CN101082052A - Acroleic acid production by biological catalysis - Google Patents

Abstract

The present invention discloses biologically catalytic process of producing acrylic acid. By using the microbe enzyme obtained through fermenting culture of Brevibacterium casei CGMCC No. 0887 and possessing nitrile hydrolyzing activity as the catalyst, acrylonitrile as the initial material is hydrolyzed in certain condition to obtain product acrylic acid. The process of the present invention has the features of mild reaction condition, simple technological path, less pollution and capacity of realizing continuous industrial production.

Description

Acroleic acid production by biological catalysis

Technical field

The invention belongs to technical field of biochemical industry, be specifically related to a kind of method of acroleic acid production by biological catalysis.

Background technology

Vinylformic acid is a kind of important organic raw material, the macromolecular materials such as multipolymer that are used to produce polyacrylic ester, polyacrylic acid and salt thereof or form with other monomers, these materials are widely used in fields such as synthetic resins, synthetic rubber, synthon and coating, latex, tackiness agent, tanning, papermaking, washing composition.

The suitability for industrialized production acrylic acid is a lot, but the most frequently used method mainly comprises: (1) acrylonitrile hydrolysis method, this method depends on chemical hydrolysis, under the catalysis of strong acid vinyl cyanide at first hydrolysis generate acrylamide, the latter further is hydrolyzed to vinylformic acid, and this method operational path is simple, and facility investment is less relatively, shortcoming is seriously polluted, the acid ammonium salt difficult treatment of by product.(2) PROPENE IN GAS PHASE OXIDATION method, propylene is oxidized to propenal under the metal catalyst effect, be oxidized to vinylformic acid again, because petroleum industry can provide cheap propylene in a large number, and acrylic acid yield height in the reaction, this method becomes most economical production acrylic acid at present, it also is the prefered method of scale operation, the throughput of this method accounts for more than 85% of world's overall throughput, but the shortcoming of this method is that investment is higher, need high temperature high voltage resistant equipment, particularly bring a lot of troubles for the purification of product owing to produce impurity such as acetate, formic acid.

Because the common severe reaction conditions of chemical method, environmental pollution is big, and side reaction is many, is difficult to satisfy the demand of market to the high-purity propylene acid product.Utilizing the biocatalysis method to produce its advantage of organic acid is the reaction conditions gentleness, and because the biological catalyst selectivity is higher, does not produce unwanted impurity usually.Utilize microorganism inherent cyan-hydrolysis enzyme catalysis acrylonitrile hydrolysis to produce ammonium acrylate, existing many pertinent literature report examples, as at Appl.Microbail.Biotechnol.1990, the Rhodococcus rhodochrous J1 Pseudomonas of describing among 34:322-324 and the United States Patent (USP) U.S.5135858; Disclosed Comamonas testosteroni 5-MGAM-4D Pseudomonas among the patent WO 03066872 (being equal to U.S.6,670,158); United States Patent (USP) U.S.6, disclosed Rhodococcusrhodochrous NCIMB 40757 or NCIMB 40833 Pseudomonas in 361,981 and U.S.6,162,624 and U.S.5,998,180; Journal of Shandong university (natural science edition) 1994,29 (2): the Pseudomonadaceae Pseudomonas of describing among the 217-223; J.Chem.Soc., Perkin Trans.1,1997, the Rhodococcus sp.AJ270 Pseudomonas of describing among the 1099-1104; Appl.Environ.Microbiol., 1976,31 (6), the Nocardia rhodochrous LL100-21 Pseudomonas of describing among the 900-906; J.Bacteriol., 1990,172 (9), the Rhodococcus Rhodochrous K22 Pseudomonas of describing among the 4807-4815.

The shown superiority that goes out of these enzymatic methods of conversion, for the instead of chemical method has been showed good prospect, but in above-mentioned these disclosed product cyan-hydrolysis enzyme microorganisms is not to have enzymatic activity high significantly, these reaction cumulative production concentrations are also not high, state of the art does not satisfy industrial production requirement, to such an extent as to can't be applied to industrial practice.

Summary of the invention

The objective of the invention is to overcome the above-mentioned deficiency of prior art, thereby a kind of method of acroleic acid production by biological catalysis is provided.

