocculated cells of the Bacillus subtilis (the preservation number is CGMCC No.3242) as a biocatalyst, catalyzing substrate lactonitrile to obtain L-lactic acid, dehydrating the reaction solution in a falling film evaporator, distilling and dehydrating by a molecular still to obtain the L-lactic acid with the product purity of over 95% and the product yield of over 90%. The invention has the advantages of simple technique operation, mild reaction condition, high utilization degree of the substrate, low energy consumption, high product purity, safety and environment protection, and wide application potential.

Description

One bacillus subtilis and the application in biocatalytic production of L-lactic acid thereof

Technical field

The invention belongs to biological chemical field, relate to a bacillus subtilis and the application in biocatalytic production of L-lactic acid thereof.

Background technology

(Lactic Acid LA), has another name called alpha-hydroxypropionic acid to lactic acid, and molecular formula is: CH 3CH (OH) COOH is a kind of natural organic acids, is one of three big organic acids.Contain a unsymmetrical carbon in the lactic acid molecules, thus rotational isomerism had, the left-handed L-lactic acid that is called, the dextral D-lactic acid that is called, both blended then are called D, L-lactic acid.Wherein have only L-lactic acid to be absorbed and metabolism, have good biological activity by human body.Reinforcement along with environmental consciousness, the world is increasing to the demand of biodegradable plastic, the polymkeric substance (poly(lactic acid)) that is obtained by the processing of L-lactic acid monomer is current one of the macromolecular material of future that has most, it had both had the similar glossiness of polystyrene, the transparency and processing characteristics, has the favorable biological degradability energy again, demand is huge, has stimulated the demand growth of L-lactic acid.The L-lactic acid of China only accounts for 2~5% of lactic acid production, and needs tens thousand of tons of fine L-of import lactic acid every year, and as seen the market development of this product has a extensive future, and has much potentiality to be exploited.

The production of lactic acid at present mainly contains chemical synthesis and biological fermentation process.Chemical synthesis production lactic acid can be undertaken by number of ways, and what wherein have realistic meaning is the lactonitrile method.This method is that acetaldehyde and prussic acid generate lactonitrile through the basic catalyst effect, and again through over-churning, the distillation back makes lactic acid with acid hydrolysis.This method by product is more, and product mainly is D, and L-lactic acid needs the expensive chemical reagent of process to split just can make and has optically active L-lactic acid, and purity is on the low side, and environmental stress is big.

Biological fermentation process is the main flow technology of producing lactic acid in the world at present, and starchy material such as adopt that mostly corn, rice, potato are done through liquefaction, makes with microbial fermentation after the saccharification.The bacterial classification that industrial fermentation is produced lactic acid also mainly is Bacterium lacticum, rhizopus and streptomycete etc. at present.For different bacterial strains, its pathways metabolism and enzyme system form also variant, and the final product of formation is also different.In China, the industrialized developing and the applied research of L-lactic acid-producing bacterial classification also are in the starting stage, and the product of traditional zymotic gained still is racemic modification lactic acid mostly.At present, comparatively advanced in the world L-lactic acid-producing technology is to be carbon source with cereals such as corns, employing be bacterium (thermophilic lactobacillus), adopt the anaerobic high temperature fermentation pattern.Biological fermentation process is produced lactic acid than the chemical synthesis mild condition, operational safety, but also exist the cycle long, and the fermentation sewage quantity is big, and Lactic Acid from Fermentation Broth content is low, and impurity is many, problems such as the purifying cost height in downstream.

The method of lactic acid purifying is a lot, and traditional lactic acid separation method adopts calcium lactate-acidolysis process mostly.With strong acid lactic acid is cemented out,, thereby obtain product, but the shortcoming of this method is that the calcium lactate crystalline particle is little that crystallisation process is wayward, produce a large amount of waste residue and liquid etc. again through activated carbon decolorizing and ion-exchange purification.Straight run distillation needs higher vacuum tightness, otherwise lactic acid is decomposed, and product yield descends.Esterification process need be handled high-concentration waste water, and production cost is higher and energy consumption is bigger.

Summary of the invention

The objective of the invention is for overcoming the limitation of above technology, the new subtilis of a strain (Bacillussubtilis) KR2 is provided, another object of the present invention provides the application of this bacterium in biocatalytic production of L-lactic acid.

