Appl Biochem Biotechnol DOI 10.1007/s12010-014-1460-7
High-level Production of Creatine Amidinohydrolase from Arthrobacter nicotianae 23710 in Escherichia coli Jun Dai & Linpei Zhang & Zhen Kang & Jian Chen & Guocheng Du
Received: 21 August 2014 / Accepted: 16 December 2014 # Springer Science+Business Media New York 2014
Abstract In the present study, the gene encoding creatinase was amplified from Arthrobacter nicotianae 23710 (CICC) and functionally overexpressed in Escherichia coli. By applying a two-stage temperature control strategy, the production of creatinase was increased up to 61.3 U/mL in 3-L fermentor with a high productivity of 6.1 U/mL/h. The recombinant creatinase shows excellent resistance to the chelating agent EDTA, the surfactants (Tween 20, Tween 80, and Triton X-100) and the common preservative NaN3 (20 mM). High-level expression of the recombinant creatinase will contribute to its application in clinical diagnosis of renal function. Keywords Creatine amidinohydrolase . Arthrobacter nicotianae . Escherichia coli . Two-stage control strategy
Introduction Creatinine is an intermediate product of creatine metabolism in human body and measurement of its concentration in biological fluids is used for clinical diagnosis of renal function [1, 2]. Although determination based on the Jaffe’s reaction has been widely used, the substrate specificity is low which cannot meet the precision measurement [3]. In contrast, the enzymatic J. Dai : L. Zhang : Z. Kang The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China e-mail: [email protected] J. Dai : L. Zhang : Z. Kang (*) : J. Chen : G. Du School of Biotechnology, Jiangnan University, Wuxi 214122, China e-mail: [email protected] J. Chen National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China G. Du The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
Appl Biochem Biotechnol
method using three enzymes (creatininase, EC 3.5.2.10; creatinase, EC 3.5.3.3; sarcosine oxidase, EC 1.5.3.1) has attracted intensive attention because of its high substrate specificity. In the enzymatic measurement of creatinine, creatinase is an important medical enzyme for catalyzing the hydrolysis of creatine to sarcosine and urea [4–6]. In 1950, Roche et al. found that two Pseudomonas strains (Pseudomonas eisenbergii and Pseudomonas ovalis) decompose creatine into urea and sarcosine, which firstly suggest that there should be creatinase present in Pseudomonas strains [7]. Appleyard et al. subsequently explained the phenomenon by characterizing the creatine catabolism pathway in P. ovalis [8] and Yoshimoto et al. successfully purified and crystallized the creatinase in Pseudomonas putida [9]. Then, creatinase was also purified and characterized from other bacteria, such as Alcaligenes sp. [10], Flavobacterium sp. [11], and Bacillus sp. [12]. To broaden its application, screening, and identification of novel creatinase with good properties (for instance high resistance to sodium azide) is attractive and beneficial to clinical applications. Recently, Zhi et al. purified and characterized a novel creatinase from a new isolated strain Arthrobacter nicotianae 02181 [4]. However, low expression level by this native strain with addition of expensive inducers would debase its potential application [13]. Consequently, it is imperative to construct robust recombinant cell factories for its high-level production. Escherichia coli, the most well-known model microorganism that possesses many advantages (such as well-characterized genetics, rapid growth on cheap substrates, well-equipped toolkit, and easy to operate) has always dominated the bacterial expression systems and remains to be the first choice for investigation at laboratory level [14–16]. In fact, many different creatinase genes from Pseudomonas sp., Bacillus sp. and Flavobacterium sp. have been cloned, heterologously expressed, and characterized in E. coli [11–13, 17, 18]. Nevertheless, low productivity or dissatisfactory properties hindered its practical application. In the present work, we successfully cloned a creatinase gene from A. nicotianae 23710 and achieved its high-level expression in E. coli. After optimization of the fermentation parameters, the titer of the recombinant creatinase was increased to 61.3 U/mL with a high productivity of 6.1 U/mL/h. To the best of our knowledge, this was the highest creatinase production reported until now.
Materials and Methods Cloning and Analysis of a Creatinase Gene from A. nicotianae 23710 Molecular cloning and manipulation of plasmids were done with E. coli JM109 (Invitrogen). The creatinase gene creJ was amplified from A. nicotianae 23710 (CICC) with the designed oligonucleotides creJ-F (5′-GAAAGAACATATGACTACCGCCAACATCGCCACCA-3′) and creJ-R (5′-TTCTCTCGAGTTACGCGTCGATGATGTTGTTCTCC-3′) according to the published creatinase sequence [4]. The PCR products were firstly ligated into the cloning vector pMD19. After sequence verification, the creatinase gene creJ was cut with NdeI and XhoI, and the digested fragment was subcloned into pET20b(+) with the same restriction sites to generate pET20b-creJ. Subsequently, the resulting vectors pET20b-creJ was transformed into E. coli BL21 (DE3) to generate the recombinant strain E. coli BL21J. Medium and Culture Conditions LB medium (g/L, peptone 10, yeast extract 5, NaCl 10, pH 7.2) was used for construction of plasmids and recombinant strains. TB medium (g/L, peptone 12, yeast extract 24, glycerol 4, KH2PO4 2.32, K2HPO4 ·3H2O 16.43) was used for all flask cultivations. Modified TB medium
Appl Biochem Biotechnol
