Biotechnol Lett (2014) 36:2325–2330 DOI 10.1007/s10529-014-1614-2
ORIGINAL RESEARCH PAPER
Purification and characterization of a novel cis-epoxysuccinate hydrolase from Klebsiella sp. that produces L(+)-tartaric acid Yongqing Cheng • Li Wang • Haifeng Pan • Wenna Bao • Weirong Sun • Zhipeng Xie • Jianguo Zhang • Yuhua Zhao
Received: 23 April 2014 / Accepted: 3 July 2014 / Published online: 22 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A strain of Klebsiella sp. BK-58 that produces cis-epoxysuccinate was screened and identified. This novel enzyme hydrolyzes cis-epoxysuccinate to L(?)-tartaric acid and was purified to homogeneity. Its molecular mass was 30.1 kDa determined by MALDI–TOF–MS analysis. It was stable up to 50 °C and from pH 5 to 11 with optima at 50 °C and pH 8.5. The enzyme was metal-independent and was strongly inhibited by 1 mM Cu2? and Ag?, and 1 % (w/v) SDS. The Km, Vmax and turnover number (kcat) values for cis-epoxysuccinate were 19.3, 2.24 mM min-1 and 220 s-1, respectively.
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1614-2) contains supplementary material, which is available to authorized users. Y. Cheng L. Wang H. Pan Z. Xie J. Zhang College of Life Science, Institute of Biochemistry, Zhejiang University, Hangzhou 310058, Zhejiang, People’s Republic of China e-mail: [email protected] H. Pan W. Bao W. Sun Z. Xie J. Zhang Hangzhou Bioking Biochemical Engineering Co., Ltd., Hangzhou 311106, Zhejiang, People’s Republic of China Y. Zhao (&) College of Life Science, Institute of Microbiology, Zhejiang University, Hangzhou 310058, Zhejiang, People’s Republic of China e-mail: [email protected]
Keywords cis-Epoxysuccinate hydrolase Epoxide hydrolase L(?)-Tartaric acid Klebsiella sp.
Introduction Epoxide hydrolases (EHs; EC 3.3.2.3) are a group of functionally related enzymes that can catalyze the enantioselective hydrolysis of an epoxide to the corresponding product (Steinreiber and Faber 2001). Although EHs are found in a variety of sources, those from microorganisms have gained attention owing to their efficient catalysis for the production of fine organic chemicals (de Vires and Janssen 2003). cisEpoxysuccinate hydrolase (ESH) catalyzes the asymmetric hydrolysis of cis-epoxysuccinic acid or its salts to the corresponding tartaric acid or tartrate (Willaert and de Vuyst 2006). L(?)-Tartaric acid is widely used in the food, building, pharmaceutical and cement industries (Bucˇko et al. 2005). Traditionally, L(?)tartaric acid, which is abundant in grapes, is extracted from winery waste. However, production of L(?)tartaric acid is strongly influenced by the yield and quality of grapes, which are dependent on the climate. Currently, the microbial method is considered to be more suitable for the production of L(?)-tartaric acid. cis-Epoxysuccinic acid has been hydrolyzed by ESHs from various microbial species, including Acetobacter (Tsurumi and Fujioka 1978), Nocardia (Wang et al. 2012), Rhodococcus (Liu et al. 2007a), Rhizobium and
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Pseudomonas (Kamatani et al. 1977). In these microorganisms, ESH is used to perform the biotransformation. Only two ESHs for L(?)-tartaric acid production have been purified and characterized: from Rhodococcus opacus (Liu et al. 2007a, b) and from Nocardia tartaricans (Wang et al. 2012). However, the amino acid sequences of both enzymes are identical (Liu et al. 2007b; Wang et al. 2012). The production of L(?)-tartaric acid still requires the use of free cells or immobilized cells instead of pure ESH because the thermal and pH stability of the purified ESHs are not ideal. Considering this information, it is necessary to identify new, more stable ESHs for L(?)-tartaric acid production. In this study, we have isolated a new ESHproducing strain identified as Klebsiella sp. BK-58. The ESH was purified and indentified to be a novel ESH for L(?)-tartaric acid production. Also, the characteristics of the purified enzyme are reported.
