KR20210001722A - A D-lactate dehydrogenase with high substrate preference for phenylpyruvate - Google Patents

Abstract

D-lactate dehydrogenase catalyzes the reduction of pyruvic acid into D-lactic acid, and thus generally shows a higher substrate preference for pyruvic acid than for phenylpyruvic acid. Unlike the existing D-lactate dehydrogenase, however, D-lactate dehydrogenase derived from Pediococcus claussenii, which has been identified through the present invention, shows a high preference for phenylpyruvic acid rather than pyruvic acid, and thus a catalytic efficiency (kcat/Km) ratio is about 1.7 times higher. In addition, in the case of applying D-lactate dehydrogenase derived from Oenococcus oeni, Weissella confuse and Weissella koreensis under in vitro conditions, it has been confirmed that a conversion rate is up to 60% based on the 12-hour enzymatic reaction, whereas D-lactate dehydrogenase derived from Pediococcus claussenii, identified through the present invention, can produce D-3-phenyllactic acid at 100% conversion rate from phenylpyruvic acid based on the same reaction time.

Description

D-lactate dehydrogenase with high substrate preference for phenylpyruvate and its gene {A D-lactate dehydrogenase with high substrate preference for phenylpyruvate}

The present invention is derived from Pediococcus claussenii showing a high substrate preference for phenylpyruvate D-lactate dehydrogenase (D-lactate dehydrogenase) and D-3-phenyl lactic acid (D-3- phenyllactate).

Hydroxy acid has various applications such as synthesis of high value-added compounds, cosmetics, fuel additives, and pharmaceutical intermediates. Among them, D-3-phenyllactate is a monomer raw material for antibiotics and cardiovascular disease treatment Danshensu, anthelmintic PF1022A, blood glucose lowering agent Englitazone, and hydroxy acid-based biopolyester compound as a monomer raw material. It can be usefully used in the field. 3-phenyllactic acid can be biosynthesized from phenylpyruvate through a reduction reaction through a catalytic action of D-lactic acid dehydrogenase. However, since the main substrate of D-lactic acid dehydrogenase is pyruvate, it generally exhibits higher catalytic activity and substrate preference for pyruvate than phenylpyruvate. Therefore, the discovery of D-lactic acid dehydrogenase, which shows higher substrate preference to phenylpyruvate than pyruvic acid, has an important meaning in increasing the efficiency of the biosynthetic process for producing D-3-phenyllactic acid from phenylpyruvate.

An object of the present invention is to discover a novel D-lactic acid dehydrogenase that exhibits high substrate preference and activity for phenyl lactic acid and to increase the biosynthetic efficiency of producing D-3-phenyl lactic acid from phenyl pyruvic acid using this.

D-3-phenyllactic acid is a kind of metabolite found in the metabolic pathway of lactic acid bacteria, and D-lactic acid dehydrogenase is a kind of 2-hydroxyacid dehydrogenase. Therefore, in the present invention, synthetic genes of D-lactic acid dehydrogenase candidates derived from four types of lactic acid bacteria ( Oenococcus oeni , Weissella confusa , Weissella koreensis and Pediococcus claussenii ) estimated based on amino acid sequence similarity were obtained, and synthetic genes derived from each strain E. coli recombinant protein was expressed in the form of recombinant protein and purified, and then the enzyme activity, kinetic parameters and conversion rate of the synthesis reaction were compared to show high catalytic activity and conversion efficiency for the production of D-3-phenyllactic acid. A new type D-lactic acid dehydrogenase was discovered.

