CN104212738A - Bacillus amyloliquefaciens WZZ002 and application - Google Patents

Abstract

The invention discloses a new bacterial strain-bacillus amyloliquefaciens WZZ002 and an application thereof in preparation of Boc-D-amino acid ester and Boc-L-amino acid through catalysis of racemized Boc-DL-amino acid ester. The enzyme in the invention is strong in regioselectivity, is high in conversion rate of a reaction, is simple in downstream separation, is low in energy consumption, is less in environmental pollution and is suitable for industrialized production.

Description

Bacillus amyloliquefaciens WZZ002 and application

(1) technical field

The present invention relates to a kind of enzyme process divides racemic Boc-DL-amino acid ester to prepare the method for chiral intermediate, carries out stereo selective hydrolysis Reactive Synthesis optical purity Boc-D-amino acid ester and the amino acid whose method of Boc-L-under the ester hydrolase effect particularly produced at bacillus amyloliquefaciens WZZ002 (CCTCC M 2013366).

(2) background technology

N-tertbutyloxycarbonyl-D-amino acid ester (being called for short Boc-D-amino acid ester) and N-tertbutyloxycarbonyl-L-amino acid (being called for short Boc-L-amino acid) are a kind of important biochemical reagents, for Peptide systhesis and amino acid protection monomer.Boc-D-amino acid ester and Boc-L-amino acid ester can obtain corresponding D-amino acid and L-amino acid by chemical hydrolysis.D-amino acid is as alpha-non-natural amino acid, and at medicine and pesticide synthesis, food, the aspect tools such as feed have been widely used.Wherein a kind of important medicine intermediate of D-alanine, be widely used in the fields such as medicine and foodstuff additive, be mainly used in synthesizing new sweeting agent alitame (L-aspartoyl-N-(2,2,4,4-tetramethyl--3-thietanyl)-D-alanimamides) and the raw material of some chirality pharmaceutical intermediate compound.Wherein L-norvaline is the important intermediate of hypertension class medicine perindopril.C4H9NO2 is the important intermediate of antiepileptic drug Levetiracetam.

At present, the synthesis Boc-D-alanine ester of bibliographical information is obtain after substrate B oc replaces esterification by D-alanine.The wherein synthetic method of D-alanine mainly chemical resolution method.Chemical resolution process routes also exists the problems such as yield is low, cost is high, step is various, product purity is lower, seriously polluted.And enzyme process is prepared D-alanine method and is mainly contained: (1) for substrate, utilizes glycolylurea lyase to prepare with the 5-methyl hydantoin of chemosynthesis; (2) acetyl of chemosynthesis or phenylacetyl-DL-Alanine are substrate, utilize L-Aminoacylase or penicillin acidylate to split; (3) take DL-Alanine as substrate, microbial degradation method; (4) be substrate with pyruvic acid, utilize transaminase to prepare.The problems such as it is not high that current Biological preparation D-alanine also exists enzyme selectivity, and production cost is high, and precursor substrate synthesis is difficult.

(3) summary of the invention

The present invention seeks to by screening yielding lipase microorganism, provide the producing microbial enzyme bacterial strain of a strain Cheap highly effective, with racemize Boc-DL-amino acid ester for reaction substrate, L-isomer in catalysis stereo selective hydrolysis Boc-DL-amino acid ester thus preparation Boc-D-amino acid ester and the amino acid whose method of Boc-L-.

The technical solution used in the present invention is:

The invention provides a strain new strains--bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WZZ002, be preserved in China typical culture collection center, preservation address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC M 2013366, preservation date on August 8th, 2013.

The invention still further relates to a kind of described bacillus amyloliquefaciens WZZ002 and split the application in N-tertbutyloxycarbonyl-DL-amino acid ester, concrete described being applied as: with the dry mycelium of bacillus amyloliquefaciens WZZ002 after the wet thallus freeze-drying that fermentation culture obtains for catalyzer, with racemize N-tertbutyloxycarbonyl-DL-amino acid ester (i.e. Boc-DL-amino acid methyl ester) for substrate, be in the phosphoric acid buffer of 6 ~ 9 (preferable ph is 7.0) in pH value, conversion reaction is carried out under 20 ~ 50 DEG C (preferably 30 DEG C), after reaction terminates, by reaction solution aftertreatment, obtain N-tertbutyloxycarbonyl-D-amino acid ester (Boc-D-amino acid ester) and N-tertbutyloxycarbonyl-L-amino acid (Boc-L-amino acid).

