- Email: [email protected]
Identification of a l -Lactate dehydrogenase with 3,4-dihydroxyphenylpyruvic reduction activity for l -Danshensu production
Process Biochemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Identification of a L-Lactate dehydrogenase with 3,4dihydroxyphenylpyruvic reduction activity for L-Danshensu production ⁎
⁎
Huan Lua, Yajun Baib, Tai-ping Fanb,c, Ye Zhaob, , Xiaohui Zhengb, , Yujie Caia,
⁎
a
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China College of Life Sciences, Northwest University, Xi’an, Shanxi, 710069, China c Department of Pharmacology, University of Cambridge, Cambridge, CB2 1T, UK b
A R T I C LE I N FO
A B S T R A C T
Keywords: Danshensu 3,4-Dihydroxyphenylpyruvate L-lactate dehydrogenase Lactobacillus fermentum Glucose dehydrogenase Whole cell biocatalysis
Danshensu (DSS), also known as 3,4-dihydroxyphenyllactate, is an herbal preparation with prominent pharmacological activities. It has lactic acid structure and may be obtained by reducing 3,4-dihydroxyphenylpyruvate with lactate dehydrogenase. We screened a coenzyme-aspecific L-lactate dehydrogenase (LF-L-LDH0845) from lactobacillus fermentum for the bioconversion of 3,4-dihydroxyphenylpyruvate to optically pure (ee ≥ 99.99%) L-DSS. LF-L-LDH0845 has an approximate molecular weight of 33.65 kDa, exhibits wide substrate scope for 2-keto-carboxylic acids. Values of Km, Kcat, and Kcat/Km for LF-L-LDH0845 with 3,4-dihydroxy-phenylpyruvate substrate were 11.37 mM, 0.2931 s−1, and 0.0258 mM−1 s−1, respectively. LF-L-LDH0845 was most active and stable at pH 6.0, the optimum temperature was 25 °C, stability decreased with increasing temperature, and activity was lost completely at 50 °C. K+ stimulated while Fe2+ and Cu2+ inhibited the enzyme activity significantly. Glucose dehydrogenase gene was coexpressed with lf-l-ldh0845 in E. coli to regenerate cofactors by oxidising glucose, which efficiently reduced 3,4-dihydroxyphenylpyruvate to L-DSS with 95.45% isolation yield.
1. Introduction Danshensu (DSS), a major hydrophilic phenolic acid extracted from the dried root of Salvia miltiorrhiza Bunge (Danshen), is also known as 3(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid [1]. DSS possesses anti-inflammatory, anti-anxiety, microcirculation amelioration, apoptosis suppression, and various other activities [2–6]. To date, L-DSS has been less well studied than the naturally occurring D-DSS enantiomer. However, L-DSS has unique pharmacological activities that D-DSS does not possess, and L-DSS can also be used as a precursor for the synthesis of polyphenolic acids such as rosmarinic acid [7]. Synthesis of DSS with high enantioselectivity is difficult due to chemical instability and its chiral structures [8]. Rao et al. synthesis LDSS using L-2-hydroxyishexanoic acid dehydrogenase (L-HicDH) and regenerates cofactor (NADH) with formate dehydrogenase (FDH) and ammonium formate [7]. Findrik et al. established a mathematical model based on enzymatic kinetics to synthesise D-DSS catalysed by Dlactate dehydrogenase from Lactobacillus leishmannii with NADH as a coenzyme coupled with NADH regeneration using formate dehydrogenase [9]. Transformation by enzymatic catalysis is sometimes faster and less
⁎
costly than using the Embden Meyerhof pathway starting with glucose [10]. In order to prepare suitable enzymes for the bioreduction of 3,4dihydroxyphenylpyruvate to L-DSS, we screened a batch of Lactobacillus for L-DSS production and identified a L-lactate dehydrogenase gene from Lactobacillus fermentum with good activity. A whole-cell catalyst from E. coli BL21 co-expressing LDH and GDH was constructed. 2. Materials and methods 2.1. Strains, plasmids, reagents and culture conditions We screened 20 Lactobacillus strains for L-DSS production, including L. fermentum (5 strains), L. plantarum (4 strains), L. delbrueckii (3 strains), L. reuteri (1 strain), L. johnsonii (3 strains), L. casei (2 strains), and L. coryniformis (2 strains). All strains were inoculated into MRS medium and cultured at 30 °C without shaking for 24 h. The host strain for gene cloning was E. coli DH5α, while the host strain for protein expression was E. coli BL21, and both were inoculated in LB medium and cultured at 200 rpm and 37 °C for 12 h. The plasmid pColdII was used as a cloning vector. All strains and vectors listed above were screened and preserved in our laboratory.
Corresponding authors. E-mail addresses: [email protected] (Y. Zhao), [email protected] (X. Zheng), [email protected] (Y. Cai).
https://doi.org/10.1016/j.procbio.2018.06.011 Received 18 March 2018; Received in revised form 7 May 2018; Accepted 15 June 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Lu, H., Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.06.011
Process Biochemistry xxx (xxxx) xxx–xxx
H. Lu et al.
Pyruvate, phenylpyruvate, α-ketobutyrate, 4-methyl-2-oxopentanoic acid, benzoylformic acid, α-ketoglutaric acid, 4-hydroxyphenylpyruvate, glucose, NADH and NADPH were acquired from Sigma-Aldrich (Shanghai, China). 3,4-dihydroxyphenylpyruvate was synthesised according to the method described previously [11]. Protein assay kit, DNA extraction and purification kits were purchased from TaKaRa (Otsu, Japan). Other molecular reagents and enzymes were purchased from Sangon Biotech (Shanghai China). The organic reagents used were domestic analysis of pure.
added as a control. The residual activity was determined under optimal conditions and the relative enzyme activities were calculated with respect to the control assay (100%). Eight α-ketoacids (pyruvate, α-ketobutyric acid, α-ketoglutaric acid, 4-methyl-2-oxobutanoic acid, phenylpyruvate, 3,4-dihydroxyphenylpyruvate, 4-hydroxy-phenylpyruvate, benzoylformic acid) were chosen to determine the substrate specificity of LF-L-LDH0845. 2.2.4. Construction of coexpression plasmid and biotransformation by whole cells The plasmid pColdII-ldh0845-T7-gdh was constructed as follows: The glucose dehydrogenase gene (GenBank accession no. EF626962.1) from Bacillus subtilis with T7 promoter and RBS was synthesized by Synbio Technologies (Suzhou, China), then amplified with primers (shown in Supplementary data) designed two restriction sites EcoR I and Pst I to the forward and reverse end respectively, and ligated to pColdII-ldh0845 vector that had been digested with the same restriction enzymes. The resulting recombinant plasmids (pColdII-ldh0845-T7gdh) were transformed into E. coli BL21(DE3) for protein expression and harvested for bioconversion. The 50 mL bioconversion reaction containing 20 mM phosphate buffer (pH 6.0), 10 g L−1 wet cells, 70 mM 3,4-dihydroxyphenylpyruvate and 70 mM glucose was incubated at 25 °C with shaking at 100 rpm. The concentration and enantioselectivity of L-DSS were quantitatively analyzed by HPLC at 2 h intervals.