(g/L, peptone 12, yeast extract 24, glycerol 10, KH2PO4 2.32, K2HPO4 ·3H2O 16.43) was used in batch fermentation. Ampicillin was added to maintain selective pressure at a final concentration of 100 μg/mL during seed preparation and fermentation. Specifically, 5 g/L creatine as inducer was supplemented in the medium when cultivating A. nicotianae 23710. Flask cultivations were performed in 250-mL Erlenmeyer flasks containing 20-mL TB medium. One percent (v/v) of seed culture was inoculated into the medium, and the mixture was incubated at 37 °C with an agitation of 220 rpm. When OD600 reached 0.6, isopropyl β-D1-thiogalactopyranoside (IPTG) was added to 0.6 mM for induction at 30 °C for 8 h. Batch fermentation was carried out in a 3-L fermentor (BioFlo115, New Brunswick Scientific Co.) containing 1.5-L modified TB medium. A 3 % (v/v) inoculum from an overnight culture was used. To induce the expression of creatinase, IPTG was artificially added with a final concentration of 0.6 mM when the value of OD600 reached 4.5. After induction, the temperature was converted from 37 to 30 °C. Throughout the whole fermentation period, pH was controlled at 7.0 with 4 M NaOH, and the dissolved oxygen was monitored and maintained above 40 % saturation. Assay of Creatinase and Protein Creatinase activity was measured by the amount of urea released from hydrolyzed creatine. At the end of fermentation, cells were harvested and disrupted by ultrasonication. Then cell-free extracts were centrifuged to obtain the supernatant as the crude enzyme. The reaction system that consists of 0.1-mL enzyme solution and 0.9-mL 50 mM phosphate buffer solution (0.01 M creatine, pH 7.0) was kept at 37 °C for 10 min. Then, 2-mL dimethylsulfoxide solution containing 2 % p-dimethylaminobenzaldehyde and 1 M HCl was added to stop the reaction. The mixture was incubated at 25 °C for 20 min; then, absorbance at 435 nm was measured. One enzyme unit was defined as the amount of enzyme forming 1 μmol urea per minute under the conditions described before. Purification of Recombinant Creatinase Cells were suspended in buffer A (50 mM phosphate buffer solution, pH 7.0) and ruptured by ultrasonic disintegration. The suspension was centrifuged (Centrifuge 5424, Eppendorf Co.) at 10,000 rpm for 10 min, and the supernatant was used as crude enzyme. Then, the crude enzyme was subjected to ammonium sulfate fractionation. Ammonium sulfate fraction of 60– 80 % were centrifuged at 12,000 rpm for 15 min, and the precipitates obtained were redissolved in buffer A and dialyzed against the same buffer overnight. The dialyzed sample was injected into an AKTA purifier (GE Healthcare) through a HiPrep 16/10 Q_XL column (GE Healthcare) equilibrated with buffer A. After removing unbound proteins with buffer A, the elution was performed with a linear gradient from 0 to 100 % buffer B (50 mM phosphate buffer solution containing 1 M NaCl). Creatinase-containing fractions were pooled and dialyzed. To further purify, the resulting solution was subjected to gel filtration on a HiLoad 16/60 superdex 200 column (GE Healthcare). Purification was detected by activity assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All purification steps were carried out below 4 °C. SDS-PAGE Gel Electrophoresis The purity and relative molecular mass of creatinase were determined by SDS-PAGE. The soluble enzyme mixed with sample buffer was boiled for 10 min, and 15 μL of mixture was
Appl Biochem Biotechnol
subjected to a 12 % gel. After electrophoresis on a vertical mini gel apparatus at 90 V for 2 h, the gel was stained with 1 % Coomassie brilliant blue (R-250) and destained with a mixture of methanol, water, and acetic acid (5:4:1) for 1 h. Characterization of Enzymatic Properties To determine the effect of temperature, enzyme assays were carried out at temperatures ranging from 25 to 60 °C. The stability against heat inactivation was analyzed by incubating the purified enzyme at different temperatures (25–60 °C) for 30 min. Then, samples were chilled on ice, and subsequently, the enzyme activity was determined at 37 °C. Residual activity was calculated by comparing the activity to an unheated control. The optimum pH of the purified creatinase was determined at different pH values in a range of 5.7–8.0 by using 50 mM phosphate buffer. To determine the effect of pH on creatinase stability, enzyme samples were incubated in a pH range of 3.0–11.0 for 6 h using the following buffers: citrate buffer solution (pH 3.0–6.0), phosphate buffer (pH 6.0–8.0), borate buffer (pH 8.0–9.0), and glycine-NaOH buffer (pH 9.0– 11.0). The kinetic parameters (Km and Vmax) were measured in 50 mM phosphate buffer using creatine concentrations ranging from 5 to 120 mM. All experiments were repeated three times, and the kinetic parameters were evaluated by the doublereciprocal plot method. For inhibition studies, the purified enzyme was preincubated with different inhibitors for 30 min in phosphate buffer (pH 7.0) and the residual creatinase activity was assayed.
Results and Discussion Functional Expression and Purification of Creatinase Applying the designed primers creJ-F and creJ-R, we successfully cloned a potential creatinase gene (designated creJ, accession no. KM027338) from A. nicotianae 23710. To further confirm and characterize this potential creatinase, we subcloned the creJ gene and controllably overexpressed it in E. coli B21 (DE3). As shown in Fig. 1a, the positive control strain A. nicotianae 23710 accumulated creatinase to 190.2 U/g dry cell weight (DCW) while no creatinase activity was detected in the wild-type E. coli BL21 (DE3). In comparison, the recombinant strain E. coli BL21J produced more creatinase (1834.1 U/g DCW) which was about 9.6-fold of that of A. nicotianae 23710, confirming that creJ is a creatinase gene and suitable for heterologous expression in E. coli. Moreover, the recombinant creatinase was successfully purified through a series of operation including ammonium sulfate precipitation, anion exchange chromatography (Fig. 1b), and gel filtration chromatography (Fig. 1c) as described in “Materials and methods.” Table 2 shows the specific activity of the fractions obtained in each purification step. The purified enzyme (about 46.4 kDa, Fig. 1d) exhibited 14.1-fold increase in specific activity (comparing with the crude enzyme after ultrasonic disintegration) with a value of 20.3 U/mg protein which was lower than that of the creatinase from A. nicotianae 02181 [4]. However, sequence alignment analysis results (Fig. 2) showed that these two creatinases have a high sequence similarity (95 %). The results indicated that although the motifs containing the putative active sites H232, N249, A260, H324, E358, and H376 are highly conserved in the creatinases [19, 20], changes of few residues might
Appl Biochem Biotechnol
Fig. 1 Functional expression and purification of the recombinant creatinase. a The amount of creatinase accumulated in different strains; b Creatinase purification by anion-exchange chromatography; c Creatinase purification by gel filtration chromatography. The brown and blue lines indicate the conductivity and the absorbance at 280 nm (A280). The arrow indicates the active fraction; d SDS-PAGE analysis of the purified creatinase. Lane M, molecular weight marker; lane 1, supernatant of the control strain E. coli BL21 (pET20b); lane 2, supernatant of the recombinant E. coli BL21J; lane 3, 60–80 % ammonium sulfate precipitation; lane 4, purified creatinase from the recombinant E. coli BL21J