The specific embodiment of the present invention is as follows:

To be the member of biochemical industry study group of our company obtain from polluted by nitrile compounds to screen the insecticide factory's soil that reaches more than 15 years subtilis involved in the present invention (Bacillus subtilis) KR2 (CGMCC No.3242).Its screening method is as follows:

Get surplus the mud sample 100 batch laggard row filter of enrichment culture and evaluation in batches from Nanjing first insecticide factory's sewage lagoon different angles:

Take by weighing 10 gram mud, add sterilized water 50mL, be inoculated in the 45mL enrichment medium, place on 30 ℃ of shaking tables 120rpm shaking culture 3 days with getting the 5mL suspension liquid after the granulated glass sphere concassation.Get this nutrient solution afterwards and carry out gradient dilution, get 10 -6, 10 -7, 10 -8Gradient dilution liquid be applied on the flat board that contains the primary dcreening operation solid medium, put 30 ℃ of constant incubators and cultivated 3~6 days.Single colony inoculation that picking grows enlarged culturing to the new primary dcreening operation solid medium, culture condition is the same.Treat to scrape after bacterium grows and get 3~5 rings and be inoculated in the liquid fermentation medium and cultivate.Shaking culture is 3 days under 120rpm on 30 ℃ of shaking tables, gets centrifugal 10min under the 15mL nutrient solution 3000rpm, goes to precipitate thalline with isopyknic normal saline flushing after the supernatant liquor, continues the centrifugal thalline that gets.The gained thalline added in the PBS damping fluid that contains 1% (%:g/100mL) iminodiacetonitrile to transforming 2 hours, cell concentration is 0.5g/L in the reaction system.Get that centrifugal 10min gets supernatant liquor under the 5mL conversion fluid 3000rpm, the HPLC method is clear liquid analytically.Illustrate that this bacterium has conversion capability to iminodiacetonitrile if conversion fluid contains iminodiethanoic acid, select the wherein the highest strain of iminodiacetic acid conversion, by " simple and clear uncle's outstanding Bacteria Identification handbook the 8th edition is accredited as subtilis.With this bacillus subtilis called after KR2, be preserved in Chinese microbial preservation management committee common micro-organisms center (CGMCC), preserving number CGMCC No.3242, preservation date are on August 19th, 2009.

Subtilis KR2 (CGMCCNo.3242) among the present invention can be simultaneously respectively with lactonitrile, vinyl cyanide, cigarette nitrile, hydroxyacetonitrile, iminodiacetonitrile is to grow on the solid primary dcreening operation substratum of only nitrogen source, the substrate scope is wide, degraded nitrile compounds ability is strong, is suitable as the production application of L-lactic acid among the present invention.

It is as follows respectively that described liquid enrichment medium in the screening method, solid primary dcreening operation substratum, liquid sieve substratum again:

Enrichment medium (g/100mL): glucose: 3, yeast extract paste: 0.5, peptone: 0.5, sodium-chlor: 0.1, potassium primary phosphate: 0.1, dipotassium hydrogen phosphate: 0.1, sal epsom: 0.05, pH 7.

Solid primary dcreening operation substratum (g/100mL): glucose: 3, iminodiacetonitrile (or vinyl cyanide or cigarette nitrile or lactonitrile or hydroxyacetonitrile): 1, sodium-chlor: 0.1, potassium primary phosphate: 0.1, dipotassium hydrogen phosphate: 0.1, sal epsom: 0.05, agar: 2, pH:7.

Liquid fermentation medium (g/100mL): glucose: 3, yeast extract paste: 0.5, peptone: 0.5, sodium-chlor: 0.1, potassium primary phosphate: 0.1, dipotassium hydrogen phosphate: 0.1, sal epsom: 0.05, urea: 0.5, pH:7.

Subtilis (Bacillus subtilis) KR2 (CGMCC No.3242) has following microbial characteristic:

1. morphological feature

Circular slick single bacterium colony appears after cultivating 48 hours on the solid plate substratum, canescence, and opaque, surface wettability, the edge is smooth, whole bacterium colony projection, easily picking; It is shaft-like that microscopy becomes, size is long to be (0.5~0.8um) * (1.5~2.5um), give birth in the gemma.

2. cultivate and learn feature

This bacterial strain 30 ℃ of cultural characteristics of cultivating after 48~96 hours down in following 3 kinds of substratum see Table 1.

The cultural characteristic of table 1 subtilis KR2 (CGMCC No.3242) on 3 kinds of substratum

3. physiological and biochemical property

Table 2. subtilis KR2 (CGMCC No.3242) Physiology and biochemistry character

(annotate: "+" expression positive in the table, "-" expression is negative.)