Since D-lactic acid dehydrogenase catalyzes the reaction of reducing pyruvic acid to D-lactic acid, it generally shows higher substrate preference for pyruvate than phenylpyruvate. However, the D-lactate dehydrogenase derived from Pediococcus claussenii discovered through the present invention shows a higher preference for phenylpyruvate than pyruvate, unlike the existing D-lactate dehydrogenase, and the catalytic efficiency (kcat/Km) ratio is based. It showed about 1.7 times higher characteristics. In addition, in the case of applying the D-lactic acid dehydrogenase derived from Oenococcus oeni , Weissella confusa, and Weissella koreensis in in vitro conditions, the conversion rate was up to 60% based on the 12 hour enzymatic reaction, whereas the D derived from Pediococcus claussenii discovered through the present invention It was confirmed that the type-lactic acid dehydrogenase can produce D-3-phenyllactic acid at 100% conversion rate from phenylpyruvate based on the same reaction time.

Figure 1 is a comparison of the relative specific activity of D-lactic acid dehydrogenase. (Top), pyruvate, wcHADH activity 428 U mg ^-1 ; (Bottom), phenylpyruvate, pcHADH activity 591 U mg ^-1 .
Figure 2 is an enzyme conversion of phenylpyruvate to D-phenyllactic acid. ■, pcHADH; ▼, wcHADH; ●, ooHADH.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol)%.

The present invention aims to produce phenyl lactic acid by reducing phenyl pyruvic acid. Accordingly, in the present invention, a novel D-lactate dehydrogenase was screened to discover a D-lactate dehydrogenase derived from Pediococcus claussenii, which has a higher substrate preference for phenylpyruvate than pyruvate, and the activity of phenylpyruvate on the reduction reaction was measured.

Specifically, the D-lactic acid dehydrogenase of the present invention is characterized in that it comprises the amino acid sequence of SEQ ID NO: 1.

[SEQ ID NO: 1]

MKITAYGIRE DERPYLNEWQ EKNNIEVQAV SELLDSETLE LAKGSDGVVA FQQKPYTDDI

FSKMNRFGIH AFSLRNVGVD NLSFNALKEN NVMLSNVPAY SPNAIAELSV TQLMALIRRI

PDFQAKMKRG DFRWEPTIAL ELNQMTVGVI GTGRIGRAAI DIFKGFGAKV ICYDVFLNPE

LEKEGAYVDT VEELYKSVDV VTLHVPALKD NYHMLDDKAF NSMKDGVFVL NYSRGSLIDT

AALIRGLDSG KIAGVGLDTY ENEVGIFEID HEDQPIDDEM FNNLNARRNV MITPHAAFYT

TNAVKNMVQV ALDNNRSLIE NGTSQNQVDL G

In addition, the present invention is characterized in that the gene encoding the D-lactic acid dehydrogenase is also within the scope of the invention, and particularly includes the gene sequence of SEQ ID NO: 2. Sugar gene sequences were registered in the NCBI database. (MH920335)

[SEQ ID NO: 2]

