J. Microbiol. Biotechnol. (2016), 26(9), 1586–1592 http://dx.doi.org/10.4014/jmb.1602.02030 Research Article Review jmb

Functional Study of Lysine Decarboxylases from Klebsiella pneumoniae in Escherichia coli and Application of Whole Cell Bioconversion for Cadaverine Production Jung-Ho Kim1, Hyun Joong Kim1, Yong Hyun Kim1, Jong Min Jeon1, Hun Suk Song1, Junyoung Kim1, So-Young No1, Ji-Hyun Shin2, Kwon-Young Choi3, Kyung Moon Park2*, and Yung-Hun Yang1*

1Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea 2Department of Biological and Chemical Engineering, Hongik University, Sejong City 30016, Republic of Korea 3Department of Environmental Engineering, Ajou University, Suwon 16499, Republic of Korea

Received: February 17, 2016 Revised: June 3, 2016 Klebsiella pneumoniae is a gram-negative, non-motile, rod-shaped, and encapsulated bacterium Accepted: June 7, 2016 in the normal flora of the intestines, mouth, skin, and food, and has decarboxylation activity, which results in generation of diamines (cadaverine, agmatine, and putrescine). However, there is no specific information on the exact mechanism of decarboxylation in K. pnuemoniae. First published online Specifically lysine decarboxylases that generate cadaverine with a wide range of applications June 10, 2016 has not been shown. Therefore, we performed a functional study of lysine decarboxylases. *Corresponding authors Enzymatic characteristics such as optimal pH, temperature, and substrates were examined by Y. H. Yang overexpressing and purifying CadA and LdcC. CadA and LdcC from K. pneumoniae had a Phone: +82-2-450-3936; o Fax: +82-2-3437-8360; preference for L-lysine, and an optimal reaction temperature of 37 C and an optimal pH of 7. E-mail: [email protected] Although the activity of purified CadA from K. pneumoniae was lower than that of CadA from K. Park Phone: +82-44-860-2429; E. coli, the activity of K. pneumoniae CadA in whole cell bioconversion was comparable to that Fax: +82-44-868-6941; of E. coli CadA, resulting in 90% lysine conversion to cadaverine with pyridoxal 5’-phosphate E-mail: [email protected] L-lysine. pISSN 1017-7825, eISSN 1738-8872 Keywords: Klebsiella pneumoniae, biotransformation, lysine decarboxylase, cadaverine, Copyright© 2016 by The Korean Society for Microbiology optimization and Biotechnology

Introduction damage [11]. The relative polyamine concentrations may vary between species and can reach the millimolar range Polyamines have an aliphatic group and two primary [26]. The most common polyamines are putrescine and amino groups [32], and they are found in almost all bacterial triamine spermidine, whereas cadaverine is much less cells and affect many cellular processes, such as transcription abundant [32]. However, owing to its industrial importance, and translation. Polyamines have protected Escherichia coli the biosynthesis of cadaverine has been well documented against oxidative and acidic stress conditions, and the [10, 24, 25]. Bioconversion of cadaverine from lysine is levels of enzymes [7, 8, 10] responsible for polyamine economical, and the production of bio-polyamides from synthesis increase under these conditions, resulting in an cheap starting materials could be an alternative to increased intracellular polyamine pool [40]. production of polyamides from petrochemicals [7, 8, 13, Polyamines participate in the biosynthesis of siderophores 39]. Polyamides can be used for making functional [5] and protect against oxygen toxicity such as superoxide products, such as fungicides, pharmaceuticals, oil and fuel stress [15]. They are also involved in plaque biofilm additives, chelating agents, and fabric softeners/surfactants formation, and they play a role in cellular differentiation [20, 32]. Several studies have aimed to identify lysine signaling [14, 34], stabilize chromatin, and prevent DNA decarboxylases [35, 37].