Materials and methods Microorganisms and culture Klebsiella sp. BK-58 was used. It was isolated from soil. After incubation in sterilized enrichment medium (5 g NaCl, 5 g peptone, 5 g yeast extract, 5 g sodium cis-epoxysuccinate and 1 g K2HPO4 per liter, the pH was adjusted to 7) at 30 °C for 48 h, a loop of culture was plated out onto a petri dish with the enrichment medium solidified with 2 % (w/v) agar. Colonies were inoculated into fermentation medium (5 g NaCl, 5 g peptone, 5 g yeast extract, 10 g sodium cis-epoxysuccinate, 1 g K2HPO4 per liter, pH was adjusted to 7). L(?)-Tartaric acid in the fermentation products was detected by HPLC. The strain with the highest L(?)tartaric acid yield, BK-58, was assigned to Klebsiella and named Klebsiella sp. BK-58 by examining its morphological, physiological and biochemical properties and comparing its 16S rDNA gene sequence (Supplementary Data File 1). It was inoculated into 3 l production medium (10 g NaCl, 10 g peptone, 5 g yeast extract, 10 g sodium cis-epoxysuccinate, 1 g K2HPO4 per liter, pH was adjusted to 7) in a 5 l jar fermenter (BioTech, Shanghai, China) for cultivation at 35 °C for 24 h. The cells were collected for the crude enzyme preparation.
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Enzyme extraction and purification All purification steps were carried out at 4 °C. After centrifugation, the harvested cells were washed twice with 0.9 % (w/v) NaCl, resuspended in 0.1 M pH 8 phosphate buffer (buffer A), and then disrupted by ultrasonication. After centrifugation, the supernatant was fractionated by the slow addition of solid ammonium sulfate. The precipitate between 40 and 70 % (w/ v) ammonium sulfate saturation was collected, redissolved, and dialyzed against buffer A for 24 h. The solution was then loaded onto a pre-equilibrated DEAE Sepharose Fast Flow column (16 9 100 mm). Some impure proteins were absorbed on the column, while the ESH which was not absorbed on the column was washed out with buffer A and then dialyzed against 25 mM pH 8 phosphate buffer (buffer B) containing 1 M KCl for 24 h. The dialyzed enzyme solution was then loaded onto a phenyl Sepharose CL4B column (10 9 100 mm) and eluted with a linear gradient of KCl (1 M–0) and ethylene glycol (0–50 %, v/v) in buffer B. The fractions showing ESH activity were pooled and dialyzed against buffer A for 24 h. The enzyme solution was then loaded onto a Sephadex G100 column (10 9 150 mm) and eluted with buffer A. The fractions containing ESH were combined and used for the subsequent research studies. Analysis of purified enzyme The purified enzyme was checked by SDS-PAGE on a 12 % (w/v) gel. It was subjected to MALDI–TOF–MS to determine the accurate size of the subunit. The enzyme was then digested by trypsin and desalted. The desalted peptides were analyzed using a 4700 Proteomics Analyzer (Applied Biosystems, CA, USA). The data analysis was performed using GPS and MASCOT with the NCBI database. ESH activity assay and protein measurement ESH activity was determined by the determination of the amount of L(?)-tartaric acid from cis-epoxysuccinate. The reaction mixture in 1 ml contained 20 ll enzyme solution and 0.2 M disodium cis-epoxysuccinate in buffer A. The reactions were carried out at 37 °C for 20 min and were stopped by adding 400 ml 1 M H2SO4 to the mixture. One unit (1 U) of ESH activity was defined as the amount of enzyme capable
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Table 1 Purification and properties of ESH from Klebsiella sp. BK-58 Purification steps
Total protein (mg)
Total activity (U)
Specific activity (U mg-1)
Purification enrichment (fold)
Yielda (%)
Crude enzyme
1,837
31,811
17.3
1
100 84.8
(NH)2SO4
1,050
26,986
25.7
1.5
DEAE-Sepharose
257
15,933
61.9
3.6
50.1
Phenyl-Sepharose
67.3
13,185
196
11.3
41.4
Sephadex G100
21
5,114
243
24.1
16.1
a
The activity of the crude enzyme was considered to be 100 % for the calculation of enzyme recovery yield
of generating 1 lmol L(?)-tartaric acid per min at pH 8 and 37 °C. Specific activity was defined as the number of units per mg protein. L(?)-Tartaric acid was detected by HPLC using a chiral column Chirex 3126 (D)-penicillamine (4.6 9 50 mm) and an evaporative light scattering detector at 280 nm. The mobile phase contained 88 % (v/v) 1 mM copper acetate (pH 4.5) and 12 % (v/v) 2-propanol. The column was eluted by 1 mM CuSO4 at 30 °C at 1 ml min-1. Chromatographically pure L(?)-tartaric acid was used as external standard. Protein was quantitatively determined by the Bradford method with bovine serum albumin as the standard. Characterization of ESH The effect of temperature, pH, metal ions and other reagents on ESH activity was performed. Kinetic parameters were determined by assaying purified enzyme at increasing substrate concentrations ranging from 10 to 100 mM for triplicate determinations. Km, Vmax and kcat were determined using Lineweaver– Burk plots. The enantioselectivity (EE) value was determined by HPLC at 30 °C on a chiral column Chirex 3126 (D)-penicillamine with a sample volume of 20 ll.
