CN101215533A - Hydantoinase and carbamoyl hydrolase producing strain, bienzyme gene and application thereof for preparing L-amino acid - Google Patents

Hydantoinase and carbamoyl hydrolase producing strain, bienzyme gene and application thereof for preparing L-amino acid Download PDF

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CN101215533A
CN101215533A CNA2007101919687A CN200710191968A CN101215533A CN 101215533 A CN101215533 A CN 101215533A CN A2007101919687 A CNA2007101919687 A CN A2007101919687A CN 200710191968 A CN200710191968 A CN 200710191968A CN 101215533 A CN101215533 A CN 101215533A
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何冰芳
欧阳平凯
梅艳珍
柏中中
姜岷
周华
韦萍
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Nanjing Tech University
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Abstract

The invention relates to a new microbe Bacillus fordii MH602 strain which can simultaneously produce hydantoinase and L-N-carbamyl hydrolase, hydantoinase produced by strain, L-N-carbamyl hydrolase gene and an application for preparing serial optical pure L-amino acid through the strain. The hydantoinase and L-N-carbamyl hydrolase that are produced by Bacillus fordii MH602 strain can overcome the defect of worse activity and heat stability of hydantoinase and L-N-carbamyl hydrolase in the L-amino acid procedure prepared by prior biologically inverted DL-5 substitution hydantoin or N-hydantoin amino acid, which is all provided with wide substrate spectrum, can catalyze a plurality of substrates and has better substrate selectivity for aromatic hydantoin. The applying operation for preparing L-amino acid by Bacillus fordii MH602 is simple and stable in operation, good in repeatability, which realizes stable and cheap preparation of L-amino acid and is beneficial to do large-scale preparation.

Description

One strain glycolylurea enzyme and carbamyl hydrolysis enzyme produce bacterium, two enzyme gene and the amino acid whose application of preparation L-
Technical field
The invention belongs to the using microbe technical field, be specifically related to glycolylurea enzyme and the L-N-carbamyl hydrolysis enzyme gene that a kind of new microbe Bacillus fordii MH602 bacterial strain, this bacterial strain that produces glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme simultaneously produced and utilize this bacterial strain to prepare the amino acid whose application of serial optical purity L-.
Background technology
Natural L-amino acid and non-natural L-amino acid and derivative thereof are in the research that is widely used of industries such as food, medicine, agricultural.Glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme are one of crucial biological catalysts of producing in natural, non-natural L-amino acid and derivative thereof, and being specially adapted to human body that conventional fermentation method is difficult to produce must amino acid and alpha-non-natural amino acid.In recent years, non-natural L-amino acid can be used as the composition of functional protein inhibitor, and prevention HIV infects, the polypeptide ligand of fibrinogen deceptor, tachykinin antagenists, neuron receptors ligand etc.Alpha-non-natural amino acid is widely used in the synthesis of biologically active peptide, as the substitute of natural amino acid.To bring many benefits to be applied to polypeptide synthetic because of the hydrophobic side chain of its change: stop that hydrophobic pocket diminishes, lipophilicity height, stability are high, proteolytic enzyme is difficult to degraded.
There are notable difference in glycolylurea enzyme that different microorganisms produces or L-N-carbamyl hydrolysis enzyme together at aspects such as substrate specificity and thermostabilitys, thereby there are significant differences in the glycolylurea enzyme or the L-N-carbamyl hydrolysis enzyme that cause different microorganisms to produce aspect industrial application.Only transforming DL-5-methylthio ethyl glycolylurea as Bacillus and Arthrobacter is the L-methionine(Met), and Pseudomonas can transform DL-para hydroxybenzene glycolylurea and generates the D-D-pHPG.In producing the relevant amino acid whose technology of L-, glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme thermostability are lower, particularly L-N-carbamyl hydrolysis enzyme activity is low, poor stability, play the effect of " bottleneck ", as the L-N-carbamyl hydrolysis enzyme less stable from Agrobacterium, 30 ℃ of transformation period only are lO minute; Though the glycolylurea enzyme that belongs to from thermophilic archeobacteria Methanococcus has thermostability preferably, the transformation period is 100 minutes at 90 ℃, and this bacterium only produces the glycolylurea enzyme and only glycolylurea had catalytic effect preferably.
There are very big-difference equally in glycolylurea enzyme that the different microorganisms Pseudomonas is produced or L-N-carbamyl hydrolysis enzyme on gene order, the Hydantoinase gene sequence homology of being reported in the data only is 20～40%, it is 40～90% that identical bacterium microorganism species produce glycolylurea enzyme homology, and L-N-carbamyl hydrolysis enzyme homology is lower, homology is no more than 40%, as L-N-carbamyl hydrolysis enzyme (GeneBank reception AF425838) from Bacillus kaustophilus, the L-N-carbamyl hydrolysis enzyme of Arthrobater aurescens (GeneBank reception AF071221) is from L-N-carbamyl hydrolysis enzyme (GeneBank reception D90469) of Pseudomona sp.NS671 etc.And the homology of D-carbamyl hydrolysis enzyme and L-carbamyl hydrolysis enzyme is lower than 15%.
In recent years, glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme are used to research and develop related products, have characteristics such as technology is simple, optical purity height, study more active.Therefore seeking high stability and highly active glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme generation bacterium and studying the glycolylurea enzyme is that gene sequence characteristic is significant.
Summary of the invention
The purpose of this invention is to provide a strain and produce the bacterial strain of glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme simultaneously, glycolylurea enzyme that this bacterial strain produces and L-N-carbamyl hydrolysis enzyme can overcome present bio-transformation DL-5-and replace activity and the relatively poor shortcoming of thermostability that glycolylurea or N-carboxamide amino acid prepare glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme in the L-amino acid process, and this bacterial strain is successfully applied on the L-amino acid High-efficient Production.
For realizing above purpose, the technical solution used in the present invention is mainly divided following three parts:
One, the present invention screens the bacterial strain that a strain can be produced glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme simultaneously with DL-5-replacement glycolylurea as only nitrogen source from soil, classification called after Bacillus fordii MH602, and its preservation registration number is CCTCCNo:M 206144.
The present invention identifies Bacillus fordii MH602 through BIOLOG automatic bacterial assessing instrument, show that this bacterial strain MH602 and bacillus sp. similarity (SIM) are 0.52, and the 16SrDNA sequential analysis is identified and to be shown that being higher than 98% bacterial strain with this sequence similarity degree is Bacillus and belongs to bacterial strain, is 98.6% with bacterial strain BacillusfordiiAY443039 similarity wherein.