Further, the quality consumption of described dry mycelium is 1 ~ 50g/L damping fluid, preferably 10 ~ 20g/L, and described initial substrate concentration is 1 ~ 200g/L damping fluid, preferably 50 ~ 100g/L.

Further, preferred described substrate is N-tertbutyloxycarbonyl-DL-Alanine ester (Boc-DL-alanine ester), N-tertbutyloxycarbonyl-DL-2-aminobutyric acid ester (Boc-DL-2-aminobutyric acid ester), N-tertbutyloxycarbonyl-DL-norvaline ester (Boc-DL-norvaline ester), N-tertbutyloxycarbonyl-DL-methionine ester (Boc-DL-methionine ester), N-tertbutyloxycarbonyl-DL-proline ester (Boc-DL-proline ester), N-tertbutyloxycarbonyl-DL-serine ester (Boc-DL-serine ester), N-tertbutyloxycarbonyl-DL-LEUCINE ester (Boc-DL-leucine ester), N-tertbutyloxycarbonyl-DL-Isoleucine ester (Boc-DL-Isoleucine ester), N-tertbutyloxycarbonyl-DL-Threonine ester (Boc-DL-Threonine ester), N-tertbutyloxycarbonyl-DL-Trp ester (Boc-DL-tryptophane ester) or N-tertbutyloxycarbonyl-DL-phenylalanine ester (Boc-DL-phenylalanine ester).

Further, catalyzer of the present invention is prepared as follows:

(1) slant culture

Bacillus amyloliquefaciens WZZ002 is seeded to slant medium, cultivates 24h at 30 DEG C, obtain inclined-plane bacterium colony; Slant medium final concentration forms: NaNO ₃3.0g/L, KH ₂pO ₄1.0g/L, MgSO ₄7H ₂o0.5g/L, KCl0.5g/L, FeSO ₄7H ₂o0.01g/L, Boc-DL-alanine ester 10mmol/L, solvent is deionized water, pH7.0;

(2) seed culture

The inclined-plane colony inoculation that step (1) is obtained in seed culture medium, 30 DEG C, 200r/min cultivates 24h, obtains seed liquor; Seed culture medium final concentration forms: peptone 5g/L, extractum carnis 5g/L, yeast extract paste 1g/L, maltose 5g/L, NaCl0.5g/L, agar 23g/L, and solvent is deionized water, pH7.0;

(3) fermentation culture

Aseptically, seed liquor is inoculated in (250mL triangular flask, liquid amount is 50mL) in fermention medium with the inoculum size of volumetric concentration 5 ~ 20%, 30 DEG C, 200r/min cultivates 24h, obtain fermented liquid, fermented liquid is centrifugal, abandon supernatant liquor, obtain bacillus amyloliquefaciens WZZ002 wet thallus, wet thallus is suspended in the phosphate buffered saline buffer of pH7.0,0.2mol/L, stir, lyophilize under-40 DEG C of conditions, obtains dry mycelium; Fermention medium consists of: peptone 5g/L, extractum carnis 5g/L, yeast extract paste 1g/L, MgSO ₄1mmol/L, solvent is deionized water, pH7.0.

The method of reaction solution aftertreatment of the present invention is: after conversion reaction completely, conversion reaction liquid is crossed and filters thalline, filtrate is with organic solvent extraction (preferably 2 times), obtain organic phase and aqueous phase respectively, organic phase underpressure distillation, except desolventizing, obtains the N-tertbutyloxycarbonyl-D-alanine methyl esters product of high-optical-purity; Described organic solvent is normal hexane, hexanaphthene or ethyl acetate; (preferably add hydrochloric acid to regulate) after aqueous phase adjust ph to 2.0, with extraction agent extraction, get the organic phase after extraction and obtain Boc-L-amino acid, described extraction agent is normal hexane, hexanaphthene or ethyl acetate.