Results and discussion Purification and analysis of the ESH from Klebsiella sp. BK-58 The ESH from Klebsiella sp. BK-58 was purified to homogeneity (24.1-fold purification) with an overall
Fig. 1 Purification of ESH from Klebsiella sp. BK-58. Samples were taken at different stages of ESH purification (Table 1) and subjected to 12 % SDS-PAGE (lane 1 molecular weight standards; lane 2 crude enzyme; lane 3 40–70 % saturated ammonium sulfate precipitate; lane 4 pooled fractions after the DEAE-Sepharose step; lane 5 pooled fractions after the PhenylSepharose step; lane 6 pooled fractions after the Sephadex G100 step)
yield of 16.1 % and a specific activity increase from 17.3 to 243 U mg-1 (Table 1). The fractions with the maximum ESH activity eluted from the Sephadex G100 column were collected and examined for purity by SDS-PAGE. The final enzyme fractions were essentially pure with a single band representing ESH. Its molecular mass was between 25 and 35 kDa as shown in Fig. 1, which was consistent with the molecular mass (30.1 kDa) estimated by MALDI–TOF–MS analysis (Fig. 2). The protein identification result showed that its peptide mass fingerprinting did not fit any of the other peptides in the database. Thus, we can be confident that the ESH from Klebsiella sp. BK-58 is a novel ESH.
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Fig. 2 MALDI–TOF mass spectrum of ESH from Klebsiella sp. BK-58
Characterization of ESH As shown in Fig. 3a, the ESH was most active between 45 and 60 °C and was stable up to 50 °C. However, only approximately 44 % of the initial activity remained after incubating the enzyme at 55 °C. Therefore, the optimum temperature of the enzyme was 50 °C when considering the reaction rate and protein stability. As shown in Fig. 3b, ESH showed the highest activity between pH 6.5 and 9.5 with optima at pH 8.5. Close to 100 % of the initial activity was observed from pH 5 to 11, and 56 % of the initial activity was observed even at pH 4 (Fig. 3c), which indicated that the enzyme was stable over a very broad pH range. As shown in Table 2, ESH from Klebsiella sp. BK58 still displayed high level of enzyme activity without any metal ions. So it is suggested as a metalindependent enzyme, which is similar to the ESHs from R. opacus and N. tartaricans (Table 3). It was strongly inhibited by Cu2?, Ag? and SDS, and was mildly inhibited by Ni2? (Table 2). Table 3 also showed the kinetic parameters and the EE values of the enzyme. The kcat, Km and Vmax for cisepoxysuccinate were calculated to be 220 s-1, 19.3 and 2.24 mM min-1, respectively. Interestingly, the
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ESH from Klebsiella sp. BK-58 had smaller Km and kcat values but had a larger kcat/Km value than the ESH from N. tartaricans. The Vmax of the ESH from Klebsiella sp. BK-58 did not appear to be significantly different from that of the ESHs from R. opacus and N. tartaricans. Km is associated with the enzyme affinity to the substrate. An enzyme with a lower Km requires a lower substrate concentration to achieve the same reaction velocity. kcat measures the number of substrate molecules turned over per enzyme molecule per second and kcat/Km is a measure of enzyme efficiency. All the above results suggested that the ESH from Klebsiella sp. BK-58 had a stronger affinity to cis-epoxysuccinate and a higher catalytic efficiency. HPLC analysis revealed that the ESH from Klebsiella sp. BK-58 could enantioselectively hydrolyze cis-epoxysuccinate to L(?)-tartaric acid with an EE value higher than 99.9 %.
Conclusions This study is the first to report an L(?)-tartaric acidproducing ESH from Klebsiella and some characteristics of the purified enzyme are reported. The ESH from Klebsiella sp. BK-58 was characterized as a
Biotechnol Lett (2014) 36:2325–2330
2329 Table 2 Effect of metal ions and chemicals on the activity of ESH from Klebsiella sp. BK-58 Chemicals
Concentration
Relative activitya (%)
None
–
100 ± 2.3b
Ag
?
1 mM
0
Zn2?
1 mM
91.4 ± 2.4
Ni2?
1 mM
76.4 ± 1.3
Cu2?
1 mM
22.3 ± 1.2
SDS
1 % (w/v)
0
EDTA-Na2
10 mM
90.7 ± 0.3 2?
2?