The present invention identifies that to the biological characteristic of Bacillus fordii MH602 this bacterial strain is a gram-positive microorganism, has gemma to form.Growth is after 16 hours in the meat soup plate culture medium, and the bacterium colony size is 2～2.5mm, and growth temperature range is 40～55 ℃, optimum temperuture is 45 ℃, and growth pH is 6.5～8.5, and optimal pH is 7.5, its Physiology and biochemistry shows that the superoxide enzyme test positive is grown under aerobic conditions.
Two, the present invention classifies the design degenerated primer as according to retrieving the Hydantoinase gene total order among the GeneBank.The Hydantoinase gene sequence homology that produces bacterium because of generic glycolylurea enzyme not is lower, relatively finds the back through sequence, and the Hydantoinase gene sequence homology of the bacterial strain generation of same genus is higher, so degenerated primer designs with the Hydantoinase gene sequence of same genus.Adopt the amplification of genomic walking method to obtain glycolylurea enzyme complete genome sequence, L-N-carbamyl hydrolysis enzyme gene then.
The present invention is isolated and cloned into the encoding gene of the glycolylurea enzyme of this bacterial strain, and it has the nucleotide sequence shown in the SEQ ID NO:1, can be used for making up the genetic engineering bacterium of glycolylurea enzyme, is used for researchs such as the conversion of related substrates and this enzyme molecular structure transformation.By PCR method separating clone this Hydantoinase gene, the DNA complete sequence analysis is the result show, this Hydantoinase gene total length 1428bp, 474 amino acid of encoding altogether, initiator codon is ATG, terminator codon is TGA, its aminoacid sequence is shown in SEQ ID NO:2.Glycolylurea enzyme homology is up to 70% in this glycolylurea enzyme and the database, is illustrated as a new glycolylurea enzyme.
The present invention is isolated and cloned into the encoding gene of the L-N-carbamyl hydrolysis enzyme of this bacterial strain, it has the nucleotide sequence shown in the SEQ IDNO:3, can be used for making up the genetic engineering bacterium of L-N-carbamyl hydrolysis enzyme, be used for researchs such as the conversion of related substrates and this enzyme molecular structure transformation.By PCR method separating clone the gene of L-N-carbamyl hydrolysis enzyme, the DNA complete sequence analysis is the result show, this Hydantoinase gene total length 1242bp, 412 amino acid of encoding altogether, initiator codon is ATG, and terminator codon is TGA, and its aminoacid sequence is shown in SEQ ID NO:4.L-N-carbamyl hydrolysis enzyme homology is up to 38% in this L-N-carbamyl hydrolysis enzyme and the database, is illustrated as a new L-N-carbamyl hydrolysis enzyme.
The present invention is connected the glycolylurea enzyme respectively with the L-N-carbamyl hydrolysis enzyme with carrier pET-22b, transform large intestine bar BL21 (DE3) bacterial strain, behind abduction delivering, utilizes the HPLC method to detect glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme activity.
Glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme that the Bacillus fordii MH602 bacterial strain of the present invention's screening is produced have thermostability and substrate tolerance preferably preferably, effectively avoid high density product and the substrate restraining effect to glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme.
Three, the invention provides Bacillus fordii MH602 bacterial strain in the amino acid whose application of preparation L-, relate generally to and utilize Bacillus fordii MH602 fermentation producing L-amino-acid.Its principle is as follows:
Figure S2007101919687D00031
Utilization provided by the invention comprises process in the amino acid whose application of preparation L-: thalline is replaced in the fermented liquid that glycolylurea is an inductor at DL-5-ferment; Collect thalline and make bacteria suspension and smudge cells; Add substrate glycolylurea catalyzed reaction, obtain L-amino acid.Wherein, DL-5-replacement glycolylurea solubleness is lower mostly, mainly exists with solid form in solution, along with the carrying out of reaction, the glycolylurea enzyme replaces glycolylurea with DL-5-and is converted into N-carboxamide-amino acid intermediate, and the L-N-carbamyl hydrolysis enzyme further is converted into intermediate soluble L-amino acid.Suitably improve temperature and help to increase the racemization speed that DL-5-replaces the solubleness of glycolylurea and increases glycolylurea, reaction is constantly moved to generating amino acid whose direction.The glycolylurea enzyme of thermostability and carbamyl hydrolysis enzyme help improving substrate conversion efficiency and seem particularly important.
Bacterial strain Bacillus fordii MH602 of the present invention is characterized in that in the amino acid whose application of preparation L-described L-amino acid is natural amino acid.
Bacterial strain Bacillus fordii MH602 of the present invention is characterized in that in the amino acid whose application of the natural L-of preparation described natural L-amino acid comprises phenylalanine, phenylglycocoll, D-pHPG, glycine, L-Ala, Xie Ansuan, methionine(Met).
Bacterial strain Bacillus fordii MH602 of the present invention is characterized in that in the amino acid whose application of preparation L-described L-amino acid is alpha-non-natural amino acid.
Bacterial strain Bacillus fordii MH602 of the present invention is in preparation non-natural L-amino acid whose application, it is characterized in that described non-natural L-amino acid comprise hyperphenylalaninemia, L-DOPA, silica-basedization of L-3-amino acid, to nitro-L-phenylalanine, to chloro-L-phenylalanine.
Beneficial effect of the present invention is that the new bacterial strain Bacillus fordii MH602 that screens can produce the glycolylurea enzyme and the carbamyl hydrolysis enzyme of thermostability and substrate tolerance; effectively avoid high density product and substrate that restraining effect and this two kinds of enzymes of glycolylurea enzyme and carbamyl hydrolysis enzyme are all had substrate spectrum widely; but the multiple substrate of catalysis; and a fragrance bunch glycolylurea there is substrate selective preferably; for the natural amino acid and non-protein amino acid that are difficult to produce with fermentation method or chemical synthesis; and utilize Bacillus fordii MH602 simple at the application operating of L-amino acid preparation; stable; good reproducibility; realized that L-amino acid is stable; produce inexpensively, help large-scale production.
Description of drawings
Fig. 1 glycolylurea enzyme is gene amplification primer design diagram (AP is a random primer)
Fig. 2 derives from the glycolylurea enzyme of MH602 and the thermostability of carbamyl hydrolysis enzyme
Fig. 3 derives from the glycolylurea enzyme of MH602 and the substrate tolerance of carbamyl hydrolysis enzyme
The microbial preservation date of the present invention is on December 31st, 2006, and depositary institution's full name is Chinese typical culture collection center, is called for short CCTCC, deposit number: CCTCC No:M 206144.