2.2. Methods 2.2.1. Screening of strains 20 strains of lactobacillus used for screening were cultured in MRS medium at 30 °C for 24 h. Cells were harvested by centrifugation (6790 g g, 4 °C, 10 min) and disrupted by ultrasonic, then centrifuged (12,000 g, 4 °C, 10 min) to remove cell debris, the supernatant was collected as the crude enzyme. The relative enzyme activity for reduction of 3,4-dihydroxy-phenylpyruvate was assayed by HPLC to identify the strain with the highest activity. The reaction system consisted of a total volume of 1 mL containing NADH (2 mM), 3,4-dihydroxy-phenylpyruvate (10 mM), buffer (20 mM), and an appropriate amount of enzyme solution. Reactions were maintained at 25 °C for 30 min, then terminated by boiling for 5 min. The reaction solution was extracted with three times the volume of ethylacetate, the upper layer was pipetted and dried with nitrogen gas until ethylacetate was completely evaporated. The powder was dissolved in 1 mL of ethanol, and then assayed by HPLC with a Chiralpak ID (0.46 cm × 25 cm×5 cm) (Daicel Chiral Technologies Co., Ltd.). Elution was performed with nhexane/ ethanol/ trifluoroacetic acid (80/20/0.1) at a flow rate of 1.0 mL/min at 220 nm.
3. Results 3.1. Strain screening, protein expression and enzyme activity assay After comparing the yield of L-DSS produced by the 20 strains of Lactobacilli, we screened seven strains with the ability to synthesize LDSS, and L. fermentum JN248 exhibited the highest activity. Five genes (GenBank accession nos. MG581694, MG581695, MG581696, MG581697, MG581698) were amplified from L. fermentum JN248 genomic DNA as the template and expressed in E. coli BL21. Crude and pure enzymes were analysed by SDS-PAGE, and pure enzymes ran as single bands with apparent molecular weights close to those expected based on the putative amino acid sequences (Fig. 1). The activity of the five purified enzymes was determined using a UV/vis spectrophotometer. LF-L-LDH0845 display the highest activity (relative activity of 100% corresponded to the specific activity 16.48 U/mg) for reducing 3,4-dihydroxy-phenylpyruvate to L-DSS with NADH as coenzyme followed by LF-L-LDH0372 (76%), LF-L-LDH1732 (47%) and LFL-LDH1566 (41%), whereas LF-L-LDH0611 was inactive. Further study found that LF-L-LDH0845 can also synthesize L-DSS with NADPH as coenzyme with specific activity of 15.30 U/mg.
2.2.2. Cloning, expression and purification of recombinant L-LDH Based on the lactate dehydrogenase genes in L. fermentum F-6 (GenBank accession no. CP005958.1), primers with suitable restriction enzyme sites were designed using Primer Premier 6.0 software (shown in Supplementary data). The ldhL genes were amplified from genomic DNA of L. fermentum JN248 as a template. Purified PCR products were ligated to the pColdⅡ cloning vector. The resultant plasmids were transferred into E. coli BL21(DE3), then induced with 0.2 mM IPTG at 200 rpm and 15 °C for 24h [12]. Cells were harvested, the crude enzyme solution was purified by passing through a His Trap HP column and a HiTrap desalting column. Protein concentration was measured using a BCA Protein Concentration Assay Kit, and both crude and pure enzyme solutions were analysed on 12% SDS-PAGE [13–15]. The activities of the five purified enzymes for the conversion of 3,4-dihydroxyphenylpyruvate to L-DSS were determined by measuring the rate of reduction of NADH or NADPH at 340 nm using a UV/vis spectrophotometer [16]. The enzyme with the highest activity was used for the further study of enzymatic properties.
3.2. Enzymatic properties of LF-L-LDH0845 Fig. 2 shows the pH and temperature profiles of LF-L-LDH0845 for the reduction of 3,4-dihydroxy-phenylpyruvate. LF-L-LDH0845 was the most active and stable at pH 6.0 whereas enzyme activity was unstable and completely lost at pH below 3.0 and above 9.0. The optimum temperature for LF-L-LDH0845 was 25 °C. After incubation at different temperatures for 1 h, the enzyme activity decreased sharply with increasing temperature. Enzyme activity was decreased to 49.48% at 30 °C, and all activity was lost after incubation at 50 °C. Kinetic parameters of LF-L-LDH0845 for different substrates were measured under the optimum conditions. Values of Km, Vmax, Kcat, and Kcat/Km for different substrates was shown in Table 1. It was interesting that the enzyme had a similar Km value for NADH and NADPH. After incubation for 6 h in the metal ion solutions, the effect of K+, 2+ Fe and Cu2+ on the enzyme activity of LF-L-LDH0845 was relatively significant, among which, K+ promoted the enzyme activity by 20% while Fe2+ inhibited enzyme activity by 70% and Cu2+ inhibited the
2.2.3. Enzymatic properties of LF-L-LDH0845 The optimum pH and temperature of LF-L-LDH0845 were assayed by varying the pH (4.0–11.0) and the temperature (10–65 °C) respectively. For the study of pH stability and thermostability, the enzyme solution was pre-incubated at different pH and temperatures for 1 h, then the residual enzyme activity for the reduction of 3,4-dihydroxyphenylpyruvate to L-DSS was determined under optimal conditions by HPLC. Relative activities were defined relative to the maximum activity [17]. Kinetic parameters for different substrates were assessed by varying the concentration of the corresponding substrate in 20 mM phosphate buffer (pH 6.0) at 25 °C. The kinetic data were calculated by non-linear fitting with OriginPro 8.0 software [18]. Different metal ions (Mn2+, Mg2+, Na+, Ni+, Ca2+, Co2+, Cu2+, Fe2+, K+ and Zn2+) were incubated with purified LF-L-LDH0845 solution at 25 °C for 6 h, and the same amount of deionised water was 2
Process Biochemistry xxx (xxxx) xxx–xxx
H. Lu et al.
Fig. 1. SDS-PAGE analysis of LF-L-LDHs from Lactobacillus fermentum JN248. The electrophoresis was performed with 12% agarose gel and stained with coomassie brilliant blue R250. Lane M, low molecular weight standards; lane 1, crude extract of E. coli BL21 (DE3)/ pColdII after induction; lane 2, crude extract of E. coli BL21 with LF-L-LDHs after induction; lane 3, the purified LF-L-LDHs after nickel-affinity chromatography. A–E shows the protein electrophoresis diagrams of LF-L-LDH0845, LFL-LDH1732, LF-L-LDH0611, LF-L-LDH1566 and LF-L-LDH0372, respectively.
3.3. Production of L-DSS by using the coupling system
enzyme activity by 60%. The effect of other metal ions on enzyme activity was considered to be negligible. As shown in Table 2, LF-L-LDH0845 was active toward pyruvate, phenylpyruvic acid, α-ketobutyrate, α-ketoglutaric acid, 4-methyl-2oxopentanoic acid, benzoylformic acid, 4-hydroxyphenylpyruvic acid, and 3,4-dihydroxyphenylpyruvic acid. The optimal substrate for this enzyme was pyruvate, followed by phenylpyruvate, confirming LF-LLDH0845 as an L-lactate dehydrogenase.
