CN102268398B - Pseudomonas putida, catalytic preparation of iminodiacetic acid from iminodiacetonitrile - Google Patents

Pseudomonas putida, catalytic preparation of iminodiacetic acid from iminodiacetonitrile Download PDF

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CN102268398B
CN102268398B CN 201110224760 CN201110224760A CN102268398B CN 102268398 B CN102268398 B CN 102268398B CN 201110224760 CN201110224760 CN 201110224760 CN 201110224760 A CN201110224760 A CN 201110224760A CN 102268398 B CN102268398 B CN 102268398B
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iminodiacetonitrile
pseudomonas
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pseudomonas putida
nitrilase
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CN102268398A (en
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郑裕国
柳志强
张涛
徐建妙
薛亚平
郑仁朝
沈寅初
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Zhejiang University of Technology ZJUT
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Abstract

The invention relates to Pseudomonas putida, and catalytic preparation of iminodiacetic acid from iminodiacetonitrile. The invention provides a method and strain for preparing iminodiacetic acid from iminodiacetonitrile under catalytic actions of microbes. The method comprises steps as follows: a strain capable of producing nitrilase is subjected to enzyme producing culture to obtain the nitrilase, and the nitrilase is used as a biocatalyst for biologically catalyzing iminodiacetonitrile toprepare iminodiacetic acid. The invention lays foundation for producing iminodiacetic acid by biologically catalyzing iminodiacetonitrile, and has important application prospects.

Description

Pseudomonas putida and preparing iminodiacetic acid by catalyzing iminodiacetonitrile thereof
(1) technical field
The present invention relates to a kind of method of producing the preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes of nitrilase, and the bacterial strain with nitrilase activity of screening acquisition.
(2) background technology
Iminodiethanoic acid (IDA) is a kind of important industrial chemicals, there is very strong complex ability energy and many kinds of metal ions complexing and form inner complex, can form blue huge legendary turtle compound with cupric ion, be commonly used for complexing agent and tensio-active agent, being usually used in organic synthesis, is also the important intermediate that glyphosate (Glyphosate) is produced.Owing to containing imino-and carboxyl in its molecule, chemical property is very active, is widely used in the fields such as plating, dyestuff, medicine, electronics.It is a kind of important chemical intermediate.
Prepare at present iminodiethanoic acid and generally adopt chemical process, according to the difference of raw material, mainly contain chloroactic acid method, nitrilotriacetic acid method, ammonia for chloroactic acid method, prussic acid method, monoethanolamine process, diethanolamine method etc.Traditional chemical process exists the generation cost high, seriously polluted, high to equipment requirements, or uses the hypertoxic weakness such as prussic acid.Utilize the biocatalysis iminodiacetonitrile to produce iminodiethanoic acid and compare with chemical hydrolysis, it is advantageous that: (1) reaction conditions gentleness.Chemical hydrolysis need to carry out 100 ℃ of left and right, must be equipped with heating and temperature control unit, severe reaction conditions, and energy consumption is high and high to equipment requirements.And biological catalysis is all generally to carry out at normal temperatures, the reaction conditions gentleness, equipment cost is relatively low.(2) raw material consumption significantly reduces, and does not substantially produce soluble salt.Utilize nitrilase one one-step hydrolysis to generate iminodiethanoic acid, saved alkaline hydrolysis and acidification technique, avoided a large amount of uses of NaOH and concentrated hydrochloric acid.Utilize NH in biological process 4oH regulates the pH value of transformation system, in product reclaims, adopts regeneration techniques, can effectively reduce the discharge of soluble salt.(3) discharge of wastewater is few.According to current chemical hydrolysis process, produce iminodiethanoic acid per ton and will at least produce 8 tons of waste water.After adopting biological process, with the nitrilase enzyme activity, 100,000 units calculate (reuse 5 times), and the wastewater flow rate of producing iminodiethanoic acid per ton can be controlled in below 2 tons, and do not have the difficult impurity such as salt in waste water, and biodegradability is strong.
Utilize nitrile invertase biocatalysis iminodiacetonitrile to prepare iminodiethanoic acid, can overcome the defect that chemical method is produced iminodiethanoic acid.Biocatalysis be up to now known to the mankind the most efficiently, tool catalyst system optionally, can provide many traditional chemical methods can not or to be difficult for the synthetic method of the synthetic chipal compounds obtained, demonstrate good chemo-selective, regioselectivity and stereoselectivity.And bioconversion reaction mild condition, reaction product purity be high, easily separate and purifying, in the production of pesticide intermediate, demonstrate huge development potentiality.
