KR101722842B1 - Method for manufacturing the mutants proline racemase - Google Patents
Method for manufacturing the mutants proline racemase Download PDFInfo
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- KR101722842B1 KR101722842B1 KR1020150092504A KR20150092504A KR101722842B1 KR 101722842 B1 KR101722842 B1 KR 101722842B1 KR 1020150092504 A KR1020150092504 A KR 1020150092504A KR 20150092504 A KR20150092504 A KR 20150092504A KR 101722842 B1 KR101722842 B1 KR 101722842B1
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Abstract
The present invention relates to a mutant proline leucylase, wherein the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is substituted by any one amino acid selected from the group consisting of leucine, isoleucine, valine and alanine, or the 290th amino acid is replaced by isoleucine or alanine With respect to the biotinase, the mutant proline racemizing enzyme may be used to convert L-homoproline to D-homoproline.
Description
The present invention relates to a process for preparing a mutant proline racemizing enzyme capable of converting L-homoproline to D-homoproline.
Homoproline is an artificially synthesized unnatural amino acid that is not a natural amino acid made by living organisms. It is classified into L-homoproline and D-homoproline according to their optical properties. The homoproline is used as an intermediate in the manufacture of foods, medicines and fine chemicals, and both L-homoproline and D-homoproline can form main structures in drugs such as immunosuppressants, anticancer agents and antifungal agents. These homoprolines can be produced by enzymes such as D-aminoacylase using acetyl-DL-2-amino-6-bromohexanoic acid as a substrate. However, There is a complication that needs to be synthesized.
Proline, on the other hand, is a natural amino acid, and L-proline can be converted to D-proline by D-proline racemase or D-proline can be converted to L-proline by proline racemase. However, the proline racemizing enzyme can not convert homoproline because it can not recognize homo proline, which is similar in structure to proline, as a substrate. Therefore, it is difficult to convert L-homoproline to D-homoproline by using the above-mentioned proline racemizing enzyme. Until now, no enzyme has been reported to convert L-homoproline to D-homoproline, and it is necessary to study homoproline racemase.
In order to solve the above problems, the present invention aims to provide a mutant proline racemizing enzyme.
It is another object of the present invention to provide a composition for preparing D-homoproline containing the mutant proline racemizing enzyme.
It is another object of the present invention to provide a gene coding for the mutant proline racemizing enzyme.
It is another object of the present invention to provide an expression vector containing the gene.
It is another object of the present invention to provide a method for producing the mutant proline racemizing enzyme.
The present invention also provides a method for producing D-homoproline using the mutant proline racemizing enzyme.
The present invention relates to a mutant proline leucylase, wherein the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is substituted by any one amino acid selected from the group consisting of leucine, isoleucine, valine and alanine, or the 290th amino acid is replaced by isoleucine or alanine Provide beer enzymes.
The present invention also provides a composition for preparing D-homoproline comprising the mutant proline racemizing enzyme.
The present invention also provides a gene encoding the mutant proline racemizing enzyme.
The present invention also provides an expression vector comprising the gene.
(A) transforming the expression vector into a microorganism to produce a recombinant microorganism; And (b) culturing the produced recombinant microorganism to express a mutant proline racemizing enzyme, wherein the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is leucine, isoleucine, Valine and alanine, or wherein the 290th amino acid is substituted with isoleucine or alanine. The present invention also provides a method for producing a mutant proline racemizing enzyme.
In addition, the present invention provides a method for producing D-homoproline comprising the step of converting L-homoproline to D-homoproline using the mutant proline racemizing enzyme.
The present invention can mutate selectively in the amino acid sequence of the proline racemizing enzyme to produce a mutant proline racemizing enzyme in a stable and economical manner. Such a mutant proline racemizing enzyme can inexpensively supply D-homoproline as well as L-homoproline, which is used for making musical instruments and the like, by converting L-homoproline into D-homoproline.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the reaction mechanism of a mutant proline racemizing enzyme. FIG.
Fig. 2 is an image showing the binding structure of proline and proline rachemilizing enzyme.
3 is an image showing the binding structure of a substrate analog and a proline racemizing enzyme.
4 is a graph showing the reactivity of mutant proline racemizing enzyme and homoproline according to Experimental Example 3 of the present invention.
FIG. 5 is a graph showing D-homoproline conversion rate of mutant proline racemizing enzyme according to Experimental Example 4 of the present invention over time. FIG.
Hereinafter, the present invention will be described.
<Mutant proline racemizing enzyme>
The present invention provides a mutant proline racemizing enzyme, wherein the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is substituted with any one amino acid selected from the group consisting of leucine, isoleucine, valine and alanine, Isoleucine or alanine.
The proline racemizing enzyme is an enzyme that can convert L-proline to D-proline or convert D-proline to L-proline. As shown in FIG. 2, when the proline racemizing enzyme binds to proline, the 102nd phenylalanine (Phe102), 130th cysteine (Cys130), and 220th phenylalanine (Phe220 ), 290th phenylalanine (Phe290) and 300th cysteine (Cys300) are provided as binding sites for proline.