lead to significantly altered specific activities. As a result, molecular engineering of the present creatinase with directed evolution strategies would be worthy of further studies. Enzymatic Characteristics of the Recombinant Creatinase To pave the way to clinical applications, the activities of the recombinant creatinase were measured at different temperatures and pHs. The optimum temperature was about 37 °C, and
Appl Biochem Biotechnol
Table 1 Comparison of creatinase production in various strains
Strains
Creatinase concentration References
Escherichia coli JA221 (pCR50)
0.04 U/mL
[13]
E. coli JM109 (pkls508)
1.8 U/mL
[11]
E. coli JM109 (pQE-51)
4.7 U/mL
[30]
Arthrobacter nicotianae 02181 70.0 U/g cell
[4]
E. coli BL21J
This work
61.3 U/mL (1834.0 U/g cell)
enzymatic activity sharply decreased when above 40 °C (Fig. 3a), which resembled to that isolated from A. nicotianae 02181 [4], Paracoccus sp. strain WB1 [21], and P. putida [9, 22]. At 37 °C, the optimum pH for the recombinant creatinase was 7.5, and only remained about 60 % activity at pH 8.0 (Fig. 3b). The enzyme stability under different temperatures and pHs were also investigated. This enzyme was stable below 40 °C but completely lost its activity after 30-min preincubation at 50 °C (Fig. 3c). Although this enzyme has an optimum pH at pH 7.5, it is stable in the range of pH 6.0 to 11.0 (Fig. 3d). Obviously, this recombinant creatinase has higher resistance to alkaline environment compared to that of A. nicotianae 02181 [4]. Simultaneously, the kinetic parameters Km and Vmax of this enzyme were also estimated with values of 53.2 mM and was 6.1 μM/min, respectively, which was similar with that produced by A. nicotianae 02181 [4]. In addition, the effects of inhibitors and metal irons on creatinase activity were also assessed. As shown in Table 3, Cu2+, Hg2+, and SDS inhibited almost all the activity while Zn2+ reduced the activity by 56 %. In contrast, no inhibition effect of other metal irons was observed. More importantly, it was notable that the recombinant creatinase shows excellent resistance to NaN3 (20 mM), EDTA, Tween 20, Tween 80, and Triton X-100, which was analogous to that previously reported [4, 21]. All the above results suggest that this recombinant creatinase is applicable for clinical applications. Key Fermentation Process Parameters for Creatinase Expression The initial pH and inoculum amount were optimized at 7.0 (Fig. 4a) and 3 % (v/v) (Fig. 4b). IPTG was added at 3 h when cells entering early exponential phase (Fig. 4c). The effect of temperature was further investigated and optimized to increase creatinase production. Cells were initially cultivated at 37 °C while temperature was maintained or shifted to 30 and 25 °C after induction. As shown in Fig. 4d, the temperature exhibited remarkable influence on creatinase expression. Induction at 30 °C resulted in high titer of creatinase which further confirmed previous conclusion Table 2 Purification of the recombinant creatinase from Escherichia coli BL21J Procedures
Volumes (mL)
Total activities (U)
Concentrations (mg/mL)
Crude enzyme
590.0
12,260.2
14.4
1.4
–
AS precipitation QFF
76.0 144.0
6654.6 4489.9
14.0 1.5
6.2 20.3
4.3 14.1
AS ammonium sulfate
Specific activities (U/mg protein)
Fold
Appl Biochem Biotechnol
Fig. 2 Sequence comparison of creatinase from A. nicotianae 23710 and A. nicotianae 02181. The figure was generated using the CLUSTALW 1.8 software package based on the creatinase from A. nicotianae 23710 and A. nicotianae 02181 [4]. The different residues are boxed in gray, and the predicted key catalytic residues were marked with red stars [19]
that the balance between translation and protein folding is crucial to functional overexpression [23–25]. In view of the inhibition effect on cell growth, IPTG was
Fig. 3 Properties of the recombinant creatinase toward temperature and pH. a Effect of temperature on the activity; b effect of pH on the activity; c effect of temperature on the stability; d effect of pH on the stability
Appl Biochem Biotechnol
Table 3 Effect of metal ions and chemicals on enzyme activity
Metal ions and chemicals
Concentrations
Residual activities (%)
Cu2+
1 mM
1
Hg2+ Co2+
1 mM 1 mM
1 104
Fe3+
1 mM
109
Ba2+
1 mM
109
Pb2+
1 mM
109
Mn2+
1 mM
111
Mg2+
1 mM
111
Li+
1 mM
112
Ca2+ EDTA
1 mM 1 mM
112 110
Zn2+
1 mM
44
NaN3
20 mM
101
Tween 20
1 g/L
112
Tween 80
1 g/L
104
Triton X-100
5 g/L
115
optimized with a concentration of 0.6 mM. Eventually, the creatinase production was improved to 33.3 U/mL (Fig. 4d).
Fig. 4 Identification and optimization of the key fermentation parameters. a Effect of initial pH of medium on the production of creatinase; b effect of inoculum on the production of creatinase; c cell growth curve of the recombinant strain E. coli BL21J. The arrow indicated as point for addition of the inducer IPTG; d effect of IPTG concentrations and temperature on the production of creatinase
Appl Biochem Biotechnol
Fig. 5 Batch fermentation of the recombinant creatinase in 3-L fermentor. Before induction, cells were cultivated at 37 °C. After induction, the temperature was decreased to 30 °C
Batch fermentation of creatinase with a two-stage temperature control strategy Batch fermentation of the recombinant creatinase was carried out in 3-L fermentor with a two-stage temperature control strategy. Specifically, temperature was switched from 37 to 30 °C after induction. Glycerol as carbon source was supplied with a final concentration of 10 g/L according to preliminary experiments (data not shown). At 10 h, cell biomass was accumulated to 31.2 OD600 (corresponding to 12.1 g/L DCW) and the production of creatinase was significantly increased to 61.3 U/mL (Fig. 5a) with a productivity of 6.1 U/mL/h. In parallel, the production of the recombinant creatinase was also monitored by SDS-PAGE analysis. Obviously, the protein bands corresponding to the enzyme increased in intensity with prolonged fermentation time (Fig. 5b), which was consistent with the above activity analysis results. In addition, it could be found that accumulation of creatinase is coupled with cell growth, indicating high-cell-density fermentation [26, 27] should be an alternative approach for further improving the production in future.