Other metal ions (1 mM Mn , Ca , Al3?, Mg2?, Co2?, Ba2?, and Fe2?) and reagents (10 mM DTT, 1 % (v/v) Tween 80 and Triton X-100) tested had no measurable effect on enzyme activity a The activity under standard reaction conditions (37 °C, pH 8) without adding chemical reagent or metal ion was defined as 100 % (47 U mg-1) b
Values are means of three replications ± SD
Table 3 Comparison of biochemical properties and kinetic parameters of three ESHs for L(?)-tartaric acid producing from different strains
Fig. 3 Effects of temperature and pH on the activity and stability of ESH from Klebsiella sp. BK-58. a The optimum temperature was assayed with temperature varied from 4 to 70 °C. Thermal stability was obtained after pre-incubation of the enzyme solution at different temperatures for 30 min. b The optimal pH was assayed with pH varied from 3.6 to 11. c pH stability was obtained after pre-incubation of the enzyme at pH varied from 3.6 to 12 for 30 min. The buffers (b, c) used were 0.1 M citric acid–sodium citrate buffer (pH 3.6–6.5), 0.1 M phosphate buffer (pH 5–9), 0.1 M glycine–sodium hydroxide buffer (pH 8.5–11) and 0.1 M Na2HPO4–NaOH buffer (pH 11–12). Overlaps were obtained when buffers were changed. The activity under standard reaction conditions (37 °C, pH 8) without pre-incubation at different temperatures or pH values was defined as 100 % (47 U mg-1). Values are means of three replications ± SD
Strains
R. opacusa
N. tartaricans
Klebsiella sp. BK-58
Molecular weight of native protein (kDa)
28
28
30.1
Optimum temp. (°C)
NR
37
50
Optimal pH
NR
8
8.5
Thermal stability
Stable below 40 °C
Stable below 40 °C
Stable up to 50 °C
pH stability (stable pH range)
7–8
7–8.5
5–11
Metal-dependence
No
No
No
kcat (s-1)
NR
260
220 ± 8.9b 19.3 ± 1.8
Km (mM)
45
35.7
kcat/Km (mM-1 s-1)
NR
7.3
11.4 ± 0.7
Vmax (mM min-1)
2.24
2.65
2.24 ± 0.3
EE value (%)
NR
NR
99.9 ± 1.2
Reference
Liu et al. (2007b)
Wang et al. (2012)
This study
NR not reported a
The amino acid sequence of the ESH from N. tartaricans is identical to that of the ESH from R. opacus
b
Values are means of three replications ± SD
novel metal-independent enzyme with a molecular mass of 30.1 kDa. It was stable up to 50 °C and from pH 5 to 11 with optima at 50 °C and pH 8.5.
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Production conditions of L(?)-tartaric acid could be widened due to its good thermal and pH stabilities. It produced L(?)-tartaric acid from cis-epoxysuccinate with an EE value higher than 99.9 %. In addition, it had a smaller Km (19.3 mM) and a larger kcat/Km (11.4 mM-1 s-1) than the ESH from N. tartaricans, which revealed that it had a stronger affinity to cisepoxysuccinate and a higher catalytic efficiency. The findings showed that the ESH from Klebsiella sp. BK58 could be of significant importance in the industrial production of L(?)-tartaric acid. Acknowledgments This project is supported by the National Natural Science Foundation of China (31300661, 31070079; 41271335), the High Technology Research and Development Program of China (863 Program) (2012AA06A203), the National Key Technology Rand D Program (2012BAC 17B04), the Science and Technology Project of Zhejiang Province (2011C13016; 2013C3303), and the Environmental Science Project of Zhejiang Province (2012B006).
References Bucˇko M, Vikartovska´ A, Lacı´k I, Kolla´rrikova´ G, Gemeiner P, Pa¨toprsty V, Brygin M (2005) Immobilization of a whole-
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Biotechnol Lett (2014) 36:2325–2330 cell epoxide-hydrolyzing biocatalyst in sodium alginate– cellulose sulfate–poly(methylene-co-guanidine) capsules using a controlled encapsulation process. Enzyme Microb Technol 36:118–126 de Vires EJ, Janssen DB (2003) Biocatalytic conversion of epoxides. Curr Opin Biotechnol 14:414–420 Kamatani Y, Okazaki H, Imai K, Fujita N, Yamazaki Y, Ogino K (1977) Production of L(?)-tartaric acid. USA Patent 4011135 Liu ZQ, Li Y, Ping LF, Xu YY, Cui FJ, Xue YP, Zheng YG (2007a) Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production. Proc Biochem 42:889–894 Liu ZQ, Li Y, Ping LF, Xu YY, Zheng YG (2007b) Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme. Appl Microbiol Biotechnol 74:99–106 Steinreiber A, Faber K (2001) Microbial epoxide hydroxylases for preparative biotransformations. Curr Opin Biotechnol 12:552–558 Tsurumi Y, Fujioka T (1978) Process for the manufacture of L(?)-tartaric acid or salts thereof. USA Patent 4092220 Wang ZQ, Wang YS, Su ZG (2012) Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli. Appl Microbiol Biotechnol 97:2433–2441 Willaert R, de Vuyst L (2006) Continuous production of L(?)tartaric acid from cis-epoxysuccinate using a membrane recycle reactor. Appl Microbiol Biotechnol 71:155–163
ORIGINAL RESEARCH PAPER
Purification and characterization of a novel cis-epoxysuccinate hydrolase from Klebsiella sp. that produces L(+)-tartaric acid Yongqing Cheng • Li Wang • Haifeng Pan • Wenna Bao • Weirong Sun • Zhipeng Xie • Jianguo Zhang • Yuhua Zhao
Received: 23 April 2014 / Accepted: 3 July 2014 / Published online: 22 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract A strain of Klebsiella sp. BK-58 that produces cis-epoxysuccinate was screened and identified. This novel enzyme hydrolyzes cis-epoxysuccinate to L(?)-tartaric acid and was purified to homogeneity. Its molecular mass was 30.1 kDa determined by MALDI–TOF–MS analysis. It was stable up to 50 °C and from pH 5 to 11 with optima at 50 °C and pH 8.5. The enzyme was metal-independent and was strongly inhibited by 1 mM Cu2? and Ag?, and 1 % (w/v) SDS. The Km, Vmax and turnover number (kcat) values for cis-epoxysuccinate were 19.3, 2.24 mM min-1 and 220 s-1, respectively.