J. Microbiol. Biotechnol. Bioconversion of CadA from K. pneumoniae in E. coli 1587

Klebsiella pneumoniae is a gram-negative, non-motile, rod- sonicated using an ultrasonicator (Vibra Cell, Soncis Scientific, shaped, and encapsulated bacterium [23]. It can also utilize USA) for 5 min on ice (10 sec on, 15 sec off). The cell lysates were lactose in anaerobic facultative fermentation. It is found in centrifuged at 1,146 ×g. The supernatant was collected and applied the intestines, skin, food, and normal flora of the mouth. to Ni-NTA beads. After a 2 h binding reaction, the Ni-NTA beads were washed three times with the 50 mM NaH PO , 0.05% NaCl, K. pneumoniae can generate CO with decarboxylases [9], 2 4 2 0.05% Tween-20 (pH 8.0) buffer containing 20 mM imidazole. and K. pneumoniae is used in the production of 2,3-butanediol Then, His-tagged CadA was eluted three times from the column and 1,3-propanediol, by means of glucose fermentation using 250 mM imidazole buffer. The reaction with 1 M L-lysine, [36]. Although K. pneumoniae has decarboxylase activity for 0.1 mM PLP, 500 mM sodium acetate buffer (pH 6.0), and 20 μl of one or more generated amines [9], the mechanism of the whole cell was performed in a 37oC water bath. The reaction decarboxylation is unclear, and no functional studies of was stopped by heating at 100oC for 5 min. We defined one unit lysine decarboxylase production by this bacterium have (mmol/cell dry weight (mg)/min)) of activity as the amount of been performed in E. coli. Therefore, we identified and enzyme producing 1 mmol of cadaverine per minute at 37oC [2, 30]. characterized lysine decarboxylases in K. pneumoniae and applied them in a whole-cell reaction system to produce a Derivatization and High-Performance Liquid Chromatography high yield of cadaverine. Analysis Three hundred microliters of 50 mM sodium borate buffer (pH 9), 100 μl of methanol, and 47 μl of distilled water were added Materials and Methods to 50 μl of target sample and 3 μl of DEEMM [1, 18]. The o Chemicals derivatization reaction was performed at 70 C for 2 h to derivatize The reagents used for reaction substrates, culture, and analysis, lysine and cadaverine. High-performance liquid chromatography such as sodium acetate anhydrate, cadaverine, isopropyl β-D-1- (HPLC; YL-9100, Korea) was used after derivatization to detect thiogalactopyranoside (IPTG), pyridoxal-5-phosphate (PLP), sodium derivatized lysine and cadaverine. A reverse-phase Agilent borate, L-ornithine monohydrochloride, L-arginine, L-lysine ZORBAX SB-C18 column was used (4.6 × 250 mm, 5 μm particle monohydrochloride, and 2,6-diaminopimelic acid (DAP) were size). The column temperature was maintained at 35°C. The mobile purchased from Sigma-Aldrich Co. Diethyl ethoxymethylenemalonate phase was composed of 100% acetonitrile (A) and 25 mM sodium (DEEMM) was purchased from Fluka Co. (Japan) for the acetate buffer (pH 4.8, B). A 1 ml/min flow rate was used with the derivatization reaction. The enzyme purification reagent Ni-NTA following gradient: 0–2 min, 20–25% A; 2–32 min, 25–60% A; 32– was purchased from Qiagen. 40 min, 60–20% A. The rest of the percentage was charged by the buffer B. Detection was carried out at 284 nm using a UV detector. Bacterial Strains and Media Genomic DNA of K. pneumoniae and E. coli K12 MG1655 were Lysine Decarboxylase Amino Acid Sequence Alignment used for cloning of each lysine decarboxylase gene, cadA and ldcC. Lysine decarboxylase sequences were obtained from the NCBI To amplify cadA, we constructed and used primers Kpn_cadA_F (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for comparing and alignment (5’-CGTCGTGGATCCATGAACGTTATTGCAATATTAATCACA-3’) of protein sequences. A number of alignments were carried out and Kpn_cadA_R (5’-ATATAAGCTTTCCCGCGATTTTTAGGACTCG- using Clustal W ver. 2 from EBI with a 0.5 transition weight. 3’) for PCR. The PCR product was inserted into the pET24ma Phylogenetic trees were constructed in MEGA version 6.06 using vector using the restriction enzymes, HindIII and BamHI. The the neighbor-joining and unweighted pair-group methods with plasmid of pET24ma::cadA from E. coli was used from a previous the arithmetic mean algorithm [31]. study [17, 21]. The plasmids cloned with each lysine decarboxylase gene were transformed into E. coli BL21 (DE3) competent cells for Results and Discussion protein expression. A single colony of transformant E. coli from the agar plate was pre-cultured in 3ml of LB medium, prior to Lysine Decarboxylases from K. pneumoniae and Functional incubating for 14 h with 200 rpm agitation at 37oC. The pre-culture Expression and Purification in E. coli (1 ml) was transferred to 50 ml of LB medium (50 μg/ml kanamycin, Biogenic amines have been related to food poisoning, o 0.1 mM IPTG) and grown at 30 C. Then, the cells were harvested and Klebsiella is found in food and produces biogenic at 4oC by centrifugation and the cell pellet was washed and stored. amines by amino acid decarboxylation [4, 33]. Based on database searches, we found that Klebsiella pneumoniae has Enzyme Purification and Enzyme Reaction CadA and LdcC from K. pneumoniae were purified and subjected multiple amino acid decarboxylases involved in biogenic to enzymatic characterization and kinetic analysis. E. coli amine production. These enzymes were classified as lysine BL21(DE3) (50 ml) transformed with pET24ma::cadA was cultured decarboxylases by phylogenetic analysis (Fig. 1). Klebsiella and induced by the addition of 0.1 M IPTG. The cells were has two lysine decarboxylases; one is cadA, 2,148bp in