On account of the solubility of DSS less than 80 mM, the initial concentration of substrate 3,4-dihydroxyphenylpyruvate in the reaction system was set to 70 mM. In this coupling system, LF-L-LDH0845 and GDH were successfully coexpressed in E. coli BL21 (DE3). After 12 h of whole-cell catalytic reaction, the substrate 3,4-dihydroxyphenylpyruvate was almost completely converted to L-DSS in the presence of Fig. 2. pH and temperature profiles of purified recombinant LF-L-LDH0845. (A) Effect of pH on enzyme activity. The enzyme activity was measured in buffers with various pH (4.0–11.0) at 30 °C. The maximum enzyme activity at pH 6.0 was defined as 100%. (B) pH stability of LFL-LDH0845. The enzyme activity was measured after preincubation at different pH (4.0–11.0) for 1 h. (C) Effect of temperature on enzyme activity. The enzyme activity was assayed in disodium phosphate-citrate buffer (pH 6.0) at 10–65 °C. The maximum enzyme activity at 25 °C was defined as 100%. (D) Thermostability of LF-L-LDH0845. The enzyme activity was measured in disodium phosphatecitrate buffer (pH 6.0) after preincubation at different temperature (10–65 °C) for 1 h.
3
Process Biochemistry xxx (xxxx) xxx–xxx
H. Lu et al.
Table 1 Kinetic parameters of LF-L-LDH0845 for various substrates. Substrate
Km (mM)
Vmax (mM min−1 mg−1)
Kcat (S−1)
Kcat/Km (mM−1 S−1)
3,4-Dihydroxyphenylpyruvate Pyruvate NADH NADPH
11.3709 2.4662 0.1234 0.1976
0.5226 × 10−3 0.0222 0.1149 0.0185
0.2931 12.4498 64.4257 10.3748
0.0258 5.0481 521.9192 52.5173
This result is consistent with a previous study in which the Km of LLDHs for 2-ketocarboxylic acids increased with substrates containing longer aliphatic chains [27]. The catalytic efficiency (Kcat/Km) of LF-LLDH0845 toward 3,4-dihydroxyphenylpyruvate 0.0258 mM−1 S−1 was lower than that toward pyruvic acid 5.0481 mM−1 S−1, suggesting that the catalytic capacity of LF-L-LDH0845 toward 3,4-dihydroxy-phenylpyruvic acid could be improved by optimising the enzyme structure in the future. LF-L-LDH0845 exhibited high substrate specificity for most α-keto acids with particular preference for pyruvate and phenylpyruvate. Since LF-L-LDH0845 is coenzyme non-specific, GDH used to construct coupling system with LF-L-LDH0845 can also regenerate both NADH and NADPH [28]. By using the whole cells containing a coupling system as biocatalyst, the coenzymes (NAD and NADP) in cells are fully utilized, moreover, the double -enzyme system is provided with a more stable catalytic environment for the industrial-scale preparation of LDSS. However, the solubility of the substrate 3,4-dihydroxyphenylpyruvate in water is low, how to increase the solubility of 3,4-dihydroxyphenylpyruvate and the yield of L-DSS is still in progress.
Table 2 The substrate specificity of LF-L-LDH0845. Substrate (10 mM)
Specific activity (U/ mg)
Relative activity (%)
Pyruvate α-Ketobutyrate α-Ketoglutaric acid 4-Methyl-2-oxopentanoic acid Phenylpyruvic acid Benzoylformic acid 4-Hydroxyphenylpyruvic acid 3,4-Dihydroxyphenylpyruvic acid
144.81 16.63 21.17 37.46 69.11 29.73 16.08 12.30
100 11.48 14.62 25.87 47.42 20.53 11.10 8.49
Conflict of interest The authors declare that they have no conflict of interests. Author contributions Huan Lu, Xiaohui Zheng and Yujie Cai conceived and designed the experimental scheme. Huan Lu and Yajun Bai carried out the experiments. Taiping Fan, Xiaohui Zheng and Yujie Cai guided and supervised the experiment. Huan Lu, Yajun Bai and Yujie Cai analyzed and processed the experimental data. Huan Lu and Yujie Cai wrote the manuscript. All authors were involved in the revision of the manuscript.
Fig. 3. Time course of the production of L-DSS by coupling system. The biocatalyst by the whole-cell E. coli BL21(DE3) harboring pColdII-ldh0845-T7-gdh was carried out at 25 °C with shaking. ■, the conversion rate of 3,4-dihydroxyphenylpyruvate to L-DSS over time; ●, the ee% of L-DSS.
Acknowledgements
glucose, resulting in a 95.45% conversion rate and high enantioselectivity (enantiomeric excess ≥99.99%) (Fig. 3).
We gratefully thank for the financial support of this work by the National Key Scientific Instrument and Equipment Development Project of China (2013YQ17052504), Program for Changjiang Scholars and Innovative Research Team in the University of Ministry of Education of China (IRT_15R55), The Key Project of Research and Development Plan of Shanxi (2017ZDCXL-SF-01-02-01), Program for Changjiang Scholars and Innovative Research in University (IRT_15R55) and the seventh group of Hundred-Talent Program of Shanxi Province (2015).
4. Discussion LDH has been studied extensively for the synthesis of phenyllactic acid and lactic acid, two structural analogues of DSS. The conversion of 3,4-dihydroxyphenylpyruvate to L-DSS by L-LDHs has not previously been reported. We screened a non-specific coenzyme-dependent LF-LLDH0845 for the bioconversion of 3,4-dihydroxyphenylpyruvate to optically pure (ee ≥ 99.99%) L-DSS. The optimum temperature of LF-LLDH0845 for catalytic reduction of 3,4-dihydroxyphenylpyruvate was 25 °C, which is lower than that of other L-LDHs for the reduction of phenylpyruvic acid [19–21]. The thermostability of LF-L-LDH0845 declined at elevated temperatures, and dropped to zero at 50 °C, consistent with other L-LDHs in previous studies [22]. The optimum pH of LF-L-LDH0845 was 6.0, which is slightly higher than the optimum pH of 5.0 for L-LDH from L. helveticus 53/7 for the production of lactic acid [23]. LF-L-LDH0845 tolerated a pH between 5.0 and 7.0. The Km of LFL-LDH0845 for 3,4-dihydroxyphenylpyruvate was 11.37 mM, which is higher than that of other L-LDHs for the reduction of phenylpyruvate or pyruvate, indicating a lower affinity of LF-L-LDH0845 for 3,4-dihydroxyphenylpyruvate than phenylpyruvate or pyruvic acid [24–26].
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2018.06.011. References [1] S. Seetapun, J. Yaoling, Y. Wang, Y.Z. Zhu, Neuroprotective effect of Danshensu derivatives as anti-ischaemia agents on SH-SY5Y cells and rat brain, Biosci. Rep. 33 (4) (2013). [2] K. Chan, S.H. Chui, D.Y. Wong, W.Y. Ha, C.L. Chan, R.N. Wong, Protective effects of Danshensu from the aqueous extract of Salvia miltiorrhiza (Danshen) against homocysteine-induced endothelial dysfunction, Life Sci. 75 (26) (2004) 3157–3171. [3] J.Y. Han, Y. Horie, J.Y. Fan, K. Sun, J. Guo, S. Miura, T. Hibi, Potential of 3,4-
