CN102994429B - Method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester through microorganism catalysis and bacterial strain - Google Patents

Method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester through microorganism catalysis and bacterial strain Download PDF

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CN102994429B
CN102994429B CN201210516645.1A CN201210516645A CN102994429B CN 102994429 B CN102994429 B CN 102994429B CN 201210516645 A CN201210516645 A CN 201210516645A CN 102994429 B CN102994429 B CN 102994429B
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ethyl
oxygen
acetic acid
acid ester
bacillus cereus
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CN102994429A (en
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汪钊
郑建永
鄢洪德
章银军
宣磊
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Zhejiang University of Technology ZJUT
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Abstract

The invention provides a cheap and efficient microorganism enzyme producing bacteria, namely, bacillus cereus (Bacillus cereus) WZZoo1, and provides a method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester by taking racemic alpha-ethyl-2-oxygen-1-pyrrolidine acetic acid ester as reaction substrate and catalyzing stereoselectivity hydrolysis(R)-alpha-ethyl-2-oxygen-1-pyrrolidine acetic acid ester through carboxylic ester hydrolase generated by the bacterium. The bacterial strain is preserved in China Center For Type Culture Collection (CCTCC) on October 14, 2012, the preservation serial number is CCTCC No: M2012403. The beneficial effect of the method mainly reflects in that the carboxylic ester hydrolase generated by the bacterial strain has strong area selectivity and high reaction conversion rate, downstream separation is simple, energy consumption is low, and environment pollution is light, so that the method is applicable to industrial production.

Description

Method and the bacterial strain of microorganism catalysis preparation (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid ester
(1) technical field
The present invention relates to bacillus cereus (Bacillus cereus) WZZ001 and prepare levetiracetam intermediate at microorganism catalysis---the application in (S)-α-ethyl shown in formula II-2-oxygen-1-pyrrolidine acetic acid ester, and a kind of microorganism catalysis prepares the method for (S)-α-ethyl shown in formula II-2-oxygen-1-pyrrolidine acetic acid ester, this intermediate can synthesize Levetiracetam by aminolysis reaction.
(2) background technology
Levetiracetam (levetiracetam, LEV, trade(brand)name Keppra) chemical name is (S)-α-ethyl-2-oxygen-1-pyrrolidine acetamide, is piracetam derivative, is a kind of new antiepileptic drugs thing (AED).Within 1999, ratify through U.S. FDA, be approved at first the assisting therapy of 4 years old above (comprising 4 years old) children's partial seizures for be grown up partial seizures .2005 its oral tablet in June and solution.On March 10th, 2007 is in Discussion on Chinese Listed (trade(brand)name Levetiracetam).Levetiracetam has the unique effect mechanism that is different from other antiepileptic drugs, it is the antiepileptic drug that synaptic vesicle Protein S V2A is combined in presynaptic nerve teminal of only confirmation so far, is also that at present report unique has the antiepileptic drug that prevention epilepsy is had an effect.Research shows, Levetiracetam and major metabolite thereof within the scope of certain therapeutic dose, neither the inhibitor of human liver's Cytochrome P450, epoxidation lytic enzyme or uridine diphosphate (UDP)-heteroside enzyme, neither they there is the substrate of high affinity, therefore, be not prone to pharmacokinetic interaction.For Adult Refractory partial seizures, Levetiracetam is that the first-selection for the treatment of epilepsy partial seizures adds with medicine; For children's partial seizures, also have good unusual effect, and side effect is slight, and clinical treatment is had to reference value.This drug effect is rapid, shows good anti-epileptic curative effect and tolerance, security, and except the assisting therapy for intractable epilepsy, its indication also expands to the single therapy of new diagnosis epilepsy gradually.
At present, the method for the synthetic left-handed Etiracetam of bibliographical information is mainly chemical resolution method and utilizes the synthetic method of chiral amino acid as initial substrate.The former, take pyrrolidone as raw material, react with racemic modification (±)-2-bromo-butyric acid methyl esters in process, again with (R)-Alpha-Methyl benzylamine salify, then obtains levo-enantiomer through reactions such as ammonia solutions; The latter, mainly take methionine(Met) as raw material, obtains left-handed product by polystep reactions such as esterification, amidation, closed loop and desulfurization.These traditional technology approach exist the problems such as yield is low, cost is high, step is various, product purity is lower, seriously polluted.