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1614-2) contains supplementary material, which is available to authorized users. Y. Cheng L. Wang H. Pan Z. Xie J. Zhang College of Life Science, Institute of Biochemistry, Zhejiang University, Hangzhou 310058, Zhejiang, People’s Republic of China e-mail: [email protected] H. Pan W. Bao W. Sun Z. Xie J. Zhang Hangzhou Bioking Biochemical Engineering Co., Ltd., Hangzhou 311106, Zhejiang, People’s Republic of China Y. Zhao (&) College of Life Science, Institute of Microbiology, Zhejiang University, Hangzhou 310058, Zhejiang, People’s Republic of China e-mail: [email protected]
Keywords cis-Epoxysuccinate hydrolase Epoxide hydrolase L(?)-Tartaric acid Klebsiella sp.
Introduction Epoxide hydrolases (EHs; EC 3.3.2.3) are a group of functionally related enzymes that can catalyze the enantioselective hydrolysis of an epoxide to the corresponding product (Steinreiber and Faber 2001). Although EHs are found in a variety of sources, those from microorganisms have gained attention owing to their efficient catalysis for the production of fine organic chemicals (de Vires and Janssen 2003). cisEpoxysuccinate hydrolase (ESH) catalyzes the asymmetric hydrolysis of cis-epoxysuccinic acid or its salts to the corresponding tartaric acid or tartrate (Willaert and de Vuyst 2006). L(?)-Tartaric acid is widely used in the food, building, pharmaceutical and cement industries (Bucˇko et al. 2005). Traditionally, L(?)tartaric acid, which is abundant in grapes, is extracted from winery waste. However, production of L(?)tartaric acid is strongly influenced by the yield and quality of grapes, which are dependent on the climate. Currently, the microbial method is considered to be more suitable for the production of L(?)-tartaric acid. cis-Epoxysuccinic acid has been hydrolyzed by ESHs from various microbial species, including Acetobacter (Tsurumi and Fujioka 1978), Nocardia (Wang et al. 2012), Rhodococcus (Liu et al. 2007a), Rhizobium and
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Pseudomonas (Kamatani et al. 1977). In these microorganisms, ESH is used to perform the biotransformation. Only two ESHs for L(?)-tartaric acid production have been purified and characterized: from Rhodococcus opacus (Liu et al. 2007a, b) and from Nocardia tartaricans (Wang et al. 2012). However, the amino acid sequences of both enzymes are identical (Liu et al. 2007b; Wang et al. 2012). The production of L(?)-tartaric acid still requires the use of free cells or immobilized cells instead of pure ESH because the thermal and pH stability of the purified ESHs are not ideal. Considering this information, it is necessary to identify new, more stable ESHs for L(?)-tartaric acid production. In this study, we have isolated a new ESHproducing strain identified as Klebsiella sp. BK-58. The ESH was purified and indentified to be a novel ESH for L(?)-tartaric acid production. Also, the characteristics of the purified enzyme are reported.
