A new cold-active and alkaline pectate lyase from Antarctic bacterium with high catalytic efficiency
- 161 Downloads
Cold-active enzymes have become attractive biocatalysts in biotechnological applications for their ability to retain high catalytic activity below 30 °C, which allows energy reduction and cost saving. Here, a 1041 bp gene pel1 encoding a 34.7 KDa pectate lyase was cloned from a facultatively psychrophilic Antarctic bacterium Massilia eurypsychrophila and heterologously expressed in Escherichia coli. PEL1 presented the highest 66% identity to the reported mesophilic pectate lyase PLXc. The purified PEL1 exhibits the optimum temperature and pH of 30 °C and 10 toward polygalacturonic acid, respectively. PEL1 is a cold-active enzyme that can retain 60% and 25% relative activity at 10 °C and 0 °C, respectively, while it loses most of activity at 40 °C for 10 min. PEL1 has the highest specific activity (78.75 U mg−1) than all other reported cold-active pectinase, making it a better choice for use in industry. Based on the detailed sequence and structure comparison between PEL1 and PLXc and mutation analysis, more flexible structure and some loop regions may contribute to the cold activity and thermal instability of PEL1. Our investigations of the cold-active mechanism of PEL1 might guide the rational design of PEL1 and other related enzymes.
KeywordsPectate lyase Enzymatic assay Cold-active Specific activity Mutation analysis
We thank Prof. Peng Fang for providing M. eurypsychrophila for our research. We thank Dr. Zhao Jing (Tianjin Institute of Industrial Biotechnology) for helpful discussions.
This work was supported by Technical innovation special fund of Hubei Province (2017ACA171), Hubei Provincial Natural Science Foundation (2018CFA042, 2018CFB319), and 2016 Wuhan Yellow Crane Talents (Science) Program.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors. All authors confirm that ethical principles have been followed in the research as well as in manuscript preparation, and approved this submission.
Conflict of interest
The authors declare that they have no conflict of interest.
- Bruhlmann F (1995) Purification and characterization of an extracellular pectate lyase from an Amycolata sp. Appl Environ Microbiol 61(10):3580–3585Google Scholar
- Kobayashi T, Hatada Y, Higaki N, Lusterio DD, Ozawa T, Koike K, Kawai S, Ito S (1999) Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate. Biochim Biophys Acta 1427(2):145–154. https://doi.org/10.1016/S0304-4165(99)00017-3 CrossRefGoogle Scholar
- Ouattara HG, Reverchon S, Niamke SL, Nasser W (2010) Biochemical properties of pectate lyases produced by three different Bacillus strains isolated from fermenting cocoa beans and characterization of their cloned genes. Appl Environ Microbiol 76(15):5214–5220. https://doi.org/10.1128/aem.00705-10 CrossRefGoogle Scholar
- Santiago M, Ramirez-Sarmiento CA, Zamora RA, Parra LP (2016) Discovery, molecular mechanisms, and industrial applications of cold-active enzymes. Front Microbiol 7(1408). https://doi.org/10.3389/fmicb.2016.01408
- Sarmiento F, Peralta R, Blamey JM (2015) Cold and hot extremozymes: industrial relevance and current trends. Front Bioeng Biotechnol 3(148). https://doi.org/10.3389/fbioe.2015.00148
- Shirai T, Igarashi K, Ozawa T, Hagihara H, Kobayashi T, Ozaki K, Ito S (2007) Ancestral sequence evolutionary trace and crystal structure analyses of alkaline alpha-amylase from Bacillus sp. KSM-1378 to clarify the alkaline adaptation process of proteins. Proteins 66(3):600–610. https://doi.org/10.1002/prot.21255 CrossRefGoogle Scholar
- Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433. https://doi.org/10.1146/annurev.biochem.75.103004.142723 CrossRefGoogle Scholar
- Solbak AI, Richardson TH, McCann RT, Kline KA, Bartnek F, Tomlinson G, Tan X, Parra-Gessert L, Frey GJ, Podar M, Luginbuhl P, Gray KA, Mathur EJ, Robertson DE, Burk MJ, Hazlewood GP, Short JM, Kerovuo J (2005) Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric. J Biol Chem 280(10):9431–9438. https://doi.org/10.1074/jbc.M411838200 CrossRefGoogle Scholar
- Xiao Z, Bergeron H, Grosse S, Beauchemin M, Garron ML, Shaya D, Sulea T, Cygler M, Lau PC (2008) Improvement of the thermostability and activity of a pectate lyase by single amino acid substitutions, using a strategy based on melting-temperature-guided sequence alignment. Appl Environ Microbiol 74(4):1183–1189. https://doi.org/10.1128/aem.02220-07 CrossRefGoogle Scholar
- Yuan P, Meng K, Wang Y, Luo H, Shi P, Huang H, Tu T, Yang P, Yao B (2012) A low-temperature-active alkaline pectate lyase from Xanthomonas campestris ACCC 10048 with high activity over a wide pH range. Appl Biochem Biotechnol 168(6):1489–1500. https://doi.org/10.1007/s12010-012-9872-8 CrossRefGoogle Scholar
- Zhang C, Yao J, Zhou C, Mao L, Zhang G, Ma Y (2013) The alkaline pectate lyase PEL168 of Bacillus subtilis heterologously expressed in Pichia pastoris is more stable and efficient for degumming ramie fiber. BMC Biotechnol 13(26). https://doi.org/10.1186/1472-6750-13-26
- Zhao Y, Zhang Y, Cao Y, Qi J, Mao L, Xue Y, Gao F, Peng H, Wang X, Gao GF, Ma Y (2011) Structural analysis of alkaline b-Mannanase from alkaliphilic Bacillus sp. N16-5: implications for adaptation to alkaline conditions. PLoS ONE 6(1):e14608. https://doi.org/10.1371/journal.pone.0014608.t001 CrossRefGoogle Scholar