4
Process Biochemistry xxx (xxxx) xxx–xxx
H. Lu et al.
[4]
[5]
[6]
[7] [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] K. Schersch, O. Betz, P. Garidel, S. Muehlau, S. Bassarab, G. Winter, Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins, part 2: stability during storage at elevated temperatures, J. Pharm. Sci. 101 (7) (2012) 2288–2306. [17] K. Min, Y.J. Yeon, Y. Um, Y.H. Kim, Novel NAD-independent D-lactate dehydrogenases from Acetobacter aceti and Acidocella species MX-AZ02 as potential candidates for in vitro biocatalytic pyruvate production, Biochem. Eng. J. 105 (2016) 358–363. [18] C. Jun, Y.S. Sa, S.-A. Gu, J.C. Joo, S. Kim, K.-J. Kim, Y.H. Kim, Discovery and characterization of a thermostable d-lactate dehydrogenase from Lactobacillus jensenii through genome mining, Process Biochem. 48 (1) (2013) 109–117. [19] S.A. Gu, C. Jun, J.C. Joo, S. Kim, S.H. Lee, Y.H. Kim, Higher thermostability of llactate dehydrogenases is a key factor in decreasing the optical purity of d-lactic acid produced from Lactobacillus coryniformis, Enzyme Microb. Technol. 58–59 (2014) 29–35. [20] Q. Zhou, W.L. Shao, Molecular genetic characterization of the thermostable L-lactate dehydrogenase gene (ldhL) of Thermoanaerobacter ethanolicus JW200 and biochemical characterization of the enzyme, Biochemistry (Mosc.) 75 (4) (2010) 526–530. [21] Chao Gao, Tianyi Jiang, Peipei Dou, Cuiqing Ma, Lixiang Li, Jian Kong, Ping Xu, NAD-independent L-lactate dehydrogenase Is required for L-lactate utilization in Pseudomonas stutzeri SDM, PLoS One 7 (5) (2012) e36519. [22] Ellen I. Garvie, Bacterial lactate dehydrogenases, Microbiol. Rev. 44 (1) (1980). [23] Kirsi Savijoki, Airi Palva, Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme, Appl. Environ. Microbiol. 63 (7) (1997) 2850–2856. [24] Jianghua Jia, Wanmeng Mu, Tao Zhang, B. Jiang, Bioconversion of phenylpyruvate to phenyllactate: gene cloning, expression, and enzymatic characterization of Dand L1-lactate dehydrogenases from Lactobacillus plantarum SK002, Appl. Biochem. Biotechnol. 162 (2009) 242–251. [25] S. Yeswanth, Y. Nanda Kumar, U. Venkateswara Prasad, V. Swarupa, V. Koteswara Rao, P. Venkata Gurunadha Krishna Sarma, Cloning and characterization of l-lactate dehydrogenase gene of Staphylococcus aureus, Anaerobe 24 (2013) 43–48. [26] Limin Wang, Yumeng Cai, Lingfeng Zhu, Honglian Guo, Bo Yu, Major role of NADdependent lactate dehydrogenases in the production of L-lactic acid with high optical purity by the thermophile Bacillus coagulans, Appl. Environ. Microbiol. 80 (23) (2014) 7134–7141. [27] H. Schütte, W. Hummel, M.-R. Kula, L-2-hydroxyisocaproate dehydrogenase—a new enzyme from Lactobacillus confusus for the stereospecific reduction of 2-ketocarboxylic acids, Appl. Microbiol. Biotechnol. 19 (1984) 167–176. [28] V. Kaswurm, W.V. Hecke, K.D. Kulbe, R. Ludwig, Guidelines for the application of NAD(P)H regenerating glucose dehydrogenase in synthetic processes, Adv. Synth. Catal. 355 (9) (2013) 1709–1714.
dihydroxy-phenyl lactic acid for ameliorating ischemia-reperfusion-induced microvascular disturbance in rat mesentery, Am. J. Physiol. Gastrointest. Liver Physiol. 296 (2009) G36–G44. F. Wang, Y.Y. Liu, L.Y. Liu, Q.J. Zeng, C.S. Wang, K. Sun, J.Y. Yang, J. Guo, J.Y. Fan, J.Y. Han, The attenuation effect of 3,4-dihydroxy-phenyl lactic acid and salvianolic acid B on venular thrombosis induced in rat mesentery by photochemical reaction, Clin. Hemorheol. Microcirc. 42 (1) (2009) 7–18. Xiaoyan Cui, Yueling Wang, Norihiro Kokudo, Dingzhi Fang, Traditional Chinese medicine and related active compounds against hepatitis B virus infection, BioScience Trends 4 (2) (2010) 39–47. G. Kwon, H.J. Kim, S.J. Park, H.E. Lee, H. Woo, Y.J. Ahn, Q. Gao, J.H. Cheong, D.S. Jang, J.H. Ryu, Anxiolytic-like effect of danshensu [(3-(3,4-dihydroxyphenyl)lactic acid)] in mice, Life Sci. 101 (1–2) (2014) 73–78. Nagaraj N. Rao, Hermann J. Roth, Enzymatic synthesis of (S)-(-)-3-(3,4-dihydroxyphenyl)lactic acid, Arch. Pharm. (Weinheim) 321 (1988) 179–180. C. Dong, Y. Wang, Y.Z. Zhu, Asymmetric synthesis and biological evaluation of Danshensu derivatives as anti-myocardial ischemia drug candidates, Bioorg. Med. Chem. 17 (9) (2009) 3499–3507. Z. Findrik, M. Poljanac, Đ. Vasić-Rački, Modelling and optimization of the (R)(+)-3,4-dihydroxyphenyllactic acid production catalyzed with D-lactate dehydrogenase from Lactobacillus leishmannii using genetic algorithm, Chem. Biochem. Eng. Q. 19 (4) (2005) 351–358. Scott P. France, Lorna J. Hepworth, Nicholas J. Turner, Sabine L. Flitsch, Constructing biocatalytic cascades: in vitro and in vivo approaches to de novo multi-enzyme pathways, ACS Catal. 7 (2017) 710–724. Y. Bai, Q. Zhang, P. Jia, L. Yang, Y. Sun, Y. Nan, S. Wang, X. Meng, Y. Wu, F. Qin, Z. Sun, X. Gao, P. Liu, K. Luo, Y. Zhang, X. Zhao, C. Xiao, S. Liao, J. Liu, C. Wang, J. Fang, X. Wang, J. Wang, R. Gao, X. An, X. Zhang, X. Zheng, Improved process for pilot-scale synthesis of Danshensu (( ± )-DSS) and its enantiomer derivatives, Org. Process Res. Dev. 18 (12) (2014) 1667–1673. J. Zhang, Z. Cui, H. Chang, X. Fan, Q. Zhao, W. Wei, Conversion of glycerol to 1,3dihydroxyacetone by glycerol dehydrogenase co-expressed with an NADH oxidase for cofactor regeneration, Biotechnol. Lett. 38 (9) (2016) 1559–1564. Zhaojuan Zheng, Cuiqing Ma, Chao Gao, Fengsong Li, Jiayang Qin, Haiwei Zhang, Kai Wang, P. Xu, Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM, PLoS One 6 (4) (2011) e19030. O. Hari Prasad, Y. Nanda Kumar, O.V. Reddy, A. Chaudhary, P.V. Sarma, Cloning, expression, purification and characterization of UMP kinase from Staphylococcus aureus, Protein J. 31 (4) (2012) 345–352. U.V. Prasad, D. Vasu, Y.N. Kumar, P.S. Kumar, S. Yeswanth, V. Swarupa, B.V. Phaneendra, A. Chaudhary, P.V. Sarma, Cloning, expression and characterization of NADP-dependent isocitrate dehydrogenase from Staphylococcus aureus, Appl. Biochem. Biotechnol. 169 (3) (2013) 862–869.