On the other hand, when the proline racemizing enzyme binds to a substrate analogue similar in structure to proline, as shown in FIG. 3, the 102nd phenylalanine (Phe102), the 220th phenylalanine (Phe220) and Binding with substrate analogues is inhibited by 290th phenylalanine (Phe290). This is because even if the substrate analogue is similar in structure to proline, the molecular weight may be different, and it may collide with the amino acid residue of the proline racemizing enzyme.
Accordingly, in the present invention, in order to convert homoproline having a structure similar to proline by using the proline racemizing enzyme, it is possible to substitute the amino acid of the proline racemizing enzyme which inhibits the binding with the substrate analog, A mutant proline racemizing enzyme can be produced.
Thus, the mutant proline racemizing enzyme can be converted to D-homoproline by binding to L-homoproline by replacing the 102nd amino acid or 290th amino acid in the amino acid sequence of the proline racemizing enzyme with an amino acid residue having a smaller molecular weight than the existing phenylalanine can do. The amino acid sequence of the proline racemizing enzyme is a nucleotide sequence of a proline racemizing enzyme derived from Trypanosoma cruzi, which is preferably Sequence Listing 1.
The mutant proline racemizing enzyme of the present invention is preferably that the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is substituted with any one amino acid selected from the group consisting of leucine, isoleucine, valine and alanine, It is more preferable that the 102nd amino acid of the racemizing enzyme is substituted with valine.
In addition, the mutant proline racemizing enzyme of the present invention is preferably such that the 290th amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is substituted with isoleucine or alanine.
In addition, the mutant proline racemizing enzyme of the present invention may be such that the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is substituted with valine and the 290th amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is replaced by isoleucine or alanine .
≪ Composition for preparing D-homoproline >
The present invention provides a composition for preparing D-homoproline comprising the mutant proline racemizing enzyme.
The composition for preparing D-homoproline of the present invention can be used for converting L-homoproline to D-homoproline.
<Gene>
The present invention provides a gene coding for the mutant proline racemizing enzyme.
The gene of the present invention is preferably any one selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 2 to 9, and more preferably the nucleotide sequence of SEQ ID NO: 4.
≪ Expression vector &
The present invention provides an expression vector containing the gene.
The expression vector has a promoter that regulates the expression of the inserted gene. The expression vector is not particularly limited, but may be pET-22T (+) having a T7 / lac promoter and expressing the inserted gene by adding IPTG.
The expression vector of the present invention may be a recombinant expression vector into which any one of the nucleotide sequences selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 2 to 9 is introduced into pET-22T (+).
≪ Preparation of a mutant proline racemizing enzyme >
(A) transforming the expression vector into a microorganism to produce a recombinant microorganism; And (b) culturing the produced recombinant microorganism to express a mutant proline racemizing enzyme, wherein the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is leucine, isoleucine, Valine and alanine, or wherein the 290th amino acid is substituted with isoleucine or alanine. The present invention also provides a method for producing a mutant proline racemizing enzyme.
The microorganism of the present invention is not particularly limited, but Escherichia coli that is easy to obtain and the like can be mentioned.
Conditions for culturing the recombinant microorganism of the present invention are not particularly limited, but it is preferable that the temperature is 25 to 37 占 폚 and the pH condition is
≪ Production method of D-homoproline >
In addition, the present invention provides a method for producing D-homoproline comprising the step of converting L-homoproline to D-homoproline using the mutant proline racemizing enzyme.
The L-homoproline of the present invention is not particularly limited, but L-homoproline, L-homoproline sodium, L-homoproline potassium, And the like.
The temperature at which the L-homoproline is converted to D-homoproline may be 25-80 ° C to indicate optimal activity of the mutant proline racemizing enzyme. Preferably 30 to 65 ° C, and more preferably 35 to 45 ° C. When the conversion temperature is increased by 10 DEG C, the rate of conversion of L-homoproline to D-homoproline is doubled, but when the conversion temperature exceeds 65 DEG C, the stability of the mutant proline racemizing enzyme is unstable The conversion speed and the conversion rate can be reduced.
The pH condition for converting the L-homoproline to D-homoproline is not particularly limited, but it is preferably pH 5.5 to 6.5.
On the other hand, the mutant proline racemizing enzyme of the present invention can not only convert L-homoproline into D-homoproline but also convert D-homoproline into L-homoproline. Therefore, the present invention relates to a composition for producing L-homoproline comprising the mutant proline racemizing enzyme and a method for converting L-homoproline to L-homoproline using the mutant proline racemizing enzyme, Can be provided.
Hereinafter, the present invention will be described concretely with reference to Examples. However, the following Examples are intended to illustrate one embodiment of the present invention, but the scope of the present invention is not limited by the following Examples.
[Examples 1 to 8] Preparation of recombinant Escherichia coli
The gene encoding the mutant proline racemizing enzyme is the 102nd and / or 290th phenylalanine (SEQ ID NO: 1) in the amino acid sequence of a trypanosoma cruzi-derived proline racemizing enzyme (GenBank, ACCESSION: 1W61_A, GI: 90108457) phenylalanine) is substituted with the amino acid shown in Table 1, and the NdeI restriction endonuclease sequence at the N-terminus of the base sequence and the histidine tag and the XhoI restriction endonuclease sequence at the C-terminus are included. The gene encoding the mutant proline racemizing enzyme has the nucleotide sequence of SEQ ID NOS: 2 to 9, respectively.