ATGAAAATTA CCGCGTATGG CATCCGTGAA GATGAACGCC CGTACCTGAA TGAATGGCAG

GAGAAAAACA ATATTGAAGT GCAAGCCGTT AGTGAACTGC TGGATTCCGA AACCCTGGAA

CTGGCGAAAG GCTCAGACGG TGTGGTTGCC TTTCAGCAAA AACCGTATAC GGATGACATT

TTCTCGAAAA TGAATCGTTT TGGCATCCAT GCGTTCAGTC TGCGCAACGT TGGTGTCGAT

AACCTGTCCT TTAATGCGCT GAAAGAAAAC AATGTTATGC TGTCAAACGT CCCGGCCTAC

TCGCCGAATG CAATTGCTGA ACTGTCAGTG ACCCAGCTGA TGGCGCTGAT TCGTCGCATC

CCGGATTTTC AAGCCAAAAT GAAACGTGGC GACTTCCGCT GGGAACCGAC GATCGCGCTG

GAACTGAATC AGATGACCGT GGGCGTTATT GGCACGGGTC GTATCGGTCG CGCGGCCATT

GATATCTTTA AAGGCTTCGG TGCAAAAGTG ATTTGCTATG ACGTTTTCCT GAACCCGGAA

CTGGAAAAAG AAGGTGCTTA TGTCGATACC GTGGAAGAAC TGTACAAAAG CGTCGACGTC

GTGACGCTGC ATGTTCCGGC ACTGAAAGAT AACTATCACA TGCTGGATGA CAAAGCTTTT

AATTCTATGA AAGACGGCGT CTTCGTGCTG AACTACAGCC GTGGTTCTCT GATTGATACC

GCAGCTCTGA TCCGCGGCCT GGACTCCGGT AAAATCGCGG GCGTGGGTCT GGATACGTAT

GAAAACGAAG TTGGTATCTT CGAAATCGAT CATGAAGACC AGCCGATTGA TGACGAAATG

TTCAACAATC TGAACGCCCG TCGCAATGTG ATGATCACCC CGCACGCGGC CTTTTACACC

ACGAACGCAG TTAAAAATAT GGTTCAGGTC GCTCTGGATA ACAATCGTAG CCTGATTGAA

AACGGCACCT CTCAGAATCA AGTGGATCTG GGT

Furthermore, in the present invention, the recombinant plasmid containing the gene is also within the scope of the present invention.

Hereinafter, an embodiment of the present invention will be described.

Example

reagent

Pediococcus claussenii, Oenococcus oeni, Weissella confusa, and Weissella koreensis D-Lactate Dehydrogenase Genes (hereinafter referred to as pcHADH, ooHADH, wcHADH, wkHADH) were codon-optimized, including C-terminal His-tag, and GenScript (New Jersey, USA). ) Was commercially synthesized. Restriction enzymes (NdeI, XhoI) were purchased from Enzynomics (Daejeon, Korea). Accepted Escherichia coli Top10 and E. coli BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA, USA). Gene sequencing was performed at Cosmogenetech (Seoul, Korea). Phenylpyruvate salt (sodium phenylpyruvate) was purchased from Tokyo Chemical Industry (Tokyo, Japan). All other reagents were purchased from Sigma-Aldrich (St Louis, USA).

Expression and purification of enzymes

-D-1-thiogalactopyranoside (IPTG) 로 20 °C 에서 24시간 동안 유도하였다. 단백질 발현을 유도한 세포는 Ni-NTA 컬럼을 이용하여 정제하였으며, 순도는 12% SDS-PAGE로 측정하였다. 효소 농도는 Bradford 분석법 (Bradford, 1976)을 이용하여 측정하였다.For protein expression, each gene was transformed with E. coli BL21 (DE3) for protein expression, incubated at 37 °C with 100 μg/ml ampicillin in lysogeny broth for 16 hours and 0.8 mM isopropyl

-D-1-thiogalactopyranoside (IPTG) was induced at 20 °C for 24 hours. Cells induced protein expression were purified using a Ni-NTA column, and the purity was measured by 12% SDS-PAGE. The enzyme concentration was measured using the Bradford assay (Bradford, 1976).

Enzyme activity and kinetics analysis

To analyze the in vitro activity of the four enzymes, the reduction of pyruvate and phenylpyruvate was monitored by measuring the oxidation rate of NADH using a 340 nm UV spectrophotometer (Molecular Devices, USA) at 30 °C. In 50 mM sodium acetate (pH 5.5) with 15.0 nM enzyme and 0.200 mM NADH, specific activity was measured using 1.25 mM pyruvic acid or phenylpyruvate as a substrate, and K _M and k _cat values for each substrate were measured. I did.