September 2016 ⎪ Vol. 26⎪ No. 9 1588 Kim et al.

Fig. 1. Amino acid sequence alignment and UPGMA bootstrap (100) phylogenetic analysis of lysine decarboxylases similar to that of Klebsiella pneumoniae and their most similar strains according to NCBI (BLASTn). 1. Klebsiella pneumoniae ornithine decarboxylase. 2. Klebsiella pneumoniae arginine decarboxylase. 3. Klebsiella pneumoniae lysine decarboxylase LdcC 4. Klebsiella pneumoniae lysine decarboxylase CadA. 5. Klebsiella pneumoniae diaminopimelate decarboxylase. 6. Escherichia coli K12 MG1655 CadA. 7. Escherichia coli K12 MG1655 LdcC. Phylogenetic trees were developed based on the maximum composite likelihood method using MEGA ver. 6.06. length; and the other is ldcC, 2,190 bp in length. The LdcC and LdcC from E. coli [12, 22], CadA could be produced protein showed 82% similarity to CadA. K. pneumoniae has more easily than LdcC (data not shown). The CadA and a lysine decarboxylase system similar to those of most LdcC overexpressed in E. coli and purified were subjected enterobacteria; that is, one constitutive and one inducible to enzymatic characterization and kinetic analysis. lysine decarboxylase (Fig. 1). After PCR amplification of the K. pneumoniae genome, the Characterization of CadA and LdcC cadA and ldcC genes were inserted into pET24ma vectors Although CadA and LdcC are lysine decarboxylases, and overexpressed in E. coli BL21. His-tag purification they have similarly broad substrate specificities [6, 38]. resulted in a high yield of the CadA protein. Like CadA Among arginine, ornithine, DAP, and lysine (Fig. 2A),

