Biotechnological Aspects of Cold-Active Enzymes

  • Mário Barroca
  • Gustavo Santos
  • Charles Gerday
  • Tony CollinsEmail author


Cold-adapted enzymes produced by organisms inhabiting permanently low temperature environments are typically characterized by a high activity at low to moderate temperatures and a poor thermal stability. Such characteristics make these enzymes highly attractive for various applications where they can enable more efficient, cost-effective, and environmentally friendlier processes than higher temperature-adapted enzymes. In this chapter, the biotechnological aspects of cold-adapted enzymes and their application in industry are reviewed and discussed with a focus on cleaning/detergents, food and beverages, molecular biology, biomedicine, pharmaceuticals, cosmetics, textiles, biofuels, and materials applications.



T.C. is supported by the Fundação para a Ciência e a Tecnologia (FCT), the European Social Fund, the Programa Operacional Potencial Humano and the Investigador FCT Programme (IF/01635/2014). M.B. acknowledges the FCT for grant PD/BD/113810/2015 within the Doctoral Program in Applied and Environmental Microbiology. This work was funded by the European Regional Development Fund (ERDF) through project EcoAgriFood (NORTE-01-0145-FEDER-000009) via the North Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement. The FCT is thanked for their funding through EngXyl (EXPL/BBB-BIO/1772/2013-FCOMP-01-0124-FEDER-041595) and the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569). All the technical staff at the CBMA are thanked for their skillful technical assistance.


  1. Adapa V, Ramya LN, Pulicherla KK, Sambasiva Rao KRS (2014) Cold active pectinases: advancing the food industry to the next generation. Appl Biochem Biotechnol 172(5):2324–2337. doi: 10.1007/s12010-013-0685-1 CrossRefPubMedGoogle Scholar
  2. Akila G, Chandra TS (2010) Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures. J Appl Microbiol 108(1):204–213. doi: 10.1111/j.1365-2672.2009.04412.x CrossRefPubMedGoogle Scholar
  3. Antranikian G, Breves R, Janßen F, Qoura FM (2004) Pullulanases from psychrophilic organisms. Patent Application WO2006032477A1, DE102004046116A1Google Scholar
  4. Awazu N, Shodai T, Takakura H, Kitagawa M, Mukai H, Kato I (2011) Microorganism-derived psychrophilic endonuclease. Granted Patent US8034597B2Google Scholar
  5. Balabanova LA, Bakunina IY, Nedashkovskaya OI, Makarenkova ID, Zaporozhets TS, Besednova NN, Zvyagintseva TN, Rasskazov VA (2010) Molecular characterization and therapeutic potential of a marine bacterium Pseudoalteromonas sp. KMM 701 α-galactosidase. Mar Biotechnol 12(1):111–120. doi: 10.1007/s10126-009-9205-2 CrossRefPubMedGoogle Scholar
  6. Barroca M, Santos G, Johansson B, Gillotin F, Feller G, Collins T (2017) Deciphering the factors defining the pH-dependence of a commercial glycoside hydrolase family 8 enzyme. Enzym Microb Technol 96:163–169. doi: 10.1016/j.enzmictec.2016.10.011 CrossRefGoogle Scholar
  7. BCC Research (2017) BCC research report. Global markets for enymes in industrial applications. BIO030J, Jan 2017. BCC Research LLC, USAGoogle Scholar
  8. Bjarnason JB, Benediktsson B (2010) Protein hydrolysates produced with the use of marine proteases. Granted Patents CA2421058C, DE60007655D1/T2, EP1227736B1, US7070953B1Google Scholar
  9. Blamey JM, Fischer F, Meyer H-P, Sarmiento F, Zinn M (2017) Enzymatic biocatalysis in chemical transformations: a promising and emerging field in green chemistry practice. In: Brahmachari G, Demain AL, Adrio JL (eds) Biotechnology of microbial enzymes, production, biocatalysis and industrial applications. Academic, New York, pp 347–403. doi: 10.1016/B978-0-12-803725-6.00014-5 Google Scholar