In vitro Phenyllactic acid conversion enzyme reaction and HPLC analysis

The conversion of phenylpyruvate to phenyllactic acid was 8.59 nM D-lactic acid dehydrogenase, 20 mM phenylpyruvate, 1,5 mM NADH, 30 mM sodium formate, 1 unit Candida boidinii formate dehydrogenase in 50 mM sodium acetate buffer ( pH6.5) for 12 hours. After obtaining at predetermined time intervals, the enzyme was inactivated at 99 °C, centrifuged, filtered with a 0.2 μM PVDF filter (GE Healthcare, Pittsburgh, USA) and analyzed by HPLC. HPLC analysis was performed on a YL9100 HPLC system (YL instruments, Anyang, Korea) equipped with a Bio-Rad Aminex® HPX-87H Ion exclusion column (300 mm x 7.8 mm), and 5 mM sulfuric acid at a rate of 0.6 ml min ^-1 It was used as a mobile phase. Phenylpyruvate and phenyllactic acid were detected at 37 °C and 210 nm UV detector at 19.42 and 41.28 minutes, respectively. In addition, using a Daicel Chiralcel OJ-H column (250 mm x 0.46 mm, 5 μm) and a mobile phase with a 90:10:0.1 (v:v) ratio of hexane, isopropanol, and trifluoroacetic acid, the optical of phenyllactic acid in a 261 nm UV detector. Purity was measured. In this case, D-phenyllactic acid was detected at 30 minutes, L-phenyllactic acid was detected at 34 minutes, and phenylpyruvate at 38 minutes, respectively.

Enzyme activity screening

To screen the activities of pcHADH, ooHADH, wcHADH, and wkHADH, specific activities of each were measured using pyruvic acid and phenylpyruvate as substrates (Figure 1). As a result, pcHADH, ooHADH, and wcHADH except wkHADH showed catalytic activity for each substrate, and wcHADH for pyruvate showed the highest specific activity at 428 U mg ^-1 , whereas pcHADH for phenylpyruvate showed 591 U mg ^-1 . It showed the highest specific activity among the four enzymes.

Enzyme kinetics analysis

As a result of calculating the values of K _M and k _cat for the kinetic analysis of each enzyme, as shown in Table 1, pcHADH showed the highest catalytic efficiency (1,348 s ^-1 mM ^-1 ), and the previously known D-type lactate dehydrogenase Showed higher efficiency than those (Table 2). In particular, pcHADH shows a higher substrate preference (165%) for phenylpyruvate than pyruvate, so it is expected that pyruvate will convert phenylpyruvate more efficiently in a whole cell reaction capable of competitive inhibition.

2-HADH Kinetic parameters for substrates

(%)

(%) Pyruvate PPA k _cat (s ^-1 ) K _m (mM) k _cat / K _m
(s ^-1 mM ^-1 ) k _cat (s ^-1 ) K _m (mM) k _cat / K _m
(s ^-1 mM ^-1 ) ooHADH 674 ± 22 1.12 ± 0.04 602 ± 1.43 77.8 ± 5.36 15.6 ± 0.53 5.00 ± 0.18 0.83 wcHADH 1,434 ± 240 1.28 ± 0.24 1,124 ± 23.6 457 ± 15.1 20.3 ± 0.60 22.5 ± 0.07 2.00 pcHADH 459 ± 30 0.56 ± 0.01 813 ± 46.1 472 ± 33.8 0.35 ± 0.05 1,348 ± 99.2 165

Bacterial origin of
enzymes Pyruvate PPA Reference k _cat
(s ^-1 ) K _m
(mM) k _cat / K _m
(s ^-1 mM ^-1 ) k _cat
(s ^-1 ) K _m
(mM) k _cat / K _m
(s ^-1 mM ^-1 ) L. bulgaricus ATCC11842 NR NR NR 11.3 11.4 1.00 Zheng, 2013 P. pentosaceus ATCC25745 320 0.49 658 173 1.73 100 Yu, 2012 S. inulinus CASD 8.85 3.40 22.60 4.94 3.32 1.49 Wang, 2016 P. acidilactici DSM20284 287 0.09 3,157 305 2.92 105 Mu, 2012 L. plantarum SK002 NR 0.06 NR NR 5.40 NR Jia, 2010 B. coagulans SDM 23.6 2.20 11 16.5 4.40 3.90 Zheng, 2011 L. fermentum JN248 NR NR NR 123 1.68 73.0 Chen, 2017 Lactobacillus sp. CGMCC 9967 NR NR NR 47.3 0.82 57.7 Xu, 2016 W. fluorescens TK1 NR NR NR 150 0.40 380 Fujii, 2011 L. pentosus JCM1558 321 0.12 2,675 40 0.80 50.0 Tokuda, 2003