Embodiment
Embodiment 1
The source of this description of test bacterial classification and screening, qualification program.
Screening culture medium (gL -1): DL-5-replaces glycolylurea 20, sucrose 20, potassium primary phosphate 3, sodium-chlor 1, sal epsom 0.25, yeast extract paste 0.1, and pH 7.5;
Phenol red plate culture medium (gL -1): glycolylurea or methyl hydantoin 3, peptone 5, yeast extract paste 3, sodium-chlor 3, phenol red 0.05, agar 20, pH 7.7;
Fermention medium (gL -1): methyl hydantoin 3, sucrose 20, yeast extract paste 1, sodium-chlor 3, corn steep liquor 20mL (the about 4g of dry weight), potassium primary phosphate 3, sal epsom 0.25, pH 7.5;
Meat soup plate culture medium (gL -1): peptone 10, extractum carnis 5, sodium-chlor 5, agar 20, pH 7.5.
Primary dcreening operation:
Gather about 1500 of soil sample from occurring in nature, get to add about 3～4 kinds of each 1g of soil sample and contain to go into to have in the test tube of 10mL screening culture medium, in 45 ℃, 200r.m -1Enrichment culture was transferred in fresh screening and culturing after three to four days under the condition, transferred after three to four times, and the nutrient solution that takes a morsel suitably dilutes, and evenly coats on the phenol red plate culture medium.After cultivating 16h, observe the variable color circle of periphery of bacterial colonies, will have the bacterial classification of obvious variable color circle evolve single from, and be inoculated in the meat soup plate culture medium and cultivate.
Multiple sieve:
Scrape from broth culture and to get a ring thalline, be seeded to the shaking in the bottle of fermention medium is housed.45C, 200r.m -1After cultivating 18h under the condition, centrifugal, the gained thalline is divided into two parts adds respectively and contain the substrate phosphoric acid buffer (pH 8.0) of 2% benzyl glycolylurea and contain in the substrate phosphoric acid buffer (pH 8.0) of 2% different third glycolylurea conversion 1h.Supernatant after conversion fluid is centrifugal is used to detect intermediate and product amino acid.
Identify:
The present invention identifies Bacillus fordii MH602 through BIOLOG automatic bacterial assessing instrument, show that this bacterial strain MH602 and bacillus sp similarity (SIM) are 0.52, and the 16SrDNA sequential analysis is identified and to be shown that being higher than 98% bacterial strain with this sequence similarity degree is Bacillus and belongs to bacterial strain, is 98.6% with bacterial strain BacillusfordiiAY443039 similarity wherein.
Embodiment 2
The biological property of this description of test Bacillus fordii MH602.
The present invention identifies that to the biological characteristic of Bacillus fordii MH602 this bacterial strain is a gram-positive microorganism, has gemma to form.Growth is after 16 hours in the meat soup plate culture medium, and the bacterium colony size is 2～2.5mm, and growth temperature range is 40～55 ℃, optimum temperuture is 45 ℃, and growth pH is 6.5～8.5, and optimal pH is 7.5, its Physiology and biochemistry shows that the superoxide enzyme test positive is grown under oxygen condition.
Embodiment 3
The separating clone program of this description of test glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme gene.
Adopt the total DNA of phenol-chloroform method extracting thalline.According to the Hydantoinase gene sequence homology, the design degenerated primer, amplification Hydantoinase gene partial sequence further adopts genomic walking method its flanking sequence that increases.Obtain the encoding sequence of key enzyme glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme.The PCR fragment electrophoresis that contains the maturing enzyme encoding sequence reclaims rear clone to pMDl 8-T carrier, carries out sequential analysis.The result shows, the reading frame that it is 1428bp and 1242bp that this fragment has a total length, and the amino acid number of encoding mature glycolylurea enzyme and carbamyl hydrolysis enzyme is 474 and 412 respectively.Its amplification procedure as shown in Figure 1.
Part Hydantoinase gene fragment H1 amplimer
Homology analysis design degenerated primer P1 and P2 amplification according to Hydantoinase gene obtain Hydantoinase gene partial sequence H1, see Fig. 1.
P1(sense)：5′GTN CGN GAA GAN GAN TGT GT 3′
P2(antisense)：5’CCN GTC GCY TCN CCT TC 3’
The PCR reaction parameter is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec; 55 ℃ of annealing 30sec; 72 ℃ are extended 1min; After circulation 30 is taken turns, 72 ℃ of insulation 10min.According to this reaction conditions, the H1 fragment of about 0.9kb that increased.
The amplimer of sequence before and after the gene H1 fragment
At the about 100-120bp of the segmental afterbody of the Hydantoinase gene H1 place design special primer P3 that has increased, nested PCR primer is P3-1 and P3-2.P3 respectively with four AP random primer amplified fragments H2, P3-1 and P3-2 are used to verify whether amplified fragments H2 is the target gene fragment.The H3 fragment that in like manner increases and H4 fragment.
P3 (sense): 5 ' biotin labeling-ACG GTG GTC TAG TGAT 3 '
P3-1(sense)：5’ACG GTG GTC TAG TGATGG T 3’
P3-2(antisense)：5’ACG GGT CAA GGC ATG ATA G 3’
P4 (antisense): 5 ' biotin labeling-ATG ACA TCG GTG GTT C 3 '
P4-1(sense)：5’TTG AAG GTC AGG GAA GAA G 3’
P4-2(antisense)：5’TCC ATGACATCG GTG GTT C 3’
P5 (sense): 5 ' biotin labeling-CAC CTT GAC AGC AGAT 3 '
P5-1(sense)：5‘CAC CTT GAC AGC AGA TAT C 3’
P5-2(antisense)：5’AAT GAC GAT AAT GAA GGC TG 3’
Step is moved primer
Random primer AP series:
AP 1.5’CTA ATACGACTCACTATAGGGNNNNATGC 3’
AP 2.5’CTA ATACGACTCACTATAGGGNNNNGATC 3’
AP 3.5’CTA ATACGACTCACTATAGGGNNNNTAGC 3’
AP 4.5’CTA ATACGACTCACTATAGGGNNNNCTAG 3’
Embodiment 4
This description of test glycolylurea enzyme and the active measuring method of L-N-carbamyl hydrolysis enzyme.