The cultural method of subtilis KR2 (CGMCC No.3242) is as follows:

(1) seed liquor preparation: carry out the preparation of subtilis KR2 (CGMCC No.3242) inclined-plane seed earlier, subtilis KR2 (CGMCC No.3242) is inoculated into behind the sterilized inclined-plane cultivated 3~7 days down at 20~35 ℃, scrape and get 3~5 ring bacterial classification inoculations in the triangular flask that sterilized liquid nutrient medium is housed, in temperature is 20~35 ℃, rotating speed is a shaking culture 30~120 hours under the condition of 100~300rpm, promptly can be used as subtilis KR2 (CGMCCNo.3242) first order seed.Under the same culture condition, first order seed is equipped with in the triangular flask of same substratum by second batch of the inoculum size switching of 3~10% (%:v/v), similarity condition is cultivated after 30~72 hours as subtilis KR2 (CGMCC No.3242) secondary seed down.

(2) enlarged culturing: subtilis KR2 (CGMCC No.3242) secondary seed is inoculated into the inoculum size of 3~10% (%:v/v) carries out fermentation culture in 7~300L fermentor tank, culture condition is: liquid amount: 60~80% (%:v/v), air flow: 0.1~1 (v/v.min), tank pressure: 0.02~0.05MPa, temperature: 20~35 ℃, rotating speed: 100~400rpm, fermentation time: 30~120 hours.With the fermented liquid in the fermentor tank in 3000rpm centrifugal 10 minutes, abandon supernatant after fermentation is finished, 3000rpm is centrifugal 10 minutes after the gained precipitate with deionized water flushing one time, abandons supernatant, must precipitate wet thallus.

Related medium component is as follows:

Slant medium (g/L): glucose: 10~30, yeast extract paste: 2~10, peptone: 2~10, sodium-chlor: 1~5, potassium primary phosphate: 0.5~3, dipotassium hydrogen phosphate: 0.5~3, sal epsom: 0.2~2, agar: 10~20, pH:5~9,121 ℃ sterilization 20min.

One-level, secondary liquid seed culture medium (g/L): glucose: 10~30, yeast extract paste: 2~10, peptone: 2~10, sodium-chlor: 1~5, potassium primary phosphate: 0.5~3, dipotassium hydrogen phosphate: 0.5~3, sal epsom: 0.2~2, hexanolactam: 0.5~10, isovaleronitrile: 0.1~5, cobalt chloride: 5~50ppm, pH:5~9.

Fermention medium (g/L): glucose: 10~50, yeast extract paste: 2~10, peptone: 2~10, sodium-chlor: 1~5, potassium primary phosphate: 0.5~3, dipotassium hydrogen phosphate: 0.5~3, sal epsom: 0.2~2, hexanolactam: 0.5~10, isovaleronitrile: 0.1~5, cobalt chloride: 5~50ppm, pH:5~9,121 ℃ sterilization 20min.

The application of subtilis (Bacillus subtilis) KR2 (CGMCC No.3242) in biocatalytic production of L-lactic acid.

Described subtilis KR2 (CGMCC No.3242) can be free cell, immobilized cell or flocculation cell, preferred immobilized cell or flocculation cell.

The method that a kind of subtilis KR2 (CGMCC No.3242) immobilized cell biological catalysis is produced L-lactic acid is:

The immobilized cell that obtains is joined in the phosphate buffered saline buffer of deionized water or pure water or pH7.0~8.0, the concentration of immobilized cell maintains 1~10% (w/v), temperature of reaction is controlled between 20~50 ℃, and stirring velocity is controlled between 50~500rpm.Reactive mode is batch formula reaction or successive reaction, and substrate can disposablely add in batch formula reaction, perhaps intermittently adds several times, and perhaps stream adds, and concentration of substrate is controlled at 10~50% (g/100ml).In the successive reaction, substrate can disposablely add, and perhaps intermittently adds several times, and perhaps stream adds.The add-on that remains substrate in this process makes concentration of substrate≤50% in the reaction solution, can finish reaction when concentration of substrate is lower than 30ppm, disposablely goes out to irritate.Reaction finishes to filter behind the static 10~30min of afterreaction liquid.The filtrate that filters out is earlier through the falling film type film evaporator processed, make the lactic acid concn in the filtrate not hang down 50%, it is 40~50 ℃ in preheating temperature then, pressure enters molecular distillation apparatus and carries out distillation dehydration under the condition of 0.1~1KPa, the distillation temperature of system is 50~80 ℃, vacuum tightness 10~100Pa, input speed 120~180mL/h, the rotating speed 100~130rpm of blade applicator rotor.