5
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Identification of a L-Lactate dehydrogenase with 3,4dihydroxyphenylpyruvic reduction activity for L-Danshensu production ⁎
⁎
Huan Lua, Yajun Baib, Tai-ping Fanb,c, Ye Zhaob, , Xiaohui Zhengb, , Yujie Caia,
⁎
a
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China College of Life Sciences, Northwest University, Xi’an, Shanxi, 710069, China c Department of Pharmacology, University of Cambridge, Cambridge, CB2 1T, UK b
A R T I C LE I N FO
A B S T R A C T
Keywords: Danshensu 3,4-Dihydroxyphenylpyruvate L-lactate dehydrogenase Lactobacillus fermentum Glucose dehydrogenase Whole cell biocatalysis
Danshensu (DSS), also known as 3,4-dihydroxyphenyllactate, is an herbal preparation with prominent pharmacological activities. It has lactic acid structure and may be obtained by reducing 3,4-dihydroxyphenylpyruvate with lactate dehydrogenase. We screened a coenzyme-aspecific L-lactate dehydrogenase (LF-L-LDH0845) from lactobacillus fermentum for the bioconversion of 3,4-dihydroxyphenylpyruvate to optically pure (ee ≥ 99.99%) L-DSS. LF-L-LDH0845 has an approximate molecular weight of 33.65 kDa, exhibits wide substrate scope for 2-keto-carboxylic acids. Values of Km, Kcat, and Kcat/Km for LF-L-LDH0845 with 3,4-dihydroxy-phenylpyruvate substrate were 11.37 mM, 0.2931 s−1, and 0.0258 mM−1 s−1, respectively. LF-L-LDH0845 was most active and stable at pH 6.0, the optimum temperature was 25 °C, stability decreased with increasing temperature, and activity was lost completely at 50 °C. K+ stimulated while Fe2+ and Cu2+ inhibited the enzyme activity significantly. Glucose dehydrogenase gene was coexpressed with lf-l-ldh0845 in E. coli to regenerate cofactors by oxidising glucose, which efficiently reduced 3,4-dihydroxyphenylpyruvate to L-DSS with 95.45% isolation yield.
1. Introduction Danshensu (DSS), a major hydrophilic phenolic acid extracted from the dried root of Salvia miltiorrhiza Bunge (Danshen), is also known as 3(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid [1]. DSS possesses anti-inflammatory, anti-anxiety, microcirculation amelioration, apoptosis suppression, and various other activities [2–6]. To date, L-DSS has been less well studied than the naturally occurring D-DSS enantiomer. However, L-DSS has unique pharmacological activities that D-DSS does not possess, and L-DSS can also be used as a precursor for the synthesis of polyphenolic acids such as rosmarinic acid [7]. Synthesis of DSS with high enantioselectivity is difficult due to chemical instability and its chiral structures [8]. Rao et al. synthesis LDSS using L-2-hydroxyishexanoic acid dehydrogenase (L-HicDH) and regenerates cofactor (NADH) with formate dehydrogenase (FDH) and ammonium formate [7]. Findrik et al. established a mathematical model based on enzymatic kinetics to synthesise D-DSS catalysed by Dlactate dehydrogenase from Lactobacillus leishmannii with NADH as a coenzyme coupled with NADH regeneration using formate dehydrogenase [9]. Transformation by enzymatic catalysis is sometimes faster and less
⁎
costly than using the Embden Meyerhof pathway starting with glucose [10]. In order to prepare suitable enzymes for the bioreduction of 3,4dihydroxyphenylpyruvate to L-DSS, we screened a batch of Lactobacillus for L-DSS production and identified a L-lactate dehydrogenase gene from Lactobacillus fermentum with good activity. A whole-cell catalyst from E. coli BL21 co-expressing LDH and GDH was constructed. 2. Materials and methods 2.1. Strains, plasmids, reagents and culture conditions We screened 20 Lactobacillus strains for L-DSS production, including L. fermentum (5 strains), L. plantarum (4 strains), L. delbrueckii (3 strains), L. reuteri (1 strain), L. johnsonii (3 strains), L. casei (2 strains), and L. coryniformis (2 strains). All strains were inoculated into MRS medium and cultured at 30 °C without shaking for 24 h. The host strain for gene cloning was E. coli DH5α, while the host strain for protein expression was E. coli BL21, and both were inoculated in LB medium and cultured at 200 rpm and 37 °C for 12 h. The plasmid pColdII was used as a cloning vector. All strains and vectors listed above were screened and preserved in our laboratory.
Corresponding authors. E-mail addresses: [email protected] (Y. Zhao), [email protected] (X. Zheng), [email protected] (Y. Cai).
https://doi.org/10.1016/j.procbio.2018.06.011 Received 18 March 2018; Received in revised form 7 May 2018; Accepted 15 June 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Lu, H., Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.06.011
Process Biochemistry xxx (xxxx) xxx–xxx
H. Lu et al.
Pyruvate, phenylpyruvate, α-ketobutyrate, 4-methyl-2-oxopentanoic acid, benzoylformic acid, α-ketoglutaric acid, 4-hydroxyphenylpyruvate, glucose, NADH and NADPH were acquired from Sigma-Aldrich (Shanghai, China). 3,4-dihydroxyphenylpyruvate was synthesised according to the method described previously [11]. Protein assay kit, DNA extraction and purification kits were purchased from TaKaRa (Otsu, Japan). Other molecular reagents and enzymes were purchased from Sangon Biotech (Shanghai China). The organic reagents used were domestic analysis of pure.
added as a control. The residual activity was determined under optimal conditions and the relative enzyme activities were calculated with respect to the control assay (100%). Eight α-ketoacids (pyruvate, α-ketobutyric acid, α-ketoglutaric acid, 4-methyl-2-oxobutanoic acid, phenylpyruvate, 3,4-dihydroxyphenylpyruvate, 4-hydroxy-phenylpyruvate, benzoylformic acid) were chosen to determine the substrate specificity of LF-L-LDH0845. 2.2.4. Construction of coexpression plasmid and biotransformation by whole cells The plasmid pColdII-ldh0845-T7-gdh was constructed as follows: The glucose dehydrogenase gene (GenBank accession no. EF626962.1) from Bacillus subtilis with T7 promoter and RBS was synthesized by Synbio Technologies (Suzhou, China), then amplified with primers (shown in Supplementary data) designed two restriction sites EcoR I and Pst I to the forward and reverse end respectively, and ligated to pColdII-ldh0845 vector that had been digested with the same restriction enzymes. The resulting recombinant plasmids (pColdII-ldh0845-T7gdh) were transformed into E. coli BL21(DE3) for protein expression and harvested for bioconversion. The 50 mL bioconversion reaction containing 20 mM phosphate buffer (pH 6.0), 10 g L−1 wet cells, 70 mM 3,4-dihydroxyphenylpyruvate and 70 mM glucose was incubated at 25 °C with shaking at 100 rpm. The concentration and enantioselectivity of L-DSS were quantitatively analyzed by HPLC at 2 h intervals.