Adopt the HPLC marker method.With DL-β-benzene Serine is internal standard substance.Chromatographic column Chromasil C18 detects N-carboxamide phenylalanine (NCPA) and phenylalanine (Phe) content; Moving phase, methyl alcohol: 20 (mM) KH 2PO 4=20: 80; Flow velocity, 1mL min -1Column temperature, 30 ℃.Phe graticule: Y=1.7899X-0.0617, R=0.99876; NCPA graticule: Y=2.0369X-0.08135, R=0.9946, X refer to the ratio of the peak area of intermediate or product and internal standard substance, and Y is corresponding product intermediate or amino acid concentration, and unit is gL -1The glycolylurea enzymic activity is to transform 0.1molL -1DL-5-benzyl glycolylurea generates the amount of NCPA and calculates, and comprises the NCPA that remains in the conversion fluid and has been converted into amino acid whose NCPA; The carbamyl hydrolysis enzyme activity is calculated to generate amino acid whose amount.Different strains glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme specific activity are as table 1.
Table 1 different strains glycolylurea enzyme activity relatively
Bacterial strain Glycolylurea enzyme/mgmL -1 Bacterial strain L-N-carbamyl hydrolysis enzyme/mgmL -1
BL21(DE3)-pET-22b 0 BL21(DE3)-pET-22b 0
BL21(DE3)-pET-Hase 4.91 BL21(DE3)-pET-Case 0.44
MH602 15.6 MH602 7.4
Embodiment 5
The measuring method of the zymologic property of this description of test glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme.
The mensuration of the thermostability of glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme
To in fermention medium, cultivate and ultrasonication after cell suspension be sub-packed in the different reaction tubess, after six hours, add substrate 20gL in 4 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ insulations -1DL-5-benzyl glycolylurea, in 45 ℃ of conversion 3h, reaction finishes back termination reaction in ice bath, measures intermediate NCPA and product P he concentration in the conversion fluid, as Fig. 2.The growing amount of product P he can be found out from figure, and the glycolylurea enzyme is all having activity below 65 ℃, and carbamyl hydrolysis enzyme is having activity below 50 ℃, glycolylurea enzyme and carbamyl hydrolysis enzyme crude enzyme liquid respectively after 50 ℃ and 45 ℃ keep 6h, active no significantly sacrificing.Two enzymes enzyme in the time of 45 ℃ is lived all more stable.Surpass 50 ℃, carbamyl hydrolysis enzyme is active obviously to descend.Two enzymes that the MH602 of this research screening produces have thermostability preferably with respect to the relevant enzyme of having reported.
The mensuration of the substrate tolerance of glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme
Substrate and production concentration too high in the reaction system often have restraining effect to enzymic activity.To in fermention medium, cultivate and ultrasonication after cell suspension be sub-packed in the different reaction tubess, add different concns substrate DL-5-benzyl glycolylurea, transform 3h in 45 ℃, reaction finishes back termination reaction in ice bath, measure intermediate NCPA and product amino acid Phe concentration in the conversion fluid, by investigating the influence of benzyl glycolylurea concentration increase, understand the influence that concentration of substrate forms product, as Fig. 3 to two enzymic activitys.As can be seen from Figure, be no more than 30gL when concentration of substrate -1The time, the almost proportional rising of intermediate NCPA growing amount, and concentration of substrate is 30～40gL -1The time, although the not proportional rising of intermediate NCPA growing amount, but still in rising trend, illustrate at 40gL -1The concentration of substrate scope in, substrate does not have obvious restraining effect to the glycolylurea enzyme.
Embodiment 6
This description of test glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme are in the application of producing on the L-amino acid.
Thalline replaces in the fermented liquid that glycolylurea is an inductor at DL-5-and ferments
Prepare fermentation culture by following weight ratio: sucrose 10g/L, corn steep liquor 20mL/L, DL-5-replaces glycolylurea 3g/L, sal epsom 0.25g/L, sodium-chlor 3g/L, potassium primary phosphate 3g/L transfers pH 7.0.Sterilization back Bacillus fordiiMH602 inoculum size is 5% (seed culture medium is identical, and incubation time is 12h), and being 45 ℃ in temperature is under the 180rpm vibration with rotating speed, fermentation 16h.
Collect thalline and make bacteria suspension and smudge cells
Thalline is collected in this bacterium fermentation back, directly the thalline reaction solution is used for ultrasonication 15min; Or adding one of following broken wall agent, the broken wall agent can be selected CTAB (cetyl trimethylammonium bromide) for use, SDS (sodium laurylsulfonate), used concentration by weight percentage 1～3%.
Add substrate glycolylurea catalyzed reaction, obtain L-amino acid
Thalline behind the broken wall is added various substrate glycolylureas (is 20g/L with the glycolylurea calculating concentration), ultrasonication 15min, 45 ℃ of oscillatory reaction 3h promptly obtain L-amino acid.
Because of adding the substrate difference, can obtain the amino acid whose productive rate of different L-and see Table 2 with relative productive rate.