The preparation of the free cell literary composition " cultural method of subtilis KR2 (CGMCC No.3242) " that sees before wherein, tired herein stating.

Wherein, subtilis KR2 (CGMCC No.3242) immobilized cell can prepare as follows: the mass ratio of sodium alginate soln in 0.5~1.5% and 8~12% polyvinyl alcohol solution is 1: 8~15 ratio, in container, both are mixed and stir, prepare solid support material solution; Then subtilis KR2 (CGMCC No.3242) wet thallus is added in the solid support material solution, prepare the microorganism mixing solutions, wherein the mass ratio of subtilis KR2 (CGMCC No.3242) wet thallus and solid support material is 1: 4~6; With this microorganism mixed solution, be added dropwise to the pH value and be 6.0~7.0 mass concentrations and be in 1~5% the calcium chloride solution and carry out the first step curing reaction, be 20~120min set time; Take out the microorganism ball after the first step is solidified, use distilled water wash 2~3 times, put into saturated metabisulfite solution again and carry out the curing of second step, be 1~4 hour set time; Take out the microorganism ball after second step solidified, use distilled water wash 2~3 times, standby, more than operation all is at room temperature to carry out.

A kind of method of utilizing subtilis KR2 (CGMCC No.3242) flocculation cell to produce L-lactic acid as biological catalyst, this method is substrate with the lactonitrile, subtilis KR2 (CGMCCNo.3242) the flocculation enchylema that adds 5~15% (v/v) is biological catalyst, in 15~35 ℃, 100~300rpm reaction obtained L-lactic acid in 15~50 hours.

Wherein, the initial concentration of described substrate lactonitrile is 1~5% (w/v g/100ml), adopts fed-batch mode to make that the ultimate density of lactonitrile is no more than 30% in the reaction system.

Wherein, flocculation cell preparation method makes the bacteria suspension that wet thallus concentration is 1~200g/L for the wet thallus with gained stirs evenly after getting off with deionized water rinsing.At 5~35 ℃, (‰: the g/1000mL) flocculation agent of ratio, 200rpm stir 10min down and make flocculation enchylema to add 0.1~15 ‰ under the condition of pH5~9 in bacteria suspension.The flocculation agent that adds can be chitosan, chitin, and polyglutamic acid, polyacrylic acid, polyacrylamide, polymeric flocculants such as polymine, preferred chitosan, addition is preferably 0.5~5 ‰ (‰: g/1000mL).

Reaction finish the back in conversion fluid, add again liquid amount 0.1~0.5 ‰ (‰: flocculation agent g/1000mL), stir evenly, leave standstill 10~30min after-filtration.Filtering the back dewaters to such an extent that concentration is not less than 50% crude lactic acid through thin-film evaporator earlier, in molecular distillation apparatus, continue distillation dehydration then, system parameter is as follows: 50~80 ℃ of distillation temperatures, vacuum tightness 10~100Pa, input speed: 120~180mL/h, the rotating speed of blade applicator rotor: 100~130rpm.

% described in the present invention and ‰ is the quality volume percent as unexplained reference.

Beneficial effect of the present invention:

The present invention looks for another way, the employing lactonitrile is a raw material, utilizes the mode of subtilis KR2 (CGMCC No.3242) biocatalysis to produce L-lactic acid, and the product purity of gained lactic acid is more than 95%, and be single optically active L-lactic acid that has, the transformation efficiency of lactonitrile surpasses 98%.Method provided by the present invention both avoided pollute in the production of chemical method big, shortcomings such as optical purity of products difference, it is big to have overcome in the biological fermentation process sewage quantity again, product concentration is low, factors such as the high unfavorable purifying of impurity.Adopt flocculation cell and the catalytic mode of immobilized cell to protect cell to avoid the restraining effect of high concentration substrate and product; help industrial amplification production; the purifying process in downstream has been simplified in the use of cell or immobilized cell of flocculating simultaneously; the simple easy handling of sepn process; the product purity height; facility investment is few, effectively raises separation efficiency, has reduced production cost.The later stage purification phase adopts the molecular distillation method separating lactic acid, and processing step is few, and product purity is higher, and color and luster is good.