  10. Bommarius AS, Paye MF (2013) Stabilizing biocatalysts. Chem Soc Rev 42:6534–6565. doi: 10.1039/c3cs60137d CrossRefPubMedGoogle Scholar
  11. Cavicchioli R, Charlton T, Ertan H, Mohd Omar S, Siddiqui KS, Williams TJ (2011) Biotechnological uses of enzymes from psychrophiles. Microb Biotechnol 4(4):449–460. doi: 10.1111/j.1751-7915.2011.00258.x CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR (2002) Low-temperature extremophiles and their applications. Curr Opin Biotechnol 13(3):253–261CrossRefPubMedGoogle Scholar
  13. Cazarin C, Lima G, da Silva J, Maróstica M (2015) Enzymes in meat processing. In: Chandrasekaran M (ed) Enzymes in food and beverage processing. CRC, Boca Raton, pp 337–351. doi: 10.1201/b19408-17 CrossRefGoogle Scholar
  14. Çelik A, Yetiş G (2012) An unusually cold active nitroreductase for prodrug activations. Bioorg Med Chem 20(11):3540-3550. doi: 10.1016/j.bmc.2012.04.004
  15. Chandrasekaran M (2015) Enzymes in food and beverage processing. CRC, Boca RatonCrossRefGoogle Scholar
  16. Collins T, Claverie P, D’Amico S, Georlette D, Gratia E, Hoyoux A, Meuwis MA, Poncin J, Sonan G, Feller G, Gerday C (2002a) Life in the cold: psychrophilic enzymes. In: Pandalai SG (ed) Recent research developments in proteins, vol 1. Transworld Research Network, Trivandrum, pp 13–26Google Scholar
  17. Collins T, Meuwis MA, Stals I, Claeyssens M, Feller G, Gerday C (2002b) A novel family 8 xylanase, functional and physicochemical characterization. J Biol Chem 277(38):35133–35139. doi: 10.1074/jbc.M204517200 CrossRefPubMedGoogle Scholar
  18. Collins T, Hoyoux A, Dutron A, Georis J, Genot B, Dauvrin T, Arnaut F, Gerday C, Feller G (2006) Use of glycoside hydrolase family 8 xylanases in baking. JCS 43:79–84CrossRefGoogle Scholar
  19. Collins T, D’Amico S, Marx J, Feller G, Gerday C (2007) Cold-adapted enzymes. In: Gerday C, Glansdorff N (eds) Physiology and biochemistry of extremophiles. ASM Press, Washington, DC, pp 165–179. doi: 10.1128/9781555815813.ch13 CrossRefGoogle Scholar
  20. Collins T, Feller G, Gerday C, Meuwis MA (2012) Family 8 enzymes with xylanolytic activity. Granted Patent US8309336B2Google Scholar
  21. Collins T, Roulling F, Florence P, Marx JC, Feller G, Gerday C, D’Amico S (2008) Fundamentals of cold-adapted enzymes. In: Margesin R, Schinner F, Marx JC, Gerday C (eds) Psychrophiles: from biodiversity to biotechnology. Springer, Berlin, pp 211–227CrossRefGoogle Scholar
  22. D’Amico S, Collins T, Marx JC, Feller G, Gerday C (2006) Psychrophilic microorganisms: challenges for life. EMBO Rep 7(4):385–389CrossRefPubMedPubMedCentralGoogle Scholar
  23. Damhus T, Kaasgaard S, Olsen HS (2013) Enzymes at work, 4th edn. Novozymes A/S, DenmarkGoogle Scholar
  24. Dornez E, Verjans P, Arnaut F, Delcour JA, Courtin CM (2011) Use of psychrophilic xylanases provides insight into the xylanase functionality in bread making. J Agric Food Chem 59(17):9553–9562. doi: 10.1021/jf201752g CrossRefPubMedGoogle Scholar
  25. Dutron A, Georis J, Genot B, Dauvrin T, Collins T, Hoyoux A, Feller G (2012) Use of family 8 enzymes with xylanolytic activity in baking. Granted Patents US8192772 (2012), EP1549147B1 (2011), CN1681392B (2010), DE60336153 D1 (2011), CA 2498014C (2011), ES2360942 (2011), DE60336153D1 (2011)Google Scholar
  26. Elleuche S, Schroder C, Sahm K, Antranikian G (2014) Extremozymes—biocatalysts with unique properties from extremophilic microorganisms. Curr Opin Biotechnol 29:116–123. doi: 10.1016/j.copbio.2014.04.003 CrossRefPubMedGoogle Scholar
  27. Festersen RM, Olsen HS, Pedersen S (2005) Alcohol product processes. Granted Patents DE04718914T1, EP1604019B1, CN1788083B, US8772001B2Google Scholar