J. Microbiol. Biotechnol. Bioconversion of CadA from K. pneumoniae in E. coli 1589

CadA and LdcC showed a preference for lysine. CadA and LdcC had some activity against arginine, unlike the E. coli CadA, which prefers ornithine [28]. The optimal temperature was 37oC (Fig. 2B), which is different from previous reports of 60oC [41]. The relative activities of LdcC and CadA were also similar at over 37oC, but CadA activity was maintained at 25oC. Further experiments used 37oC. The optimal pH of E. coli lysine decarboxylase is 6 [22]. However, that of K. pneumoniae lysine decarboxylase was pH 7 (Fig. 2C). In 500 mM sodium acetate buffer, the final pH was 8.5 after a 2 h reaction. The conversion of lysine into cadaverine was almost complete in less than 15 min when a low concentration of lysine was used. Compared

with previous data, CadA showed higher Km and lower

kcat values, resulting in a lower kcat/Km value than LdcC (Table 1). Although the reaction conditions were slightly different from those of other enzymes, K. pneumoniae CadA had lower activity than CadA from E. coli.

Optimization of Whole Cell Reaction A whole cell system is preferred for cadaverine production owing to its robustness [17], and, as a result, various whole- cell system conditions were examined. As PLP is required for decarboxylase activity [19, 29] and PLP was one of the major factors for cadaverine production, unlike growth- based production comparatively less effect of additional PLP in fermentation with a [25, 27], the effect of PLP concentration was examined in the whole cell system (Fig. 3A). In the absence of PLP, lysine consumption for conversion of 1 M L-lysine to lysine was only 20% (data not shown). Following addition of over 0.025 mM PLP, lysine consumption was about 90%, suggesting that bioconversion required PLP as a cofactor. Bioconversion by LdcC was markedly lower than that of CadA; however, LdcC showed more PLP-dependent behavior. The conversion yields of both cells containing CadA and LdcC from K. pneumoniae Fig. 2. Characterization of the purified lysine decarboxylases, increased with increasing PLP concentration (Fig. 3A). The CadA and LdcC. conversion yield of the whole cell system with CadA from (A) Substrate specificity test (ornithine, lysine, arginine, and 2,6- K. pneumoniae was 3–5% greater than that of cells containing diaminopimelic acid (DAP)). (B) Optimal temperature for enzyme CadA from E. coli. When the reaction was performed at activity. (C) Optimal pH for enzyme activity. 37oC with various substrate concentrations, 1 M of lysine

Table 1. Kinetic constants for lysine decarboxylases (CadA) from Klebsiella pneumoniae and other microorganisms -1 -1 -1 -1 Strain Km (mM ) kcat (s ) kcat/Km (s ·m·M ) Condition Reference E. coli 0.42 30 71 pH 6.5, between 4oC and 10oC [16] Selemonas ruminatium 0.96 None None pH 6.0, 30oC [38] Hafnia alvei 1.7 175 102.9 37oC[3] Klebsiella pneumoniae 7.7 0.98 0.12 pH 6.0, 37oC This study

September 2016 ⎪ Vol. 26⎪ No. 9 1590 Kim et al.

Fig. 4. Whole-cell cadaverine conversion yields. Whole-cell cadaverine conversion by lysine decarboxylase (CadA) from K. pneumoniae and E. coli (A) at pH 6 (E. coli’s optimal pH) and alkaline pH 9 and 10. (B) Conversion yield over time with or without 500 mM sodium acetate buffer.

was fully consumed during a 2 h reaction (Fig. 3B). However, substrate inhibition occurred at over 1.25 M. Moreover, addition of a greater number of cells containing CadA increased the conversion ratio of lysine into cadaverine (data not shown). Cadaverine production was similar irrespective of the presence of buffer (Fig. 3C). Although a dramatic increase in pH under 8 occurred in the buffer-free system and pH differed according to the presence of buffer, Optimization of whole cell bioconversion. Fig. 3. the overall conversion ratio was similar to that of CadA K. pneumoniae lysine decarboxylase (CadA and LdcC) activity at different from E. coli. (A) PLP concentrations and (B) substrate (lysine) concentrations. (C) Effect of 500 mM sodium acetate buffer (pH 6) for keeping the enzyme’s optimal pH from produced cadaverine’s natural pH and Application of CadA to Whole-Cell Cadaverine Conversion added 1 M lysine. Although the activity of purified CadA from K. pneumoniae