(3) summary of the invention
The object of the invention is the deficiency in order to solve aforesaid method, by screening yielding lipase microorganism, provide the microbial enzyme of a strain Cheap highly effective to produce bacterium, and provide take racemic ' alpha '-ethyl-2-oxygen-1-pyrrolidine acetic acid ester as reaction substrate, the ester hydrolase catalysis stereo selective hydrolysis (R) producing with this bacterium thereby-α-ethyl-2-oxygen-1-pyrrolidine acetic acid ester preparation (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid ester method.
The technical solution used in the present invention is:
Bacillus cereus (Bacillus cereus) WZZ001, is preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, preservation date: on October 14th, 2012, deposit number: CCTCC No:M 2012403.
This bacterial strain obtains by following program screening and separating:
(1) take wet soil sample 1 g and be suspended in 10 mL stroke-physiological saline solution, vibration mixes; Connect 3 mL suspensions in 50 mL liquid enrichment mediums, under 30 ℃, 200 r/min, cultivate after 4 ~ 5 d, aseptic technique is transferred the bacterium liquid of 1 mL muddiness to fresh liquid enrichment medium, enrichment 3 ~ 4 times continuously.In enrichment medium, take racemic ' alpha '-ethyl-2-oxygen-1-methyl pyrrolidineacetate as sole carbon source, fill a prescription as follows: NaNO 33.0 g/L, KH 2pO 41.0 g/L, MgSO 47H 2o 0.5 g/L, KCl 0.5 g/L, FeSO 47H 2o 0.01 g/L, (±)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate 10 mmol/L, pH 7.0.
(2) by bacterium liquid after enrichment through gradient dilution, coating separating plate substratum, separating for several times, obtains single bacterium colony.Separating plate substratum consists of: enrichment medium adds agar 23 g/L, pH 7.0.
(3) picking separates the single bacterium colony obtaining and is successively inoculated in slant medium and the seed culture medium of 50 mL, and 30 ℃, 200 r/min are cultivated 24 h, obtain seed liquor; Under aseptic condition, to get 3 mL seed liquor and be inoculated in the culture medium of 50 mL, 30 ℃, 200 r/min are cultivated 24 h, obtain fermented liquid.Slant medium composition is consistent with separating plate substratum; Seed culture based formulas is: peptone 5 g/L, extractum carnis 5 g/L, yeast extract paste 1 g/L, glucose 5 g/L, MgSO 45 g/L, K 2hPO 41.5 g/L, KH 2pO 40.5 g/L, solvent is water, pH 7.0; Culture medium consists of: peptone 10 g/L, glucose 5 g/L, MgSO 45 g/L, K 2hPO 41.5 g/L, KH 2pO 40.5 g/L, sweet oil 5 g/L, solvent is water, pH 7.0.
(4) get 10 mL fermented liquids centrifugal 15 min of 6000 rpm in 10 mL centrifuge tubes, abandon supernatant liquor, the phosphate buffered saline buffer that adds 2 mL 0.2 mol/L pH 7.2, vibration mixes, then adds 5.66 g/L substrate (±)-α-ethyls-2-oxygen-1-methyl pyrrolidineacetate.Under 30 ℃, 200 r/min, transform 24h.Take out centrifuge tube, add 2 mL ethyl acetate, fully vibration, centrifugal 15 min of 6000 rpm.Get upper organic phase 1 mL, dewater with a small amount of anhydrous sodium sulfate drying, with the enantiomeric excess value (e.e.) of gas chromatographic detection (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate, and detecting the specific rotatory power of asymmetric hydrolysis product with polarimeter, screening obtains described microorganism strains-WZZ001 bacterium.
Colony morphology characteristic: at 30 ℃, cultivate 32h, bacterium colony is rounded, and edge is decomposite leaf shape, central protuberance, oyster white, dry, tarnish.