  28. Fields PA, Dong Y, Meng X, Somero GN (2015) Adaptations of protein structure and function to temperature: there is more than one way to ‘skin a cat’. J Exp Biol 218(12):1801–1811. doi: 10.1242/jeb.114298 CrossRefPubMedGoogle Scholar
  29. Fornbacke M, Clarsund M (2013) Cold-adapted proteases as an emerging class of therapeutics. Infect Dis Ther 2(1):15–26. doi: 10.1007/s40121-013-0002-x CrossRefPubMedPubMedCentralGoogle Scholar
  30. Gerday C (2013) Psychrophily and catalysis. Biology 2(2):719–741. doi: 10.3390/biology2020719 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Gerday C (2014) Fundamentals of cold-active enzymes. In: Buzzini P, Margesin R (eds) Cold-adapted yeasts: biodiversity, adaptation strategies and biotechnological significance. Springer, Berlin, pp 325–350. doi: 10.1007/978-3-642-39681-6_15 CrossRefGoogle Scholar
  32. Ghosh M, Pulicherla KK, Rekha VP, Raja PK, Sambasiva Rao KR (2012) Cold active beta-galactosidase from Thalassospira sp. 3SC-21 to use in milk lactose hydrolysis: a novel source from deep waters of Bay-of-Bengal. World J Microbiol Biotechnol 28(9):2859–2869. doi: 10.1007/s11274-012-1097-z CrossRefPubMedGoogle Scholar
  33. Gohel V, Duan G (2012) No-cook process for ethanol production using indian broken rice and pearl millet. Int J Microbiol 2012:680232. doi: 10.1155/2012/680232 CrossRefPubMedPubMedCentralGoogle Scholar
  34. He H, Chen X, Li J, Zhang Y, Gao P (2004) Taste improvement of refrigerated meat treated with cold-adapted protease. Food Chem 84(2):307–311. doi: 10.1016/S0308-8146(03)00242-5 CrossRefGoogle Scholar
  35. Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F, Francois JM, Baise E, Feller G, Gerday C (2001) Cold-adapted beta-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis. Appl Environ Microbiol 67(4):1529–1535CrossRefPubMedPubMedCentralGoogle Scholar
  36. Huston AL (2008) Biotechnological aspects of cold-adapted enzymes. In: Margesin R, Schinner F, Marx JC, Gerday C (eds) Psychrophiles: from biodiversity to biotechnology. Springer, Berlin, pp 347–364CrossRefGoogle Scholar
  37. Ji L, Yang J, Fan H, Yang Y, Li B, Yu X, Zhu N, Yuan H (2014) Synergy of crude enzyme cocktail from cold-adapted Cladosporium cladosporioides Ch2-2 with commercial xylanase achieving high sugars yield at low cost. Biotechnol Biofuels 7(1):130. doi: 10.1186/s13068-014-0130-x CrossRefPubMedPubMedCentralGoogle Scholar
  38. Junpei Z, Rui Z, Zunxi H, Zhifeng S, Xianghua T, Junjun L, Qian W (2016) Low-temperature salt-tolerant product-inhibition-resistant beta-N-acetyl glucosamine enzyme JB10NagA. Patent Application CN105483101AGoogle Scholar
  39. Karan R, Capes MD, Dassarma S (2012) Function and biotechnology of extremophilic enzymes in low water activity. Aquat Biol 8(1):4. doi: 10.1186/2046-9063-8-4 CrossRefGoogle Scholar
  40. Karasova-Lipovova P, Strnad H, Spiwok V, Mala S, Kralova B, Russell NJ (2003) The cloning, purification and characterisation of a cold-active [beta]-galactosidase from the psychrotolerant Antarctic bacterium Arthrobacter sp. C2-2. Enzym Microb Technol 33(6):836–844CrossRefGoogle Scholar
  41. Kirk O, Christensen MW (2002) Lipases from Candida antarctica: unique biocatalysts from a unique origin. Org Process Res Dev 6(4):446–451. doi: 10.1021/op0200165 CrossRefGoogle Scholar
  42. Kobori H, Sullivan CW, Shizuya H (1984) Heat-labile alkaline phosphatase from Antarctic bacteria: rapid 5’ end labelling of nucleic acids. Proc Natl Acad Sci USA 81:6691–6695CrossRefPubMedPubMedCentralGoogle Scholar
  43. Lanes O, Leiros I, Smalas AO, Willassen NP (2002) Identification, cloning, and expression of uracil-DNA glycosylase from Atlantic cod (Gadus morhua): characterization and homology modeling of the cold-active catalytic domain. Extremophiles 6(1):73–86CrossRefPubMedGoogle Scholar