Conclusions In the present study, we cloned an A. nicotianae creatinase gene creJ and firstly overexpressed it in E. coli. As we expected, the recombinant creatinase shows satisfactory properties in particular with excellent resistance to the common preservative NaN3. By applying a temperature two-phase control strategy, the production of the recombinant creatinase was improved to 61.3 U/mL with a high productivity of 6.1 U/ mL/h. To the best of our knowledge (Table 1), this is the highest reported creatinase production and productivity until now. The results obtained in this work will be useful for its industrial production and practical applications. Further research should be focused on improving its specific activity by molecular engineering with directed evolution strategies [28, 29]. Acknowledgments This work was financially supported by the National High Technology Research and Development Program of China (863 Program, 2011AA100905), Program for Changjiang Scholars and Innovative Research Team in University (no. IRT1135), the National Science Foundation for Post-doctoral Scientists of China (2013 M540414), the Jiangsu Planned Projects for Postdoctoral Research Funds (1301010B), and the 111 Project.
Appl Biochem Biotechnol
References 1. Kim, E. J., Haruyama, T., Yanagida, Y., Kobatake, E., & Aizawa, M. (1999). Analytica Chimica Acta, 394, 225–231. 2. Lad, U., Khokhar, S., & Kale, G. M. (2008). Analytical Chemistry, 80, 7910–7917. 3. Serafin, V., Hernandez, P., Agui, L., Yanez-Sedeno, P., & Pingarron, J. M. (2013). Electrochimica Acta, 97, 175–183. 4. Zhi, Q., Kong, P. Y., Zang, J. T., Cui, Y. H., Li, S. H., Li, P., Yi, W. J., Wang, Y., Chen, A., & Hu, C. M. (2009). Process Biochemistry, 44, 460–465. 5. Schumacher, G., Hilscher, W., Mollering, H., Siedel, J., & Buckel, P. (1993). Annales Biologie Clinique, 51, 815–819. 6. Goren, M. P., Osborne, S., & Wright, R. K. (1986). Clinical Chemistry, 32, 548–551. 7. Roche, J., Lacombe, G., & Girard, H. (1950). Biochimica Biophysica Acta, 6, 210–216. 8. Appleyard, G., & Woods, D. D. (1956). Journal of General Microbiology, 14, 351–365. 9. Yoshimoto, T., Oka, I., & Tsuru, D. (1976). Journal of Biochemistry, 79, 1381–1383. 10. Matsuda, Y., Wakamatsu, N., Inouye, Y., Uede, S., Hashimoto, Y., Asano, K., & Nakamura, S. (1986). Chemical & Pharmaceutical Bulletin, 34, 2155–2160. 11. Koyama, Y., Kitao, S., Yamamoto-Otake, H., Suzuki, M., & Nakano, E. (1990). Agricultural and Biological Chemistry, 54, 1453–1457. 12. Suzuki, K., Sagai, H., Sugiyama, M., & Imamura, S. (1993). Journal of Fermentation and Bioengineering, 76, 77–81. 13. Chang, M. C., Chang, C. C., & Chang, J. C. (1992). Applied and Environmental Microbiology, 58, 3437– 3440. 14. Kang, Z., Wang, Q., Zhang, H., & Qi, Q. (2008). Applied Microbiology and Biotechnology, 79, 203–208. 15. Kang, Z., Zhang, C., Du, G., & Chen, J. (2014). Applied Biochemistry and Biotechnology, 172, 2012–2021. 16. Rosano, G. L., & Ceccarelli, E. A. (2014). Frontiers in Microbiology, 5, 172. 17. Hong, M. C., Chang, J. C., Wu, M. L., & Chang, M. C. (1998). Biochemical Genetics, 36, 407–415. 18. Suzuki, K., Sagai, H., Imamura, S., & Sugiyama, M. (1994). Journal of Fermentation and Bioengineering, 77, 231–234. 19. Hoeffken, H. W., Knof, S. H., Bartlett, P. A., Huber, R., Moellering, H., & Schumacher, G. (1988). Journal of Molecular Biology, 204, 417–433. 20. Coll, M., Knof, S. H., Ohga, Y., Messerschmidt, A., Huber, R., Moellering, H., Russmann, L., & Schumacher, G. (1990). Journal of Molecular Biology, 214, 597–610. 21. Wang, Y. Y., Ma, X. H., Zhao, W. F., Jia, X. M., Kai, L., & Xu, X. H. (2006). Process Biochemistry, 41, 2072–2077. 22. Yoshimoto, T., Oka, I., & Tsuru, D. (1976). Archives of Biochemistry and Biophysics, 177, 508–515. 23. Zhang, X., Lian, J. Z., Kai, L., Huang, L., Cen, P. L., & Xu, Z. N. (2014). Enzyme and Microbial Technology, 55, 26–30. 24. Gopal, G. J., & Kumar, A. (2013). The Protein Journal, 32, 419–425. 25. Hu, S., Wang, M., Cai, G., & He, M. (2013). The Journal of Biological Chemistry, 288, 30855–30861. 26. Yegane-Sarkandy, S., Farnoud, A. M., Shojaosadati, S. A., Khalilzadeh, R., Sadeghyzadeh, M., Ranjbar, B., & Babaeipour, V. (2009). Biotechnology and Applied Biochemistry, 54, 31–39. 27. Seo, M. J., Choi, H. J., Chung, K. H., & Pyun, Y. R. (2011). Journal of Microbiology and Biotechnology, 21, 1053–1056. 28. Socha, R. D., & Tokuriki, N. (2013). Febs Journal, 280, 5582–5595. 29. Sen, S., Dasu, V. V., & Mandal, B. (2007). Applied Biochemistry and Biotechnology, 143, 212–223. 30. Hsu, W. F., Ng, I. S., Tsai, S. W., & Chang, M. C. (2003). Journal of The Chinese Institute of Chemical Engineers, 34, 305–310.