The application of table 2 MH602 on various L-amino acid are produced
Substrate The glycolylurea enzyme The L-N-carbamyl hydrolysis enzyme Corresponding amino acid
Product a b Product a b
/g·L -1 /％ ％ /g·L -1 /％ /％
Natural amino acid benzyl glycolylurea 20.3 98 100 11.8 72 100 Phenylalanine
The benzene glycolylurea 19.0 98 100 10.7 71 95 Phenylglycocoll
The para hydroxybenzene glycolylurea 16.2 77 79 7.7 67 65 D-pHPG
Glycolylurea 9.5 82 97 4.5 60 85 Glycine
Methyl hydantoin 12.2 93 95 5.6 61 89 L-Ala
Different third glycolylurea 7.01 44 45 2.9 24 35 Xie Ansuan
The methylthio ethyl glycolylurea 4.5 24 24 0.63 4 6 Methionine(Met)
Alpha-non-natural amino acid DL-styroyl glycolylurea 18.3 91 92 9.9 78 91.2 Hyperphenylalaninemia
DL-5-3,4-dihydroxy benzyl glycolylurea 15.9 70 100 10.2 70 92.5 The L-DOPA
DL-5-replaces silica-basedization glycolylurea 16.1 77 91 8.8 64 77.1 Silica-basedization of L-3-amino acid
To the nitro glycolylurea 9.75 56 89 5.2 45 67.0 To nitro-L-phenylalanine
The p-chlorobenzyl glycolylurea 10.1 81 70 6.3 55 86.9 To chloro-L-phenylalanine
Annotate: a, molar yield; B, the relative reactivity of enzyme
Sequence table
＜110〉Nanjing University of Technology
＜120〉a strain glycolylurea enzyme and carbamyl hydrolysis enzyme produce bacterium, two enzyme gene and the amino acid whose application of preparation L-
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<170>PatentIn version 3.3
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＜213〉Bacillus fordii MH602 glycolylurea enzyme
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atg aaa aaa atc att aaa gat gga aca gt t gta acc tcc aca gat gtc 48
Lys Lys Ile Ile Lys Asp Gly Thr Val Val Thr Ser Thr Asp Val
1 5 10 15
tat acc gca gat gta tta att gaa gat gaa aaa att cag gca atc ggt 96
Tyr Thr Ala Asp Val Leu Ile Glu Asp Glu Lys Ile Gln Ala Ile Gly
20 25 30
caa aac ata agc gat ata tat gct gaa gtg atc gat gcc tcc ggt tat 144
Gln Asn Ile Ser Asp Ile Tyr Ala Glu Val Ile Asp Ala Ser Gly Tyr
35 40 45
tat gtg atg cca gga ggt att gat ccg cat aca cat atg gat atg ccg 192
Tyr Val Met Pro Gly Gly Ile Asp Pro His Thr His Met Asp Met Pro
50 55 60
ttc ggt gga act gtt aca gca gat gat ttt gaa act ggt tcc ata gca 240
Phe Gly Gly Thr Val Thr Ala Asp Asp Phe Glu Thr Gly Ser Ile Ala
65 70 75
gcg gca tgc gga gga aca acg aca atc att gat ttt tgc ttg acg gga 288
Ala Ala Cys Gly Gly Thr Thr Thr Ile Ile Asp Phe Cys Leu Thr Gly
80 85 90 95
aaa gga aaa tca tta aaa agc gct ttg gaa aaa tgg cat gcc aaa tca 336
Lys Gly Lys Ser Leu Lys Ser Ala Leu Glu Lys Trp Hi s Ala Lys Ser
100 105 110
gcg ggt aaa gct gtg att gac tat gga ttt cac ttg cag atc gcg gaa 384
Ala Gly Lys Ala Val Ile Asp Tyr Gly Phe His Leu Gln Ile Ala Glu
115 120 125
gca aat gaa gaa gta ttt atg gaa atg cct cag ata att gaa gaa gaa 432
Ala Asn Glu Glu Val Phe Met Glu Met Pro Gln Ile Ile Glu Glu Glu
130 135 140
gga gtt act tct ttt aaa ata ttc atg gct tat aag gat gta tta caa 480
Gly Val Thr Ser Phe Lys Ile Phe Met Ala Tyr Lys Asp Val Leu Gln
145 150 155
gcg gat gat gag acc ctt ttc cag aca ttg gtc acg gcc agg gaa cac 528
Ala Asp Asp Glu Thr Leu Phe Gln Thr Leu Val Thr Ala Arg Glu His
160 165 170 175
ggt ggt cta gtg atg gtg cat gcg gaa aat ggg gat gtc att aac tat 576
Gly Gly Leu Val Met Val His Ala Glu Asn Gly Asp Val Ile Asn Tyr
180 185 190
ttg aca gaa aaa gcc tta aag gaa gga aat acg gaa cct atc tat cat 624
Leu Thr Glu Lys Ala Leu Lys Glu Gly Asn Thr Glu Pro Ile Tyr His
195 200 205
gcc ttg acc cgt ccg cca gag ctt gag gga gaa gcg act gga cgt gcc 672
Ala Leu Thr Arg Pro Pro Glu Leu Glu Gly Glu Ala Thr Gly Arg Ala
210 215 220
gca agg ctc act gcg ctg gcg gat tct caa ttg tat gtg gtt cat gtg 720
Ala Arg Leu Thr Ala Leu Ala Asp Ser Gln Leu Tyr Val Val His Val
225 230 235
acg tgc aag gaa gca gtt gaa caa att gct gaa gca cgc aga agg gga 768
Thr Cys Lys Glu Ala Val Glu Gln Ile Ala Glu Ala Arg Arg Arg Gly
240 245 250 255
tat cgt gtt ttt gga gag acg tgc cca cag tat ctt gtt ctt gat caa 816
Tyr Arg Val Phe Gly Glu Thr Cys Pro Gln Tyr Leu Val Leu Asp Gln
260 265 270
act tac tta gaa agg ccg gat ttt gag ggg gca aaa tat gtc tgg tcg 864
Thr Tyr Leu Glu Arg Pro Asp Phe Glu Gly Ala Lys Tyr Val Trp Ser
275 280 285
cct cca tta cga gag cgt tca aat cag gaa gtt tta tgg aat gca ttg 912
Pro Pro Leu Arg Glu Arg Ser Asn Gln Glu Val Leu Trp Asn Ala Leu
290 295 300
aaa agc gga gat ctg cag gcg ata ggc tct gac cat tgt tct ttc aat 960
Lys Ser Gly Asp Leu Gln Ala Ile Gly Ser Asp His Cys Ser Phe Asn
305 310 315
ttt aga ggt caa aaa gaa ttg ggg aaa aat gat ttt tct aaa ata ccg 1008
Phe Arg Gly Gln Lys Glu Leu Gly Lys Asn Asp Phe Ser Lys Ile Pro
320 325 330 335