  44. Liszka MJ, Clark ME, Schneider E, Clark DS (2012) Nature versus nurture: developing enzymes that function under extreme conditions. Annu Rev Chem Biomol Eng 3:77–102. doi: 10.1146/annurev-chembioeng-061010-114239 CrossRefPubMedGoogle Scholar
  45. Littlechild JA (2015) Enzymes from extreme environments and their industrial applications. Front Bioeng Biotechnol 3:161. doi: 10.3389/fbioe.2015.00161 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Margesin R, Fauster V, Fonteyne PA (2005) Characterization of cold-active pectate lyases from psychrophilic Mrakia frigida. Lett Appl Microbiol 40(6):453–459CrossRefPubMedGoogle Scholar
  47. Margesin R, Feller G, Gerday C, Russell NJ (2003) Cold-adapted microorganisms: adaptation strategies and biotechnological potential. In: Britton G (ed) Encyclopedia of environmental microbiology. Wiley, New York. doi: 10.1002/0471263397.env150 Google Scholar
  48. Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 40(6):1451–1463. doi: 10.1016/j.enzmictec.2007.01.018 CrossRefGoogle Scholar
  49. Muller-Greven JC, Post MA, Kubu CJ (2012) Recombinant Colwellia psychrerythraea alkaline phosphatase and uses thereof. Granted Patents US8486665B2, US8129168B2Google Scholar
  50. Naga Padma P, Anuradha K, Reddy G (2011) Pectinolytic yeast isolates for cold-active polygalacturonase production. Innov Food Sci Emerg Technol. 12(2):178–181. doi: 10.1016/j.ifset.2011.02.001 CrossRefGoogle Scholar
  51. Nakagawa T, Ikehata R, Myoda T, Miyaji T, Tomizuka N (2007) Overexpression and functional analysis of cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2. Protein Expr Purif 54(2):295–299. doi: 10.1016/j.pep.2007.03.010 CrossRefPubMedGoogle Scholar
  52. Nilsen I, Overbö K, Lanes O (2008) Shrimp alkaline phosphatase. Granted Patents US 7323325B2, DE60130567D1, DE60130567T2, EP1326890B1Google Scholar
  53. Pan X, Tu T, Wang L, Luo H, Ma R, Shi P, Meng K, Yao B (2014) A novel low-temperature-active pectin methylesterase from Penicillium chrysogenum F46 with high efficiency in fruit firming. Food Chem 162:229–234. doi: 10.1016/j.foodchem.2014.04.069 CrossRefPubMedGoogle Scholar
  54. Pawar R, Vasudeo Z, Siddhivinayak B, Govind P (2009) Application of protease isolated from Bacillus sp. 158 in enzymatic cleansing of contact lenses. Biotechnology 8(2):276–280. doi: 10.3923/biotech.2009.276.280 CrossRefGoogle Scholar
  55. Pawlak-Szukalska A, Wanarska M, Popinigis AT, Kur J (2014) A novel cold-active β-d-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp. 32cB—Gene cloning, purification and characterization. Process Biochem 49(12):2122–2133. doi: 10.1016/j.procbio.2014.09.018 CrossRefGoogle Scholar
  56. Rina M, Pozidis C, Mavromatis K, Tzanodaskalaki M, Kokkinidis M, Bouriotis V (2000) Alkaline phosphatase from the Antarctic strain TAB5. Properties and psychrophilic adaptations. Eur J Biochem 267(4):1230–1238CrossRefPubMedGoogle Scholar
  57. Santiago M, Ramírez-Sarmiento CA, Zamora RA, Parra LP (2016) Discovery, molecular mechanisms, and industrial applications of cold-active enzymes. Front Microbiol 7:1408. doi: 10.3389/fmicb.2016.01408 PubMedPubMedCentralGoogle Scholar
  58. Sarmiento F, Peralta R, Blamey JM (2015) Cold and hot extremozymes: industrial relevance and current trends. Front Bioeng Biotechnol 3:148. doi: 10.3389/fbioe.2015.00148 CrossRefPubMedPubMedCentralGoogle Scholar
  59. Schmidt M, Stougaard P (2010) Identification, cloning and expression of a cold-active beta-galactosidase from a novel Arctic bacterium, Alkalilactibacillus ikkense. Environ Technol 31(10):1107–1114. doi: 10.1080/09593331003677872 CrossRefPubMedGoogle Scholar