High-level Production of Creatine Amidinohydrolase from Arthrobacter nicotianae 23710 in Escherichia coli Jun Dai & Linpei Zhang & Zhen Kang & Jian Chen & Guocheng Du
Received: 21 August 2014 / Accepted: 16 December 2014 # Springer Science+Business Media New York 2014
Abstract In the present study, the gene encoding creatinase was amplified from Arthrobacter nicotianae 23710 (CICC) and functionally overexpressed in Escherichia coli. By applying a two-stage temperature control strategy, the production of creatinase was increased up to 61.3 U/mL in 3-L fermentor with a high productivity of 6.1 U/mL/h. The recombinant creatinase shows excellent resistance to the chelating agent EDTA, the surfactants (Tween 20, Tween 80, and Triton X-100) and the common preservative NaN3 (20 mM). High-level expression of the recombinant creatinase will contribute to its application in clinical diagnosis of renal function. Keywords Creatine amidinohydrolase . Arthrobacter nicotianae . Escherichia coli . Two-stage control strategy
Introduction Creatinine is an intermediate product of creatine metabolism in human body and measurement of its concentration in biological fluids is used for clinical diagnosis of renal function [1, 2]. Although determination based on the Jaffe’s reaction has been widely used, the substrate specificity is low which cannot meet the precision measurement [3]. In contrast, the enzymatic J. Dai : L. Zhang : Z. Kang The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China e-mail: [email protected] J. Dai : L. Zhang : Z. Kang (*) : J. Chen : G. Du School of Biotechnology, Jiangnan University, Wuxi 214122, China e-mail: [email protected] J. Chen National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China G. Du The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
Appl Biochem Biotechnol
method using three enzymes (creatininase, EC 3.5.2.10; creatinase, EC 3.5.3.3; sarcosine oxidase, EC 1.5.3.1) has attracted intensive attention because of its high substrate specificity. In the enzymatic measurement of creatinine, creatinase is an important medical enzyme for catalyzing the hydrolysis of creatine to sarcosine and urea [4–6]. In 1950, Roche et al. found that two Pseudomonas strains (Pseudomonas eisenbergii and Pseudomonas ovalis) decompose creatine into urea and sarcosine, which firstly suggest that there should be creatinase present in Pseudomonas strains [7]. Appleyard et al. subsequently explained the phenomenon by characterizing the creatine catabolism pathway in P. ovalis [8] and Yoshimoto et al. successfully purified and crystallized the creatinase in Pseudomonas putida [9]. Then, creatinase was also purified and characterized from other bacteria, such as Alcaligenes sp. [10], Flavobacterium sp. [11], and Bacillus sp. [12]. To broaden its application, screening, and identification of novel creatinase with good properties (for instance high resistance to sodium azide) is attractive and beneficial to clinical applications. Recently, Zhi et al. purified and characterized a novel creatinase from a new isolated strain Arthrobacter nicotianae 02181 [4]. However, low expression level by this native strain with addition of expensive inducers would debase its potential application [13]. Consequently, it is imperative to construct robust recombinant cell factories for its high-level production. Escherichia coli, the most well-known model microorganism that possesses many advantages (such as well-characterized genetics, rapid growth on cheap substrates, well-equipped toolkit, and easy to operate) has always dominated the bacterial expression systems and remains to be the first choice for investigation at laboratory level [14–16]. In fact, many different creatinase genes from Pseudomonas sp., Bacillus sp. and Flavobacterium sp. have been cloned, heterologously expressed, and characterized in E. coli [11–13, 17, 18]. Nevertheless, low productivity or dissatisfactory properties hindered its practical application. In the present work, we successfully cloned a creatinase gene from A. nicotianae 23710 and achieved its high-level expression in E. coli. After optimization of the fermentation parameters, the titer of the recombinant creatinase was increased to 61.3 U/mL with a high productivity of 6.1 U/mL/h. To the best of our knowledge, this was the highest creatinase production reported until now.
Materials and Methods Cloning and Analysis of a Creatinase Gene from A. nicotianae 23710 Molecular cloning and manipulation of plasmids were done with E. coli JM109 (Invitrogen). The creatinase gene creJ was amplified from A. nicotianae 23710 (CICC) with the designed oligonucleotides creJ-F (5′-GAAAGAACATATGACTACCGCCAACATCGCCACCA-3′) and creJ-R (5′-TTCTCTCGAGTTACGCGTCGATGATGTTGTTCTCC-3′) according to the published creatinase sequence [4]. The PCR products were firstly ligated into the cloning vector pMD19. After sequence verification, the creatinase gene creJ was cut with NdeI and XhoI, and the digested fragment was subcloned into pET20b(+) with the same restriction sites to generate pET20b-creJ. Subsequently, the resulting vectors pET20b-creJ was transformed into E. coli BL21 (DE3) to generate the recombinant strain E. coli BL21J. Medium and Culture Conditions LB medium (g/L, peptone 10, yeast extract 5, NaCl 10, pH 7.2) was used for construction of plasmids and recombinant strains. TB medium (g/L, peptone 12, yeast extract 24, glycerol 4, KH2PO4 2.32, K2HPO4 ·3H2O 16.43) was used for all flask cultivations. Modified TB medium
Appl Biochem Biotechnol