NR: Not Reported

Enzymatic conversion of phenylpyruvate to phenyllactic acid

After separating and purifying the three enzymes (pcHADH, ooHADH, wcHADH) showing activity against phenylpyruvate, each reaction proceeded with an in vitro enzymatic reaction in which a NADH regeneration system was introduced using phenylpyruvate as a substrate. It was confirmed that type-phenyllactic acid was produced (Figure 2). After the reaction for 4 hours, more than 99% of D-phenyllactic acid was enzymatically converted by pcHADH, but for wcHADH and ooHADH, the conversion rate was only about 60% and 25%, respectively, up to 12 hours. Through this, it was confirmed that pcHADH had high catalytic activity and conversion efficiency for D-phenyllactic acid.

<110> Industry-University Cooperation Foundation Sogang University <120> A D-lactate dehydrogenase with high substrate preference for phenylpyruvate <130> PN190220 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 331 <212> PRT <213> Unknown <220> <223> Pediococcus claussenii <400> 1 Met Lys Ile Thr Ala Tyr Gly Ile Arg Glu Asp Glu Arg Pro Tyr Leu 1 5 10 15 Asn Glu Trp Gln Glu Lys Asn Asn Ile Glu Val Gln Ala Val Ser Glu 20 25 30 Leu Leu Asp Ser Glu Thr Leu Glu Leu Ala Lys Gly Ser Asp Gly Val 35 40 45 Val Ala Phe Gln Gln Lys Pro Tyr Thr Asp Asp Ile Phe Ser Lys Met 50 55 60 Asn Arg Phe Gly Ile His Ala Phe Ser Leu Arg Asn Val Gly Val Asp 65 70 75 80 Asn Leu Ser Phe Asn Ala Leu Lys Glu Asn Asn Val Met Leu Ser Asn 85 90 95 Val Pro Ala Tyr Ser Pro Asn Ala Ile Ala Glu Leu Ser Val Thr Gln 100 105 110 Leu Met Ala Leu Ile Arg Arg Ile Pro Asp Phe Gln Ala Lys Met Lys 115 120 125 Arg Gly Asp Phe Arg Trp Glu Pro Thr Ile Ala Leu Glu Leu Asn Gln 130 135 140 Met Thr Val Gly Val Ile Gly Thr Gly Arg Ile Gly Arg Ala Ala Ile 145 150 155 160 Asp Ile Phe Lys Gly Phe Gly Ala Lys Val Ile Cys Tyr Asp Val Phe 165 170 175 Leu Asn Pro Glu Leu Glu Lys Glu Gly Ala Tyr Val Asp Thr Val Glu 180 185 190 Glu Leu Tyr Lys Ser Val Asp Val Val Thr Leu His Val Pro Ala Leu 195 200 205 Lys Asp Asn Tyr His Met Leu Asp Asp Lys Ala Phe Asn Ser Met Lys 210 215 220 Asp Gly Val Phe Val Leu Asn Tyr Ser Arg Gly Ser Leu Ile Asp Thr 225 230 235 240 Ala Ala Leu Ile Arg Gly Leu Asp Ser Gly Lys Ile Ala Gly Val Gly 245 250 255 Leu Asp Thr Tyr Glu Asn Glu Val Gly Ile Phe Glu Ile Asp His Glu 260 265 270 Asp Gln Pro Ile Asp Asp Glu Met Phe Asn Asn Leu Asn Ala Arg Arg 275 280 285 Asn Val Met Ile Thr Pro His Ala Ala Phe Tyr Thr Thr Asn Ala Val 290 295 300 Lys Asn Met Val Gln Val Ala Leu Asp Asn Asn Arg Ser Leu Ile Glu 305 310 315 320 Asn Gly Thr Ser Gln Asn Gln Val Asp Leu Gly 325 330 <210> 2 <211> 993 <212> DNA <213> Unknown <220> <223> Pediococcus claussenii <400> 2 atgaaaatta ccgcgtatgg catccgtgaa gatgaacgcc cgtacctgaa tgaatggcag 60 gagaaaaaca atattgaagt gcaagccgtt agtgaactgc tggattccga aaccctggaa 120 ctggcgaaag gctcagacgg tgtggttgcc tttcagcaaa aaccgtatac ggatgacatt 180 ttctcgaaaa tgaatcgttt tggcatccat gcgttcagtc tgcgcaacgt tggtgtcgat 240 aacctgtcct ttaatgcgct gaaagaaaac aatgttatgc tgtcaaacgt cccggcctac 300 tcgccgaatg caattgctga actgtcagtg acccagctga tggcgctgat tcgtcgcatc 360 ccggattttc aagccaaaat gaaacgtggc gacttccgct gggaaccgac gatcgcgctg 420 gaactgaatc agatgaccgt gggcgttatt ggcacgggtc gtatcggtcg cgcggccatt 480 gatatcttta aaggcttcgg tgcaaaagtg atttgctatg acgttttcct gaacccggaa 540 ctggaaaaag aaggtgctta tgtcgatacc gtggaagaac tgtacaaaag cgtcgacgtc 600 gtgacgctgc atgttccggc actgaaagat aactatcaca tgctggatga caaagctttt 660 aattctatga aagacggcgt cttcgtgctg aactacagcc gtggttctct gattgatacc 720 gcagctctga tccgcggcct ggactccggt aaaatcgcgg gcgtgggtct ggatacgtat 780 gaaaacgaag ttggtatctt cgaaatcgat catgaagacc agccgattga tgacgaaatg 840 ttcaacaatc tgaacgcccg tcgcaatgtg atgatcaccc cgcacgcggc cttttacacc 900 acgaacgcag ttaaaaatat ggttcaggtc gctctggata acaatcgtag cctgattgaa 960 aacggcacct ctcagaatca agtggatctg ggt 993