In sum, technological operation of the present invention is simple, the reaction conditions gentleness, and substrate utilization degree height, energy consumption is low, the product purity height, safety and environmental protection has broad application prospects.

The preservation of the biological material specimens that relates to

The present invention subtilis required for protection (Bacillus subtilis) KR2 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; the address is a Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica; preserving number is CGMCC NO.3242, and preservation date is on August 19th, 2009.

Embodiment

The preparation of embodiment 1 free cell

In aseptic Bechtop, scrape and get subtilis KR2 (CGMCC No.3242) slant strains 3 ring with transfering loop, be inoculated in the level liquid seed culture medium (glucose: 15, yeast extract paste: 5, peptone: 5, sodium-chlor: 2, potassium primary phosphate: 1, dipotassium hydrogen phosphate: 1, sal epsom: 0.5, hexanolactam: 5, isovaleronitrile: 1, cobalt chloride: 10ppm, pH:7.0, unit: g/L).In temperature is 25 ℃, rotating speed be under the 150rpm shaking culture got final product in 48 hours one-level subtilis KR2 (CGMCC No.3242) seed liquor, this primary seed solution is transferred to be equipped with proceeding in the sterilized substratum of sample ingredient according to 10% inoculum size cultivates, can get subtilis KR2 (CGMCC No.3242) secondary seed solution after 48 hours.

Subtilis KR2 (CGMCC No.3242) secondary seed solution is inoculated in the sterilized liquid fermentation medium of 35L according to 10% inoculum size, (glucose: 30, yeast extract paste: 10, peptone: 10, sodium-chlor: 2, potassium primary phosphate: 1, dipotassium hydrogen phosphate: 1, sal epsom: 0.5, isovaleronitrile: 1, hexanolactam: 5, cobalt chloride: 10ppm, pH:7, unit: g/L), the fermentor tank volume is 50L, adopt blowing air to control dissolved oxygen and be not less than 10% with the mode that stirs coupling, air flow: 0.2v/v.min, tank pressure: 0.03MPa, temperature: 25 ℃, initial rotating speed 100rpm, according to the adjusting in steps of dissolved oxygen of fermentation liquid level, maximum speed of revolution is no more than 400rpm in the fermenting process, ferments to make subtilis KR2 (CGMCC NO.3242) cell fermentation liquid after 72 hours.

The preparation of embodiment 2 immobilized cells

Sodium alginate soln in 1.1%: the mass ratio of 11% polyvinyl alcohol solution is 1: 10 a ratio, and in container, sodium alginate soln with 1.1% and 11% polyvinyl alcohol solution mix and stir, and prepare solid support material solution.Then subtilis KR2 (CGMCC NO.3242) wet thallus is added in the solid support material solution, prepare the microorganism mixing solutions.Subtilis KR2 (CGMCC NO.3242) wet thallus: the mass ratio of solid support material is 1: 5.To mass concentration is to be added dropwise to the microorganism mixed solution for preparing in 4% the calcium chloride solution (regulating the pH value with sodium hydroxide solution is 6.6), carries out the first step curing reaction of microorganism ball, and the curing reaction time is 0.5 hour.Microorganism ball after pulling the first step out and solidify with net with distilled water wash 3 times, is put into saturated metabisulfite solution earlier again, carries out the second step curing reaction, and the curing reaction time is 3 hours.Pull the second microorganism ball that goes on foot after solidifying out with net, use distilled water wash 2 times, standby.

The preparation of embodiment 3 flocculation enchylema

Get the centrifugal 10min of 3L subtilis KR2 (CGMCC NO.3242) fermented liquid 3000rpm and must precipitate thalline, continue the centrifugal 10min of 3000rpm, abandon supernatant and must precipitate wet thallus with behind the 3L deionized water rinsing one time.Subtilis KR2 (CGMCC NO.3242) precipitation wet thallus is weighed afterwards with deionized water it to be washed and is got off to make the bacteria suspension that concentration is 35g/L.30 ℃ of downward modulation bacteria suspension pH are 7, and (‰: the g/1000mL) chitosan of ratio, the 200rpm lower magnetic force stirs the flocculation enchylema that 10min can make subtilis KR2 (CGMCC NO.3242) to add 2 ‰ in bacteria suspension.