Cell morphological characteristic: cell is straight or bending rod-short, size is [(0.4 ~ 0.6) μ m × (1.2 ~ 1.6) μ m], single dispersed arrangement.
Physiological and biochemical property: WZZ001 bacterium is gram-positive microorganism, and obligate is aerobic, growth changes insensitive to pH.Utilize inorganic nitrogen-sourced ability poor.Growth and product enzyme are subject to Cu 2+, Mn 2+inhibition, be subject to Mg 2+, Fe 3+, Ca 2+deng promotion.
After sequencing, WZZ001 16S, RDNA sequence is as follows:follows:GTGCTATACATGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACT CTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCA TGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCA AGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTG CAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGT GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACA CCCGAAGTCGGTGGGGTAACCTTT
According to physio-biochemical characteristics and molecular biology identification, this bacterial strain is accredited as bacillus cereus.
The invention still further relates to the application of described bacillus cereus WZZ001 in (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid ester shown in microorganism catalysis preparation (II).
The invention still further relates to a kind of microorganism catalysis and prepare the method for (S)-α-ethyl shown in formula II-2-oxygen-1-pyrrolidine acetic acid ester, described method comprises: take the racemic ' alpha '-ethyl shown in formula I-2-oxygen-1-pyrrolidine acetic acid ester as reaction substrate, under the effect of microorganism ester hydrolase, carry out selective hydrolysis and make described (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid ester; Described microorganism ester hydrolase from bacillus cereus CCTCC No:M 2012403 containing enzyme somatic cells;
Figure BDA0000253033201
In formula I, (II), the alkyl that R is C1 ~ C4.
The reaction formula relating to is as follows:
Figure BDA0000253033202
Described microorganism ester hydrolase can be prepared as follows: bacillus cereus WZZ001 is seeded to culture medium, under 30 ~ 37 ℃, rotating speed 150 ~ 200r/min condition, cultivate 1 ~ 2 day, centrifuging obtains wet thallus, and drying must contain the dry mycelium of described microorganism ester hydrolase; Described culture medium final concentration consists of: peptone 10 g/L, glucose 5 g/L, MgSO 45 g/L, K 2hPO 41.5 g/L, KH 2pO 40.5 g/L, sweet oil 5 g/L, solvent is water, pH 7.0.
Conventionally, bacterial strain needs to carry out seed enlarged culturing before cultivating producing enzyme, and method is as follows: picking inclined-plane inoculation is in seed culture medium, and 30 ℃, 200 r/min are cultivated 24 h, obtain seed liquor, seed liquor is seeded to culture medium with 5 ~ 10% volume ratios again; Seed culture based formulas is: peptone 5 g/L, extractum carnis 5 g/L, yeast extract paste 1 g/L, glucose 5 g/L, MgSO 45 g/L, K 2hPO 41.5 g/L, KH 2pO 40.5 g/L, solvent is water, pH 7.0;
Described selective hydrolysis carries out in 10 ~ 50 ℃, the damping fluid of pH6.0 ~ 8.0.
Preferably, described selective hydrolysis carries out in the phosphate buffered saline buffer of pH7.2.
In damping fluid, initial substrate concentration is 5 ~ 100g/L, is that 1 ~ 50g/L(is with somatic cells dry weight basis containing enzyme somatic cells addition).
Concrete, described method can be as follows: by adding in the buffered soln that contains reaction substrate racemic ' alpha '-ethyl-2-oxygen-1-pyrrolidine acetic acid ester containing enzyme somatic cells of bacillus cereus WZZ001, substrate final concentration is 5 ~ 100g/L, thalline add-on is 1 ~ 50g/L, at 20 ~ 50 ℃, react 2 ~ 60 hours, reaction solution obtains described (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate through separation and purification.
For improving reaction efficiency, in described damping fluid, also can be added with the solubility promoter that volume is damping fluid volume 10 ~ 20%, described solubility promoter is one of following: acetone, acetonitrile or dimethyl sulfoxide (DMSO).