The present invention obtains nitrilase by fermentation culture brevibacterium casei (Brevibacterium casei) CGMCC No.0887 (this bacterial strain has been submitted China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation that is positioned at the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City on January 20th, 2003), hydrolysis reaction by nitrilase catalyzing propone nitrile obtains product vinylformic acid again, and reaction formula is as follows:

The specific implementation process of production acrylic acid of the present invention is as follows:

One, preparation biological catalyst nitrilase

(1) is that brevibacterium casei (Brevibacterium casei) bacterial strain of CGMCC No.0887 carries out shake-flask culture or the top fermentation jar is cultivated to preserving number, obtains fermented liquid;

Shake-flask culture: an amount of substratum (10-70%) of in triangular flask, packing into, a certain amount of bacterial classification (1-10%) is inserted in the sterilization back, under 20-40 ℃, cultivates 30-120 hour in the rotary shaking table (100-300rpm), obtains fermented liquid, and it is standby to remove agar.

Wherein, shake-flask culture base composition (%) comprising: glucose: 1-2.0; Yeast extract paste: 0.2-1; NaCl:0.05-0.15; K ₂HPO ₄: 0.05-0.3; MgSO ₄7H ₂O:0.01-0.3; Urea: 0.1-1, pH are 7.0-7.5.

Last jar of fermentation culture: an amount of seed culture medium (10-70%) of in seeding tank, packing into, a certain amount of bacterial classification (1-10%) is inserted in the sterilization back, at temperature 20-40 ℃, cultivates 30-120 hour under the condition of mixing speed 100-300rpm, obtain seed liquor, at 50L-20m ³Fermentor tank in an amount of fermention medium (40-70%) of packing into, the seed liquor of 2-7% is inserted in real jar of sterilization back, at air flow 1: 0.2-1: 1, mixing speed 50-400rpm, tank pressure 0.03-0.06MPa, under temperature 20-40 ℃ the condition, fermentation culture 30-200 hour, acquisition contained the fermented liquid of enzyme.

Wherein, last jar of fermentation culture composition comprises:

Seed culture medium is formed (%): glucose: 0.5-2.0; Yeast extract paste: 0.2-1; NaCl:0.05-0.15; K ₂HPO ₄: 0.05-0.3; MgSO ₄7H ₂O:0.01-0.3; Urea 0.1-1; PH7.0-7.5.

Fermention medium is formed and is comprised: glucose: 1.0-2.5; Yeast extract paste: 0.2-1; Urea 0.1-2; K ₂HPO ₄: 0.03-0.1; KH ₂PO ₄: 0.03-0.1; MgSO ₄7H ₂O:0.03-0.1; PH6.5-7.5.

(2) fermented liquid carries out the enzyme cell harvesting by centrifugal or membrane filtration system again, prepares biological catalyst then;

The enzyme cell harvesting: fermented liquid is centrifugal under the condition of separation factor 〉=5000, abandoning supernatant, and cell is centrifugal again after with deionized water wash, collects spissated wet cell liquid; Perhaps fermented liquid carries out the membrane sepn filtration with membrane filtration system, and adds the deionized water wash of 2 times of fermentating liquid volumes, collects spissated wet cell liquid, and membrane filtration system wherein can be hollow-fibre membrane or rolled film or ceramic membrane or ultra-filtration membrane.

Preparation biological catalyst: the containing the enzyme cell and can directly be used as catalyzer of collection; Or by being used as catalyzer after the immobilizations such as ordinary method such as diatomite adsorption; Or the cytoclasis liquid that cell is obtained after with fragmentations such as ordinary method such as ultrasonic wave is as catalyzer; Or the centrifugal enzyme clear liquid of removing behind the cell debris of cytoclasis liquid is directly as catalyzer; Maybe the zymoprotein that thick enzyme that will obtain through purifying or purity are higher is as catalyzer.