Escherichia coli BL21 (DE3) was transformed into the vector pET-22b (+) obtained by digesting with restriction enzymes NdeI and XhoI, respectively, and the recombinant Escherichia coli was transformed into Escherichia coli BL21 .
[Comparative Example 1] Production of recombinant Escherichia coli
Recombinant E. coli was prepared in the same manner as in Example 1, except that the amino acid sequence of SEQ ID NO: 1 was used instead of the nucleotide sequence of SEQ ID NO: 2 as a gene in Example 1.
[Experimental Example 1] Confirmation of virtual binding structure with proline racemizing enzyme
A substrate analogue with a proline or proline-like structure was used to confirm the binding structure with the proline racemizing enzyme. Docking simulation of proline or substrate analogues was performed on proline racemizing enzyme (PDB code: 1W61) using the SYBYL-X (Tripos, ver. 2.1) program. The results are shown in FIG. 2 and FIG.
As shown in FIG. 2, when the proline and the proline racemizing enzyme are combined, the 102nd phenylalanine (Phe102), the 130th cysteine (Cys130), the 220th phenylalanine (Phe220) , 290th phenylalanine (Phe290), and 300th cysteine (Cys300) bind to proline.
As shown in FIG. 3, when the substrate analogue and the proline racemizing enzyme were bound, it was confirmed that docking with the substrate analogue was inhibited by the 102nd phenylalanine, the 220th phenylalanine and the 290th phenylalanine of the proline racemizing enzyme .
Therefore, when the homoproline and the proline racemizing enzyme are combined, since the molecular weight of homoproline (129) is larger than that of proline (115), the 102nd, 220th and 290th amino acid residues of the proline racemizing enzyme are homozygous It could be predicted that it will collide with proline and hinder docking.
[Experimental Example 2] Measurement of enzyme activity on L-homoproline
The recombinant Escherichia coli (Examples 1 to 8 and Comparative Example 1) prepared by substituting the 102nd and 290th amino acids of the proline racemizing enzyme predicted in Experimental Example 1 with an amino acid other than phenylalanine were used to prepare L-homoproline Enzyme activity was measured. Examples 1 to 8 and Comparative Example 1 were cultured at 37 占 폚 in LB medium containing ampicillin (1 mg / ml). The expression of the mutant proline racemizing enzyme was induced by administering 0.1 M IPTG to the culture, followed by further culturing for 16 hours in a shaking incubator at 200 rpm. The culture solution was centrifuged to recover the recombinant E. coli, and the recombinant E. coli was resuspended in a disruption buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole) and disrupted with an ultrasonic mill. A histidine-labeled mutant proline racemizing enzyme was selectively separated using a Ni-NTA column in the crushed cell solution.
0.5 mg / L of the separated mutant proline racemizing enzyme was added to a reaction solution containing 10 mM L-homoproline and 300 mM sodium acetate buffer (pH 6.0). After 168 hours of reaction, 10 μl of 10% H 2 SO 4 (v / v) was added to terminate the reaction. The resulting D-homoproline was loaded on a CHIROBIOTIC T column (25 cm x 4.6 mm, 5 um) and the activity of the enzyme was measured by HPLC (Agilent Technologies 1200 Series LC system, USA). The results are shown in Table 2 below.
As shown in the above Table 2, the mutant proline racemizing enzyme expressed in Examples 1 to 8 is superior in activity to the proline racemizing enzyme expressed in Comparative Example 1, and in particular, the mutant proline racemizing enzyme expressed in Example 3 It was confirmed that the enzyme had the highest activity.
On the other hand, the mutant proline racemizing enzyme expressed in Examples 7 and 8 was obtained by substituting both the 102nd amino acid and the 290th amino acid, indicating that the enzymatic activity was increased as compared with the enzyme substituted with the 290th amino acid (Examples 5 and 6) I could confirm. Therefore, it was confirmed that the binding of L-homoproline to the mutant proline racemizing enzyme is important for the 102nd amino acid residue in the amino acid sequence of the proline racemizing enzyme.
[Experimental Example 3] Measurement of reactivity between mutant proline racemizing enzyme and homoproline
The reactivity of the enzymes expressed in Examples 1 to 8 to homoproline was measured. D-homoproline converted from L-homoproline and L-homoproline converted from D-homoproline were measured in the same manner as in Experimental Example 1, and the results are shown in FIG.
As shown in Fig. 4, the mutant proline racemizing enzymes expressed in Examples 1 to 8 were enzymatically reactive to D-homoproline as well as L-homoproline, and in particular, the mutant proline racemization expressed in Example 3 It was confirmed that the enzyme had the highest enzyme reactivity.