Materials and methods Microorganisms and culture Klebsiella sp. BK-58 was used. It was isolated from soil. After incubation in sterilized enrichment medium (5 g NaCl, 5 g peptone, 5 g yeast extract, 5 g sodium cis-epoxysuccinate and 1 g K2HPO4 per liter, the pH was adjusted to 7) at 30 °C for 48 h, a loop of culture was plated out onto a petri dish with the enrichment medium solidified with 2 % (w/v) agar. Colonies were inoculated into fermentation medium (5 g NaCl, 5 g peptone, 5 g yeast extract, 10 g sodium cis-epoxysuccinate, 1 g K2HPO4 per liter, pH was adjusted to 7). L(?)-Tartaric acid in the fermentation products was detected by HPLC. The strain with the highest L(?)tartaric acid yield, BK-58, was assigned to Klebsiella and named Klebsiella sp. BK-58 by examining its morphological, physiological and biochemical properties and comparing its 16S rDNA gene sequence (Supplementary Data File 1). It was inoculated into 3 l production medium (10 g NaCl, 10 g peptone, 5 g yeast extract, 10 g sodium cis-epoxysuccinate, 1 g K2HPO4 per liter, pH was adjusted to 7) in a 5 l jar fermenter (BioTech, Shanghai, China) for cultivation at 35 °C for 24 h. The cells were collected for the crude enzyme preparation.
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Enzyme extraction and purification All purification steps were carried out at 4 °C. After centrifugation, the harvested cells were washed twice with 0.9 % (w/v) NaCl, resuspended in 0.1 M pH 8 phosphate buffer (buffer A), and then disrupted by ultrasonication. After centrifugation, the supernatant was fractionated by the slow addition of solid ammonium sulfate. The precipitate between 40 and 70 % (w/ v) ammonium sulfate saturation was collected, redissolved, and dialyzed against buffer A for 24 h. The solution was then loaded onto a pre-equilibrated DEAE Sepharose Fast Flow column (16 9 100 mm). Some impure proteins were absorbed on the column, while the ESH which was not absorbed on the column was washed out with buffer A and then dialyzed against 25 mM pH 8 phosphate buffer (buffer B) containing 1 M KCl for 24 h. The dialyzed enzyme solution was then loaded onto a phenyl Sepharose CL4B column (10 9 100 mm) and eluted with a linear gradient of KCl (1 M–0) and ethylene glycol (0–50 %, v/v) in buffer B. The fractions showing ESH activity were pooled and dialyzed against buffer A for 24 h. The enzyme solution was then loaded onto a Sephadex G100 column (10 9 150 mm) and eluted with buffer A. The fractions containing ESH were combined and used for the subsequent research studies. Analysis of purified enzyme The purified enzyme was checked by SDS-PAGE on a 12 % (w/v) gel. It was subjected to MALDI–TOF–MS to determine the accurate size of the subunit. The enzyme was then digested by trypsin and desalted. The desalted peptides were analyzed using a 4700 Proteomics Analyzer (Applied Biosystems, CA, USA). The data analysis was performed using GPS and MASCOT with the NCBI database. ESH activity assay and protein measurement ESH activity was determined by the determination of the amount of L(?)-tartaric acid from cis-epoxysuccinate. The reaction mixture in 1 ml contained 20 ll enzyme solution and 0.2 M disodium cis-epoxysuccinate in buffer A. The reactions were carried out at 37 °C for 20 min and were stopped by adding 400 ml 1 M H2SO4 to the mixture. One unit (1 U) of ESH activity was defined as the amount of enzyme capable
Biotechnol Lett (2014) 36:2325–2330
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Table 1 Purification and properties of ESH from Klebsiella sp. BK-58 Purification steps
Total protein (mg)
Total activity (U)
Specific activity (U mg-1)
Purification enrichment (fold)
Yielda (%)
Crude enzyme
1,837
31,811
17.3
1
100 84.8
(NH)2SO4
1,050
26,986
25.7
1.5
DEAE-Sepharose
257
15,933
61.9
3.6
50.1
Phenyl-Sepharose
67.3
13,185
196
11.3
41.4
Sephadex G100
21
5,114
243
24.1
16.1
a
The activity of the crude enzyme was considered to be 100 % for the calculation of enzyme recovery yield
of generating 1 lmol L(?)-tartaric acid per min at pH 8 and 37 °C. Specific activity was defined as the number of units per mg protein. L(?)-Tartaric acid was detected by HPLC using a chiral column Chirex 3126 (D)-penicillamine (4.6 9 50 mm) and an evaporative light scattering detector at 280 nm. The mobile phase contained 88 % (v/v) 1 mM copper acetate (pH 4.5) and 12 % (v/v) 2-propanol. The column was eluted by 1 mM CuSO4 at 30 °C at 1 ml min-1. Chromatographically pure L(?)-tartaric acid was used as external standard. Protein was quantitatively determined by the Bradford method with bovine serum albumin as the standard. Characterization of ESH The effect of temperature, pH, metal ions and other reagents on ESH activity was performed. Kinetic parameters were determined by assaying purified enzyme at increasing substrate concentrations ranging from 10 to 100 mM for triplicate determinations. Km, Vmax and kcat were determined using Lineweaver– Burk plots. The enantioselectivity (EE) value was determined by HPLC at 30 °C on a chiral column Chirex 3126 (D)-penicillamine with a sample volume of 20 ll.