(3) summary of the invention
The present invention seeks to the cultivation of production by biological enzyme and obtain the preparation method that nitrilase catalysis iminodiacetonitrile prepares iminodiethanoic acid.The present invention another purpose be to provide the microorganism of the product nitrilase that obtains of screening.
The technical solution used in the present invention is:
Pseudomonas putida (Pseudomonas putia) ZJB09135, be preserved in Chinese Typical Representative culture collection center, address: China. Wuhan. and Wuhan University, 430072, deposit number CCTCC No:M 209055, preservation date on March 18th, 2009.
Above-mentioned bacterial strains is separated and obtains from soil by bio-engineering research institute of Zhejiang Polytechnical University.
The invention still further relates to the method for utilizing aforementioned strain microorganism preparing iminodiacetic acid by catalyzing iminodiacetonitrile, described method is to utilize the bacterial strain that produces nitrilase, the multiparity enzyme is cultivated and to be obtained nitrilase, take this enzyme to prepare iminodiethanoic acid as biological catalyst iminodiacetonitrile.The process that the biocatalysis iminodiacetonitrile is produced iminodiethanoic acid is as follows:
Figure BDA0000081435860000031
The method for preparing iminodiethanoic acid of the present invention is specific as follows:
(A) described pseudomonas putida CCTCC No:M 209055 is seeded to culture medium, adds inductor a to carry out fermentation culture 2～5d under 100～200r/min, 20～35 ℃ of conditions; Described inductor a is n-Butyronitrile, isopropyl cyanide or hexanolactam; Described culture medium final concentration consists of: glycerine 8g/L, yeast extract paste 6g/L, NaCl 1g/L, K 2hPO 45g/L, MgSO 40.2g/L solvent is water, pH value 7.0; The final concentration of described inductor a is 5g/L;
(B) get the aqueous solution that iminodiacetonitrile is mixed with mass concentration 0.5%～6%, adjusting initial pH is 7.0～7.5, as the enzymic catalytic reaction substrate solution; The crude enzyme liquid that the wet thallus that the step (A) of usining fermentation obtains, cytoclasis obtain or the immobilized cell obtained through immobilization are counted 0.1～10g wet thallus/10mL substrate solution as ，Mei source, enzyme source add-on with wet thallus; Controlling temperature of reaction and be 30 ℃, reaction pH is 6.0～9.0, carries out conversion reaction 8～16h, and after reaction finishes, reaction solution obtains iminodiethanoic acid through separation and purification.
The described fermentation culture of above-mentioned steps (A) is preferably carried out fermentation culture 3d under 30 ℃ of conditions; The described control temperature of reaction of step (B) is preferably 30 ℃, carries out conversion reaction 10h.
The microorganism of product nitrilase of the present invention is screened acquisition by the following method, and described method comprises primary dcreening operation and multiple sieve:
(1) primary dcreening operation: inoculation to be screened, to the primary dcreening operation substratum, is cultivated to 1～4d for 25～37 ℃, produce the bacterial strain of yellow variable color circle in picking colony, be inoculated into the LB slant medium, get bacterial strain after 25～30 ℃ of cultivation 2～5d and carry out multiple sieve; Described primary dcreening operation substratum final concentration is composed as follows: imido grpup diacetonitrile 5g/L, yeast extract paste 6g/L, NaCl1g/L, K 2hPO 41g/L, MgSO 41g/L, bromine potassium phenol violet 1g/L, agar powder 20g/L, solvent is water, pH 7.0; Be that the every 1L of described primary dcreening operation substratum prepares by following composition: imido grpup diacetonitrile 5g, yeast extract paste 6g, NaCl 1g, K 2hPO 41g, MgSO 41g, bromine potassium phenol violet 0.1g, agar powder 20g, solvent is water, pH 7.0;
28～30 ℃ of the described culture temperature of preferred steps (1) primary dcreening operation, cultivate 3～4d.