(g/L, peptone 12, yeast extract 24, glycerol 10, KH2PO4 2.32, K2HPO4 ·3H2O 16.43) was used in batch fermentation. Ampicillin was added to maintain selective pressure at a final concentration of 100 μg/mL during seed preparation and fermentation. Specifically, 5 g/L creatine as inducer was supplemented in the medium when cultivating A. nicotianae 23710. Flask cultivations were performed in 250-mL Erlenmeyer flasks containing 20-mL TB medium. One percent (v/v) of seed culture was inoculated into the medium, and the mixture was incubated at 37 °C with an agitation of 220 rpm. When OD600 reached 0.6, isopropyl β-D1-thiogalactopyranoside (IPTG) was added to 0.6 mM for induction at 30 °C for 8 h. Batch fermentation was carried out in a 3-L fermentor (BioFlo115, New Brunswick Scientific Co.) containing 1.5-L modified TB medium. A 3 % (v/v) inoculum from an overnight culture was used. To induce the expression of creatinase, IPTG was artificially added with a final concentration of 0.6 mM when the value of OD600 reached 4.5. After induction, the temperature was converted from 37 to 30 °C. Throughout the whole fermentation period, pH was controlled at 7.0 with 4 M NaOH, and the dissolved oxygen was monitored and maintained above 40 % saturation. Assay of Creatinase and Protein Creatinase activity was measured by the amount of urea released from hydrolyzed creatine. At the end of fermentation, cells were harvested and disrupted by ultrasonication. Then cell-free extracts were centrifuged to obtain the supernatant as the crude enzyme. The reaction system that consists of 0.1-mL enzyme solution and 0.9-mL 50 mM phosphate buffer solution (0.01 M creatine, pH 7.0) was kept at 37 °C for 10 min. Then, 2-mL dimethylsulfoxide solution containing 2 % p-dimethylaminobenzaldehyde and 1 M HCl was added to stop the reaction. The mixture was incubated at 25 °C for 20 min; then, absorbance at 435 nm was measured. One enzyme unit was defined as the amount of enzyme forming 1 μmol urea per minute under the conditions described before. Purification of Recombinant Creatinase Cells were suspended in buffer A (50 mM phosphate buffer solution, pH 7.0) and ruptured by ultrasonic disintegration. The suspension was centrifuged (Centrifuge 5424, Eppendorf Co.) at 10,000 rpm for 10 min, and the supernatant was used as crude enzyme. Then, the crude enzyme was subjected to ammonium sulfate fractionation. Ammonium sulfate fraction of 60– 80 % were centrifuged at 12,000 rpm for 15 min, and the precipitates obtained were redissolved in buffer A and dialyzed against the same buffer overnight. The dialyzed sample was injected into an AKTA purifier (GE Healthcare) through a HiPrep 16/10 Q_XL column (GE Healthcare) equilibrated with buffer A. After removing unbound proteins with buffer A, the elution was performed with a linear gradient from 0 to 100 % buffer B (50 mM phosphate buffer solution containing 1 M NaCl). Creatinase-containing fractions were pooled and dialyzed. To further purify, the resulting solution was subjected to gel filtration on a HiLoad 16/60 superdex 200 column (GE Healthcare). Purification was detected by activity assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All purification steps were carried out below 4 °C. SDS-PAGE Gel Electrophoresis The purity and relative molecular mass of creatinase were determined by SDS-PAGE. The soluble enzyme mixed with sample buffer was boiled for 10 min, and 15 μL of mixture was
Appl Biochem Biotechnol
subjected to a 12 % gel. After electrophoresis on a vertical mini gel apparatus at 90 V for 2 h, the gel was stained with 1 % Coomassie brilliant blue (R-250) and destained with a mixture of methanol, water, and acetic acid (5:4:1) for 1 h. Characterization of Enzymatic Properties To determine the effect of temperature, enzyme assays were carried out at temperatures ranging from 25 to 60 °C. The stability against heat inactivation was analyzed by incubating the purified enzyme at different temperatures (25–60 °C) for 30 min. Then, samples were chilled on ice, and subsequently, the enzyme activity was determined at 37 °C. Residual activity was calculated by comparing the activity to an unheated control. The optimum pH of the purified creatinase was determined at different pH values in a range of 5.7–8.0 by using 50 mM phosphate buffer. To determine the effect of pH on creatinase stability, enzyme samples were incubated in a pH range of 3.0–11.0 for 6 h using the following buffers: citrate buffer solution (pH 3.0–6.0), phosphate buffer (pH 6.0–8.0), borate buffer (pH 8.0–9.0), and glycine-NaOH buffer (pH 9.0– 11.0). The kinetic parameters (Km and Vmax) were measured in 50 mM phosphate buffer using creatine concentrations ranging from 5 to 120 mM. All experiments were repeated three times, and the kinetic parameters were evaluated by the doublereciprocal plot method. For inhibition studies, the purified enzyme was preincubated with different inhibitors for 30 min in phosphate buffer (pH 7.0) and the residual creatinase activity was assayed.
Results and Discussion Functional Expression and Purification of Creatinase Applying the designed primers creJ-F and creJ-R, we successfully cloned a potential creatinase gene (designated creJ, accession no. KM027338) from A. nicotianae 23710. To further confirm and characterize this potential creatinase, we subcloned the creJ gene and controllably overexpressed it in E. coli B21 (DE3). As shown in Fig. 1a, the positive control strain A. nicotianae 23710 accumulated creatinase to 190.2 U/g dry cell weight (DCW) while no creatinase activity was detected in the wild-type E. coli BL21 (DE3). In comparison, the recombinant strain E. coli BL21J produced more creatinase (1834.1 U/g DCW) which was about 9.6-fold of that of A. nicotianae 23710, confirming that creJ is a creatinase gene and suitable for heterologous expression in E. coli. Moreover, the recombinant creatinase was successfully purified through a series of operation including ammonium sulfate precipitation, anion exchange chromatography (Fig. 1b), and gel filtration chromatography (Fig. 1c) as described in “Materials and methods.” Table 2 shows the specific activity of the fractions obtained in each purification step. The purified enzyme (about 46.4 kDa, Fig. 1d) exhibited 14.1-fold increase in specific activity (comparing with the crude enzyme after ultrasonic disintegration) with a value of 20.3 U/mg protein which was lower than that of the creatinase from A. nicotianae 02181 [4]. However, sequence alignment analysis results (Fig. 2) showed that these two creatinases have a high sequence similarity (95 %). The results indicated that although the motifs containing the putative active sites H232, N249, A260, H324, E358, and H376 are highly conserved in the creatinases [19, 20], changes of few residues might
Appl Biochem Biotechnol
Fig. 1 Functional expression and purification of the recombinant creatinase. a The amount of creatinase accumulated in different strains; b Creatinase purification by anion-exchange chromatography; c Creatinase purification by gel filtration chromatography. The brown and blue lines indicate the conductivity and the absorbance at 280 nm (A280). The arrow indicates the active fraction; d SDS-PAGE analysis of the purified creatinase. Lane M, molecular weight marker; lane 1, supernatant of the control strain E. coli BL21 (pET20b); lane 2, supernatant of the recombinant E. coli BL21J; lane 3, 60–80 % ammonium sulfate precipitation; lane 4, purified creatinase from the recombinant E. coli BL21J