In addition, it was confirmed that the mutant proline racemizing enzyme expressed in Example 3 was substituted with valine at the 102nd amino acid, and was more excellent in enzyme reactivity than the enzyme in which the 102nd amino acid was substituted with alanine (Example 4). Thus, the optimal mutant proline racemizing enzyme is characterized in that the molecular weight of the 102nd amino acid residue is less than phenylalanine (molecular weight 165), but greater than alanine (molecular weight 89), the 102nd amino acid residue in the amino acid sequence of the proline racemizing enzyme is valine 117).
[Experimental Example 4] Measurement of D-homoproline conversion rate with time
The D-homoproline conversion rates were measured with the mutant proline racemizing enzyme expressed in Examples 3, 7 and 8, which were shown to be reactive with L-homoproline in Experimental Example 3, over time. The resulting D-homoproline was measured in the same manner as in Experimental Example 1, and the results are shown in FIG.
As shown in FIG. 5, it was confirmed that the mutant proline racemizing enzyme expressed in Examples 3, 7 and 8 converted L-homoproline to D-homoproline according to the reaction time. In addition, it was confirmed that the mutant proline racemizing enzyme expressed in Example 3 is superior to the mutant proline racemizing enzyme expressed in Examples 7 and 8 in the conversion of D-homoproline.
<110> Kwangwoon University Industry-Academic Collaboration Foundation <120> METHOD FOR MANUFACTURING THE MUTANTS PROLINE RACEMASE <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 414 <212> PRT <213> Trypanosoma cruzi <400> 1 Met Gly Gln Glu Lys Leu Leu Phe Asp Gln Lys Tyr Lys Ile Ile Lys 1 5 10 15 Gly Glu Lys Lys Glu Lys Lys Lys Asn Gln Arg Ala Asn Arg Arg Glu 20 25 30 His Gln Gln Lys Arg Glu Ile Met Arg Phe Lys Lys Ser Phe Thr Cys 35 40 45 Ile Asp Met His Thr Glu Gly Glu Ala Ala Arg Ile Val Thr Ser Gly 50 55 60 Leu Pro His Ile Pro Gly Ser Asn Met Ala Glu Lys Lys Ala Tyr Leu 65 70 75 80 Gln Glu Asn Met Asp Tyr Leu Arg Arg Gly Ile Met Leu Glu Pro Arg 85 90 95 Gly His Asp Asp Met Phe Gly Ala Phe Leu Phe Asp Pro Ile Glu Glu 100 105 110 Gly Ala Asp Leu Gly Ile Val Phe Met Asp Thr Gly Gly Tyr Leu Asn 115 120 125 Met Cys Gly His Asn Ser Ile Ala Ala Val Thr Ala Ala Val Glu Thr 130 135 140 Gly Ile Val Ser Val Pro Ala Lys Ala Thr Asn Val Pro Val Val Leu 145 150 155 160 Asp Thr Pro Ala Gly Leu Val Arg Gly Thr Ala His Leu Gln Ser Gly 165 170 175 Thr Glu Ser Glu Val Ser Asn Ala Ser Ile Ile Asn Val Ser Ser Phe 180 185 190 Leu Tyr Gln Gln Asp Val Val Val Val Leu Pro Lys Pro Tyr Gly Glu 195 200 205 Val Arg Val Asp Ile Ala Phe Gly Gly Asn Phe Phe Ala Ile Val Pro 210 215 220 Ala Glu Gln Leu Gly Ile Asp Ile Ser Val Gln Asn Leu Ser Arg Leu 225 230 235 240 Gln Glu Ala Gly Glu Leu Leu Arg Thr Glu Ile Asn Arg Ser Val Lys 245 250 255 Val Gln His Pro Gln Leu Pro His Ile Asn Thr Val Asp Cys Val Glu 260 265 270 Ile Tyr Gly Pro Pro Thr Asn Pro Glu Ala Asn Tyr Lys Asn Val Val 275 280 285 Ile Phe Gly Asn Arg Gln Ala Asp Arg Ser Pro Cys Gly Thr Gly Thr 290 295 300 Ser Ala Lys Met Ala Thr Leu Tyr Ala Lys Gly Gln Leu Arg Ile Gly 305 310 315 320 Glu Thr Phe Val Tyr Glu Ser Ile Leu Gly Ser Leu Phe Gln Gly Arg 325 330 335 Val Leu Gly Glu Glu Arg Ile Pro Gly Val Lys Val Pro Val Thr Lys 340 345 350 Asp Ala Glu Glu Gly Met Leu Val Val Thr Ala Glu Ile Thr Gly Lys 355 360 365 Ala Phe Ile Met Gly Phe Asn Thr Met Leu Phe Asp Pro Thr Asp