Enzyme is lived and is defined as: at 30 DEG C, the enzyme amount needed for BOC-DL-alanine ester of per minute catalytic hydrolysis 1 μm of ol.Enzyme activity determination conditions method is: get 0.2g dry mycelium, phosphate buffer solution (pH value the is 7.0) 10ml containing final concentration 5g/L Boc-DL-amino acid ester is added in thalline, under 30 DEG C of conditions, mixing speed 200rpm, after reaction 20min, cross and filter enzyme, get the content of filtrate with gas chromatography determination Boc-D-alanine ester, Boc-L-alanine ester, Boc-D-L-Ala and Boc-L-L-Ala.

The present invention with the Boc-DL-amino acid ester shown in formula I for reaction substrate, at catalyzer (the i.e. dry mycelium of bacillus amyloliquefaciens WZZ002 fermentation culture acquisition, this dry mycelium produces microorganism ester hydrolase) under effect, carry out selective hydrolysis and obtain described Boc-D-amino acid ester (II) and Boc-L-amino acid (III):

In formula I, (II) and (III), R group is the alkyl of C1 ~ C4, R ₁group is the side-chain radical of a-amino acid, is preferably methyl.

The invention provides a strain new strains--bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WZZ002 and prepare the application in Boc-D-amino acid ester and Boc-L-amino acid at catalysis racemize Boc-DL-amino acid ester, the regioselectivity that described bacillus amyloliquefaciens WZZ002 produces enzyme is strong, reaction conversion ratio is high, downstream separation is simple, energy consumption is low, environmental pollution is little, is applicable to suitability for industrialized production.

(4) accompanying drawing explanation

Fig. 1 racemic modification Boc-DL-alanine methyl ester standard substance gas chromatogram;

The gas chromatogram of Fig. 2 Boc-L-alanine methyl ester standard substance;

The gas chromatogram of Fig. 3 microorganism catalysis product B oc-D-alanine methyl ester.

Fig. 4 is bacterial strain WZZ002 phylogenetic evolution tree.

(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: the screening of bacterial strain and qualification

1, the screening of bacterial strain WZZ002

Taking wet soil sample 1g (obtaining soil near Shang Tang river, Hangzhou, Zhejiang province city) is suspended in 10mL stroke-physiological saline solution, vibration mixing; Connect 3mL suspension in 50mL liquid enrichment medium, 30 DEG C, cultivate 4 ~ 5d under 200r/min after, the bacterium liquid of aseptic technique switching 1mL muddiness to fresh liquid enrichment medium, continuous enrichment 3 ~ 4 times.In enrichment medium, with Boc-DL-alanine ester for sole carbon source, fill a prescription as follows: NaNO ₃3.0g/L, KH ₂pO ₄1.0g/L, MgSO ₄7H ₂o0.5g/L, KCl0.5g/L, FeSO ₄7H ₂o0.01g/L, Boc-DL-alanine ester 10mmol/L, solvent is deionized water, pH7.0.By bacterium liquid after enrichment through physiological saline gradient dilution, coating separating plate substratum, separating for several times, obtains single bacterium colony.Separating plate substratum consists of: enrichment medium adds agar 23g/L (final concentration), pH7.0.

Picking is separated single colony inoculation of obtaining in slant medium, 24h is cultivated at 30 DEG C, from slant medium picking one transfering loop colony inoculation to seed culture medium (liquid amount is 50ml), 30 DEG C, 200r/min cultivates 24h, aseptically, getting 3mL seed liquor is inoculated in the fermention medium of 50mL, 30 DEG C, 200r/min cultivates 24h, obtains fermented liquid.Slant medium composition is consistent with separating plate substratum; Seed culture based formulas is: peptone 5g/L, extractum carnis 5g/L, yeast extract paste 1g/L, maltose 5g/L, NaCl0.5g/L, and solvent is deionized water, pH7.0; Fermention medium consists of: peptone 5g/L, extractum carnis 5g/L, yeast extract paste 1g/L, MgSO ₄1mmol/L, solvent is deionized water, pH7.0.