aat gga ggc ccc ttt gtc gaa gac cga ttt agc gtc tta ttt tca gaa 1056
Asn Gly Gly Pro Phe Val Glu Asp Arg Phe Ser Val Leu Phe Ser Glu
340 345 350
ggt gtg aaa aaa ggc cgg att tca atc cat gat ttt gtc aat att ata 1104
Gly Val Lys Lys Gly Arg Ile Ser Ile His Asp Phe Val Asn Ile Ile
355 360 365
tcg aca cag gca gcc aaa ttg ttt gga ttg ttc cca aga aag ggg acg 1152
Ser Thr Gln Ala Ala Lys Leu Phe Gly Leu Phe Pro Arg Lys Gly Thr
370 375 380
ata gcc ccg ggc agt gac gct gat att gtt ata ttt gat cca aat gtt 1200
Ile Ala Pro Gly Ser Asp Ala Asp Ile Val Ile Phe Asp Pro Asn Val
385 390 395
gaa cgg atc att tct gtt gag act cat cat atg aat gtg gat tat agc 1248
Glu Arg Ile Ile Ser Val Glu Thr His His Met Asn Val Asp Tyr Ser
400 405 410 415
gca ctt gaa ggg ttg aag att atc gga gaa ccg att aca gtg ctc tca 1296
Ala Leu Glu Gly Leu Lys Ile Ile Gly Glu Pro Ile Thr Val Leu Ser
420 425 430
cgt gga aat tat gtg gtg aag gat aaa gaa ttc gtc gga aag cct ggg 1344
Arg Gly Asn Tyr Val Val Lys Asp Lys Glu Phe Val Gly Lys Pro Gly
435 440 445
aaa ggg aaa ttt tta aaa tgc aac aaa ttc aat cac gat ttg tat aag 1392
Lys Gly Lys Phe Leu Lys Cys Asn Lys Phe Asn His Asp Leu Tyr Lys
450 455 460
gaa act ggt caa ttg gag ggt ttg tta aaa ggg tga 1428
Glu Thr Gly Gln Leu Glu Gly Leu Leu Lys Gly
465 470
<210>2
<211>474
<212>PRT
＜213〉Bacillus fordii MH602 glycolylurea enzyme
<400>2
Lys Lys Ile Ile Lys Asp Gly Thr Val Val Thr Ser Thr Asp Val Tyr
1 5 10 15
Thr Ala Asp Val Leu Ile Glu Asp Glu Lys Ile Gln Ala Ile Gly Gln
20 25 30
Asn Ile Ser Asp Ile Tyr Ala Glu Val Ile Asp Ala Ser Gly Tyr Tyr
35 40 45
Val Met Pro Gly Gly Ile Asp Pro His Thr His Met Asp Met Pro Phe
50 55 60
Gly Gly Thr Val Thr Ala Asp Asp Phe Glu Thr Gly Ser Ile Ala Ala
65 70 75 80
Ala Cys Gly Gly Thr Thr Thr Ile Ile Asp Phe Cys Leu Thr Gly Lys
85 90 95
Gly Lys Ser Leu Lys Ser Ala Leu Glu Lys Trp His Ala Lys Ser Ala
100 105 110
Gly Lys Ala Val Ile Asp Tyr Gly Phe His Leu Gln Ile Ala Glu Ala
115 120 125
Asn Glu Glu Val Phe Met Glu Met Pro Gln Ile Ile Glu Glu Glu Gly
130 135 140
Val Thr Ser Phe Lys Ile Phe Met Ala Tyr Lys Asp Val Leu Gln Ala
145 150 155 160
Asp Asp Glu Thr Leu Phe Gln Thr Leu Val Thr Ala Arg Glu His Gly
165 170 175
Gly Leu Val Met Val His Ala Glu Asn Gly Asp Val Ile Asn Tyr Leu
180 185 190
Thr Glu Lys Ala Leu Lys Glu Gly Asn Thr Glu Pro Ile Tyr His Ala
195 200 205
Leu Thr Arg Pro Pro Glu Leu Glu Gly Glu Ala Thr Gly Arg Ala Ala
210 215 220
Arg Leu Thr Ala Leu Ala Asp Ser Gln Leu Tyr Val Val His Val Thr
225 230 235 240
Cys Lys Glu Ala Val Glu Gln Ile Ala Glu Ala Arg Arg Arg Gly Tyr
245 250 255
Arg Val Phe Gly Glu Thr Cys Pro Gln Tyr Leu Val Leu Asp Gln Thr
260 265 270
Tyr Leu Glu Arg Pro Asp Phe Glu Gly Ala Lys Tyr Val Trp Ser Pro
275 280 285
Pro Leu Arg Glu Arg Ser Asn Gln Glu Val Leu Trp Asn Ala Leu Lys
290 295 300
Ser Gly Asp Leu Gln Ala Ile Gly Ser Asp His Cys Ser Phe Asn Phe
305 310 315 320
Arg Gly Gln Lys Glu Leu Gly Lys Asn Asp Phe Ser Lys Ile Pro Asn
325 330 335
Gly Gly Pro Phe Val Glu Asp Arg Phe Ser Val Leu Phe Ser Glu Gly
340 345 350
Val Lys Lys Gly Arg Ile Ser Ile His Asp Phe Val Asn Ile Ile Ser
355 360 365
Thr Gln Ala Ala Lys Leu Phe Gly Leu Phe Pro Arg Lys Gly Thr Ile
370 375 380
Ala Pro Gly Ser Asp Ala Asp Ile Val Ile Phe Asp Pro Asn Val Glu
385 390 395 400
Arg Ile Ile Ser Val Glu Thr His His Met Asn Val Asp Tyr Ser Ala
405 410 415
Leu Glu Gly Leu Lys Ile Ile Gly Glu Pro Ile Thr Val Leu Ser Arg
420 425 430
Gly Asn Tyr Val Val Lys Asp Lys Glu Phe Val Gly Lys Pro Gly Lys
435 440 445
Gly Lys Phe Leu Lys Cys Asn Lys Phe Asn Hi s Asp Leu Tyr Lys Glu
450 455 460
Thr Gly Gln Leu Glu Gly Leu Leu Lys Gly
465 470
<210>3
<211>1242
<212>DNA
＜213〉Bacillus fordii MH602 L-N-carbamyl hydrolysis enzyme
<220>
<221>CDS
<222>(4)..(1239)
<400>3
atg gaa aaa caa aaa gtg ctt atc aat ggg gaa aga cta aag gat aca 48
Glu Lys Gln Lys Val Leu Ile Asn Gly Glu Arg Leu Lys Asp Thr
1 5 10 15
ata gaa gaa ttc gct gat ttc ggc agg act gaa aaa aat ggg gtt gcc 96
Ile Glu Glu Phe Ala Asp Phe Gly Arg Thr Glu Lys Asn Gly Val Thr
20 25 30
cgt ttg gca tta tcc gat gtt gat gtt aaa gca aga aga cat ttt caa 144
Arg Leu Ala Leu Ser Asp Val Asp Val Lys Ala Arg Arg His Phe Gln
35 40 45
agc tta tgt gaa cag cta ggc atg tcg gtt gtg tgg gac gat atg gga 192
Ser Leu Cys Glu Gln Leu Gly Met Ser Val Val Trp Asp Asp Met Gly
50 55 60
aat atg tat gca aaa ttg ccg gga att gat aat gat cag cct ccg gtg 240
Asn Met Tyr Ala Lys Leu Pro Gly Ile Asp Asn Asp Gln Pro Pro Val
65 70 75
gtt atc gga tcc cat ttg gat tcg gtg aaa aaa ggc gga cgg ttt gat 288
Val Ile Gly Ser His Leu Asp Ser Val Lys Lys Gly Gly Arg Phe Asp
80 85 90 95