Two, biocatalysis hydrolysis reaction:

The biocatalysis hydrolysis reaction of the vinyl cyanide aqueous solution: in water or phosphoric acid buffer or water-organic medium or little water organic reaction medium, add biological catalyst, react after adding the substrate vinyl cyanide, pH is controlled between the 1.0-10.0, temperature of reaction is controlled between 0-70 ℃, and stirring velocity is controlled between the 50-500rpm.Reactive mode can be batch formula reaction or successive reaction, substrate can disposablely add in batch formula reaction, perhaps intermittently add several times, perhaps stream adds, the add-on that all must guarantee substrate at any time can not make concentration of substrate 〉=50% in the reaction solution, reaction times decides according to the particular case of reaction, reaction should guarantee that concentration of substrate is low as far as possible when finishing, this can prolong the reaction times by stopping reinforced back, or reaching this requirement by adding a small amount of biological catalyst reaction for some time, reaction finishes the back and separates by centrifugal or membrane filtration system and obtain reacting clear liquid; In successive reaction, substrate can disposablely add, perhaps intermittently add several times, perhaps stream adds, the add-on that all must guarantee substrate at any time can not make concentration of substrate 〉=50% in the reaction solution, when the production concentration accumulation reaches certain value 〉=10% (in vinylformic acid), and can be disposable by membrane filtration system, perhaps several times off and on, perhaps distribute the reaction clear liquid continuously, simultaneously can be disposable, perhaps intermittently several times, perhaps add reaction medium and substrate continuously, and according to the situation of reacting, can be disposable, perhaps intermittently several times, perhaps quantitatively add new biological catalyst continuously, can carry out continuously to guarantee reaction.

Embodiment

Further specified the present invention in order to illustrate better below by embodiment.

In the listed percentage concentration, except that indicating especially, be the quality concentration of volume percent in following examples.The starting material that below relate to are all commercially available obtaining.

Enzyme is lived and defined: the vinyl cyanide with a microgram in 1 hour is converted into the needed enzyme amount of vinylformic acid, is enzyme unit (U) alive.

The shake flask fermentation of embodiment 1, brevibacterium casei (Brevibacterium casei) CGMCC No.0887

Prepare shake flask fermentation culture medium A, B, C, D, E respectively, wherein the substratum composition comprises:

A: glucose 1.5%, yeast extract paste 0.5%, NaCl0.1%, K ₂HPO ₄0.05%, MgSO ₄7H ₂O0.05%, urea 0.5%, pH7.2;

B: glucose 1.8%, yeast extract paste 0.8%, NaCl0.15%, K ₂HPO ₄0.2%, MgSO ₄7H ₂O0.2%, urea 1%, pH7.4;

C: glucose 2.0%, yeast extract paste 1.0%, NaCl0.1%, K ₂HPO ₄0.30%, MgSO ₄7H ₂O0.01%, urea 0.2%, pH7.0;

D: glucose 1.0%, yeast extract paste 0.2%, NaCl0.15%, K ₂HPO ₄0.15%, MgSO ₄7H ₂O0.15%, urea 0.8%, pH7.5;

E: glucose 1.2%, yeast extract paste 0.4%, NaCl0.05%, K ₂HPO ₄0.1%, MgSO ₄7H ₂O0.3%, urea 0.1%, pH7.1;

Pour above prepared culture medium into 250mL shaking in the bottle with the 60mL portion respectively, 120 ℃ of autoclavings 20 minutes.Inoculate brevibacterium casei (Brevibacterium casei) CGMCC No.0887 after the cooling respectively in each substratum, in 28 ℃, 250rpm rotating speed concussion was down cultivated 72 hours.

After cultivating end, collect fermented liquid respectively, get 10ml, add 1mL vinyl cyanide (folding hundred), reaction is 10 minutes under the mixing speed of 40 ℃ and 200rpm, adds the 5M hydrochloric acid soln, termination reaction, reaction solution is measured acrylic acid amount with high pressure liquid chromatography quantitative analysis method HPLC (Tianjin, island LC-10A of company), calculates enzyme as follows and lives, and the results are shown in table 1.

C: production concentration, mg/mL (mg/ml);

V ₁: reaction solution volume, mL (milliliter);

V ₂: used fermentating liquid volume, mL (milliliter);

T: the reaction times, hr (hour).