Get 10mL fermented liquid in 10mL centrifuge tube, the centrifugal 15min of 6000rpm, abandons supernatant liquor, adds the phosphate buffered saline buffer of 2mL0.2mol/L, pH7.0, vibration mixing, then adds final concentration 5.26g/L substrate B OC-DL-alanine methyl ester.30 DEG C, under 200r/min, transform 2h.Take out centrifuge tube, add 2mL ethyl acetate, fully vibrate, the centrifugal 15min of 6000rpm.Get upper organic phase 1mL, dewater with a small amount of anhydrous sodium sulfate drying, with the gas phase adsorption peak of gas chromatographic detection asymmetric hydrolysis product B oc-D-alanine methyl ester and Boc-L-L-Ala, and calculate enantiomeric excess value (e.e.), screening enantiomeric excess value reaches the bacterial strain of 98%, namely obtains bacterial strain WZZ002.The gas chromatogram of Boc-DL-alanine methyl ester and Boc-L-alanine methyl ester standard substance and converted product Boc-D-alanine methyl ester is shown in shown in Fig. 1-Fig. 3.

Enantiomeric excess value calculation formula:

${e.e.}_s=\frac{A_{D1}-A_{L1}}{A_{D1}+A_{L1}}\×100\%$ Formula (1)

${e.e.}_p=\frac{A_{L2}{-A}_{D2}}{A_{L2}+A_{D2}}\×100\%$ Formula (2)

Wherein, e.e.s represents the enantiomeric excess value of Boc-D-alanine methyl ester, e.e. _prepresent the enantiomeric excess value of Boc-L-L-Ala, A _d1, A _l1, A _l2and A _d2be respectively the peak area of gas chromatographic detection of Boc-D-alanine ester, Boc-L-alanine ester, Boc-L-L-Ala, Boc-D-L-Ala.

Substrate conversion efficiency calculation formula:

$c=\frac{{e.e.}_s}{{e.e.}_s+{e.e.}_p}\×100\%$ Formula (3)

In formula (3), c is substrate conversion efficiency.

Analytical conditions for gas chromatography: adopt Agilent6890 type gas chromatograph; Chiral gas chromatography post BGB-174 (0.25mm × 30m); Carrier gas N ₂(70kPa), splitting ratio 50:1, injector temperature 250 DEG C, detector temperature 250 DEG C; Column temperature rise program: initial temperature 100 DEG C, keep 3min, 5 DEG C/min is warming up to 200 DEG C, keeps 2min; Sample size 1.5 μ L, air flow quantity: 300mL/min; Make-up gas flow: N ₂25mL/min; Hydrogen ion flame detector.Boc-D-alanine methyl ester and Boc-L-alanine methyl ester go out peak at 13.9min and 14.2min respectively, and hydrolysate Boc-D-L-Ala and Boc-L-L-Ala go out peak at 22.2min and 22.5min respectively.

2, the qualification of bacterial strain WZZ002

(1) physiological and biochemical property: bacterial strain WZZ002 is gram-positive microorganism, obligate aerobic, grow pH change more responsive, optimal pH is 7.0.Utilize inorganic nitrogen-sourced ability poor.Growth and product enzyme are subject to Cu ²⁺, Mn ²⁺, Zn ²⁺deng suppression, be subject to Mg ²⁺, K ⁺deng promotion.

(2) 16S rDNA sequence

Through the order-checking qualification of student on commission's work biotechnology (Shanghai) limited-liability company, the 16S rDNA sequence of bacterial strain WZZ002 is for shown in SEQ ID NO.1:

GCGCTTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTGTCGTCAGCTCGTGTCATGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGAAA

According to physio-biochemical characteristics and molecular biology identification, and bacterial strain WZZ002 phylogenetic evolution tree (Fig. 4), bacterial strain WZZ002 is accredited as bacillus amyloliquefaciens (Bacillus amyloliquefaciens), called after bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WZZ002, be preserved in China typical culture collection center (address China, Wuhan, Wuhan University, 430072), deposit number CCTCC M 2013366, preservation date on August 8th, 2013.

Embodiment 2: the preparation of catalyzer

(1) slant culture

Bacillus amyloliquefaciens WZZ002 is seeded to slant medium, cultivates 24h at 30 DEG C, obtain inclined-plane bacterium colony.Slant medium composition is with embodiment 1.

(2) seed culture

The inclined-plane colony inoculation that step (1) is obtained in the seed culture medium of 50mL, 30 DEG C, 200r/min cultivates 24h, obtains seed liquor.Seed culture medium composition is with embodiment 1.