(2) multiple sieve: the inoculation that primary dcreening operation is obtained is to sieving again substratum, under 100～200r/min, 25～35 ℃, cultivates 2～7d, collects the growing amount that thalline is measured iminodiethanoic acid, collects and generates the microorganism that the iminodiacetic acid (salt) acid content is greater than 10%; Described to sieve again the substratum final concentration composed as follows: glycerine 8g/L, yeast extract paste 6g/L, NaCl 1g/L, K 2hPO 45g/L, MgSO 40.2g/L, n-Butyronitrile 5g, solvent is water, and pH 7.0, and the described every 1L of substratum that sieves again prepares by following composition: glycerine 8g, yeast extract paste 6g, NaCl 1g, K 2hPO 45g, MgSO 40.2g, inductor b 5g, solvent is water, pH 7.0.
It is 30～32 ℃ that preferred steps (2) is sieved described culture temperature again.
What inductor a of the present invention and inductor b were represented is all the inductor in microbial cultivation process, and a and b just appear at different step in order to distinguish inductor here.
Beneficial effect of the present invention is mainly reflected in: provide a kind of bacterial strain multiparity enzyme that produces nitrilase that utilizes to cultivate, obtain nitrilase, take this enzyme prepares the method for iminodiethanoic acid as biological catalyst iminodiacetonitrile, but High-efficient Production iminodiethanoic acid, the present invention also provides the screening method that can produce the microorganism of nitrilase, and obtained the bacterial strain of multiple product nitrilase, provide the foundation for adopting the biocatalysis iminodiacetonitrile to produce iminodiethanoic acid, there is the important application prospect.
(4) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the primary dcreening operation that produces the nitrilase bacterial classification
The every 1L of primary dcreening operation substratum prepared by following the composition: yeast extract paste 6g, NaCl 1g, K 2hPO 41g, MgSO 41g, agar powder 20g, pH 7.0 water complement to 1000mL, after sterilizing, are cooled to 50 ℃ to add 5g iminodiacetonitrile and 1g purpurum bromocresolis, mix flat board.
Collect bacterium and screened, coat flat board upper, 28～30 ℃ of temperature, cultivate 3～4d, produce the bacterial strain of yellow variable color circle in picking colony, be inoculated into LB slant culture (25～28 ℃);
The microorganism strains of yellow variable color circle has: edge pseudomonas ZJB09121 (Pseudomonas marginalis ZJB09121), pseudomonas putida ZJB09134 (Pseudomonas putia ZJB09134), pseudomonas putida ZJB09129 (Pseudomonas putia ZJB09129), pseudomonas putida ZJB09135 (Pseudomonas putia ZJB09135) (being CCTCC No:M 209055), Pseudomonas fluorescens ZJB09137 (Pseudomonas fluorescens ZJB09137), acid-producing Klebsiella bacterium ZJB09123 (Kelbsiella ocytoca ZJB09123), Fu Shi rhodococcus ZJB09124 (Rhodococcus wratislaviensis ZJB09124), have a liking for pyridine rhodococcus ZJB09126 (Rhodococcus pyridinivorans ZJB09126), prunosus red coccus ZJB09125 (Rhodococcus rhodochrous ZJB09125), Rhodococcus ruber ZJB09127 (Rhodococcus rubber ZJB09127), micrococcus luteus ZJB09131 (Micrococcus luteus ZJB09131), brevibacterium halotolerans ZJB09132 (Brevibacterium halotoerans ZJB09132), quick acidovorax facilis ZJB09122 (Acidovorax facilis ZJB09122), Bacillus foecalis alkaligenes ZJB09138 (Alcaligenes faecalis ZJB09138), Arthrobacter (Arthrobacter mtroguajacolicus) ZJUTB06-99.
Embodiment 2: the multiple sieve that produces the nitrilase bacterial classification
By produced metachromasia and the large bacterial strain of yellow variable color circle screened arranged in embodiment 1, be inoculated in multiple sieve substratum 30～32 ℃, 100～200r/min, 500mL triangular flask liquid amount 150mL, fermentation culture 3d, centrifugal crude enzyme liquid or the wet thallus of obtaining of fermented liquid.Respectively get the 1g wet thallus, be placed in the 500ml triangular flask, add the reaction substrate iminodiacetonitrile aqueous solution of 50ml 2% (w/w), react 60min under 30 ℃ of shaking table 150r/min conditions, centrifugal removal thalline, the growing amount of mensuration iminodiethanoic acid, calculate transformation efficiency, the results are shown in Table 1.