The enzyme of table 1, shake flask fermentation liquid is lived

The shake flask fermentation substratum	Fermented liquid after shake flask fermentation finishes
			pH	Enzyme U/mL alive
	A	5.4			0.86×10 4
B			5.8	0.93×10 4

C	5.7	0.91×10 4
			D	5.5	0.78×10 4
E	6.0	1.01×10 4

Embodiment 2, brevibacterium casei (Brevibacterium casei) CGMCC No.0887 ferment tank

Prepare seed culture medium A, B, C, D respectively, wherein the substratum composition comprises:

A: glucose 2.0%, yeast extract paste 0.5%, NaCl0.05%, K ₂HPO ₄0.05%, MgSO ₄7H ₂O0.1%, urea 0.5%, pH7.2;

B: glucose 0.5%, yeast extract paste 0.2%, NaCl0.1%, K ₂HPO ₄0.25%, MgSO ₄7H ₂O0.01%, urea 0.1%, pH7.5;

C: glucose 1.5%, yeast extract paste 1.0%, NaCl0.15%, K ₂HPO ₄0.15%, MgSO ₄7H ₂O0.2%, urea 0.8%, pH7.0;

D: glucose 1.0%, yeast extract paste 0.8%, NaCl0.1%, K ₂HPO ₄0.3%, MgSO ₄7H ₂O0.3%, urea 1.0%, pH7.1.

Prepare fermention medium again, its composition (%) comprising: glucose: 1.8%; Yeast extract paste: 0.5%; Urea 0.5%; K ₂HPO ₄: 0.05%; KH ₂PO ₄: 0.05%; MgSO ₄7H ₂O:0.05%; PH7.0.

The seed culture medium 50% of in the 5L seeding tank, packing into, 1% bacterial classification is inserted in the sterilization back, 30 ℃ of temperature, cultivates 48 hours under the condition of mixing speed 200rpm, obtain seed liquor, the fermention medium 60% of in the fermentor tank of 50L, packing into, 8% seed liquor was inserted in real jar of sterilization back, air flow 1: 0.5, mixing speed 300rpm, tank pressure 0.05Mpa, under the condition that temperature is 30 ℃, fermentation culture 96 hours.

After fermentation culture finishes, collect fermented liquid respectively and measure enzyme (measuring method is with embodiment 1) alive, the results are shown in table 2.

The enzyme of table 2, ferment tank liquid is lived

Seed culture medium	After the last jar fermentation ends fermented liquid
			pH	Enzyme U/mL alive
	A	5.4			0.86×10 4
B			5.8	0.93×10 4
	C	5.7			0.91×10 4
D			5.5	0.78×10 4

Embodiment 3, brevibacterium casei (Brevibacterium casei) CGMCC No.0887 ferment tank

The preparation seed culture medium, it consists of and comprises: glucose 1.5%, yeast extract paste 0.5%, NaCl0.1%, K ₂HPO ₄0.05%, MgSO ₄7H ₂O0.05%, urea 0.5%, pH7.2;

Prepare fermention medium A, B, C, D more respectively, substratum is formed and is comprised:

A: glucose: 1.8%; Yeast extract paste: 0.5%; Urea 0.5%; K ₂HPO ₄: 0.05%; KH ₂PO ₄: 0.05%; MgSO ₄7H ₂O:0.1%; PH6.5;

B: glucose: 0.5%; Yeast extract paste: 0.2%; Urea 0.1%; K ₂HPO ₄: 0.03%; KH ₂PO ₄: 0.08%; MgSO ₄7H ₂O:0.08%; PH6.8;

C: glucose: 1.1%; Yeast extract paste: 0.8%; Urea 1.3%; K ₂HPO ₄: 0.08%; KH ₂PO ₄: 0.03%; MgSO ₄7H ₂O:0.05%; PH7.2;

D: glucose: 2.0%; Yeast extract paste: 1.0%; Urea 2.0%; K ₂HPO ₄: 0.1%; KH ₂PO ₄: 0.1%; MgSO ₄7H ₂O:0.03%; PH7.5;

The seed culture medium 40% of in the 5L seeding tank, packing into, 1.5% bacterial classification is inserted in the sterilization back, 28 ℃ of temperature, cultivates 40 hours under the condition of mixing speed 250rpm, obtain seed liquor, in the fermentor tank of 50L, be respectively charged into fermention medium A, B, C, D each 50%, 6% seed liquor was inserted in real jar of sterilization back, air flow 1: 0.6, mixing speed 250rpm, tank pressure 0.04Mpa, under the condition that temperature is 28 ℃, fermentation culture 100 hours.