Pro 370 375 380 Phe Lys Asn Gly Phe Thr Leu Lys Gln Tyr Ile Trp Ser Ser Ser Val 385 390 395 400 Asp Lys Leu Ala Ala Ala Leu Glu His His His His His 405 410 <210> 2 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> F102L <400> 2 catatgcgca aaagcgtttg ccccaagcag aaattcttct ttagcgcatt tccattcttc 60 tttttctttt gtgtttttcc gctgattagt cgtacaggtc aggaaaaact gctgtttgat 120 caaaaatata aaattataaa gggtgaaaaa aaagaaaaaa aaaaaaatca gagggcgaat 180 cgtcgggaac atcagcaaaa acgtgagatt atgcgtttca aaaaatcttt tacgtgtatt 240 gatatgcaca ccgagggcga agccgcacgt atcgtcactt ctggtctgcc gcacatacca 300 ggctctaata tggccgaaaa aaaggcatac ctgcaggaaa acatggatta tctgcgtcga 360 ggtattatgc tggaaccgcg gggtcatgac gatatgctcg gagcctttct gttcgatccg 420 attgaggagg gtgcagacct ggggatggtc tttatggata cgggtggtta cctgaatatg 480 tgtggtcaca atagcattgc tgccgttaca gccgcagttg agactgggat cgtgagcgtt 540 ccggcaaagg caaccaacgt cccggtggta ttggatacgc cggcaggtct ggttcgcggg 600 accgcacatt tgcaaagcgg tacagaaagc gaggttagca acgccagcat catcaatgta 660 ccgagctttc tgtatcaaca ggatgtggtt gttgttttac ccaaaccgta tggcgaagta 720 agagttgaca tagccttcgg tggtaacttt tttgcgatag tcccagccga gcagctgggt 780 atcgatatat ccgtgcagaa tctaagccgt cttcaggaag caggtgaact gctgcgtact 840 gaaattaatc gtagcgtgaa ggtccaacat ccgcagctcc ctcatatcaa tacagtcgat 900 tgcgttgaaa tttatggtcc tcccaccaac ccggaggcaa actacaaaaa cgtagttatt 960 tttggtaatc gtcaggctga ccgctcccca tgcggtaccg gtacgagcgc gaaaatggca 1020 acactgtatg cgaaagggca gctgcggatt ggcgagacgt ttgtttatga gagtatttta 1080 ggtagtcttt tccaaggtcg tgtcctcggt gaagaacgta tccctggtgt taaggtacct 1140 gttactaaag acgcggaaga aggcatgctg gtggtgactg cagaaattac cggcaaagcg 1200 tttatcatgg ggtttaatac catgctgttt gacccaaccg atccgttcaa gaacggtttt 1260 accctgaagc agctcgag 1278 <210> 3 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> F102I <400> 3 catatgcgca aaagcgtttg ccccaagcag aaattcttct ttagcgcatt tccattcttc 60 tttttctttt gtgtttttcc gctgattagt cgtacaggtc aggaaaaact gctgtttgat 120 caaaaatata aaattataaa gggtgaaaaa aaagaaaaaa aaaaaaatca gagggcgaat 180 cgtcgggaac atcagcaaaa acgtgagatt atgcgtttca aaaaatcttt tacgtgtatt 240 gatatgcaca ccgagggcga agccgcacgt atcgtcactt ctggtctgcc gcacatacca 300 ggctctaata tggccgaaaa aaaggcatac ctgcaggaaa acatggatta tctgcgtcga 360 ggtattatgc tggaaccgcg gggtcatgac gatatgatag gagcctttct gttcgatccg 420 attgaggagg gtgcagacct ggggatggtc tttatggata cgggtggtta cctgaatatg 480 tgtggtcaca atagcattgc tgccgttaca gccgcagttg agactgggat cgtgagcgtt 540 ccggcaaagg caaccaacgt cccggtggta ttggatacgc cggcaggtct ggttcgcggg 600 accgcacatt tgcaaagcgg tacagaaagc gaggttagca acgccagcat catcaatgta 660 ccgagctttc tgtatcaaca ggatgtggtt gttgttttac ccaaaccgta tggcgaagta 720 agagttgaca tagccttcgg tggtaacttt tttgcgatag tcccagccga gcagctgggt 780 atcgatatat ccgtgcagaa tctaagccgt cttcaggaag caggtgaact gctgcgtact 840 gaaattaatc gtagcgtgaa ggtccaacat ccgcagctcc ctcatatcaa tacagtcgat 900 tgcgttgaaa tttatggtcc tcccaccaac ccggaggcaa actacaaaaa cgtagttatt 960 tttggtaatc gtcaggctga ccgctcccca tgcggtaccg gtacgagcgc gaaaatggca 1020 acactgtatg cgaaagggca gctgcggatt ggcgagacgt ttgtttatga gagtatttta 1080 ggtagtcttt tccaaggtcg tgtcctcggt gaagaacgta tccctggtgt taaggtacct 1140 gttactaaag acgcggaaga aggcatgctg gtggtgactg cagaaattac cggcaaagcg 1200 tttatcatgg ggtttaatac catgctgttt gacccaaccg atccgttcaa gaacggtttt 1260 accctgaagc agctcgag 1278 <210> 4 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> F102V <400> 4 catatgcgca aaagcgtttg ccccaagcag aaattcttct ttagcgcatt tccattcttc 60 tttttctttt