Results and discussion Purification and analysis of the ESH from Klebsiella sp. BK-58 The ESH from Klebsiella sp. BK-58 was purified to homogeneity (24.1-fold purification) with an overall
Fig. 1 Purification of ESH from Klebsiella sp. BK-58. Samples were taken at different stages of ESH purification (Table 1) and subjected to 12 % SDS-PAGE (lane 1 molecular weight standards; lane 2 crude enzyme; lane 3 40–70 % saturated ammonium sulfate precipitate; lane 4 pooled fractions after the DEAE-Sepharose step; lane 5 pooled fractions after the PhenylSepharose step; lane 6 pooled fractions after the Sephadex G100 step)
yield of 16.1 % and a specific activity increase from 17.3 to 243 U mg-1 (Table 1). The fractions with the maximum ESH activity eluted from the Sephadex G100 column were collected and examined for purity by SDS-PAGE. The final enzyme fractions were essentially pure with a single band representing ESH. Its molecular mass was between 25 and 35 kDa as shown in Fig. 1, which was consistent with the molecular mass (30.1 kDa) estimated by MALDI–TOF–MS analysis (Fig. 2). The protein identification result showed that its peptide mass fingerprinting did not fit any of the other peptides in the database. Thus, we can be confident that the ESH from Klebsiella sp. BK-58 is a novel ESH.
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Fig. 2 MALDI–TOF mass spectrum of ESH from Klebsiella sp. BK-58
Characterization of ESH As shown in Fig. 3a, the ESH was most active between 45 and 60 °C and was stable up to 50 °C. However, only approximately 44 % of the initial activity remained after incubating the enzyme at 55 °C. Therefore, the optimum temperature of the enzyme was 50 °C when considering the reaction rate and protein stability. As shown in Fig. 3b, ESH showed the highest activity between pH 6.5 and 9.5 with optima at pH 8.5. Close to 100 % of the initial activity was observed from pH 5 to 11, and 56 % of the initial activity was observed even at pH 4 (Fig. 3c), which indicated that the enzyme was stable over a very broad pH range. As shown in Table 2, ESH from Klebsiella sp. BK58 still displayed high level of enzyme activity without any metal ions. So it is suggested as a metalindependent enzyme, which is similar to the ESHs from R. opacus and N. tartaricans (Table 3). It was strongly inhibited by Cu2?, Ag? and SDS, and was mildly inhibited by Ni2? (Table 2). Table 3 also showed the kinetic parameters and the EE values of the enzyme. The kcat, Km and Vmax for cisepoxysuccinate were calculated to be 220 s-1, 19.3 and 2.24 mM min-1, respectively. Interestingly, the
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ESH from Klebsiella sp. BK-58 had smaller Km and kcat values but had a larger kcat/Km value than the ESH from N. tartaricans. The Vmax of the ESH from Klebsiella sp. BK-58 did not appear to be significantly different from that of the ESHs from R. opacus and N. tartaricans. Km is associated with the enzyme affinity to the substrate. An enzyme with a lower Km requires a lower substrate concentration to achieve the same reaction velocity. kcat measures the number of substrate molecules turned over per enzyme molecule per second and kcat/Km is a measure of enzyme efficiency. All the above results suggested that the ESH from Klebsiella sp. BK-58 had a stronger affinity to cis-epoxysuccinate and a higher catalytic efficiency. HPLC analysis revealed that the ESH from Klebsiella sp. BK-58 could enantioselectively hydrolyze cis-epoxysuccinate to L(?)-tartaric acid with an EE value higher than 99.9 %.
Conclusions This study is the first to report an L(?)-tartaric acidproducing ESH from Klebsiella and some characteristics of the purified enzyme are reported. The ESH from Klebsiella sp. BK-58 was characterized as a
Biotechnol Lett (2014) 36:2325–2330
2329 Table 2 Effect of metal ions and chemicals on the activity of ESH from Klebsiella sp. BK-58 Chemicals
Concentration
Relative activitya (%)
None
–
100 ± 2.3b
Ag
?
1 mM
0
Zn2?
1 mM
91.4 ± 2.4
Ni2?
1 mM
76.4 ± 1.3
Cu2?
1 mM
22.3 ± 1.2
SDS
1 % (w/v)
0
EDTA-Na2
10 mM
90.7 ± 0.3 2?
2?