Embodiment 4

In the 50L stirring tank, concentration be housed in advance be 4% lactonitrile aqueous solution 30L, in temperature is 20 ℃, mixing speed is to wherein adding the 300g wet thallus under the 150rpm, the reaction 30min after gradually to the lactonitrile solution that wherein drips high density, finally make the cumulative concentration of substrate be no more than 18%, react after 38 hours that to detect concentration of substrate content be 25ppm, termination reaction.Blanking after static half an hour is emitted the back press filtration with reaction solution, and the gained cenobium continues cover and uses down in the batch reaction.Filtrate is used earlier the falling film type film evaporator processed, make the lactic acid concn in the filtrate be not less than 50%, be preheating to 45 ℃ then, pressure is to enter molecular distillation apparatus under the condition of 900Pa to carry out distillation dehydration, the distillation temperature of system is 50 ℃, vacuum tightness 100Pa, input speed: 180mL/h, the rotating speed of blade applicator rotor: 130rpm.Lactonitrile 2.5kg is added in test altogether by the gross, finally gathers in the crops purity and be 98.7% L-lactic acid 2.95kg, and yield reaches 91.9%.

Embodiment 5

In the 50L stirring tank, concentration be housed in advance be 4% lactonitrile aqueous solution 30L, be 35 ℃ in temperature, mixing speed is to wherein adding the 600g immobilized cell under the 150rpm.Gradually to the lactonitrile solution that wherein drips high density, the cumulative concentration that finally makes substrate is 40% behind the reaction 30min, and react after 40 hours detection concentration of substrate content is 26ppm, termination reaction.Static half an hour, blanking.The immobilized cell continuation cover that filtration obtains is used down in the batch reaction.Filtrate is used earlier the falling film type film evaporator processed, make the lactic acid concn in the filtrate be not less than 50%, be preheating to 45 ℃ then, pressure is to enter molecular distillation apparatus under the condition of 800Pa to carry out distillation dehydration, the distillation temperature of system is 70 ℃, vacuum tightness 50Pa, input speed: 150mL/h, the rotating speed of blade applicator rotor: 120rpm.Lactonitrile 4kg is added in test altogether by the gross, finally gathers in the crops purity and be 97.8% L-lactic acid 4.77kg, and yield reaches 92.0%.

The immobilized cell continuation cover that filtration obtains is used down in the batch reaction, fed intake by the above-mentioned parameter that provides and react and collection.Add lactonitrile 3.5kg altogether in the test by the gross, finally gather in the crops purity and be 97.3% L-lactic acid 4.23kg, yield reaches 92.8%.

Embodiment 6

In the 50L stirring tank, it is the lactonitrile aqueous solution 30L of 4% (w/v) that concentration is housed in advance, is 50 ℃ in temperature, and mixing speed is to wherein adding 3L flocculation cenobium (embodiment 3 preparations) under the 150rpm.Gradually to the lactonitrile solution that wherein drips high density, the concentration that finally makes substrate is 30% behind the reaction 30min, and react after 30 hours detection concentration of substrate content is 25ppm, termination reaction.As the chitosan that adds 0.02 ‰ in the reaction solution again, blanking after static half an hour behind the stirring 20min.Reaction solution is emitted the back press filtration, filtrate is used earlier the falling film type film evaporator processed, make the lactic acid concn in the filtrate be not less than 50%, be preheating to 45 ℃ then, pressure is to enter molecular distillation apparatus under the condition of 200Pa to carry out distillation dehydration, and the distillation temperature of system is 80 ℃, vacuum tightness 20Pa, input speed: 120mL/h, the rotating speed of blade applicator rotor: 100rpm.Add lactonitrile 3kg altogether in the test by the gross, finally gather in the crops purity and be 96.8% L-lactic acid 3.65kg, yield reaches 92.9%.

The part that the present invention does not relate to prior art that maybe can adopt all same as the prior art is realized.

Claims (10)