gtgtttttcc gctgattagt cgtacaggtc aggaaaaact gctgtttgat 120 caaaaatata aaattataaa gggtgaaaaa aaagaaaaaa aaaaaaatca gagggcgaat 180 cgtcgggaac atcagcaaaa acgtgagatt atgcgtttca aaaaatcttt tacgtgtatt 240 gatatgcaca ccgagggcga agccgcacgt atcgtcactt ctggtctgcc gcacatacca 300 ggctctaata tggccgaaaa aaaggcatac ctgcaggaaa acatggatta tctgcgtcga 360 ggtattatgc tggaaccgcg gggtcatgac gatatggtgg gagcctttct gttcgatccg 420 attgaggagg gtgcagacct ggggatggtc tttatggata cgggtggtta cctgaatatg 480 tgtggtcaca atagcattgc tgccgttaca gccgcagttg agactgggat cgtgagcgtt 540 ccggcaaagg caaccaacgt cccggtggta ttggatacgc cggcaggtct ggttcgcggg 600 accgcacatt tgcaaagcgg tacagaaagc gaggttagca acgccagcat catcaatgta 660 ccgagctttc tgtatcaaca ggatgtggtt gttgttttac ccaaaccgta tggcgaagta 720 agagttgaca tagccttcgg tggtaacttt tttgcgatag tcccagccga gcagctgggt 780 atcgatatat ccgtgcagaa tctaagccgt cttcaggaag caggtgaact gctgcgtact 840 gaaattaatc gtagcgtgaa ggtccaacat ccgcagctcc ctcatatcaa tacagtcgat 900 tgcgttgaaa tttatggtcc tcccaccaac ccggaggcaa actacaaaaa cgtagttatt 960 tttggtaatc gtcaggctga ccgctcccca tgcggtaccg gtacgagcgc gaaaatggca 1020 acactgtatg cgaaagggca gctgcggatt ggcgagacgt ttgtttatga gagtatttta 1080 ggtagtcttt tccaaggtcg tgtcctcggt gaagaacgta tccctggtgt taaggtacct 1140 gttactaaag acgcggaaga aggcatgctg gtggtgactg cagaaattac cggcaaagcg 1200 tttatcatgg ggtttaatac catgctgttt gacccaaccg atccgttcaa gaacggtttt 1260 accctgaagc agctcgag 1278 <210> 5 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> F102A <400> 5 catatgcgca aaagcgtttg ccccaagcag aaattcttct ttagcgcatt tccattcttc 60 tttttctttt gtgtttttcc gctgattagt cgtacaggtc aggaaaaact gctgtttgat 120 caaaaatata aaattataaa gggtgaaaaa aaagaaaaaa aaaaaaatca gagggcgaat 180 cgtcgggaac atcagcaaaa acgtgagatt atgcgtttca aaaaatcttt tacgtgtatt 240 gatatgcaca ccgagggcga agccgcacgt atcgtcactt ctggtctgcc gcacatacca 300 ggctctaata tggccgaaaa aaaggcatac ctgcaggaaa acatggatta tctgcgtcga 360 ggtattatgc tggaaccgcg gggtcatgac gatatggcgg gagcctttct gttcgatccg 420 attgaggagg gtgcagacct ggggatggtc tttatggata cgggtggtta cctgaatatg 480 tgtggtcaca atagcattgc tgccgttaca gccgcagttg agactgggat cgtgagcgtt 540 ccggcaaagg caaccaacgt cccggtggta ttggatacgc cggcaggtct ggttcgcggg 600 accgcacatt tgcaaagcgg tacagaaagc gaggttagca acgccagcat catcaatgta 660 ccgagctttc tgtatcaaca ggatgtggtt gttgttttac ccaaaccgta tggcgaagta 720 agagttgaca tagccttcgg tggtaacttt tttgcgatag tcccagccga gcagctgggt 780 atcgatatat ccgtgcagaa tctaagccgt cttcaggaag caggtgaact gctgcgtact 840 gaaattaatc gtagcgtgaa ggtccaacat ccgcagctcc ctcatatcaa tacagtcgat 900 tgcgttgaaa tttatggtcc tcccaccaac ccggaggcaa actacaaaaa cgtagttatt 960 tttggtaatc gtcaggctga ccgctcccca tgcggtaccg gtacgagcgc gaaaatggca 1020 acactgtatg cgaaagggca gctgcggatt ggcgagacgt ttgtttatga gagtatttta 1080 ggtagtcttt tccaaggtcg tgtcctcggt gaagaacgta tccctggtgt taaggtacct 1140 gttactaaag acgcggaaga aggcatgctg gtggtgactg cagaaattac cggcaaagcg 1200 tttatcatgg ggtttaatac catgctgttt gacccaaccg atccgttcaa gaacggtttt 1260 accctgaagc agctcgag 1278 <210> 6 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> F290L <400> 6 catatgcgca aaagcgtttg ccccaagcag aaattcttct ttagcgcatt tccattcttc 60 tttttctttt gtgtttttcc gctgattagt cgtacaggtc aggaaaaact gctgtttgat 120 caaaaatata aaattataaa gggtgaaaaa aaagaaaaaa aaaaaaatca gagggcgaat 180 cgtcgggaac atcagcaaaa acgtgagatt atgcgtttca aaaaatcttt tacgtgtatt 240 gatatgcaca ccgagggcga agccgcacgt atcgtcactt ctggtctgcc gcacatacca 300 ggctctaata tggccgaaaa aaaggcatac ctgcaggaaa