Sieving again the every 1L of substratum prepares by following composition: glycerine 8g, yeast extract paste 6g, NaCl 1g, K 2hPO 45g, MgSO 40.2g, n-Butyronitrile 5g, pH value 7.0, water complements to 1000mL; The inductor n-Butyronitrile also can isopropyl cyanide or hexanolactam replacement.
Table 1: the multiple sieve result of producing the nitrilase bacterial classification
Sequence number Strain name Transformation efficiency (%)
1 Edge pseudomonas (Pseudomonas marginalis) ZJB09121 26.5
2 Quick acidovorax facilis (Acidovorax flcilis) ZJB09122 21.9
3 Acid-producing Klebsiella bacterium (Kelbsiella ocytoca) ZJB09123 22.3
4 Fu Shi rhodococcus (Rhodococcus wratislaviensis) ZJB09124 23.6
5 Prunosus red coccus (Rhodococcus rhodochrous) ZJB09125 24.7
6 Have a liking for pyridine rhodococcus (Rhodococcus pyridinivorans) ZJB09126 30.0
7 Rhodococcus ruber (Rhodococcus rubber) ZJB09127 23.3
8 Pseudomonas putida (Pseudomonas putia) ZJB09129 24.4
9 Pseudomonas putida (Pseudomonas putia) ZJB09135 23,9
10 Micrococcus luteus (Micrococcus luteus) ZJB09131 24.7
11 Brevibacterium halotolerans (Brevibacterium halotoerans) ZJB09132 25.8
12 Pseudomonas putida (Pseudomonas putia) ZJB09134 13.1
13 Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09137 14.3
14 Bacillus foecalis alkaligenes (Pseudomonas aeruginosa) ZJB09138 54.8
15 Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99 26.4
Embodiment 3: cultivation and the biocatalysis of producing the nitrilase microorganism prepare iminodiethanoic acid
The every 1L of culture medium prepared by following the composition: glycerine 8g, yeast extract paste 6g, NaCl 1g, K 2hPO 45g, MgSO 40.2g water complements to 1000mL; Separately add inductor n-Butyronitrile 5g.
Culture medium is sub-packed in the 500mL triangular flask, liquid amount 100mL/ bottle, and with Autoclave heat sterilization 20min, temperature is 121 ℃.
By the bacterial strain obtained in embodiment 1, inoculate respectively bacterial classification that 1 ring cultivates through slant activation in the culture medium that has added inductor, at the shaking flask rotating speed, be 200r/min, temperature is under 30 ℃, cultivates 3d, collects wet thallus as the thick enzyme of nitrilase.Add the reaction that is hydrolyzed of 5g wet thallus in the iminodiacetonitrile aqueous solution of 100mL 3% (w/w), control 30 ℃ of temperature, mixing speed 200r/min, the ammoniacal liquor auto-feeding is regulated pH 7.0, measure the productive rate of product iminodiethanoic acid and calculate transformation efficiency after hydrolysis 10h, the results are shown in Table 2.
Table 2: the productive rate and the transformation efficiency that produce nitrilase bacterial classification iminodiethanoic acid
Sequence number Strain name Transformation efficiency (%)
1 Edge pseudomonas (Pseudomonas marginalis) ZJB09121 23.7
2 Quick acidovorax facilis (Acidovorax facilis) ZJB09122 25.1
3 Acid-producing Klebsiella bacterium (Kelbsiella ocytoca) ZJB09123 20.8
4 Fu Shi rhodococcus (Rhodococcus wratislaviensis) ZJB09124 24.5
5 Prunosus red coccus (Rhodococcus rhodochrous) ZJB09125 26.2
6 Have a liking for pyridine rhodococcus (Rhodococcus pyridinivorans) ZJB09126 30.7
7 Rhodococcus ruber (Rhodococcus rubber) ZJB09127 23.9
8 Pseudomonas putida (Pseudomonas putia) ZJB09129 23.1
9 Pseudomonas putida (Pseudomonas putia) ZJB09135 24.2
10 Micrococcus luteus (Micrococcus luteus) ZJB09131 24.8
11 Brevibacterium halotolerans (Brevibacterium halotoerans) ZJB09132 25.7
12 Pseudomonas putida (Pseudomonas putia) ZJB09134 13.0
13 Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09137 14.7
14 Bacillus foecalis alkaligenes (Pseudomonas aeruginosa) ZJB09138 54.9
15 Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99 26.5
Embodiment 4: the free cell biocatalysis is produced iminodiethanoic acid
In order to investigate the stability of the nitrilase that bacterial isolates produces obtained in embodiment 1, press the method for embodiment 3, the bacterial isolates obtained in embodiment 1 is carried out to free cell enzymic hydrolysis test in batches repeatedly, measure the content of iminodiethanoic acid after hydrolysis 10h, and centrifugal collection thalline, the thalline of centrifugal collection is used further to hydrolysis reaction, 3 times repeatedly.Test-results is as table 3.