1. a subtilis (Bacillus subtilis) KR2 is preserved in Chinese microbial preservation management committee common micro-organisms center, and preserving number is CGMCC No.3242.
2. the application of the described subtilis CGMCC of claim 1 No.3242 in biological catalysis production L-lactic acid.
3. application according to claim 2, the method that it is characterized in that described subtilis CGMCC No.3242 biological catalysis production L-lactic acid is in the 0.2mol/L phosphate buffered saline buffer of deionized water or pure water or pH7.0~8.0, subtilis CGMCC No.3242 immobilized cell with 1~10% is a catalyzer, at 20~50 ℃, 50~500rpm catalytic substrate lactonitrile obtains L-lactic acid.
4. application according to claim 3, the concentration that it is characterized in that described substrate lactonitrile is 10~50%.
5. application according to claim 3, the preparation method who it is characterized in that described immobilized cell is: the mass ratio of sodium alginate soln in 0.5~1.5% and 8~12% polyvinyl alcohol solution is 1: 8~15 ratio, in container, both are mixed and stir, prepare solid support material solution; Then subtilis CGMCC No.3242 wet thallus is added in the solid support material solution, prepare the microorganism mixing solutions, wherein the mass ratio of subtilis CGMCC No.3242 wet thallus and solid support material is 1: 4~6; With this microorganism mixed solution, be added dropwise to the pH value and be 6.0~7.0 mass concentrations and be in 1~5% the calcium chloride solution, carry out the first step curing reaction, be 20~120min set time; Take out the microorganism ball after the first step is solidified, use distilled water wash 2~3 times, put into saturated metabisulfite solution again, carry out the curing of second step, be 1~4 hour set time; Take out the microorganism ball after second step solidified, use distilled water wash 2~3 times, standby.
6. application according to claim 3, it is characterized in that described reaction finishes to filter behind the static 10~30min of afterreaction liquid, the filtrate that filters out is earlier through the falling film type film evaporator processed, make the lactic acid concn in the filtrate be not less than 50%, it is 40~50 ℃ in preheating temperature then, pressure enters molecular distillation apparatus and carries out distillation dehydration under the condition of 0.1~1KPa, the distillation temperature of system is 50~80 ℃, vacuum tightness 10~100Pa, input speed: 120~180mL/h, the rotating speed of blade applicator rotor: 100~130rpm.
7. application according to claim 2, it is characterized in that method that described subtilis CGMCC No.3242 biological catalysis produces L-lactic acid is for being substrate with the lactonitrile, subtilis KR2 (CGMCC No.3242) the flocculation enchylema of interpolation 5~15% is biological catalyst, in 15~35 ℃, 100~300rpm reaction obtained L-lactic acid in 15~50 hours.
8. application according to claim 7, the initial concentration that it is characterized in that preparing described substrate lactonitrile is 1~5%, adopts fed-batch mode to make that the ultimate density of lactonitrile is no more than 30% in the reaction system.
9. application according to claim 7, it is characterized in that preparing the used flocculation agent of described flocculation cell is chitosan, polyglutamic acid, polyacrylic acid, polyacrylamide or polymine, preferred chitosan.The flocculation agent addition is 0.1~15 ‰.
10. the application of subtilis KR2 according to claim 7 (CGMCC No.3242) in the synthetic L-lactic acid of biological catalysis, it is characterized in that the flocculation agent that described reaction finishes to add 0.1~0.5 ‰ concentration earlier in the back flocculates again, filtering the back dewaters to such an extent that concentration is not less than 50% crude lactic acid through thin-film evaporator earlier, in molecular distillation apparatus, continue distillation dehydration then, system parameter is as follows: 50~80 ℃ of distillation temperatures, vacuum tightness 10~100Pa, input speed: 120~180mL/h, the rotating speed of blade applicator rotor: 100~130rpm.
CN2009102643033A 2009-12-18 2009-12-18 Bacillus subtilis and application thereof in biocatalytic production of L-lactic acid CN101724592B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN2009102643033A CN101724592B (en) 2009-12-18 2009-12-18 Bacillus subtilis and application thereof in biocatalytic production of L-lactic acid

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN2009102643033A CN101724592B (en) 2009-12-18 2009-12-18 Bacillus subtilis and application thereof in biocatalytic production of L-lactic acid

Publications (2)

Publication Number Publication Date
CN101724592A true CN101724592A (en) 2010-06-09
CN101724592B CN101724592B (en) 2012-06-27

Family

ID=42446147

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2009102643033A CN101724592B (en) 2009-12-18 2009-12-18 Bacillus subtilis and application thereof in biocatalytic production of L-lactic acid

Country Status (1)

Country Link
CN (1) CN101724592B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974458A (en) * 2010-09-30 2011-02-16 江南大学 Vacillus subtilis strain and application thereof
CN102628066A (en) * 2012-04-17 2012-08-08 山西大学 Preparation method and application of microbial flocculant
CN104450585A (en) * 2014-12-16 2015-03-25 四川省农业科学院农产品加工研究所 Bacillus subtilis for preparing L-lactic acid with high yield by taking corn steep liquor as only nitrogen source
CN107190029A (en) * 2016-03-14 2017-09-22 浦项工科大学校产学协力团 Use the preparation of the D type lactic acid of novel enzyme