J. Microbiol. Biotechnol. Bioconversion of CadA from K. pneumoniae in E. coli 1591

was slightly lower than that of CadA from E. coli [17], 3. Beier H, Fecker LF, Berlin J. 1987. Lysine decarboxylase from cadaverine production in a whole cell system is affected by Hafnia alvei: purification, molecular data and preparation of various factors. Therefore, cadaverine production using polyclonal antibodies. Z. Naturforsch. C 42: 1307-1312. CadA from E. coli and K. pneumoniae in a whole cell system 4. Bover-Cid S, Hugas M, Izquierdo-Pulido M, Vidal-Carou MC. was compared. The reaction was carried out using whole 2001. Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages. Int. J. Food Microbiol. 66: 185-189. cells with 1 M L-lysine and 0.1 mM PLP in 500 mM sodium 5. Brickman TJ, Armstrong SK. 1996. The ornithine decarboxylase acetate buffer at different pH values for 2 h. The conversion gene odc is required for alcaligin siderophore biosynthesis in yields of K. pneumoniae CadA and E. coli CadA were similar Bordetella spp.: putrescine is a precursor of alcaligin. J. irrespective of pH (Fig. 4A). At an initial pH of 6, the Bacteriol. 178: 54-60. conversion yield of the whole cell system with CadA from 6. Burrell M, Hanfrey CC, Kinch LN, Elliott KA, Michael AJ. K. pneumoniae was 80% and that with CadA from E. coli was 2012. Evolution of a novel lysine decarboxylase in siderophore 86%. The conversion yield at initial pH values of 9 and 10 biosynthesis. Mol. Microbiol. 86: 485-499. was over 75%. The conversion yields of whole cell systems 7. Buschke N, Becker J, Schäfer R, Kiefer P, Biedendieck R, with E. coli CadA and K. pneumoniae CadA were similar Wittmann C. 2013. Systems metabolic engineering of xylose irrespective of the buffer system. The final cadaverine yield - utilizing Corynebacterium glutamicum for production of 1,5- from lysine was 94% in both the presence and absence of diaminopentane. Biotechnol. J. 8: 557-570. sodium acetate buffer, indicating no difference in the 8. Buschke N, Schröder H, Wittmann C. 2011. Metabolic engineering of Corynebacterium glutamicum for production of whole cell systems incorporating CadA from E. coli and 1,5-diaminopentane from hemicellulose. Biotechnol. J. 6: 306-317. K. pneumoniae (Fig. 4B). 9. Cowan ST, Steel KJ, Shaw C, Duguid JP. 1960. A classification In conclusion, we compared lysine decarboxylases of the Klebsiella group. Microbiology 23: 601-612. purified from K. pneumoniae with that from E. coli using an 10. Gale EF, Epps HMR. 1944. Studies on bacterial amino-acid E. coli whole cell system. Although the activity of CadA decarboxylases: 1. L (+)-lysine decarboxylase. Biochem. J. 38: 