, Volume 22, Issue 3, pp 473–484 | Cite as

Identification and characterization of a novel cold-tolerant extracellular protease from Planococcus sp. CGMCC 8088

  • Kun Chen
  • Qingshan Mo
  • Huan Liu
  • Feiyan Yuan
  • Haonan Chai
  • Fuping Lu
  • Huitu Zhang
Original Paper


A psychrophilic extracellular protease was isolated from the marine bacterium Planococcus sp. M7 found in the deep-sea mud of the Southern Indian Ocean. The mature protease is about 43 kDa and contains 389 amino acids. Sequence alignment revealed that the protease whose catalytic triad was comprised of Ser224, Lys249, and Gln253 contains a catalytic module belonging to the serralysin-type protease family 41, and displays 46.55% identity with the experimentally verified serine protease from Bacillus subtilis str. 168. The enzyme displayed an alkaline mesophilic preference with an optimum pH of 10.0 and an optimum temperature of 35 °C. The enzyme retained its activity from 5 to 35 °C and was resistant to repeated freezing and thawing, but was completely inactivated at 55 °C. Calcium ions had a protective effect against thermal denaturation. More than 60% of the maximum activity was retained at pH values in the range of 5.0–11.0. Almost no activity loss was detected after 1 h of incubation at pH 8.0–10.0 and 20 °C, or with 1.0% SDS. Most important, this protease also showed good stability and compatibility with the standard enzyme-free detergent, which indicates its special interest for applications in detergent industry.


Cold-tolerant protease Psychrophile Planococcus sp. Commercial processes 



This research was supported by the National High Technology Research and Development Program of China (Grant number: 2013AA102803; Task number: 2013AA102803C), the National Key Research and Development Program of China (Grant number: 2017YFB0308401), and the National Natural Science Foundation of China (Grant number: 81373309).