ggt acg ctt ggt gtg ttg act gga ctt gaa gtt gtc agg acg atg gtt 336
Gly Thr Leu Gly Val Leu Thr Gly Leu Glu Val Val Arg Thr Met Val
100 105 110
gaa aac ggc atc aag cct gag ata cct atc att gtt gct aat att acg 384
Glu Asn Gly Ile Lys Pro Glu Ile Pro Ile Ile Val Ala Asn Ile Thr
115 120 125
aat gaa gaa ggt gcg cgg ttt gaa cct tcg ttg atg gct tca ggt gtt 432
Asn Glu Glu Gly Ala Arg Phe Glu Pro Ser Leu Met Ala Ser Gly Val
130 135 140
ctt tcc gga cgt ttt gat aaa gca gcc atg ctg aaa tca aca gat gtg 480
Leu Ser Gly Arg Phe Asp Lys Ala Ala Met Leu Lys Ser Thr Asp Val
145 150 155
gat gga atc aca ttt gca gaa gca ctc aaa aaa agc ggc tat gaa gga 528
Asp Gly Ile Thr Phe Ala Glu Ala Leu Lys Lys Ser Gly Tyr Glu Gly
160 165 170 175
aaa aaa gag aat cgt tta aaa gaa gca gct gct ttt ttg gaa tta cat 576
Lys Lys Glu Asn Arg Leu Lys Glu Ala Ala Ala Phe Leu Glu Leu His
180 185 190
att gaa cag gga cct gtt ttg gaa agt gag gac att cag att ggt ata 624
Ile Glu Gln Gly Pro Val Leu Glu Ser Glu Asp Ile Gln Ile Gly Ile
195 200 205
gtt gag tgt gtt gtc ggc atg gtg tgt ttt gaa ata gaa gtc act gga 672
Val Glu Cys Val Val Gly Met Val Cys Phe Glu Ile Glu Val Thr Gly
210 215 220
gaa tct gac cat gca ggt act aca cca atg tca atg aga aaa gat gct 720
Glu Ser Asp His Ala Gly Thr Thr Pro Met Ser Met Arg Lys Asp Ala
225 230 235
ttg ttt gct gct aat caa ttg att tcg gaa ata cgc cag aaa atg aat 768
Leu Phe Ala Ala Asn Gln Leu Ile Ser Glu Ile Arg Gln Lys Met Asn
240 245 250 255
agg tta gat gat caa ctc gta tac acg gtt gga agg atg acg gtc agt 816
Arg Leu Asp Asp Gln Leu Val Tyr Thr Val Gly Arg Met Thr Val Ser
260 265 270
cca aat atc cat act gta ata cca aat aag gtt gtg ttc aca ata ggg 864
Pro Asn Ile His Thr Val Ile Pro Asn Lys Val Val Phe Thr Ile Gly
275 280 285
gcc agg cat caa gat gga aaa ata ata cgg cag gtg gaa gaa atc att 912
Ala Arg His Gln Asp Gly Lys Ile Ile Arg Gln Val Glu Glu Ile Ile
290 295 300
caa gga ctg ccg aat tca tcc gga aaa gaa aaa tgt aat gta aca acc 960
Gln Gly Leu Pro Asn Ser Ser Gly Lys Glu Lys Cys Asn Val Thr Thr
305 310 315
acc aaa tta tgg gat cgt cat acg gta tgg ttt aat gaa gaa atc gtg 1008
Thr Lys Leu Trp Asp Arg His Thr Val Trp Phe Asn Glu Glu Ile Val
320 325 330 335
aat aca ctg gaa aaa tcg gca aga agt ctt gga tat tcc ttt aag cgt 1056
Asn Thr Leu Glu Lys Ser Ala Arg Ser Leu Gly Tyr Ser Phe Lys Arg
340 345 350
atg gtt agt gga gcg ggg cat gat gca cag ttt att gca acg tat atc 1104
Met Val Ser Gly Ala Gly His Asp Ala Gln Phe Ile Ala Thr Tyr Ile
355 360 365
ccg acg gca atg gtc ttt gtc ccc agt atc aac ggg aaa agt cat gac 1152
Pro Thr Ala Met Val Phe Val Pro Ser Ile Asn Gly Lys Ser His Asp
370 375 380
gaa gat gag ttg aca acg tgg gaa gat tgt gaa aat gga gtg aat gtt 1200
Glu Asp Glu Leu Thr Thr Trp Glu Asp Cys Glu Asn Gly Val Asn Val
385 390 395
att tta cag aca gtt tta gat tta aca acg gat aag ttg tga 1242
Ile Leu Gln Thr Val Leu Asp Leu Thr Thr Asp Lys Leu
400 405 410
<210>4
<211>412
<212>PRT
＜213〉Bacillus fordii MH602 L-N-carbamyl hydrolysis enzyme
<400>4
Glu Lys Gln Lys Val Leu Ile Asn Gly Glu Arg Leu Lys Asp Thr Ile
1 5 10 15
Glu Glu Phe Ala Asp Phe Gly Arg Thr Glu Lys Asn Gly Val Thr Arg
20 25 30
Leu Ala Leu Ser Asp Val Asp Val Lys Ala Arg Arg His Phe Gln Ser
35 40 45
Leu Cys Glu Gln Leu Gly Met Ser Val Val Trp Asp Asp Met Gly Asn
50 55 60
Met Tyr Ala Lys Leu Pro Gly Ile Asp Asn Asp Gln Pro Pro Val Val
65 70 75 80
Ile Gly Ser His Leu Asp Ser Val Lys Lys Gly Gly Arg Phe Asp Gly
85 90 95
Thr Leu Gly Val Leu Thr Gly Leu Glu Val Val Arg Thr Met Val Glu
100 105 110
Asn Gly Ile Lys Pro Glu Ile Pro Ile Ile Val Ala Asn Ile Thr Asn
115 120 125
Glu Glu Gly Ala Arg Phe Glu Pro Ser Leu Met Ala Ser Gly Val Leu
130 135 140
Ser Gly Arg Phe Asp Lys Ala Ala Met Leu Lys Ser Thr Asp Val Asp
145 150 155 160
Gly Ile Thr Phe Ala Glu Ala Leu Lys Lys Ser Gly Tyr Glu Gly Lys
165 170 175
Lys Glu Asn Arg Leu Lys Glu Ala Ala Ala Phe Leu Glu Leu Hi s Ile
180 185 190
Glu Gln Gly Pro Val Leu Glu Ser Glu Asp Ile Gln Ile Gly Ile Val
195 200 205
Glu Cys Val Val Gly Met Val Cys Phe Glu Ile Glu Val Thr Gly Glu
210 215 220
Ser Asp His Ala Gly Thr Thr Pro Met Ser Met Arg Lys Asp Ala Leu
225 230 235 240
Phe Ala Ala Asn Gln Leu Ile Ser Glu Ile Arg Gln Lys Met Asn Arg
245 250 255
Leu Asp Asp Gln Leu Val Tyr Thr Val Gly Arg Met Thr Val Ser Pro
260 265 270
Asn Ile His Thr Val Ile Pro Asn Lys Val Val Phe Thr Ile Gly Ala
275 280 285
Arg His Gln Asp Gly Lys Ile Ile Arg Gln Val Glu Glu Ile Ile Gln
290 295 300
Gly Leu Pro Asn Ser Ser Gly Lys Glu Lys Cys Asn Val Thr Thr Thr
305 310 315 320
Lys Leu Trp Asp Arg His Thr Val Trp Phe Asn Glu Glu Ile Val Asn
325 330 335
Thr Leu Glu Lys Ser Ala Arg Ser Leu Gly Tyr Ser Phe Lys Arg Met