After fermentation culture finishes, collect fermented liquid respectively and measure enzyme (measuring method is with embodiment 1) alive, the results are shown in table 3.

The enzyme of table 3, ferment tank liquid is lived

Fermention medium	Fermented liquid after ferment tank finishes
			pH	Enzyme U/mL alive
	A	5.4			0.86×10 4
B			5.8	0.93×10 4
	C	5.7			0.91×10 4
D			5.5	0.78×10 4

Embodiment 4, fermented liquid is centrifugal or membrane filtration

Get the fermented liquid 2L that embodiment 2 and 3 obtains respectively, centrifugal with low-temperature and high-speed, frozen centrifugation 15 minutes, tubular fibre membrane filtration (flow 180mL/min, pressure 0.8Kg/cm under 6 ℃ and 15000rpm rotating speed ²), rolled film filters (flow 180mL/min, pressure 16Kg/cm ²), ceramic membrane filter (flow 180mL/min, pressure 4Kg/cm ²) or flat plate ultrafiltration membrane (Ultra-fllo) filtration (flow 180mL/min, pressure 3Kg/cm ²).Use 4L deionized water wash cell then respectively, collect concentrating cells liquid, get an amount of concentrating cells liquid and return to the original fermented solution volume, measure enzyme and live (measuring method is with embodiment 1), the results are shown in table 4 with the deionized water dilution.

The enzyme of table 4, processing secondary fermentation liquid is lived

The fermentation liquor treatment mode	Fermented liquid	After the processing	Enzyme rate of loss % alive
				Enzyme U/mL alive	Enzyme U/mL alive
	Low-temperature and high-speed is centrifugal	0.93×10 4				0.86×10 4	7.5
The tubular fibre membrane filtration			0.93×10 4	0.92×10 4	1.1

Rolled film filters	0.93×10 4	0.89×10 4	4.3
				Ceramic membrane filter	0.93×10 4	0.88×10 4	5.4
Flat plate ultrafiltration membrane filters (Ultra-flo)	0.93×10 4	0.85×10 4	8.6

Embodiment 5, biological catalyst preparation

Get the fermented liquid that embodiment 2 obtains, press the tubular fibre membrane filtering method of embodiment 4,4 times of washing concentrating, the concentrating cells that obtains is further handled by following method, obtains the multi-form biological catalyst with nitrilase activity:

1., after the sodium alginate of getting concentrating cells and 3.2% mixes, be injected into the CaCl of 0.2M ₂Carry out the gelation embedding in the solution, make immobilized cell.

2., get concentrating cells with the ultrasonic cell-break device with cytoclasis, 8 ℃ of condition 800W, temperature, each fragmentation volume 100mL, accumulative total ultrasonic time 1 hour obtains cytoclasis liquid.

3., the cytoclasis liquid got 2. is centrifugal down in 15000g, 6 ℃, collects supernatant liquor, obtains acellular enzyme liquid.

4., get 3. supernatant liquor, saltouing through 10%, 20%, 30%, 40%, 50%, 60% and 70% ammoniumsulphate soln respectively, collect the precipitation component under the 20-50% saturation ratio, place the 10mM potassium phosphate buffer of pH7.0 to dialyse 24 hours, and then in pure water, dialysed 24 hours, obtain crude enzyme liquid, promptly obtain thick enzyme powder through after the vacuum freezedrying.

5., the thick enzyme powder of getting is 4. handled with the DEAE-Sephadex post of crossing through the 10mM potassium phosphate buffer pre-equilibration of pH7.0, use the 10mM potassium phosphate buffer wash-out of the pH7.0 that contains 0.1M, 0.2M, 0.3M, 0.4M, 0.5M and 0.6M Repone K respectively, collection has the component of nitrilase activity, abundant dialysis desalting in pure water, promptly get the higher relatively enzyme solution of purity, promptly obtain the enzyme powder through after the vacuum freezedrying.

With deionized water above various multi-form biological catalysts are returned to initial fermentating liquid volume, measure enzyme and live (measuring method is with embodiment 1), the results are shown in table 5.