Claims (12)

A composition for producing D-3-phenyllactate containing D-lactate dehydrogenase (D-lactate dehydrogenase) consisting of the amino acid sequence of SEQ ID NO: 1. A microorganism for producing D-3-phenyllactate transduced with the gene represented by the nucleotide sequence of SEQ ID NO: 2. The microorganism for producing D-3- phenyllactic acid according to claim 2, wherein the gene is derived from Pediococcus claussenii . The microorganism for producing D-3-phenyllactic acid according to claim 2, wherein the microorganism is Escherichia coli . A composition for producing D-3-phenyllactic acid comprising a microorganism for producing D-3-phenyllactate or a culture solution thereof transduced with the gene represented by the nucleotide sequence of SEQ ID NO: 2. The composition for producing D-3- phenyllactic acid according to claim 5, wherein the gene is derived from Pediococcus claussenii . The composition for producing D-3-phenyllactic acid according to claim 5, wherein the microorganism is Escherichia coli . The composition for producing D-3-phenyllactic acid according to claim 5, wherein the composition further comprises phenylpyruvate as a substrate. D-3-phenyl lactic acid production method comprising a culture step of culturing a microorganism for producing D-3-phenyllactate transduced with the gene represented by the nucleotide sequence of SEQ ID NO: 2. The method of claim 9, wherein the gene is derived from Pediococcus claussenii . The method for producing D-3-phenyllactic acid according to claim 9, wherein the microorganism is Escherichia coli . The method for producing D-3-phenyllactic acid according to claim 9, wherein the culturing step is performed using phenylpyruvate as a substrate.

Images