acatggatta tctgcgtcga 360 ggtattatgc tggaaccgcg gggtcatgac gatatgtttg gagcctttct gttcgatccg 420 attgaggagg gtgcagacct ggggatggtc tttatggata cgggtggtta cctgaatatg 480 tgtggtcaca atagcattgc tgccgttaca gccgcagttg agactgggat cgtgagcgtt 540 ccggcaaagg caaccaacgt cccggtggta ttggatacgc cggcaggtct ggttcgcggg 600 accgcacatt tgcaaagcgg tacagaaagc gaggttagca acgccagcat catcaatgta 660 ccgagctttc tgtatcaaca ggatgtggtt gttgttttac ccaaaccgta tggcgaagta 720 agagttgaca tagccttcgg tggtaacttt tttgcgatag tcccagccga gcagctgggt 780 atcgatatat ccgtgcagaa tctaagccgt cttcaggaag caggtgaact gctgcgtact 840 gaaattaatc gtagcgtgaa ggtccaacat ccgcagctcc ctcatatcaa tacagtcgat 900 tgcgttgaaa tttatggtcc tcccaccaac ccggaggcaa actacaaaaa cgtagttatt 960 ctcggtaatc gtcaggctga ccgctcccca tgcggtaccg gtacgagcgc gaaaatggca 1020 acactgtatg cgaaagggca gctgcggatt ggcgagacgt ttgtttatga gagtatttta 1080 ggtagtcttt tccaaggtcg tgtcctcggt gaagaacgta tccctggtgt taaggtacct 1140 gttactaaag acgcggaaga aggcatgctg gtggtgactg cagaaattac cggcaaagcg 1200 tttatcatgg ggtttaatac catgctgttt gacccaaccg atccgttcaa gaacggtttt 1260 accctgaagc agctcgag 1278 <210> 7 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> F290V <400> 7 catatgcgca aaagcgtttg ccccaagcag aaattcttct ttagcgcatt tccattcttc 60 tttttctttt gtgtttttcc gctgattagt cgtacaggtc aggaaaaact gctgtttgat 120 caaaaatata aaattataaa gggtgaaaaa aaagaaaaaa aaaaaaatca gagggcgaat 180 cgtcgggaac atcagcaaaa acgtgagatt atgcgtttca aaaaatcttt tacgtgtatt 240 gatatgcaca ccgagggcga agccgcacgt atcgtcactt ctggtctgcc gcacatacca 300 ggctctaata tggccgaaaa aaaggcatac ctgcaggaaa acatggatta tctgcgtcga 360 ggtattatgc tggaaccgcg gggtcatgac gatatgtttg gagcctttct gttcgatccg 420 attgaggagg gtgcagacct ggggatggtc tttatggata cgggtggtta cctgaatatg 480 tgtggtcaca atagcattgc tgccgttaca gccgcagttg agactgggat cgtgagcgtt 540 ccggcaaagg caaccaacgt cccggtggta ttggatacgc cggcaggtct ggttcgcggg 600 accgcacatt tgcaaagcgg tacagaaagc gaggttagca acgccagcat catcaatgta 660 ccgagctttc tgtatcaaca ggatgtggtt gttgttttac ccaaaccgta tggcgaagta 720 agagttgaca tagccttcgg tggtaacttt tttgcgatag tcccagccga gcagctgggt 780 atcgatatat ccgtgcagaa tctaagccgt cttcaggaag caggtgaact gctgcgtact 840 gaaattaatc gtagcgtgaa ggtccaacat ccgcagctcc ctcatatcaa tacagtcgat 900 tgcgttgaaa tttatggtcc tcccaccaac ccggaggcaa actacaaaaa cgtagttatt 960 gtgggtaatc gtcaggctga ccgctcccca tgcggtaccg gtacgagcgc gaaaatggca 1020 acactgtatg cgaaagggca gctgcggatt ggcgagacgt ttgtttatga gagtatttta 1080 ggtagtcttt tccaaggtcg tgtcctcggt gaagaacgta tccctggtgt taaggtacct 1140 gttactaaag acgcggaaga aggcatgctg gtggtgactg cagaaattac cggcaaagcg 1200 tttatcatgg ggtttaatac catgctgttt gacccaaccg atccgttcaa gaacggtttt 1260 accctgaagc agctcgag 1278 <210> 8 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> F102V F290L <400> 8 catatgcgca aaagcgtttg ccccaagcag aaattcttct ttagcgcatt tccattcttc 60 tttttctttt gtgtttttcc gctgattagt cgtacaggtc aggaaaaact gctgtttgat 120 caaaaatata aaattataaa gggtgaaaaa aaagaaaaaa aaaaaaatca gagggcgaat 180 cgtcgggaac atcagcaaaa acgtgagatt atgcgtttca aaaaatcttt tacgtgtatt 240 gatatgcaca ccgagggcga agccgcacgt atcgtcactt ctggtctgcc gcacatacca 300 ggctctaata tggccgaaaa aaaggcatac ctgcaggaaa acatggatta tctgcgtcga 360 ggtattatgc tggaaccgcg gggtcatgac gatatggtgg gagcctttct gttcgatccg 420 attgaggagg gtgcagacct ggggatggtc tttatggata cgggtggtta cctgaatatg 480 tgtggtcaca atagcattgc tgccgttaca gccgcagttg agactgggat cgtgagcgtt 540 ccggcaaagg caaccaacgt cccggtggta ttggatacgc cggcaggtct ggttcgcggg 600 