340 345 350
Val Ser Gly Ala Gly His Asp Ala Gln Phe Ile Ala Thr Tyr Ile Pro
355 360 365
Thr Ala Met Val Phe Val Pro Ser Ile Asn Gly Lys Ser His Asp Glu
370 375 380
Asp Glu Leu Thr Thr Trp Glu Asp Cys Glu Asn Gly Val Asn Val Ile
385 390 395 400
Leu Gln Thr Val Leu Asp Leu Thr Thr Asp Lys Leu
405 410

Claims (10)

1. a strain can be produced the bacterial strain of glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme simultaneously, classification called after Bacillus fordiiMH602, and its preservation registration number is CCTCC No:M 206144.
2. the bacterial strain Bacillusfordii MH602 that can produce glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme simultaneously according to claim 1 is characterized in that it produces the glycolylurea enzyme and has the aminoacid sequence shown in the SEQ ID NO:2.
3. the encoding gene of glycolylurea enzyme according to claim 2, it has the nucleotide sequence shown in the SEQ ID NO:1.
4. the bacterial strain Bacillusfordii MH602 that can produce glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme simultaneously according to claim 1 is characterized in that it produces the L-N-carbamyl hydrolysis enzyme and has the aminoacid sequence shown in the SEQ ID NO:4.
5. the encoding gene of L-N-carbamyl hydrolysis enzyme according to claim 4, it has the nucleotide sequence shown in the SEQ ID NO:3.
6. the described bacterial strain Bacillus fordiiMH602 of glycolylurea enzyme and L-N-carbamyl hydrolysis enzyme that produces simultaneously of claim 1 is in the amino acid whose application of preparation L-.
7. bacterial strain Bacillus fordii MH602 according to claim 6 is characterized in that in the amino acid whose application of preparation L-described L-amino acid is natural amino acid.
8. bacterial strain Bacillus fordii MH602 according to claim 7 is characterized in that in the amino acid whose application of the natural L-of preparation described natural L-amino acid comprises phenylalanine, phenylglycocoll, D-pHPG, glycine, L-Ala, Xie Ansuan, methionine(Met).
9. bacterial strain Bacillus fordii MH602 according to claim 6 is characterized in that in the amino acid whose application of preparation L-described L-amino acid is alpha-non-natural amino acid.
10. bacterial strain Bacillus fordii MH602 according to claim 9 is in preparation non-natural L-amino acid whose application, it is characterized in that described non-natural L-amino acid comprise high L-phenylalanine, L-DOPA, silica-basedization of L-3-amino acid, to nitro-L-phenylalanine, to chloro-L-phenylalanine.
CN2007101919687A 2007-12-28 2007-12-28 Hydantoinase and carbamoyl hydrolase producing strain, bienzyme gene and application thereof for preparing L-amino acid Expired - Fee Related CN101215533B (en)

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CN103655433A (en) * 2013-04-28 2014-03-26 钱臻 Biological fermentation hair shampoo and preparation method thereof
CN109988734A (en) * 2019-05-14 2019-07-09 西南林业大学 A kind of good fortune enlightening Bacillus strain and its application
CN110396484A (en) * 2019-06-12 2019-11-01 中国科学技术大学 A kind of hydantoin enzyme and carbamyl hydrolysis enzyme producing strains and its preparation method and application
CN110699396A (en) * 2019-11-15 2020-01-17 江南大学 Method for preparing D-aromatic amino acid by cascade reaction
CN110714035A (en) * 2019-11-15 2020-01-21 江南大学 Method for preparing D-aliphatic amino acid by cascade reaction
CN111621452A (en) * 2019-02-28 2020-09-04 中国科学院微生物研究所 Bacillus subtilis for producing D-p-hydroxyphenylglycine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103655433A (en) * 2013-04-28 2014-03-26 钱臻 Biological fermentation hair shampoo and preparation method thereof
CN111621452A (en) * 2019-02-28 2020-09-04 中国科学院微生物研究所 Bacillus subtilis for producing D-p-hydroxyphenylglycine
CN109988734A (en) * 2019-05-14 2019-07-09 西南林业大学 A kind of good fortune enlightening Bacillus strain and its application
CN109988734B (en) * 2019-05-14 2022-05-20 西南林业大学 Bacillus foedii strain and application thereof
CN110396484A (en) * 2019-06-12 2019-11-01 中国科学技术大学 A kind of hydantoin enzyme and carbamyl hydrolysis enzyme producing strains and its preparation method and application
CN110699396A (en) * 2019-11-15 2020-01-17 江南大学 Method for preparing D-aromatic amino acid by cascade reaction
CN110714035A (en) * 2019-11-15 2020-01-21 江南大学 Method for preparing D-aliphatic amino acid by cascade reaction
CN110699396B (en) * 2019-11-15 2022-03-01 江南大学 Method for preparing D-aromatic amino acid by cascade reaction

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