(3) fermentation culture

Aseptically, get 3mL seed liquor and be inoculated in (250mL triangular flask, liquid amount is 50mL) in the fermention medium of 50mL, 30 DEG C, 200r/min cultivates 24h, obtains fermented liquid.By centrifugal for fermented liquid 6000rpm 15min after cultivation terminates, abandon supernatant liquor, obtain bacillus amyloliquefaciens WZZ002 wet thallus 8.5g, the phosphate buffered saline buffer adding 10ml, pH7.0,0.2mol/L stirs, lyophilize under-40 DEG C of conditions, obtains dry mycelium 0.8g.Through enzyme activity determination, this dry mycelium is 5.0U/g than enzyme activity.

Embodiment 3:

Bacillus amyloliquefaciens WZZ002 dry mycelium 0.1g embodiment 2 method prepared adds in the 10mL pH7.0 phosphoric acid buffer containing 20g/L Boc-DL-alanine methyl ester, after reacting 1h under 30 DEG C of conditions, reaction solution after reaction transforms is centrifugal, get supernatant liquor, after adding isopyknic extraction into ethyl acetate 2 times, merge organic phase, underpressure distillation, except desolventizing, obtains Boc-D-alanine methyl ester product; Aqueous phase, by after interpolation hydrochloric acid to pH2.0, is used extraction agent extraction into ethyl acetate, is got organic phase and obtain Boc-L-L-Ala.Method described in embodiment 1 is adopted to detect the enantiomeric excess value e.e.99.9% of Boc-D-alanine methyl ester, Boc-L-L-Ala e.e.99.4%, substrate conversion efficiency 50.9%.

Embodiment 4:

Bacillus amyloliquefaciens WZZ002 dry mycelium 0.2g embodiment 2 method prepared adds in 10mL, pH7.0 phosphoric acid buffer containing 100g/L Boc-DL-alanine methyl ester, after reacting 3h under 30 DEG C of conditions, reaction solution after reaction being transformed is centrifugal, other operations are with embodiment 3, the enantiomeric excess value e.e.99.6% of Boc-D-alanine methyl ester, Boc-L-L-Ala e.e.99.8%, substrate conversion efficiency 50.2%.

Embodiment 5:

Bacillus amyloliquefaciens WZZ002 dry mycelium 0.5g embodiment 2 method prepared adds in 10mL, pH7.0 phosphoric acid buffer containing 200g/L Boc-DL-alanine methyl ester, after reacting 3h under 30 DEG C of conditions, reaction solution after reaction transforms is centrifugal, other operations are with embodiment 3, the enantiomeric excess value e.e.99.9% of Boc-D-alanine methyl ester, Boc-L-L-Ala e.e.99.5%, substrate conversion efficiency 49.8%.

Embodiment 6:

Bacillus amyloliquefaciens WZZ002 dry mycelium 0.5g embodiment 2 method prepared respectively adds in 10mL, pH7.0 phosphoric acid buffer containing 100g/L Boc-DL-alanine methyl ester, Boc-DL-alanine ethyl ester, Boc-DL-L-Ala propyl ester and Boc-DL-alanine butyl ester successively, after reacting 3h under 30 DEG C of conditions, reaction solution after reaction transforms is centrifugal, other operations are with embodiment 3, obtain Boc-L-alanine ester compound respectively, result is as shown in table 1.

The different substrate conversion efficiency of table 1

Substrate title	Substrate conversion efficiency	Boc-D-alanine ester e.e.	Boc-L-L-Ala e.e.
				Boc-DL-alanine methyl ester	50.1％	99.9％	99.5％
Boc-DL-alanine ethyl ester	50.1％	99.9％	99.4％
				Boc-DL-L-Ala propyl ester	50.5％	99.6％	99.2％
Boc-DL-alanine butyl ester	50.2％	99.5％	99.0％

Embodiment 7:

Bacillus amyloliquefaciens WZZ002 dry mycelium 0.5g embodiment 2 method prepared adds in the 10mL pH7.0 phosphoric acid buffer containing 100g/L, different substrate, after reacting 5h under 30 DEG C of conditions, reaction solution after reaction transforms is centrifugal, and other operations are with embodiment 3, and reaction result is as shown in table 2.

The transformation efficiency of the different substrate of table 2

The above is only preferred embodiment of the present invention, not does any pro forma restriction to technology contents of the present invention.Every above embodiment is done according to technical spirit of the present invention any simple modification, equivalent variations and modification, all fall within the scope of protection of the present invention.