2.2. Methods 2.2.1. Screening of strains 20 strains of lactobacillus used for screening were cultured in MRS medium at 30 °C for 24 h. Cells were harvested by centrifugation (6790 g g, 4 °C, 10 min) and disrupted by ultrasonic, then centrifuged (12,000 g, 4 °C, 10 min) to remove cell debris, the supernatant was collected as the crude enzyme. The relative enzyme activity for reduction of 3,4-dihydroxy-phenylpyruvate was assayed by HPLC to identify the strain with the highest activity. The reaction system consisted of a total volume of 1 mL containing NADH (2 mM), 3,4-dihydroxy-phenylpyruvate (10 mM), buffer (20 mM), and an appropriate amount of enzyme solution. Reactions were maintained at 25 °C for 30 min, then terminated by boiling for 5 min. The reaction solution was extracted with three times the volume of ethylacetate, the upper layer was pipetted and dried with nitrogen gas until ethylacetate was completely evaporated. The powder was dissolved in 1 mL of ethanol, and then assayed by HPLC with a Chiralpak ID (0.46 cm × 25 cm×5 cm) (Daicel Chiral Technologies Co., Ltd.). Elution was performed with nhexane/ ethanol/ trifluoroacetic acid (80/20/0.1) at a flow rate of 1.0 mL/min at 220 nm.
3. Results 3.1. Strain screening, protein expression and enzyme activity assay After comparing the yield of L-DSS produced by the 20 strains of Lactobacilli, we screened seven strains with the ability to synthesize LDSS, and L. fermentum JN248 exhibited the highest activity. Five genes (GenBank accession nos. MG581694, MG581695, MG581696, MG581697, MG581698) were amplified from L. fermentum JN248 genomic DNA as the template and expressed in E. coli BL21. Crude and pure enzymes were analysed by SDS-PAGE, and pure enzymes ran as single bands with apparent molecular weights close to those expected based on the putative amino acid sequences (Fig. 1). The activity of the five purified enzymes was determined using a UV/vis spectrophotometer. LF-L-LDH0845 display the highest activity (relative activity of 100% corresponded to the specific activity 16.48 U/mg) for reducing 3,4-dihydroxy-phenylpyruvate to L-DSS with NADH as coenzyme followed by LF-L-LDH0372 (76%), LF-L-LDH1732 (47%) and LFL-LDH1566 (41%), whereas LF-L-LDH0611 was inactive. Further study found that LF-L-LDH0845 can also synthesize L-DSS with NADPH as coenzyme with specific activity of 15.30 U/mg.
2.2.2. Cloning, expression and purification of recombinant L-LDH Based on the lactate dehydrogenase genes in L. fermentum F-6 (GenBank accession no. CP005958.1), primers with suitable restriction enzyme sites were designed using Primer Premier 6.0 software (shown in Supplementary data). The ldhL genes were amplified from genomic DNA of L. fermentum JN248 as a template. Purified PCR products were ligated to the pColdⅡ cloning vector. The resultant plasmids were transferred into E. coli BL21(DE3), then induced with 0.2 mM IPTG at 200 rpm and 15 °C for 24h [12]. Cells were harvested, the crude enzyme solution was purified by passing through a His Trap HP column and a HiTrap desalting column. Protein concentration was measured using a BCA Protein Concentration Assay Kit, and both crude and pure enzyme solutions were analysed on 12% SDS-PAGE [13–15]. The activities of the five purified enzymes for the conversion of 3,4-dihydroxyphenylpyruvate to L-DSS were determined by measuring the rate of reduction of NADH or NADPH at 340 nm using a UV/vis spectrophotometer [16]. The enzyme with the highest activity was used for the further study of enzymatic properties.
3.2. Enzymatic properties of LF-L-LDH0845 Fig. 2 shows the pH and temperature profiles of LF-L-LDH0845 for the reduction of 3,4-dihydroxy-phenylpyruvate. LF-L-LDH0845 was the most active and stable at pH 6.0 whereas enzyme activity was unstable and completely lost at pH below 3.0 and above 9.0. The optimum temperature for LF-L-LDH0845 was 25 °C. After incubation at different temperatures for 1 h, the enzyme activity decreased sharply with increasing temperature. Enzyme activity was decreased to 49.48% at 30 °C, and all activity was lost after incubation at 50 °C. Kinetic parameters of LF-L-LDH0845 for different substrates were measured under the optimum conditions. Values of Km, Vmax, Kcat, and Kcat/Km for different substrates was shown in Table 1. It was interesting that the enzyme had a similar Km value for NADH and NADPH. After incubation for 6 h in the metal ion solutions, the effect of K+, 2+ Fe and Cu2+ on the enzyme activity of LF-L-LDH0845 was relatively significant, among which, K+ promoted the enzyme activity by 20% while Fe2+ inhibited enzyme activity by 70% and Cu2+ inhibited the
2.2.3. Enzymatic properties of LF-L-LDH0845 The optimum pH and temperature of LF-L-LDH0845 were assayed by varying the pH (4.0–11.0) and the temperature (10–65 °C) respectively. For the study of pH stability and thermostability, the enzyme solution was pre-incubated at different pH and temperatures for 1 h, then the residual enzyme activity for the reduction of 3,4-dihydroxyphenylpyruvate to L-DSS was determined under optimal conditions by HPLC. Relative activities were defined relative to the maximum activity [17]. Kinetic parameters for different substrates were assessed by varying the concentration of the corresponding substrate in 20 mM phosphate buffer (pH 6.0) at 25 °C. The kinetic data were calculated by non-linear fitting with OriginPro 8.0 software [18]. Different metal ions (Mn2+, Mg2+, Na+, Ni+, Ca2+, Co2+, Cu2+, Fe2+, K+ and Zn2+) were incubated with purified LF-L-LDH0845 solution at 25 °C for 6 h, and the same amount of deionised water was 2
Process Biochemistry xxx (xxxx) xxx–xxx
H. Lu et al.
Fig. 1. SDS-PAGE analysis of LF-L-LDHs from Lactobacillus fermentum JN248. The electrophoresis was performed with 12% agarose gel and stained with coomassie brilliant blue R250. Lane M, low molecular weight standards; lane 1, crude extract of E. coli BL21 (DE3)/ pColdII after induction; lane 2, crude extract of E. coli BL21 with LF-L-LDHs after induction; lane 3, the purified LF-L-LDHs after nickel-affinity chromatography. A–E shows the protein electrophoresis diagrams of LF-L-LDH0845, LFL-LDH1732, LF-L-LDH0611, LF-L-LDH1566 and LF-L-LDH0372, respectively.
3.3. Production of L-DSS by using the coupling system
enzyme activity by 60%. The effect of other metal ions on enzyme activity was considered to be negligible. As shown in Table 2, LF-L-LDH0845 was active toward pyruvate, phenylpyruvic acid, α-ketobutyrate, α-ketoglutaric acid, 4-methyl-2oxopentanoic acid, benzoylformic acid, 4-hydroxyphenylpyruvic acid, and 3,4-dihydroxyphenylpyruvic acid. The optimal substrate for this enzyme was pyruvate, followed by phenylpyruvate, confirming LF-LLDH0845 as an L-lactate dehydrogenase.