The enzyme of table 5, multi-form biological catalyst is lived

Catalyst mode	Fermented liquid	Enchylema	The fixation cell cytosol	Cytoclasis liquid	Acellular enzyme liquid	Crude enzyme liquid	Enzyme liquid
								Enzyme lives (* 10 4U/mL)	0.95	0.93	0.81	0.89	0.85	0.37	0.13

Embodiment 6, cytoclasis liquid catalytic activity

Get the cytoclasis liquid that embodiment 5 obtains, with deionized water cytoclasis liquid is returned to initial fermentating liquid volume, get 50mL respectively, each adds 2% (v/v) substrate vinyl cyanide, and [by volume 50mL counts, promptly returned to and added 1mL vinyl cyanide (folding hundred) in the aforementioned solution of initial fermentating liquid volume] by every 50mL, in differing temps, react under the same rotational speed of 200rpm, reacted 0.5 hour, with high pressure liquid chromatography (HPLC) quantitative analysis method (Tianjin, island LC-10A of company, down together) substrate vinyl cyanide and the acrylic acid amount of product in the assaying reaction liquid are the volumetric molar concentration that 50mL calculates substrate and product with the reaction solution volume then, the results are shown in table 6.

The catalytic activity of table 6, cytoclasis liquid

Temperature (℃)	Analytical results (mM)
			Vinyl cyanide	Vinylformic acid
	0	265.4			101.6
10			123.8	234.6
	20	33.7			337.4
30			19.1	344.6
	40	11.9			351.7
50			10.7	317.8
	60	140.7			153.7
70			261.7	43.5

Embodiment 7, the enzyme powder catalytic activity

Get the enzyme powder that embodiment 5 obtains, get 10mg respectively, damping fluid with different pH returns to initial fermentating liquid volume, get 20mL, [by volume 20mL counts the substrate vinyl cyanide of adding 2% (v/v), promptly returned to and added 0.4mL vinyl cyanide (folding hundred) in the aforementioned solution of initial fermentating liquid volume] by every 20mL, under 30 ℃ and 200rpm condition, react, reaction 20min, HPLC stratographic analysis reaction result is (with embodiment 6, with the reaction solution volume is the volumetric molar concentration that 20mL calculates substrate and product), the results are shown in table 7.

The catalytic activity of table 7, enzyme powder

pH	Analytical results (mM)
			Vinyl cyanide	Vinylformic acid
	2.0	350.8			14.6
3.0			309.4	57.9
	5.0	97.7			268.3
6.0			23.3	334.9
	7.0	9.4			352.4
8.0			9.3	349.8
	10.0	34.2			318.6

Embodiment 8, concentrating cells liquid catalytic activity

Get the concentrating cells liquid that embodiment 4 obtains, with deionized water enchylema is returned to initial fermentating liquid volume, get 50mL respectively, [by volume 50mL counts to add the different substrate vinyl cyanide of measuring, promptly returned in the aforementioned solution of initial fermentating liquid volume and added 0.05mL respectively by every 50mL, 0.25mL, 0.5mL, 2.5mL, 5mL, 15mL, the vinyl cyanide of 20mL and 25mL (folding hundred), the concentration of substrate (v/v) of 50mL calculating so by volume is respectively 0.1%, 0.5%, 1%, 5%, 10%, 30%, 40% and 50%], under 30 ℃ of bath temperatures and 200rpm mixing speed condition, react, reacted 0.5 hour, HPLC stratographic analysis reaction result is (with embodiment 6, with the reaction solution volume is the volumetric molar concentration that 50mL calculates substrate and product), list in table 8.

The catalytic activity of table 8, concentrating cells liquid

Concentration of substrate (v/v, %)	Analytical results (mM)
			Vinyl cyanide	Vinylformic acid
	0.1	Do not have			18.2
0.5			Do not have	91.3
	1.0	Do not have			181.0
5.0			63.8	811.6
	10.0	364.8			1286.7
30.0			3487.6	1542.3
	40.0	5496.7			917.5
50.0			8109.3	618.4

Embodiment 9, the enzyme powder catalytic activity

Get the enzyme powder that embodiment 5 obtains, return to initial fermentating liquid volume with reaction medium not, get 50mL, [by volume 50mL counts to add 2% (v/v) substrate vinyl cyanide respectively, promptly returned to and added 1mL vinyl cyanide (folding hundred) in the aforementioned solution of initial fermentating liquid volume] by every 50mL, under 30 ℃ of bath temperatures and 200rpm mixing speed condition, react, reacted 0.5 hour, HPLC stratographic analysis reaction result is (with embodiment 6, with the reaction solution volume is the volumetric molar concentration that 50mL calculates substrate and product), the results are shown in table 9.