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974458A (en) * 2010-09-30 2011-02-16 江南大学 Vacillus subtilis strain and application thereof
CN102628066A (en) * 2012-04-17 2012-08-08 山西大学 Preparation method and application of microbial flocculant
CN102628066B (en) * 2012-04-17 2013-11-20 山西大学 Preparation method and application of microbial flocculant
CN104450585A (en) * 2014-12-16 2015-03-25 四川省农业科学院农产品加工研究所 Bacillus subtilis for preparing L-lactic acid with high yield by taking corn steep liquor as only nitrogen source
CN104450585B (en) * 2014-12-16 2017-05-03 四川省农业科学院农产品加工研究所 Bacillus subtilis for preparing L-lactic acid with high yield by taking corn steep liquor as only nitrogen source
CN107190029A (en) * 2016-03-14 2017-09-22 浦项工科大学校产学协力团 Use the preparation of the D type lactic acid of novel enzyme

Also Published As

Publication number Publication date
CN101724592B (en) 2012-06-27

Similar Documents

Publication Publication Date Title
Islam et al. Strategies for cost-effective and enhanced production of bacterial cellulose
Argun et al. Bio-hydrogen production by different operational modes of dark and photo-fermentation: an overview
Tay et al. Production of L (+)‐lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor
CN102212476B (en) Method for cleanly and efficiently producing microorganism bactericide
CN100338221C (en) Preparation of lactic acid from a pentose-containing substrate
CN101864362B (en) Compound microbial bacterial preparation and application thereof
CN102080119B (en) Method for producing oil by mixed culture of yeast and alga
CN101109010B (en) Mycopremna generating gamma- polyglutamic acid and culturing method thereof
CN100473720C (en) Method for producing L-lactic acid and coagulate bacillus cereus special for the same
CN101792778B (en) Method for fermentation production of succinic acid by circulating utilization of recombinant Bacillus coli cells
CN101948785B (en) Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium
CN101240259B (en) Novel constructed high-yield fumaric acid gene engineering bacterium and method for producing fumaric acid thereby
CN103374534B (en) Yarrowia lipolytica strain and method thereof for synthesizing erythritol
CN102660487B (en) Geobacillus sp. UTM02 and application thereof
CN105154358B (en) A kind of method of bacillus and its simultaneous saccharification and fermentation production Pfansteihl
CN1807610A (en) Method for producing low temperature cellulase using microbe fermentation
CN101565683B (en) Method for preparing optically-pure R, R type 2, 3-butanedio by utilizing Bacillus polymyxa
CN102260729B (en) Bioflocculant fermentation method with mycelium pellet as vector
CN101285047B (en) D-lactic acid-producing strain with high optical purity and process for producing D-lactic acid by fermentation thereof
CN103421714B (en) Bacillus shackletonii and application thereof in fermentation production of polyhydroxybutyrate
CN103898004B (en) The method of Selective medium and fermentative production U-32070E thereof
CN102796673A (en) Feruloyl esterase production strain and method for producing feruloyl esterase by using same
CN103058476B (en) Novel treatment method for excess sludge
CN104694590B (en) The fermentation preparation of gamma-polyglutamic acid-and the bacterial strain of product gamma-polyglutamic acid-
CN101613667B (en) Method for preparing L-lactic acid and special bacteria agent thereof

Legal Events

Date Code Title Description
PB01 Publication
C06 Publication
SE01 Entry into force of request for substantive examination
C10 Entry into substantive examination
ASS Succession or assignment of patent right

Owner name: NANJING HUAZHOU PHARMACEUTICAL CO., LTD.

Free format text: FORMER OWNER: NANJING FIRST PESTICIDE GROUP CO., LTD.

Effective date: 20110922

TA01 Transfer of patent application right

Effective date of registration: 20110922

Address after: Dongfeng Road, Xi Zhen Gaochun County of Nanjing City, Jiangsu province 211308, No. 9

Applicant after: Nanjing Huazhou Pharmaceutical Co., Ltd.

Address before: 211300 Pagoda Road 269, Gaochun County, Jiangsu, Nanjing

Applicant before: Nanjing First Pesticide Group Co., Ltd.

C41 Transfer of patent application or patent right or utility model
COR Change of bibliographic data

Free format text: CORRECT: ADDRESS; FROM: 211300 NANJING, JIANGSU PROVINCE TO: 211308 NANJING, JIANGSU PROVINCE

C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20120627

Termination date: 20121218

C17 Cessation of patent right