On account of the solubility of DSS less than 80 mM, the initial concentration of substrate 3,4-dihydroxyphenylpyruvate in the reaction system was set to 70 mM. In this coupling system, LF-L-LDH0845 and GDH were successfully coexpressed in E. coli BL21 (DE3). After 12 h of whole-cell catalytic reaction, the substrate 3,4-dihydroxyphenylpyruvate was almost completely converted to L-DSS in the presence of Fig. 2. pH and temperature profiles of purified recombinant LF-L-LDH0845. (A) Effect of pH on enzyme activity. The enzyme activity was measured in buffers with various pH (4.0–11.0) at 30 °C. The maximum enzyme activity at pH 6.0 was defined as 100%. (B) pH stability of LFL-LDH0845. The enzyme activity was measured after preincubation at different pH (4.0–11.0) for 1 h. (C) Effect of temperature on enzyme activity. The enzyme activity was assayed in disodium phosphate-citrate buffer (pH 6.0) at 10–65 °C. The maximum enzyme activity at 25 °C was defined as 100%. (D) Thermostability of LF-L-LDH0845. The enzyme activity was measured in disodium phosphatecitrate buffer (pH 6.0) after preincubation at different temperature (10–65 °C) for 1 h.
3
Process Biochemistry xxx (xxxx) xxx–xxx
H. Lu et al.
Table 1 Kinetic parameters of LF-L-LDH0845 for various substrates. Substrate
Km (mM)
Vmax (mM min−1 mg−1)
Kcat (S−1)
Kcat/Km (mM−1 S−1)
3,4-Dihydroxyphenylpyruvate Pyruvate NADH NADPH
11.3709 2.4662 0.1234 0.1976
0.5226 × 10−3 0.0222 0.1149 0.0185
0.2931 12.4498 64.4257 10.3748
0.0258 5.0481 521.9192 52.5173
This result is consistent with a previous study in which the Km of LLDHs for 2-ketocarboxylic acids increased with substrates containing longer aliphatic chains [27]. The catalytic efficiency (Kcat/Km) of LF-LLDH0845 toward 3,4-dihydroxyphenylpyruvate 0.0258 mM−1 S−1 was lower than that toward pyruvic acid 5.0481 mM−1 S−1, suggesting that the catalytic capacity of LF-L-LDH0845 toward 3,4-dihydroxy-phenylpyruvic acid could be improved by optimising the enzyme structure in the future. LF-L-LDH0845 exhibited high substrate specificity for most α-keto acids with particular preference for pyruvate and phenylpyruvate. Since LF-L-LDH0845 is coenzyme non-specific, GDH used to construct coupling system with LF-L-LDH0845 can also regenerate both NADH and NADPH [28]. By using the whole cells containing a coupling system as biocatalyst, the coenzymes (NAD and NADP) in cells are fully utilized, moreover, the double -enzyme system is provided with a more stable catalytic environment for the industrial-scale preparation of LDSS. However, the solubility of the substrate 3,4-dihydroxyphenylpyruvate in water is low, how to increase the solubility of 3,4-dihydroxyphenylpyruvate and the yield of L-DSS is still in progress.
Table 2 The substrate specificity of LF-L-LDH0845. Substrate (10 mM)
Specific activity (U/ mg)
Relative activity (%)
Pyruvate α-Ketobutyrate α-Ketoglutaric acid 4-Methyl-2-oxopentanoic acid Phenylpyruvic acid Benzoylformic acid 4-Hydroxyphenylpyruvic acid 3,4-Dihydroxyphenylpyruvic acid
144.81 16.63 21.17 37.46 69.11 29.73 16.08 12.30
100 11.48 14.62 25.87 47.42 20.53 11.10 8.49
Conflict of interest The authors declare that they have no conflict of interests. Author contributions Huan Lu, Xiaohui Zheng and Yujie Cai conceived and designed the experimental scheme. Huan Lu and Yajun Bai carried out the experiments. Taiping Fan, Xiaohui Zheng and Yujie Cai guided and supervised the experiment. Huan Lu, Yajun Bai and Yujie Cai analyzed and processed the experimental data. Huan Lu and Yujie Cai wrote the manuscript. All authors were involved in the revision of the manuscript.
Fig. 3. Time course of the production of L-DSS by coupling system. The biocatalyst by the whole-cell E. coli BL21(DE3) harboring pColdII-ldh0845-T7-gdh was carried out at 25 °C with shaking. ■, the conversion rate of 3,4-dihydroxyphenylpyruvate to L-DSS over time; ●, the ee% of L-DSS.
Acknowledgements
glucose, resulting in a 95.45% conversion rate and high enantioselectivity (enantiomeric excess ≥99.99%) (Fig. 3).
We gratefully thank for the financial support of this work by the National Key Scientific Instrument and Equipment Development Project of China (2013YQ17052504), Program for Changjiang Scholars and Innovative Research Team in the University of Ministry of Education of China (IRT_15R55), The Key Project of Research and Development Plan of Shanxi (2017ZDCXL-SF-01-02-01), Program for Changjiang Scholars and Innovative Research in University (IRT_15R55) and the seventh group of Hundred-Talent Program of Shanxi Province (2015).
4. Discussion LDH has been studied extensively for the synthesis of phenyllactic acid and lactic acid, two structural analogues of DSS. The conversion of 3,4-dihydroxyphenylpyruvate to L-DSS by L-LDHs has not previously been reported. We screened a non-specific coenzyme-dependent LF-LLDH0845 for the bioconversion of 3,4-dihydroxyphenylpyruvate to optically pure (ee ≥ 99.99%) L-DSS. The optimum temperature of LF-LLDH0845 for catalytic reduction of 3,4-dihydroxyphenylpyruvate was 25 °C, which is lower than that of other L-LDHs for the reduction of phenylpyruvic acid [19–21]. The thermostability of LF-L-LDH0845 declined at elevated temperatures, and dropped to zero at 50 °C, consistent with other L-LDHs in previous studies [22]. The optimum pH of LF-L-LDH0845 was 6.0, which is slightly higher than the optimum pH of 5.0 for L-LDH from L. helveticus 53/7 for the production of lactic acid [23]. LF-L-LDH0845 tolerated a pH between 5.0 and 7.0. The Km of LFL-LDH0845 for 3,4-dihydroxyphenylpyruvate was 11.37 mM, which is higher than that of other L-LDHs for the reduction of phenylpyruvate or pyruvate, indicating a lower affinity of LF-L-LDH0845 for 3,4-dihydroxyphenylpyruvate than phenylpyruvate or pyruvic acid [24–26].
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.procbio.2018.06.011. References [1] S. Seetapun, J. Yaoling, Y. Wang, Y.Z. Zhu, Neuroprotective effect of Danshensu derivatives as anti-ischaemia agents on SH-SY5Y cells and rat brain, Biosci. Rep. 33 (4) (2013). [2] K. Chan, S.H. Chui, D.Y. Wong, W.Y. Ha, C.L. Chan, R.N. Wong, Protective effects of Danshensu from the aqueous extract of Salvia miltiorrhiza (Danshen) against homocysteine-induced endothelial dysfunction, Life Sci. 75 (26) (2004) 3157–3171. [3] J.Y. Han, Y. Horie, J.Y. Fan, K. Sun, J. Guo, S. Miura, T. Hibi, Potential of 3,4-