The catalytic activity of table 9, enzyme powder

Reaction medium	Analytical results (mM)
			Vinyl cyanide	Vinylformic acid
	Water	9.7			349.6
Phosphoric acid buffer (pH6.8)			6.3	353.4
	Water-methanol	11.3			348.2

Normal hexane	54.2	313.7
			Water-ethanol	11.4	344.6
Water-acetone	17.8	326.9

Embodiment 10, concentrating cells liquid catalytic activity

Get the concentrating cells liquid that embodiment 4 obtains, with deionized water enchylema is returned to initial fermentating liquid volume, get 1.5L in three mouthfuls of reaction flasks of 2L, [by volume 1.5L counts the vinyl cyanide of adding 0.5%, promptly returned to and added 7.5 gram vinyl cyanide (folding hundred) in the aforementioned solution of initial fermentating liquid volume] by every 1.5L, under 28 ℃ of bath temperatures and 150rpm mixing speed condition, react, intermittently add substrate, promptly analyze by the HPLC chromatogram tracking, when concentration of substrate was lower than 0.1%, the amount by 0.5% added substrate with aforementioned manner; Reacted 68 hours, it is 55 times that substrate accumulative total adds indegree, add 412.5g altogether, about 825mL, reaction back HPLC stratographic analysis reaction result (, being the volumetric molar concentration that 1.5L calculates substrate and product) with the reaction solution volume with embodiment 6, substrate acrylonitrile residue concentration＜0.05%, reaction conversion ratio＞99.0%, end product concentration 3.13M, acrylic acid yield 98.0%.

Embodiment 11, concentrating cells liquid catalytic activity

Get the concentrating cells liquid that embodiment 4 obtains, with deionized water enchylema is returned to initial fermentating liquid volume, get 2.0L in three mouthfuls of reaction flasks of 3L, pump into vinyl cyanide with peristaltic pump, the about 4-6mL/min of stream rate of acceleration, under 30 ℃ of bath temperatures and 200rpm mixing speed condition, react, the HPLC chromatogram tracking is analyzed, regulate the stream rate of acceleration according to the height of concentration of substrate, guarantee concentration of substrate≤0.5%[promptly by the amount of vinyl cyanide in the reaction solution of every 2.0L (folding hundred) smaller or equal to 10 grams, when the product cumulative concentration reaches the 3.0M left and right sides, stop to feed in raw material down together],, continuing reaction makes base consumption intact, termination reaction when substrate residual concentration＜0.05%, 56 hours entire reaction course time, HPLC stratographic analysis reaction result in reaction back is (with embodiment 6, with the reaction solution volume is the volumetric molar concentration that 2.0L calculates product), transformation efficiency＞99.0%, end product concentration 3.47M, acrylic acid yield＞98.0%.

Embodiment 12, the industrialization continuous production

The preparation seed culture medium, its composition: glucose 1.5%, yeast extract paste 0.5%, NaCl0.1%, K ₂HPO ₄0.05%, MgSO ₄7H ₂O0.05%, urea 0.5%, pH7.2.

Prepare fermention medium again, its composition (%): glucose 1.5%, yeast extract paste 0.5%, NaCl0.1%, K ₂HPO ₄0.05%, MgSO ₄7H ₂O0.05%, urea 0.5%, pH7.2.

The seed culture medium 50% of packing in the 5L seeding tank, 1% bacterial classification is inserted in the sterilization back, 30 ℃ of temperature, cultivates 48 hours under the condition of mixing speed 200rpm, obtains seed liquor, at 50L ³Fermentor tank in the fermention medium 60% of packing into, 8% seed liquor is inserted in real jar of sterilization back, air flow 1: 0.5, mixing speed 300rpm, tank pressure 0.05Mpa, under the condition that temperature is 30 ℃, fermentation culture 96 hours.