232. from K. pneumoniae was lower than that of CadA from 11. Ha HC, Sirisoma NS, Kuppusamy P, Zweier JL, Woster PM, E. coli, the overall conversion of K. pneumoniae CadA was Casero RA. 1998. The natural polyamine spermine functions comparable to that of E. coli CadA, and resulted in ~90% directly as a free radical scavenger. Proc. Nat. Acad. Sci. USA conversion of lysine to cadaverine after optimization of 95: 11140-11145. various reaction factors. 12. Herminghaus S, Schreier PH, McCarthy JEG, Landsmann J, Botterman J, Berlin J. 1991. Expression of a bacterial lysine decarboxylase gene and transport of the protein into Acknowledgments chloroplasts of transgenic tobacco. Plant Mol. Biol. 17: 475-486. 13. Ikeda N, Miyamoto M, Adachi N, Nakano M, Tanaka T, The study was supported by the National Research Kondo A. 2013. Direct cadaverine production from cellobiose Foundation of Korea (NRF) funded by the Ministry of using beta-glucosidase displaying Escherichia coli. AMB Education (NRF-2015R1A2A2A04006014) and (NRF- Express 3: 67. 2015M1A5A1037196). This work was also supported by the 14. Jacob M. 2006. Biofilms, a new approach to the microbiology R&D Program of MOTIE/KEIT (10047910, 10049674) and of dental plaque. Odontology 94: 1-9. the Energy Efficiency & Resources of the Korea Institute of 15. Jung IL, Kim IG. 2003. Thiamine protects against paraquat- Energy Technology Evaluation and Planning (KETEP) induced damage: scavenging activity of reactive oxygen grant funded by the Korea Government Ministry of Trade, species. Environ. Toxicol. Pharmacol. 15: 19-26. Industry and Energy (20133030000300). 16. Kanjee U, Gutsche I, Alexopoulos E, Zhao B, El Bakkouri M, Thibault G, et al. 2011. Linkage between the bacterial acid stress and stringent responses: the structure of the inducible References lysine decarboxylase. EMBO J. 30: 931-944. 17. Kim HJ, Kim YH, Shin J-H, Bhatia SK, Sathiyanarayanan G, 1. Alaiz M, Navarro JL, Giron J, Vioque E. 1992. Amino acid Seo H-M, et al. 2015. Optimization of direct lysine analysis by high-performance liquid chromatography after decarboxylase biotransformation for cadaverine production derivatization with diethyl ethoxymethylenemalonate. J. with whole cell biocatalysts at high substrate concentration. Chromatogr. 591: 181-186. J. Microbiol. Biotechnol. 25: 1108-1113. 2. Alberty RA, Bock RM. 1953. Alteration of the kinetic 18. Kim YH, Kim HJ, Shin J-H, Bhatia SK, Seo H-M, Kim Y-G, properties of an enzyme by the binding of buffer, inhibitor, et al. 2015. Application of diethyl ethoxymethylenemalonate or substrate. Proc. Nat. Acad. Sci. USA 39: 895. (DEEMM) derivatization for monitoring of lysine decarboxylase