lead to significantly altered specific activities. As a result, molecular engineering of the present creatinase with directed evolution strategies would be worthy of further studies. Enzymatic Characteristics of the Recombinant Creatinase To pave the way to clinical applications, the activities of the recombinant creatinase were measured at different temperatures and pHs. The optimum temperature was about 37 °C, and
Appl Biochem Biotechnol
Table 1 Comparison of creatinase production in various strains
Strains
Creatinase concentration References
Escherichia coli JA221 (pCR50)
0.04 U/mL
[13]
E. coli JM109 (pkls508)
1.8 U/mL
[11]
E. coli JM109 (pQE-51)
4.7 U/mL
[30]
Arthrobacter nicotianae 02181 70.0 U/g cell
[4]
E. coli BL21J
This work
61.3 U/mL (1834.0 U/g cell)
enzymatic activity sharply decreased when above 40 °C (Fig. 3a), which resembled to that isolated from A. nicotianae 02181 [4], Paracoccus sp. strain WB1 [21], and P. putida [9, 22]. At 37 °C, the optimum pH for the recombinant creatinase was 7.5, and only remained about 60 % activity at pH 8.0 (Fig. 3b). The enzyme stability under different temperatures and pHs were also investigated. This enzyme was stable below 40 °C but completely lost its activity after 30-min preincubation at 50 °C (Fig. 3c). Although this enzyme has an optimum pH at pH 7.5, it is stable in the range of pH 6.0 to 11.0 (Fig. 3d). Obviously, this recombinant creatinase has higher resistance to alkaline environment compared to that of A. nicotianae 02181 [4]. Simultaneously, the kinetic parameters Km and Vmax of this enzyme were also estimated with values of 53.2 mM and was 6.1 μM/min, respectively, which was similar with that produced by A. nicotianae 02181 [4]. In addition, the effects of inhibitors and metal irons on creatinase activity were also assessed. As shown in Table 3, Cu2+, Hg2+, and SDS inhibited almost all the activity while Zn2+ reduced the activity by 56 %. In contrast, no inhibition effect of other metal irons was observed. More importantly, it was notable that the recombinant creatinase shows excellent resistance to NaN3 (20 mM), EDTA, Tween 20, Tween 80, and Triton X-100, which was analogous to that previously reported [4, 21]. All the above results suggest that this recombinant creatinase is applicable for clinical applications. Key Fermentation Process Parameters for Creatinase Expression The initial pH and inoculum amount were optimized at 7.0 (Fig. 4a) and 3 % (v/v) (Fig. 4b). IPTG was added at 3 h when cells entering early exponential phase (Fig. 4c). The effect of temperature was further investigated and optimized to increase creatinase production. Cells were initially cultivated at 37 °C while temperature was maintained or shifted to 30 and 25 °C after induction. As shown in Fig. 4d, the temperature exhibited remarkable influence on creatinase expression. Induction at 30 °C resulted in high titer of creatinase which further confirmed previous conclusion Table 2 Purification of the recombinant creatinase from Escherichia coli BL21J Procedures
Volumes (mL)
Total activities (U)
Concentrations (mg/mL)
Crude enzyme
590.0
12,260.2
14.4
1.4
–
AS precipitation QFF
76.0 144.0
6654.6 4489.9
14.0 1.5
6.2 20.3
4.3 14.1
AS ammonium sulfate
Specific activities (U/mg protein)
Fold
Appl Biochem Biotechnol
Fig. 2 Sequence comparison of creatinase from A. nicotianae 23710 and A. nicotianae 02181. The figure was generated using the CLUSTALW 1.8 software package based on the creatinase from A. nicotianae 23710 and A. nicotianae 02181 [4]. The different residues are boxed in gray, and the predicted key catalytic residues were marked with red stars [19]
that the balance between translation and protein folding is crucial to functional overexpression [23–25]. In view of the inhibition effect on cell growth, IPTG was
Fig. 3 Properties of the recombinant creatinase toward temperature and pH. a Effect of temperature on the activity; b effect of pH on the activity; c effect of temperature on the stability; d effect of pH on the stability
Appl Biochem Biotechnol
Table 3 Effect of metal ions and chemicals on enzyme activity
Metal ions and chemicals
Concentrations
Residual activities (%)
Cu2+
1 mM
1
Hg2+ Co2+
1 mM 1 mM
1 104
Fe3+
1 mM
109
Ba2+
1 mM
109
Pb2+
1 mM
109
Mn2+
1 mM
111
Mg2+
1 mM
111
Li+
1 mM
112
Ca2+ EDTA
1 mM 1 mM
112 110
Zn2+
1 mM
44
NaN3
20 mM
101
Tween 20
1 g/L
112
Tween 80
1 g/L
104
Triton X-100
5 g/L
115
optimized with a concentration of 0.6 mM. Eventually, the creatinase production was improved to 33.3 U/mL (Fig. 4d).
Fig. 4 Identification and optimization of the key fermentation parameters. a Effect of initial pH of medium on the production of creatinase; b effect of inoculum on the production of creatinase; c cell growth curve of the recombinant strain E. coli BL21J. The arrow indicated as point for addition of the inducer IPTG; d effect of IPTG concentrations and temperature on the production of creatinase
Appl Biochem Biotechnol
Fig. 5 Batch fermentation of the recombinant creatinase in 3-L fermentor. Before induction, cells were cultivated at 37 °C. After induction, the temperature was decreased to 30 °C
Batch fermentation of creatinase with a two-stage temperature control strategy Batch fermentation of the recombinant creatinase was carried out in 3-L fermentor with a two-stage temperature control strategy. Specifically, temperature was switched from 37 to 30 °C after induction. Glycerol as carbon source was supplied with a final concentration of 10 g/L according to preliminary experiments (data not shown). At 10 h, cell biomass was accumulated to 31.2 OD600 (corresponding to 12.1 g/L DCW) and the production of creatinase was significantly increased to 61.3 U/mL (Fig. 5a) with a productivity of 6.1 U/mL/h. In parallel, the production of the recombinant creatinase was also monitored by SDS-PAGE analysis. Obviously, the protein bands corresponding to the enzyme increased in intensity with prolonged fermentation time (Fig. 5b), which was consistent with the above activity analysis results. In addition, it could be found that accumulation of creatinase is coupled with cell growth, indicating high-cell-density fermentation [26, 27] should be an alternative approach for further improving the production in future.