accgcacatt tgcaaagcgg tacagaaagc gaggttagca acgccagcat catcaatgta 660 ccgagctttc tgtatcaaca ggatgtggtt gttgttttac ccaaaccgta tggcgaagta 720 agagttgaca tagccttcgg tggtaacttt tttgcgatag tcccagccga gcagctgggt 780 atcgatatat ccgtgcagaa tctaagccgt cttcaggaag caggtgaact gctgcgtact 840 gaaattaatc gtagcgtgaa ggtccaacat ccgcagctcc ctcatatcaa tacagtcgat 900 tgcgttgaaa tttatggtcc tcccaccaac ccggaggcaa actacaaaaa cgtagttatt 960 ctcggtaatc gtcaggctga ccgctcccca tgcggtaccg gtacgagcgc gaaaatggca 1020 acactgtatg cgaaagggca gctgcggatt ggcgagacgt ttgtttatga gagtatttta 1080 ggtagtcttt tccaaggtcg tgtcctcggt gaagaacgta tccctggtgt taaggtacct 1140 gttactaaag acgcggaaga aggcatgctg gtggtgactg cagaaattac cggcaaagcg 1200 tttatcatgg ggtttaatac catgctgttt gacccaaccg atccgttcaa gaacggtttt 1260 accctgaagc agctcgag 1278 <210> 9 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> F102V F290V <400> 9 catatgcgca aaagcgtttg ccccaagcag aaattcttct ttagcgcatt tccattcttc 60 tttttctttt gtgtttttcc gctgattagt cgtacaggtc aggaaaaact gctgtttgat 120 caaaaatata aaattataaa gggtgaaaaa aaagaaaaaa aaaaaaatca gagggcgaat 180 cgtcgggaac atcagcaaaa acgtgagatt atgcgtttca aaaaatcttt tacgtgtatt 240 gatatgcaca ccgagggcga agccgcacgt atcgtcactt ctggtctgcc gcacatacca 300 ggctctaata tggccgaaaa aaaggcatac ctgcaggaaa acatggatta tctgcgtcga 360 ggtattatgc tggaaccgcg gggtcatgac gatatggtgg gagcctttct gttcgatccg 420 attgaggagg gtgcagacct ggggatggtc tttatggata cgggtggtta cctgaatatg 480 tgtggtcaca atagcattgc tgccgttaca gccgcagttg agactgggat cgtgagcgtt 540 ccggcaaagg caaccaacgt cccggtggta ttggatacgc cggcaggtct ggttcgcggg 600 accgcacatt tgcaaagcgg tacagaaagc gaggttagca acgccagcat catcaatgta 660 ccgagctttc tgtatcaaca ggatgtggtt gttgttttac ccaaaccgta tggcgaagta 720 agagttgaca tagccttcgg tggtaacttt gtggcgatag tcccagccga gcagctgggt 780 atcgatatat ccgtgcagaa tctaagccgt cttcaggaag caggtgaact gctgcgtact 840 gaaattaatc gtagcgtgaa ggtccaacat ccgcagctcc ctcatatcaa tacagtcgat 900 tgcgttgaaa tttatggtcc tcccaccaac ccggaggcaa actacaaaaa cgtagttatt 960 ctcggtaatc gtcaggctga ccgctcccca tgcggtaccg gtacgagcgc gaaaatggca 1020 acactgtatg cgaaagggca gctgcggatt ggcgagacgt ttgtttatga gagtatttta 1080 ggtagtcttt tccaaggtcg tgtcctcggt gaagaacgta tccctggtgt taaggtacct 1140 gttactaaag acgcggaaga aggcatgctg gtggtgactg cagaaattac cggcaaagcg 1200 tttatcatgg ggtttaatac catgctgttt gacccaaccg atccgttcaa gaacggtttt 1260 accctgaagc agctcgag 1278
Claims (15)
A mutant proline racemizing enzyme capable of converting L-homoproline to D-homoproline.
Wherein the proline racemase of SEQ ID NO: 1 is a proline racemizing enzyme derived from Trypanosoma cruzi.
A mutant proline racemizing enzyme wherein the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is substituted with valine.
Wherein the gene comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 2-9.
(b) culturing the produced recombinant microorganism to express a mutant proline racemizing enzyme
And,
Wherein the mutant proline racemizing enzyme is a mutant proline racemizing enzyme wherein the 102nd amino acid of the proline racemizing enzyme of SEQ ID NO: 1 is substituted with any one amino acid selected from the group consisting of leucine, isoleucine, valine and alanine, or the 290th amino acid is substituted with isoleucine or alanine Lt; RTI ID = 0.0 > of prolactinase < / RTI >
Wherein the culture of the recombinant microorganism is carried out at a temperature of 25 to 37 占 폚 and at a pH of 6 to 8.
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