Table 3: the free cell of product nitrilase bacterial classification is enzymic hydrolysis test-results in batches repeatedly
Figure BDA0000081435860000081
Embodiment 5: the immobilized cell biocatalysis is produced iminodiethanoic acid
In order further to investigate the stability of the nitrilase that bacterial isolates produces obtained in embodiment 1, the part bacterial strain is carried out to cell fixation, and carry out enzymic hydrolysis test in batches repeatedly.
Take the edge pseudomonas ZJB09121 (Pseudomonas marginalis ZJB09121) cultivated by embodiment 3, pseudomonas putida ZJB09134 (Pseudomonas putia ZJB09134), pseudomonas putida ZJB09129 (Pseudomonas putia ZJB09129), pseudomonas putida ZJB09135 (Pseudomonas putia ZJB09135), Pseudomonas fluorescens ZJB09137 (Pseudomonas fluorescens ZJB09137), acid-producing Klebsiella bacterium ZJB09123 (Kelbsiella ocytoca ZJB09123), Fu Shi rhodococcus ZJB09124 (Rhodococcus wratislaviensis ZJB09124), Fu Shi rhodococcus ZJB09126 (Rhodococcus pyridinivorans ZJB09126), prunosus red coccus ZJB09125 (Rhodococcus rhodochrous ZJB09125), Rhodococcus ruber ZJB09127 (Rhodococcus rubber ZJB09127), micrococcus luteus ZJB09131 (Micrococcus luteus ZJB09131), brevibacterium halotolerans ZJB09132 (Brevibacterium halotoerans ZJB09132), quick acidovorax facilis ZJB09122 (Acidovorax facilis ZJB09122, Bacillus foecalis alkaligenes ZJB09138 (Alcaligenes faecalis ZJB09138), the somatic cells of Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99, be mixed with bacteria suspension with deionized water, by concentration, it is 2% (w/w) sodium alginate heating for dissolving, the even mixed solution that after cooling, will be made into 6% (g wet thallus/ml sodium alginate soln) in somatic cells and the sodium alginate soln of wet thallus, and mixed solution is splashed into to the CaCl of 2% (w/w) 2in solution, immobilization 12h, leach particle, clean with physiological saline, above-mentioned immobilized cell uses the glutaraldehyde of 0.2% (w/v)-HCl solution (containing 30mM CaCl again 2) stir process 24h under room temperature, with distillation washing 3 times, obtain crosslinked immobilized cell afterwards.By the crosslinked immobilized cell of above-mentioned different strains, for batch hydrolysis fractionation repeatedly, 3 times repeatedly, result is as table 4.
Table 4: the free cell of product nitrilase bacterial classification is enzymic hydrolysis test-results in batches repeatedly
Figure BDA0000081435860000091
Figure BDA0000081435860000101
Embodiment 6: the resolvase biocatalysis is produced iminodiethanoic acid
By the bacterium thalline obtained through fermentation in embodiment 1, utilize 0.9% physiological saline washing 3 times, collecting thalline is resuspended in Tris-HCl pH 7.0 damping fluids, regulate cell concentration and count 10% (g/ml) with wet thallus, utilize ultrasonic wave, high pressure cracker, high pressure homogenizer to carry out fragmentation to cell, obtain the crude enzyme liquid that contains free nitrilase, utilize resolvase to carry out biocatalytic reaction.Add the reaction that is hydrolyzed of 1ml crude enzyme liquid in the iminodiacetonitrile aqueous solution of 9ml3% (w/w), control 30 ℃ of temperature, mixing speed 200r/min, the ammoniacal liquor auto-feeding is regulated pH 7.0, after hydrolysis 10h, after the centrifugal 20min of 10000rpm, measure the productive rate of product iminodiethanoic acid in supernatant liquor and calculate transformation efficiency, the results are shown in Table 5.