Other metal ions (1 mM Mn , Ca , Al3?, Mg2?, Co2?, Ba2?, and Fe2?) and reagents (10 mM DTT, 1 % (v/v) Tween 80 and Triton X-100) tested had no measurable effect on enzyme activity a The activity under standard reaction conditions (37 °C, pH 8) without adding chemical reagent or metal ion was defined as 100 % (47 U mg-1) b
Values are means of three replications ± SD
Table 3 Comparison of biochemical properties and kinetic parameters of three ESHs for L(?)-tartaric acid producing from different strains
Fig. 3 Effects of temperature and pH on the activity and stability of ESH from Klebsiella sp. BK-58. a The optimum temperature was assayed with temperature varied from 4 to 70 °C. Thermal stability was obtained after pre-incubation of the enzyme solution at different temperatures for 30 min. b The optimal pH was assayed with pH varied from 3.6 to 11. c pH stability was obtained after pre-incubation of the enzyme at pH varied from 3.6 to 12 for 30 min. The buffers (b, c) used were 0.1 M citric acid–sodium citrate buffer (pH 3.6–6.5), 0.1 M phosphate buffer (pH 5–9), 0.1 M glycine–sodium hydroxide buffer (pH 8.5–11) and 0.1 M Na2HPO4–NaOH buffer (pH 11–12). Overlaps were obtained when buffers were changed. The activity under standard reaction conditions (37 °C, pH 8) without pre-incubation at different temperatures or pH values was defined as 100 % (47 U mg-1). Values are means of three replications ± SD
Strains
R. opacusa
N. tartaricans
Klebsiella sp. BK-58
Molecular weight of native protein (kDa)
28
28
30.1
Optimum temp. (°C)
NR
37
50
Optimal pH
NR
8
8.5
Thermal stability
Stable below 40 °C
Stable below 40 °C
Stable up to 50 °C
pH stability (stable pH range)
7–8
7–8.5
5–11
Metal-dependence
No
No
No
kcat (s-1)
NR
260
220 ± 8.9b 19.3 ± 1.8
Km (mM)
45
35.7
kcat/Km (mM-1 s-1)
NR
7.3
11.4 ± 0.7
Vmax (mM min-1)
2.24
2.65
2.24 ± 0.3
EE value (%)
NR
NR
99.9 ± 1.2
Reference
Liu et al. (2007b)
Wang et al. (2012)
This study
NR not reported a
The amino acid sequence of the ESH from N. tartaricans is identical to that of the ESH from R. opacus
b
Values are means of three replications ± SD
novel metal-independent enzyme with a molecular mass of 30.1 kDa. It was stable up to 50 °C and from pH 5 to 11 with optima at 50 °C and pH 8.5.
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2330
Production conditions of L(?)-tartaric acid could be widened due to its good thermal and pH stabilities. It produced L(?)-tartaric acid from cis-epoxysuccinate with an EE value higher than 99.9 %. In addition, it had a smaller Km (19.3 mM) and a larger kcat/Km (11.4 mM-1 s-1) than the ESH from N. tartaricans, which revealed that it had a stronger affinity to cisepoxysuccinate and a higher catalytic efficiency. The findings showed that the ESH from Klebsiella sp. BK58 could be of significant importance in the industrial production of L(?)-tartaric acid. Acknowledgments This project is supported by the National Natural Science Foundation of China (31300661, 31070079; 41271335), the High Technology Research and Development Program of China (863 Program) (2012AA06A203), the National Key Technology Rand D Program (2012BAC 17B04), the Science and Technology Project of Zhejiang Province (2011C13016; 2013C3303), and the Environmental Science Project of Zhejiang Province (2012B006).
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Biotechnol Lett (2014) 36:2325–2330 cell epoxide-hydrolyzing biocatalyst in sodium alginate– cellulose sulfate–poly(methylene-co-guanidine) capsules using a controlled encapsulation process. Enzyme Microb Technol 36:118–126 de Vires EJ, Janssen DB (2003) Biocatalytic conversion of epoxides. Curr Opin Biotechnol 14:414–420 Kamatani Y, Okazaki H, Imai K, Fujita N, Yamazaki Y, Ogino K (1977) Production of L(?)-tartaric acid. USA Patent 4011135 Liu ZQ, Li Y, Ping LF, Xu YY, Cui FJ, Xue YP, Zheng YG (2007a) Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production. Proc Biochem 42:889–894 Liu ZQ, Li Y, Ping LF, Xu YY, Zheng YG (2007b) Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme. Appl Microbiol Biotechnol 74:99–106 Steinreiber A, Faber K (2001) Microbial epoxide hydroxylases for preparative biotransformations. Curr Opin Biotechnol 12:552–558 Tsurumi Y, Fujioka T (1978) Process for the manufacture of L(?)-tartaric acid or salts thereof. USA Patent 4092220 Wang ZQ, Wang YS, Su ZG (2012) Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli. Appl Microbiol Biotechnol 97:2433–2441 Willaert R, de Vuyst L (2006) Continuous production of L(?)tartaric acid from cis-epoxysuccinate using a membrane recycle reactor. Appl Microbiol Biotechnol 71:155–163