Conclusions In the present study, we cloned an A. nicotianae creatinase gene creJ and firstly overexpressed it in E. coli. As we expected, the recombinant creatinase shows satisfactory properties in particular with excellent resistance to the common preservative NaN3. By applying a temperature two-phase control strategy, the production of the recombinant creatinase was improved to 61.3 U/mL with a high productivity of 6.1 U/ mL/h. To the best of our knowledge (Table 1), this is the highest reported creatinase production and productivity until now. The results obtained in this work will be useful for its industrial production and practical applications. Further research should be focused on improving its specific activity by molecular engineering with directed evolution strategies [28, 29]. Acknowledgments This work was financially supported by the National High Technology Research and Development Program of China (863 Program, 2011AA100905), Program for Changjiang Scholars and Innovative Research Team in University (no. IRT1135), the National Science Foundation for Post-doctoral Scientists of China (2013 M540414), the Jiangsu Planned Projects for Postdoctoral Research Funds (1301010B), and the 111 Project.
Appl Biochem Biotechnol
References 1. Kim, E. J., Haruyama, T., Yanagida, Y., Kobatake, E., & Aizawa, M. (1999). Analytica Chimica Acta, 394, 225–231. 2. Lad, U., Khokhar, S., & Kale, G. M. (2008). Analytical Chemistry, 80, 7910–7917. 3. Serafin, V., Hernandez, P., Agui, L., Yanez-Sedeno, P., & Pingarron, J. M. (2013). Electrochimica Acta, 97, 175–183. 4. Zhi, Q., Kong, P. Y., Zang, J. T., Cui, Y. H., Li, S. H., Li, P., Yi, W. J., Wang, Y., Chen, A., & Hu, C. M. (2009). Process Biochemistry, 44, 460–465. 5. Schumacher, G., Hilscher, W., Mollering, H., Siedel, J., & Buckel, P. (1993). Annales Biologie Clinique, 51, 815–819. 6. Goren, M. P., Osborne, S., & Wright, R. K. (1986). Clinical Chemistry, 32, 548–551. 7. Roche, J., Lacombe, G., & Girard, H. (1950). Biochimica Biophysica Acta, 6, 210–216. 8. Appleyard, G., & Woods, D. D. (1956). Journal of General Microbiology, 14, 351–365. 9. Yoshimoto, T., Oka, I., & Tsuru, D. (1976). Journal of Biochemistry, 79, 1381–1383. 10. Matsuda, Y., Wakamatsu, N., Inouye, Y., Uede, S., Hashimoto, Y., Asano, K., & Nakamura, S. (1986). Chemical & Pharmaceutical Bulletin, 34, 2155–2160. 11. Koyama, Y., Kitao, S., Yamamoto-Otake, H., Suzuki, M., & Nakano, E. (1990). Agricultural and Biological Chemistry, 54, 1453–1457. 12. Suzuki, K., Sagai, H., Sugiyama, M., & Imamura, S. (1993). Journal of Fermentation and Bioengineering, 76, 77–81. 13. Chang, M. C., Chang, C. C., & Chang, J. C. (1992). Applied and Environmental Microbiology, 58, 3437– 3440. 14. Kang, Z., Wang, Q., Zhang, H., & Qi, Q. (2008). Applied Microbiology and Biotechnology, 79, 203–208. 15. Kang, Z., Zhang, C., Du, G., & Chen, J. (2014). Applied Biochemistry and Biotechnology, 172, 2012–2021. 16. Rosano, G. L., & Ceccarelli, E. A. (2014). Frontiers in Microbiology, 5, 172. 17. Hong, M. C., Chang, J. C., Wu, M. L., & Chang, M. C. (1998). Biochemical Genetics, 36, 407–415. 18. Suzuki, K., Sagai, H., Imamura, S., & Sugiyama, M. (1994). Journal of Fermentation and Bioengineering, 77, 231–234. 19. Hoeffken, H. W., Knof, S. H., Bartlett, P. A., Huber, R., Moellering, H., & Schumacher, G. (1988). Journal of Molecular Biology, 204, 417–433. 20. Coll, M., Knof, S. H., Ohga, Y., Messerschmidt, A., Huber, R., Moellering, H., Russmann, L., & Schumacher, G. (1990). Journal of Molecular Biology, 214, 597–610. 21. Wang, Y. Y., Ma, X. H., Zhao, W. F., Jia, X. M., Kai, L., & Xu, X. H. (2006). Process Biochemistry, 41, 2072–2077. 22. Yoshimoto, T., Oka, I., & Tsuru, D. (1976). Archives of Biochemistry and Biophysics, 177, 508–515. 23. Zhang, X., Lian, J. Z., Kai, L., Huang, L., Cen, P. L., & Xu, Z. N. (2014). Enzyme and Microbial Technology, 55, 26–30. 24. Gopal, G. J., & Kumar, A. (2013). The Protein Journal, 32, 419–425. 25. Hu, S., Wang, M., Cai, G., & He, M. (2013). The Journal of Biological Chemistry, 288, 30855–30861. 26. Yegane-Sarkandy, S., Farnoud, A. M., Shojaosadati, S. A., Khalilzadeh, R., Sadeghyzadeh, M., Ranjbar, B., & Babaeipour, V. (2009). Biotechnology and Applied Biochemistry, 54, 31–39. 27. Seo, M. J., Choi, H. J., Chung, K. H., & Pyun, Y. R. (2011). Journal of Microbiology and Biotechnology, 21, 1053–1056. 28. Socha, R. D., & Tokuriki, N. (2013). Febs Journal, 280, 5582–5595. 29. Sen, S., Dasu, V. V., & Mandal, B. (2007). Applied Biochemistry and Biotechnology, 143, 212–223. 30. Hsu, W. F., Ng, I. S., Tsai, S. W., & Chang, M. C. (2003). Journal of The Chinese Institute of Chemical Engineers, 34, 305–310.