After fermentation culture finishes, collect fermented liquid respectively, filter with flat plate ultrafiltration membrane, the twice deionized water wash, collect concentrating cells, deionized water is diluted to original volume, adds enchylema 6L, 30 ℃ of jacket water (J.W.) bath temperatures then in the reactor of the 10L that has membrane separation apparatus, mixing speed 300rpm, pump into acrylonitrile solution with peristaltic pump, the about 12-18mL/min of stream rate of acceleration, the HPLC chromatogram tracking is analyzed, regulate the stream rate of acceleration according to the height of concentration of substrate, guarantee concentration of substrate (in reaction solution volume 6L) about 0.5%, when the product cumulative concentration reaches about 15% (in reaction solution volume 6L) left and right sides, stop to feed in raw material, continuation reaction for some time makes base consumption intact, when concentration of substrate＜0.05%, leach the reaction clear liquid by membrane filtration system, add deionized water to reactor then, again current adding substrate, continue reaction, and add concentrating cells at any time according to response situation, so successive reaction constantly obtains containing the acrylic acid reaction clear liquid of product.

The present invention adopts the method for biocatalysis to produce vinylformic acid, utilize nitrilase that vinyl cyanide is converted into corresponding acid, but its operating process is simple, reaction conditions is gentle, the little industrialization continuous production of environmental pollution.

The present invention compares with existing method, have obvious characteristics and progress: the new bacterial strain of bacterium producing multi enzyme preparation for obtaining from the natural source screening, now be accredited as brevibacterium casei (Brevibacterium casei), its preserving number is CGMCC No.0887, through long-term breeding work, the fermentation broth enzyme vigor has reached 100,000 U/mL; In Catalyst Production, introduce membrane separation technique, production can be carried out in serialization; Good reaction selectivity, raw material availability are high, environmental pollution is little, production cost is low; The product cumulative concentration is high, but industrializing implementation production.

Claims (8)

1, a kind of method of acroleic acid production by biological catalysis, comprising by nitrilase comes the hydrolysis reaction of catalyzing propone nitrile to obtain vinylformic acid, it is characterized in that described nitrilase obtains by fermentation culture brevibacterium casei (Brevibacteriumcasei) CGMCC No.0887.

2, the method for claim 1 is characterized in that, the fermentation culture conditions of this brevibacterium casei is as follows:

Seed culture medium is formed: glucose: 0.5-2.0wt%, yeast extract paste: 0.2-1wt%, NaCl:0.05-0.15wt%, K ₂HPO ₄: 0.05-0.3wt%, MgSO ₄7H ₂O:0.01-0.3wt%, urea 0.1-1, pH7.0-7.5;

Fermention medium is formed: glucose: 1.0-2.5wt%, yeast extract paste: 0.2-1wt%, urea 0.1-2wt%, K ₂HPO ₄: 0.03-0.1wt%, KH ₂PO ₄: 0.03-0.1wt%, MgSO ₄7H ₂O:0.03-0.1wt%, pH6.5-7.5;

Fermentation condition: air flow 1: 0.2-1: 1, mixing speed 50-400rpm, tank pressure 0.03-0.06MPa, temperature 20-40 ℃, time: 30-200 hour.

3, the method for claim 1 is characterized in that, described nitrilase is the fermented liquid that fermentation culture obtains to be carried out the enzyme cell harvesting by centrifugal or membrane filtration system obtain.

4, method as claimed in claim 3 is characterized in that, described membrane filtration system includes but not limited to: hollow-fibre membrane or rolled film or ceramic membrane or ultra-filtration membrane.

5, the method for claim 1 is characterized in that, the reaction medium of described hydrolysis reaction includes but not limited to: water or damping fluid or water-organic medium or little aqueous organic media.

6, method as claimed in claim 5 is characterized in that, the damping fluid in the described reaction medium includes but not limited to phosphoric acid buffer, and organic medium includes but not limited to: methyl alcohol or normal hexane or ethanol or acetone.

7, the method for claim 1 is characterized in that, the condition of described hydrolysis reaction is as follows: temperature is 0-70 ℃, and pH is 1-10, and the substrate acrylonitrile concentration is controlled at 0-50wt%.

8, a kind of brevibacterium casei (Brevibacterium casei) that is used for acroleic acid production by biological catalysis is characterized in that preserving number is CGMCC No.0887.