  1. Alam SI, Dube S, Reddy GSN et al (2005) Purification and characterisation of extracellular protease produced by Clostridium sp. from Schirmacher oasis, Antarctica. Enzyme Microb Technol 36:824–831. CrossRefGoogle Scholar
  2. Baghel VS, Tripathi RD, Ramteke PW et al (2005) Psychrotrophic proteolytic bacteria from cold environment of Gangotri glacier, Western Himalaya, India. Enzyme Microb Technol 36:654–659. CrossRefGoogle Scholar
  3. Damare S, Raghukumar C, Muraleedharan UD et al (2006) Deep-sea fungi as a source of alkaline and cold-tolerant proteases. Enzyme Microb Technol 39:172–181. CrossRefGoogle Scholar
  4. Davail S, Feller G, Narinx E et al (1994) Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the Antarctic psychrophile Bacillus TA41. J Biol Chem 269:17448–17453PubMedGoogle Scholar
  5. Feller G (2013) Psychrophilic enzymes: from folding to function and biotechnology. Scientifica (Cairo) 2013:512840. Google Scholar
  6. Garsoux GE, Lamotte J, Gerday C et al (2004) Kinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis. Biochem J 384:247–253. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Jeong YJ, Baek SC, Kim H (2017) Cloning and characterization of a novel intracellular serine protease (IspK) from Bacillus megaterium with a potential additive for detergents. Int J Biol Macromol. Google Scholar
  8. Joshi S, Satyanarayana T (2013) Biotechnology of cold-active proteases. Biology (Basel) 2:755–783. PubMedCentralGoogle Scholar
  9. Kim S, Park YJ, Kim J (2016) Inverse PCR for subtyping of Acinetobacter baumannii carrying ISAba1. J Microbiol 54:376–380. CrossRefPubMedGoogle Scholar
  10. Margesin R, Dieplinger H, Hofmann J et al (2005) A cold-active extracellular metalloprotease from Pedobacter cryoconitis—production and properties. Res Microbiol 156:499–505. CrossRefPubMedGoogle Scholar
  11. Mhamdi S, Bkhairia I, Nasri R et al (2017) Evaluation of the biotechnological potential of a novel purified protease BS1 from Bacillus safensis S406 on the chitin extraction and detergent formulation. Int J Biol Macromol 104:739–747. CrossRefPubMedGoogle Scholar
  12. Mhetras NC, Bastawde KB, Gokhale DV (2009) Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207. Bioresour Technol 100:1486–1490. CrossRefPubMedGoogle Scholar
  13. Mohammadi M, Sepehrizadeh Z, Ebrahim-Habibi A et al (2016) Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis. Enzyme Microb Technol 93–94:18–28. CrossRefPubMedGoogle Scholar
  14. Mokashe N, Chaudhari B, Patil U (2017) Detergent-compatible robust alkaline protease from newly isolated halotolerant Salinicoccus sp. UN-12. J Surfactants Deterg 20:1–17. CrossRefGoogle Scholar
  15. Morgan-Kiss RM, Priscu JC, Pocock T et al (2006) Adaptation and acclimation of photosynthetic microorganisms to permanently cold environments. Microbiol Mol Biol Rev 70:222–252. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Oh KH, Seong CS, Lee SW et al (1999) Isolation of a psychrotrophic Azospirillum sp. and characterization of its extracellular protease. FEMS Microbiol Lett 174:173–178. CrossRefPubMedGoogle Scholar
  17. Óskarsson KR, Nygaard M, Ellertsson B et al (2016) A single mutation Gln142Lys doubles the catalytic activity of VPR, a cold adapted subtilisin-like serine proteinase. Biochim Biophys Acta Proteins Proteom 1864:1436–1443. CrossRefGoogle Scholar
  18. Pereira JQ, Ambrosini A, Passaglia LMP et al (2017) A new cold-adapted serine peptidase from Antarctic Lysobacter sp. A03: insights about enzyme activity at low temperatures. Int J Biol Macromol 103:854–862. CrossRefPubMedGoogle Scholar
  19. Saba I, Qazi PH, Rather SA et al (2012) Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India. World J Microbiol Biotechnol 28:1071–1079. CrossRefPubMedGoogle Scholar
  20. Salwan R, Gulati A, Kasana RC (2010) Phylogenetic diversity of alkaline protease-producing psychrotrophic bacteria from glacier and cold environments of Lahaul and Spiti, India. J Basic Microbiol 50:150–159. PubMedGoogle Scholar
  21. Santiago M, Ramírez-Sarmiento CA, Zamora RA et al (2016) Discovery, molecular mechanisms, and industrial applications of cold-active enzymes. Front Microbiol 7:1408PubMedPubMedCentralGoogle Scholar
  22. Sarmiento F, Peralta R, Blamey JM (2015) Cold and hot extremozymes: industrial relevance and current trends. Front Bioeng Biotechnol 3:148. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Scoble HA, Biller JE, Biemann K (1987) A graphics display-oriented strategy for the amino acid sequencing of peptides by tandem mass spectrometry. Fresenius’ Zeitschrift für Anal Chemie 327:239–245. CrossRefGoogle Scholar
  24. Siddiqui KS (2015) Some like it hot, some like it cold: temperature dependent biotechnological applications and improvements in extremophilic enzymes. Biotechnol Adv 33:1912–1922CrossRefPubMedGoogle Scholar
  25. Struvay C, Feller G (2012) Optimization to low temperature activity in psychrophilic enzymes. Int J Mol Sci 13:11643–11665. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Wang E, Koutsioulis D, Leiros HKS et al (2007) Crystal structure of alkaline phosphatase from the Antarctic Bacterium TAB 5. J Mol Biol 366:1318–1331. CrossRefPubMedGoogle Scholar
  27. Wang G, Luo H, Wang Y et al (2011) A novel cold-active xylanase gene from the environmental DNA of goat rumen contents: direct cloning, expression and enzyme characterization. Bioresour Technol 102:3330–3336. CrossRefPubMedGoogle Scholar
  28. Wang G, Wang Q, Lin X et al (2016a) A novel cold-adapted and highly salt-tolerant esterase from Alkalibacterium sp. SL3 from the sediment of a soda lake. Sci Rep 6:19494. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Wang Z, Sun L, Cheng J et al (2016b) The optimization of fermentation conditions and enzyme properties of Stenotrophomonas maltophilia for protease production. Biotechnol Appl Biochem 63:292–299. CrossRefPubMedGoogle Scholar
  30. Yang J, Li J, Mai Z et al (2013) Purification, characterization, and gene cloning of a cold-adapted thermolysin-like protease from Halobacillus sp. SCSIO 20089. J Biosci Bioeng 115:628–632. CrossRefPubMedGoogle Scholar
  31. Zhang DC, Yu Y, Xin YH et al (2008) Colwellia polaris sp. nov., a psychrotolerant bacterium isolated from Arctic sea ice. Int J Syst Evol Microbiol 58:1931–1934. CrossRefPubMedGoogle Scholar
  32. Zhang SC, Sun M, Li T et al (2011) Structure analysis of a new psychrophilic marine protease. PLoS One 6:e26939. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Zhang H, Mu H, Mo Q et al (2016) Gene cloning, expression and characterization of a novel cold-adapted protease from Planococcus sp. J Mol Catal B Enzym 130:1–8. CrossRefGoogle Scholar
  34. Zheng X, Chu X, Zhang W et al (2011) A novel cold-adapted lipase from Acinetobacter sp. XMZ-26: gene cloning and characterisation. Appl Microbiol Biotechnol 90:971–980. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of BioengineeringTianjin University of Science and TechnologyTianjinPeople’s Republic of China
  2. 2.College of Marine Science and EngineeringTianjin University of Science and TechnologyTianjinPeople’s Republic of China
  3. 3.Industrial Microbiology Laboratory, College of BiotechnologyTianjin University of Science and TechnologyTianjinPeople’s Republic of China

Personalised recommendations