Claims (7)

1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) WZZ002, is preserved in China typical culture collection center, preservation address: China, Wuhan, Wuhan University, postcode 430072, deposit number CCTCC M 2013366, preservation date on August 8th, 2013.

2. bacillus amyloliquefaciens WZZ002 described in a claim 1 is splitting the application in N-tertbutyloxycarbonyl-DL-amino acid ester.

3. apply as claimed in claim 2, it is characterized in that described being applied as: with the dry mycelium of bacillus amyloliquefaciens WZZ002 after the wet thallus freeze-drying that fermentation culture obtains for catalyzer, with racemize N-tertbutyloxycarbonyl-DL-amino acid ester for substrate, be in the phosphoric acid buffer of 6 ~ 9 in pH value, conversion reaction is carried out at 20 ~ 50 DEG C, after reaction terminates, by reaction solution aftertreatment, obtain N-tertbutyloxycarbonyl-D-amino acid ester and N-tertbutyloxycarbonyl-L-amino acid.

4. apply as claimed in claim 3, it is characterized in that the quality consumption of described dry mycelium is 1 ~ 50g/L damping fluid, the starting point concentration of described substrate is 1 ~ 200g/L damping fluid.

5. apply as claimed in claim 3, it is characterized in that described substrate is N-tertbutyloxycarbonyl-DL-Alanine ester, N-tertbutyloxycarbonyl-DL-2-aminobutyric acid ester, N-tertbutyloxycarbonyl-DL-norvaline ester, N-tertbutyloxycarbonyl-DL-methionine ester, N-tertbutyloxycarbonyl-DL-proline ester, N-tertbutyloxycarbonyl-DL-serine ester, N-tertbutyloxycarbonyl-DL-LEUCINE ester, N-tertbutyloxycarbonyl-DL-Isoleucine ester, N-tertbutyloxycarbonyl-DL-Threonine ester, N-tertbutyloxycarbonyl-DL-Trp ester or N-tertbutyloxycarbonyl-DL-phenylalanine ester.

6. apply as claimed in claim 3, it is characterized in that described catalyzer is prepared as follows:

(1) slant culture

Bacillus amyloliquefaciens WZZ002 is seeded to slant medium, cultivates 24h at 30 DEG C, obtain inclined-plane bacterium colony; Slant medium final concentration forms: NaNO ₃3.0g/L, KH ₂pO ₄1.0g/L, MgSO ₄7H ₂o0.5g/L, KCl0.5g/L, FeSO ₄7H ₂o0.01g/L, Boc-DL-alanine ester 10mmol/L, solvent is deionized water, pH7.0;

(2) seed culture

The inclined-plane colony inoculation that step (1) is obtained in seed culture medium, 30 DEG C, 200r/min cultivates 24h, obtains seed liquor; Seed culture medium final concentration forms: peptone 5g/L, extractum carnis 5g/L, yeast extract paste 1g/L, maltose 5g/L, NaCl0.5g/L, agar 23g/L, and solvent is deionized water, pH7.0;

(3) fermentation culture

Aseptically, seed liquor is inoculated in fermention medium with the inoculum size of volumetric concentration 5 ~ 20%, 30 DEG C, 200r/min cultivates 24h, obtains fermented liquid, and fermented liquid is centrifugal, abandon supernatant liquor, obtain bacillus amyloliquefaciens WZZ002 wet thallus, wet thallus is suspended in the phosphate buffered saline buffer of pH7.0,0.2mol/L, stirs, lyophilize, obtains dry mycelium; Fermention medium consists of: peptone 5g/L, extractum carnis 5g/L, yeast extract paste 1g/L, MgSO ₄1mmol/L, solvent is deionized water, pH7.0.

7. apply as claimed in claim 3, it is characterized in that the method for described reaction solution aftertreatment is: after conversion reaction completely, conversion reaction liquid is crossed and filters thalline, filtrate organic solvent extraction, obtain organic phase and aqueous phase respectively, organic phase underpressure distillation, except desolventizing, obtains N-tertbutyloxycarbonyl-D-amino acid ester; Described organic solvent is normal hexane, hexanaphthene or ethyl acetate; After aqueous phase adjust pH to 2.0, with extraction agent extraction, get the organic phase after extraction and obtain N-tertbutyloxycarbonyl-L-amino acid, described extraction agent is normal hexane, hexanaphthene or ethyl acetate.