  60. Shahidi F, Janak Kamil YVA (2001) Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends Food Sci Tech 12(12):435–464. doi: 10.1016/S0924-2244(02)00021-3 CrossRefGoogle Scholar
  61. Shimizu K, Cha J, Stucky GD, Morse DE (1998) Silicatein α: cathepsin L-like protein in sponge biosilica. Proc Natl Acad Sci USA 95(11):6234–6238CrossRefPubMedPubMedCentralGoogle Scholar
  62. Siddiqui KS (2015) Some like it hot, some like it cold: Temperature dependent biotechnological applications and improvements in extremophilic enzymes. Biotechnol Adv 33(8):1912–1922. doi: 10.1016/j.biotechadv.2015.11.001 CrossRefPubMedGoogle Scholar
  63. Stougaard P, Schmidt M (2012) Cold-active beta-galactosidase, a method of producing same and use of such enzyme. Granted Patents US8288143, EP2396403B1, EP2396403B8, CN102361974BGoogle Scholar
  64. Suen W-C, Zhang N, Xiao L, Madison V, Zaks A (2004) Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling. Protein Eng Des Sel 17(2):133–140. doi: 10.1093/protein/gzh017 CrossRefPubMedGoogle Scholar
  65. Sullivan CW, Shizuya H, Kobori H (1988) Heat sensitive bacterial alkaline phosphatase. Granted Patent US4720458Google Scholar
  66. The Freedonia Group (2016) World enzymes industry study with forecasts for 2020 & 2025, Study #3417.
  67. Tsuji M, Yokota Y, Shimohara K, Kudoh S, Hoshino T (2013) An application of wastewater treatment in a cold environment and stable lipase production of Antarctic basidiomycetous yeast Mrakia blollopis. PLoS One 8(3):e59376. doi: 10.1371/journal.pone.0059376 CrossRefPubMedPubMedCentralGoogle Scholar
  68. Tu T, Meng K, Bai Y, Shi P, Luo H, Wang Y, Yang P, Zhang Y, Zhang W, Yao B (2013) High-yield production of a low-temperature-active polygalacturonase for papaya juice clarification. Food Chem 141(3):2974–2981. doi: 10.1016/j.foodchem.2013.05.132 CrossRefPubMedGoogle Scholar
  69. Valentini F, Diamanti A, Palleschi G (2010) New bio-cleaning strategies on porous building materials affected by biodeterioration event. Appl Surf Sci 256(22):6550–6563. doi: 10.1016/j.apsusc.2010.04.046 CrossRefGoogle Scholar
  70. Van de Voorde I, Goiris K, Syryn E, Van den Bussche C, Aerts G (2014) Evaluation of the cold-active Pseudoalteromonas haloplanktis β-galactosidase enzyme for lactose hydrolysis in whey permeate as primary step of D-tagatose production. Process Biochem 49(12):2134–2140. doi: 10.1016/j.procbio.2014.09.010 CrossRefGoogle Scholar
  71. Venugopal V (2016) Enzymes from seafood processing waste and their applications in seafood processing. In: Se-Kwon K, Fidel T (eds) Advances in food and nutrition research, vol 78. Academic, New York, pp 47–69. doi: 10.1016/bs.afnr.2016.06.004 Google Scholar
  72. Wang X, Schloßmacher U, Wiens M, Batel R, Schröder HC, Müller WEG (2012) Silicateins, silicatein interactors and cellular interplay in sponge skeletogenesis: formation of glass fiber-like spicules. FEBS J 279(10):1721–1736. doi: 10.1111/j.1742-4658.2012.08533.x CrossRefPubMedGoogle Scholar
  73. Webster A, May E (2006) Bioremediation of weathered-building stone surfaces. Trends Biotechnol 24(6):255–260. doi: 10.1016/j.tibtech.2006.04.005 CrossRefPubMedGoogle Scholar
  74. Wen J, Ren C, Huang N, Liu Y, Zeng R (2015) Draft genome of bagasse-degrading bacteria Bacillus aryabhattai GZ03 from deep sea water. Mar Genomics 19:13–14. doi: 10.1016/j.margen.2014.11.004 CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Mário Barroca
    • 1
  • Gustavo Santos
    • 1
  • Charles Gerday
    • 2
  • Tony Collins
    • 1
    Email author
  1. 1.Department of Biology, Centre of Molecular and Environmental Biology (CBMA)University of MinhoBragaPortugal
  2. 2.Laboratory of BiochemistryInstitute of Chemistry B6, University of LiègeLiège-Sart TilmanBelgium

Personalised recommendations