Table 5: free nitrilase biocatalysis is produced the iminodiethanoic acid result
Sequence number Strain name Transformation efficiency (%)
1 Edge pseudomonas (Pseudomonas marginalis) ZJB09121 22.9
2 Quick acidovorax facilis (Acidovorax facilis) ZJB09122 26.7
3 Acid-producing Klebsiella bacterium (Kelbsiella ocytoca) ZJB09123 22.3
4 Fu Shi rhodococcus (Rhodococcus wratislaviensis) ZJB09124 24.1
5 Prunosus red coccus (Rhodococcus rhodochrous) ZJB09125 27.2
6 Have a liking for pyridine rhodococcus (Rhodococcus pyridinivorans) ZJB09126 29.1
7 Rhodococcus ruber (Rhodococcus rubber) ZJB09127 24.7
8 Pseudomonas putida (Pseudomonas putia) ZJB09129 26.5
9 Pseudomonas putida (Pseudomonas putia) ZJB09135 28.3
10 Micrococcus luteus (Micrococcus luteus) ZJB09131 31.2
11 Brevibacterium halotolerans (Brevibacterium halotoerans) ZJB09132 25.6
12 Pseudomonas putida (Pseudomonas putia) ZJB09134 20.1
13 Pseudomonas fluorescens (Pseudomonas fluorescens) ZJB09137 15.3
14 Bacillus foecalis alkaligenes (Pseudomonas aeruginosa) ZJB09138 55.9
15 Arthrobacter (Arthrobacter nitroguajacolicus) ZJUTB06-99 28.7
By above embodiment, reach a conclusion: the pseudomonas (Pseudomonas) of acquisition that the present invention screens, klebsiella (Kelbsiella), rhodococcus (Rhodococcus), micrococci (Micrococcus), bacillus (Alcaligenes), tyrothricin (Brevibacterium), Arthrobacter (Arthrobacter), bacterial strain and mutant strain thereof that acidovorax facilis (Acidovorax) etc. belong to, all effective to producing iminodiethanoic acid, free cell no matter, resolvase after cytoclasis, immobilized cell has good conversion iminodiacetonitrile to produce the effect of iminodiethanoic acid.The stability of the nitrilase that the free cell of these bacterial strains and immobilized cell produce is better, so these bacterial strains can be used for biocatalysis hydrolysis iminodiacetonitrile, and then produces iminodiethanoic acid.

Claims (4)

1. pseudomonas putida (Pseudomonas putia) ZJB09135, be preserved in Chinese Typical Representative culture collection center, deposit number CCTCC No:M 209055, preservation date on March 18th, 2009.
2. a method of utilizing the described pseudomonas putida CCTCC of claim 1 No:M 209055 preparing iminodiacetic acid by catalyzing iminodiacetonitrile, described method is as follows:
(A) described pseudomonas putida CCTCC No:M 209055 is seeded to culture medium, adds inductor a to carry out fermentation culture 2 ~ 5d under 100 ~ 200 r/min, 20 ~ 35 ℃ of conditions; Described inductor a is n-Butyronitrile, isopropyl cyanide or hexanolactam; Described culture medium final concentration consists of: glycerine 8 g/L, yeast extract paste 6 g/L, NaCl 1 g/L, K 2hPO 45 g/L, MgSO 40.2 g/L, solvent is water, pH value 7. 0; The final concentration of described inductor a is 5 g/L;
(B) get the aqueous solution that iminodiacetonitrile is mixed with mass concentration 3%, adjusting initial pH is 7.0 ~ 7.5, as the enzymic catalytic reaction substrate solution; The crude enzyme liquid that the wet thallus that the step (A) of usining fermentation obtains, cytoclasis obtain or the immobilized cell obtained through immobilization are counted 1g wet thallus/10 mL substrate solutions as ，Mei source, enzyme source add-on with wet thallus; Controlling temperature of reaction and be 30 ℃, reaction pH is 7.0, carries out conversion reaction 8 ~ 16 h, and after reaction finishes, reaction solution obtains iminodiethanoic acid through separation and purification.
3. method as claimed in claim 2, while it is characterized in that described step (B) is got the iminodiacetonitrile obtained aqueous solution with ammoniacal liquor, NaOH or HCl aqueous solution adjust pH.
4. method as claimed in claim 2, is characterized in that the described fermentation culture of described step (A) carries out fermentation culture 3d under 30 ℃ of conditions.
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