September 2016 ⎪ Vol. 26⎪ No. 9 1592 Kim et al.

activity. J. Mol. Catal. B Enzym. 115: 151-154. Kim Y-G, et al. 2015. Exopolysaccharide from psychrotrophic 19. Kind S, Jeong WK, Schröder H, Wittmann C. 2010. Systems- Arctic glacier soil bacterium Flavobacterium sp. ASB 3-3 and wide metabolic pathway engineering in Corynebacterium its potential applications. RSC Adv. 5: 84492-84502. glutamicum for bio-based production of diaminopentane. 32. Schneider J, Wendisch VF. 2011. Biotechnological production Metab. Eng. 12: 341-351. of polyamines by bacteria: recent achievements and future 20. Kroschwitz JI, Seidel A. 2004. Kirk-Othmer Encyclopedia of perspectives. Appl. Microbiol. Biotechnol. 91: 17-30. Chemical Technology. 5th Ed. Wiley-Interscience, Hoboken. 33. Stratton JE, Hutkins RW, Taylor SL. 1991. Biogenic amines 21. Lee SG, Lee JO, Yi JK, Kim BG. 2002. Production of cytidine in cheese and other fermented foods: a review. J. Food Prot. 5’-monophosphate N-acetylneuraminic acid using recombinant 54: 460-470. Escherichia coli as a biocatalyst. Biotechnol. Bioeng. 80: 516-524. 34. Sturgill G, Rather PN. 2004. Evidence that putrescine acts as 22. Lemonnier M, Lane D. 1998. Expression of the second lysine an extracellular signal required for swarming in Proteus decarboxylase gene of Escherichia coli. Microbiology 144: 751-760. mirabilis. Mol. Microbiol. 51: 437-446. 23. Matasyoh LG. 2012. Genetic variation and medicinal activity 35. Sugawara A, Matsui D, Takahashi N, Yamada M, Asano Y, in Ocimum gratissimum L. of Kenya. PhD Thesis. Jomo Isobe K. 2014. Characterization of a pyridoxal-5’-phosphate- Kenyatta University of Agriculture and Technology, Juja, dependent L-lysine decarboxylase/oxidase from Burkholderia Kenya. sp. AIU 395. J. Biosci. Bioeng. 118: 496-501. 24. Meng SY, Bennett GN. 1992. Nucleotide sequence of the 36. Syu MJ. 2001. Biological production of 2,3-butanediol. Appl. Escherichia coli cad operon: a system for neutralization of Microbiol. Biotechnol. 55: 10-18. low extracellular pH. J. Bacteriol. 174: 2659-2669. 37. Takatsuka Y, Onoda M, Sugiyama T, Muramoto K, Tomita 25. Mimitsuka T, Sawai H, Hatsu M, Yamada K. 2007. Metabolic T, Kamio Y. 1999. Novel characteristics of Selenomonas engineering of Corynebacterium glutamicum for cadaverine ruminantium lysine decarboxylase capable of decarboxylating fermentation. Biosci. Biotechnol. Biochem. 71: 2130-2135. both L-lysine and L-ornithine. Biosci. Biotechnol. Biochem. 63: 26. Miyamoto S, Kashiwagi K, Ito K, Watanabe S-I, Igarashi K. 1063-1069. 1993. Estimation of polyamine distribution and polyamine 38. Takatsuka Y, Yamaguchi Y, Ono M, Kamio Y. 2000. Gene stimulation of protein synthesis in Escherichia coli. Arch. cloning and molecular characterization of lysine decarboxylase Biochem. Biophys. 300: 63-68. from Selenomonas ruminantium delineate its evolutionary 27. Qian ZG, Xia XX, Lee SY. 2011. Metabolic engineering of relationship to ornithine decarboxylases from eukaryotes. J. Escherichia coli for the production of cadaverine: a five Bacteriol. 182: 6732-6741. carbon diamine. Biotechnol. Bioeng. 108: 93-103. 39. Tateno T, Okada Y, Tsuchidate T, Tanaka T, Fukuda H, 28. Sabo DL, Boeker EA, Byers B, Waron H, Fischer EH. 1974. Kondo A. 2009. Direct production of cadaverine from soluble Purification and physical properties of inducible Escherichia starch using Corynebacterium glutamicum coexpressing alpha- coli lysine decarboxylase. Biochemistry 13: 662-670. amylase and lysine decarboxylase. Appl. Microbiol. Biotechnol. 29. Sabo DL, Fischer EH. 1974. Chemical properties of Escherichia 82: 115-121. coli lysine decarboxylase including a segment of its pyridoxal 40. Tkachenko A, Shumkov A, Akhova A. 2009. Adaptive functions 5’-phosphate binding site. Biochemistry 13: 670-676. of Escherichia coli polyamines in response to sublethal 30. Sarciaux JM, Mansour S, Hageman MJ, Nail SL. 1999. concentrations of antibiotics. Microbiology 78: 25-32. o o o Effects of buffer composition and processing conditions on 41. Vienoz inskien J, Janus evic iu t R, Pauliukonis A, Kazlauskas aggregation of bovine IgG during freeze-drying. J. Pharm. D. 1985. Lysine decarboxylase assay by the pH-stat method. Sci. 88: 1354-1361. Anal. Biochem. 146: 180-183. 31. Sathiyanarayanan G, Yi D-H, Bhatia SK, Kim J-H, Seo HM,

J. Microbiol. Biotechnol.