Analytical conditions for gas chromatography: adopt gas chromatograph Agilent 6890 type gas chromatographs; Chiral gas chromatography post BGB-175(0.25 mm × 30 m); Carrier gas N2(70 kPa), splitting ratio 50:1,250 ℃ of injector temperatures, 250 ℃ of detector temperatures; Column temperature rise program: 120 ℃ of initial temperatures, keep 3 min, 2 ℃/min is warming up to 180 ℃, keeps 1 min; Sample size 1 μ L, air flow quantity: 450 mL/min; Make-up gas flow: He 45 mL/min; Hydrogen ion flame detector.(R)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate and (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate go out peak at 26.8min and 27.4min respectively.
(S) extraction of-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate and refining: remove by filter lipase or thalline after enzymatic conversion, after organic solvent (ethyl acetate) extraction, underpressure distillation is removed organic solvent and is obtained product, can obtain highly purified (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate product through rectifying.
Beneficial effect of the present invention is mainly reflected in: the regioselectivity of the ester hydrolase that bacterial strain of the present invention produces is strong, and reaction conversion ratio is high, and downstream separation is simple, and energy consumption is low, and environmental pollution is little, is applicable to suitability for industrialized production.
(4) accompanying drawing explanation
Fig. 1 racemic modification α-ethyl-2-oxygen-1-methyl pyrrolidineacetate reference substance gas chromatogram;
The gas chromatogram of Fig. 2 (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate standard substance;
The phase color atlas of Fig. 3 microorganism catalysis product (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate.
(5) 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:
Picking bacillus cereus WZZ001 inclined-plane colony inoculation is in the seed culture medium of 50 mL, and 30 ℃, 200 r/min are cultivated 24 h, obtain seed liquor; Under aseptic condition, to get 3 mL seed liquor and be inoculated in the culture medium of 50 mL, 30 ℃, 200 r/min are cultivated 24 h, obtain fermented liquid.After cultivation finishes, by centrifugal fermented liquid 6000 rpm 15 min, abandon supernatant liquor and obtain bacillus cereus WZZ001 wet thallus, add the phosphate buffered saline buffer of a small amount of pH 7.2 0.2 mol/L to stir, under-40 ℃ of conditions, lyophilize obtains dry mycelium.Through enzyme activity determination, this dry mycelium is 5.0 U/g than enzyme activity.
Seed culture based formulas is: peptone 5 g/L, extractum carnis 5 g/L, yeast extract paste 1 g/L, glucose 5 g/L, MgSO 45 g/L, K 2hPO 41.5 g/L, KH 2pO 40.5 g/L, solvent is water, pH 7.0; Culture medium formula is: peptone 10 g/L, glucose 5 g/L, MgSO 45 g/L, K 2hPO 41.5 g/L, KH 2pO 40.5 g/L, sweet oil 5 g/L, solvent is water, pH 7.0.
U is 1 enzyme activity unit, 1 described enzyme activity unit is the amount of the lipase of 1 μ mol amount of per minute catalytic hydrolysis-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate of measuring under defined terms, described prescribed condition is: get 0.2g bacterium powder, to the phosphate buffer solution 10ml that adds racemic ' alpha '-ethyl-2-oxygen-1-pyrrolidine acetic acid ester concentration 5 g/L in thalline, under 30 ℃ of conditions, mixing speed 200 rpm, after reaction 60min, remove by filter enzyme, extract reaction solution the content of measuring the α-ethyl-2-oxygen-1-methyl pyrrolidineacetate after transforming with gas chromatographic analysis.
Embodiment 2:
0.1g bacillus cereus WZZ001 dry mycelium adds in the 10mL pH7.2 phosphoric acid buffer that contains 5g/L racemic ' alpha '-ethyl-2-oxygen-1-methyl pyrrolidineacetate, under 30 ℃ of conditions, after reaction 24h, reaction solution after reaction transforms is centrifugal, get supernatant liquor, add isopyknic ethyl acetate extraction, obtain (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate.Adopt the enantiomeric excess value e.e. 99.5% of preceding method detection (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate, productive rate 48.9%.