4
Process Biochemistry xxx (xxxx) xxx–xxx
H. Lu et al.
[4]
[5]
[6]
[7] [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] K. Schersch, O. Betz, P. Garidel, S. Muehlau, S. Bassarab, G. Winter, Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins, part 2: stability during storage at elevated temperatures, J. Pharm. Sci. 101 (7) (2012) 2288–2306. [17] K. Min, Y.J. Yeon, Y. Um, Y.H. Kim, Novel NAD-independent D-lactate dehydrogenases from Acetobacter aceti and Acidocella species MX-AZ02 as potential candidates for in vitro biocatalytic pyruvate production, Biochem. Eng. J. 105 (2016) 358–363. [18] C. Jun, Y.S. Sa, S.-A. Gu, J.C. Joo, S. Kim, K.-J. Kim, Y.H. Kim, Discovery and characterization of a thermostable d-lactate dehydrogenase from Lactobacillus jensenii through genome mining, Process Biochem. 48 (1) (2013) 109–117. [19] S.A. Gu, C. Jun, J.C. Joo, S. Kim, S.H. Lee, Y.H. Kim, Higher thermostability of llactate dehydrogenases is a key factor in decreasing the optical purity of d-lactic acid produced from Lactobacillus coryniformis, Enzyme Microb. Technol. 58–59 (2014) 29–35. [20] Q. Zhou, W.L. Shao, Molecular genetic characterization of the thermostable L-lactate dehydrogenase gene (ldhL) of Thermoanaerobacter ethanolicus JW200 and biochemical characterization of the enzyme, Biochemistry (Mosc.) 75 (4) (2010) 526–530. [21] Chao Gao, Tianyi Jiang, Peipei Dou, Cuiqing Ma, Lixiang Li, Jian Kong, Ping Xu, NAD-independent L-lactate dehydrogenase Is required for L-lactate utilization in Pseudomonas stutzeri SDM, PLoS One 7 (5) (2012) e36519. [22] Ellen I. Garvie, Bacterial lactate dehydrogenases, Microbiol. Rev. 44 (1) (1980). [23] Kirsi Savijoki, Airi Palva, Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme, Appl. Environ. Microbiol. 63 (7) (1997) 2850–2856. [24] Jianghua Jia, Wanmeng Mu, Tao Zhang, B. Jiang, Bioconversion of phenylpyruvate to phenyllactate: gene cloning, expression, and enzymatic characterization of Dand L1-lactate dehydrogenases from Lactobacillus plantarum SK002, Appl. Biochem. Biotechnol. 162 (2009) 242–251. [25] S. Yeswanth, Y. Nanda Kumar, U. Venkateswara Prasad, V. Swarupa, V. Koteswara Rao, P. Venkata Gurunadha Krishna Sarma, Cloning and characterization of l-lactate dehydrogenase gene of Staphylococcus aureus, Anaerobe 24 (2013) 43–48. [26] Limin Wang, Yumeng Cai, Lingfeng Zhu, Honglian Guo, Bo Yu, Major role of NADdependent lactate dehydrogenases in the production of L-lactic acid with high optical purity by the thermophile Bacillus coagulans, Appl. Environ. Microbiol. 80 (23) (2014) 7134–7141. [27] H. Schütte, W. Hummel, M.-R. Kula, L-2-hydroxyisocaproate dehydrogenase—a new enzyme from Lactobacillus confusus for the stereospecific reduction of 2-ketocarboxylic acids, Appl. Microbiol. Biotechnol. 19 (1984) 167–176. [28] V. Kaswurm, W.V. Hecke, K.D. Kulbe, R. Ludwig, Guidelines for the application of NAD(P)H regenerating glucose dehydrogenase in synthetic processes, Adv. Synth. Catal. 355 (9) (2013) 1709–1714.
dihydroxy-phenyl lactic acid for ameliorating ischemia-reperfusion-induced microvascular disturbance in rat mesentery, Am. J. Physiol. Gastrointest. Liver Physiol. 296 (2009) G36–G44. F. Wang, Y.Y. Liu, L.Y. Liu, Q.J. Zeng, C.S. Wang, K. Sun, J.Y. Yang, J. Guo, J.Y. Fan, J.Y. Han, The attenuation effect of 3,4-dihydroxy-phenyl lactic acid and salvianolic acid B on venular thrombosis induced in rat mesentery by photochemical reaction, Clin. Hemorheol. Microcirc. 42 (1) (2009) 7–18. Xiaoyan Cui, Yueling Wang, Norihiro Kokudo, Dingzhi Fang, Traditional Chinese medicine and related active compounds against hepatitis B virus infection, BioScience Trends 4 (2) (2010) 39–47. G. Kwon, H.J. Kim, S.J. Park, H.E. Lee, H. Woo, Y.J. Ahn, Q. Gao, J.H. Cheong, D.S. Jang, J.H. Ryu, Anxiolytic-like effect of danshensu [(3-(3,4-dihydroxyphenyl)lactic acid)] in mice, Life Sci. 101 (1–2) (2014) 73–78. Nagaraj N. Rao, Hermann J. Roth, Enzymatic synthesis of (S)-(-)-3-(3,4-dihydroxyphenyl)lactic acid, Arch. Pharm. (Weinheim) 321 (1988) 179–180. C. Dong, Y. Wang, Y.Z. Zhu, Asymmetric synthesis and biological evaluation of Danshensu derivatives as anti-myocardial ischemia drug candidates, Bioorg. Med. Chem. 17 (9) (2009) 3499–3507. Z. Findrik, M. Poljanac, Đ. Vasić-Rački, Modelling and optimization of the (R)(+)-3,4-dihydroxyphenyllactic acid production catalyzed with D-lactate dehydrogenase from Lactobacillus leishmannii using genetic algorithm, Chem. Biochem. Eng. Q. 19 (4) (2005) 351–358. Scott P. France, Lorna J. Hepworth, Nicholas J. Turner, Sabine L. Flitsch, Constructing biocatalytic cascades: in vitro and in vivo approaches to de novo multi-enzyme pathways, ACS Catal. 7 (2017) 710–724. Y. Bai, Q. Zhang, P. Jia, L. Yang, Y. Sun, Y. Nan, S. Wang, X. Meng, Y. Wu, F. Qin, Z. Sun, X. Gao, P. Liu, K. Luo, Y. Zhang, X. Zhao, C. Xiao, S. Liao, J. Liu, C. Wang, J. Fang, X. Wang, J. Wang, R. Gao, X. An, X. Zhang, X. Zheng, Improved process for pilot-scale synthesis of Danshensu (( ± )-DSS) and its enantiomer derivatives, Org. Process Res. Dev. 18 (12) (2014) 1667–1673. J. Zhang, Z. Cui, H. Chang, X. Fan, Q. Zhao, W. Wei, Conversion of glycerol to 1,3dihydroxyacetone by glycerol dehydrogenase co-expressed with an NADH oxidase for cofactor regeneration, Biotechnol. Lett. 38 (9) (2016) 1559–1564. Zhaojuan Zheng, Cuiqing Ma, Chao Gao, Fengsong Li, Jiayang Qin, Haiwei Zhang, Kai Wang, P. Xu, Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM, PLoS One 6 (4) (2011) e19030. O. Hari Prasad, Y. Nanda Kumar, O.V. Reddy, A. Chaudhary, P.V. Sarma, Cloning, expression, purification and characterization of UMP kinase from Staphylococcus aureus, Protein J. 31 (4) (2012) 345–352. U.V. Prasad, D. Vasu, Y.N. Kumar, P.S. Kumar, S. Yeswanth, V. Swarupa, B.V. Phaneendra, A. Chaudhary, P.V. Sarma, Cloning, expression and characterization of NADP-dependent isocitrate dehydrogenase from Staphylococcus aureus, Appl. Biochem. Biotechnol. 169 (3) (2013) 862–869.
5