Embodiment 3:
0.2g bacillus cereus WZZ001 dry mycelium adds in the 10mLpH7.2 phosphoric acid buffer that contains 10g/L racemic ' alpha '-ethyl-2-oxygen-1-methyl pyrrolidineacetate, under 30 ℃ of conditions, after reaction 24h, reaction solution after reaction transforms is centrifugal, get supernatant liquor, add isopyknic ethyl acetate extraction, obtain (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate.Adopt the enantiomeric excess value e.e. 99.2% of preceding method detection (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate, productive rate 46.8%.
Embodiment 4:
0.4g bacillus cereus WZZ001 dry mycelium adds in the 10mL pH7.2 phosphoric acid buffer that contains 20g/L racemic ' alpha '-ethyl-2-oxygen-1-methyl pyrrolidineacetate, under 30 ℃ of conditions, after reaction 24h, reaction solution after reaction transforms is centrifugal, get supernatant liquor, add isopyknic ethyl acetate extraction, obtain (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate.Adopt the enantiomeric excess value e.e. 98.2% of preceding method detection (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate, productive rate 45.6%.
Embodiment 5:
0.2g bacillus cereus WZZ001 dry mycelium adds in the 10mL pH7.2 phosphoric acid buffer that contains 10g/L racemic ' alpha '-ethyl-2-oxygen-1-pyrrolidine acetic acid ethyl ester, under 30 ℃ of conditions, after reaction 24h, reaction solution after reaction transforms is centrifugal, get supernatant liquor, add isopyknic ethyl acetate extraction, obtain (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate.Adopt the enantiomeric excess value e.e. 99.2% of preceding method detection (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate, productive rate 45.8%.
Embodiment 6:
0.4g bacillus cereus WZZ001 dry mycelium adds in the 10mL pH7.2 phosphoric acid buffer that contains 20g/L racemic ' alpha '-ethyl-2-oxygen-1-pyrrolidine acetic acid ethyl ester, under 30 ℃ of conditions, after reaction 24h, reaction solution after reaction transforms is centrifugal, get supernatant liquor, add isopyknic ethyl acetate extraction, obtain (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate.Adopt the enantiomeric excess value e.e. 96.2% of preceding method detection (S)-α-ethyl-2-oxygen-1-methyl pyrrolidineacetate, productive rate 45.8%.
The above, be only preferred embodiment of the present invention, not technology contents of the present invention done to any pro forma restriction.Any simple modification, equivalent variations and modification that every foundation technical spirit of the present invention is done above embodiment, all fall within the scope of protection of the present invention.
Figure IDA00002530333000011
Figure IDA00002530333000021

Claims (7)

1. bacillus cereus (Bacillus cereus) WZZ001, is preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, preservation date: on October 14th, 2012, deposit number: CCTCC No:M2012403.
2. bacillus cereus WZZ001 as claimed in claim 1, is characterized in that the 16SrDNA sequence of this bacterial strain is as shown in SEQ ID No.1.
3. the application of bacillus cereus WZZ001 claimed in claim 1 in (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid ester shown in microorganism catalysis preparation (II);
In formula II, the alkyl that R is C1～C4.
4. a microorganism catalysis is prepared the method for (S)-α-ethyl shown in formula II-2-oxygen-1-pyrrolidine acetic acid ester, described method comprises: take the racemic ' alpha '-ethyl shown in formula I-2-oxygen-1-pyrrolidine acetic acid ester as reaction substrate, under the effect of microorganism ester hydrolase, carry out selective hydrolysis and make described (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid ester; Described microorganism ester hydrolase from bacillus cereus CCTCC No:M2012403 containing enzyme somatic cells;
In formula I, (II), the alkyl that R is C1～C4.
5. method as claimed in claim 4, is characterized in that described selective hydrolysis carries out in 10～50 ℃, the damping fluid of pH6.0～8.0.
6. method as claimed in claim 5, is characterized in that described selective hydrolysis carries out in the phosphate buffered saline buffer of pH7.2.
7. the method as described in claim 5 or 6, is characterized in that in damping fluid, initial substrate concentration is 5～100g/L, is 1～50g/L containing enzyme somatic cells addition.
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