Applied Microbiology and Biotechnology

, Volume 96, Issue 6, pp 1517–1526 | Cite as

Purification and characterization of a novel extracellular inulinase from a new yeast species Candida kutaonensis sp. nov. KRF1T

  • Bo Yuan
  • Nan Hu
  • Juan SunEmail author
  • Shi-An WangEmail author
  • Fu-Li Li
Biotechnologically relevant enzymes and proteins


A novel extracellular exoinulinase was purified and characterized from a new yeast strain KRF1T, and the gene encoding the enzyme was successfully cloned. The enzyme was stable at low pH between 3.0 and 6.5. The K m and V max values of the purified enzyme for inulin were 2.3 mg/mL and 4.8 mg/min, respectively. The optimum temperature of the inulinase was 50 °C, and the enzyme remained 78 % of activity at 60 °C for 2 h. The inulinase showed an amino acid sequence identity of 58 % to its closest homolog in Meyerozyma (Pichia) guilliermondii. In the secondary structure, the domain G (VMEVH) of the enzyme contained three unique residues (V, M, and H). Compared with previously reported inulinases, the enzyme from strain KRF1T displayed strong acid resistance, notable thermostability, and high affinity for the substrate of inulin. Based on sequence analysis of the 26S rDNA D1/D2 domain and phenotypic characterization, the yeast strain KRF1T was found to represent a novel anamorphic, ascomycetous yeast species. A complete description of the species is given and the name Candida kutaonensis sp. nov (type strain = KRF1T = AS 2.4027T = CBS 11388T) is proposed.


Candida kutaonensis sp. nov. Conserved motif Inulinase Purification Yeast 



This study was supported by grants from the National Natural Science Foundation of China (NSFC, No. 30900007), Chinese Academy of Sciences (No. KSCX2-EW-J-10), and China Agriculture Research System (No. CARS-35).

Supplementary material

253_2012_4108_MOESM1_ESM.pdf (253 kb)
ESM 1 (PDF 253 kb)


  1. Azhari R, Szlak AM, Ilan E, Sideman S, Lotan N (1989) Purification and characterization of endo- and exo-inulinase. Biotechnol Appl Biochem 11:105–117CrossRefGoogle Scholar
  2. Bai FY, Zhao JH, Takashima M, Jia JH, Boekhout T, Nakase T (2002) Reclassification of the Sporobolomyces roseus and Sporidiobolus pararoseus complexes, with the description of Sporobolomyces phaffii sp nov. Int J Syst Evol Microbiol 52:2309–2314CrossRefGoogle Scholar
  3. Basso A, Spizzo P, Ferrario V, Knapic L, Savko N, Braiuca P, Ebert C, Ricca E, Calabro V, Gardossi L (2010) Endo- and exo-inulinases: enzyme-substrate interaction and rational immobilization. Biotechnol Prog 26:397–405Google Scholar
  4. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  5. Chen HQ, Chen XM, Li Y, Wang J, Jin ZY, Xu XM, Zhao JW, Chen TX, Xie ZJ (2009) Purification and characterisation of exo- and endo-inulinase from Aspergillus ficuum JNSP5-06. Food Chem 115:1206–1212CrossRefGoogle Scholar
  6. de Souza-Motta CM, Cavalcanti MAD, Porto ALF, Moreira KA, de Lima JL (2005) Aspergillus niveus Blochwitz 4128URM: new source for inulinase production. Braz Arch Biol Technol 48:343–350CrossRefGoogle Scholar
  7. Erdal S, Canli O, Algur OF (2011) Inulinase production by Geotrichum candidum using Jerusalem artichoke as sole carbon source. Romanian Biotechnol Lett 16:6378–6383Google Scholar
  8. Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A (2000) Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol 3:1351–1371CrossRefGoogle Scholar
  9. Gill PK, Manhas RK, Singh P (2006a) Comparative analysis of thermostability of extracellular inulinase activity from Aspergillus fumigatus with commercially available (Novozyme) inulinase. Bioresour Technol 97:355–358CrossRefGoogle Scholar
  10. Gill PK, Manhas RK, Singh P (2006b) Hydrolysis of inulin by immobilized thermostable extracellular exoinulinase from Aspergillus fumigatus. J Food Eng 76:369–375CrossRefGoogle Scholar
  11. Gong F, Zhang T, Chi ZM, Sheng J, Li J, Wang XH (2008) Purification and characterization of extracellular inulinase from a marine yeast Pichia guilliermondii and inulin hydrolysis by the purified inulinase. Biotechnol Bioproc Eng 13:533–539CrossRefGoogle Scholar
  12. Goosen C, Van der Maarel MJEC, Dijkhuizen L (2008) Exo-inulinase of Aspergillus niger N402: a hydrolytic enzyme with significant transfructosylating activity. Biocatal Biotransform 26:49–58CrossRefGoogle Scholar
  13. Hellman U, Wernstedt C, Gonez J, Heldin CH (1995) Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem 224:451–455CrossRefGoogle Scholar
  14. Juliana B, Coitinho VMG, de Almeida MN, Falkoski DL, de Queiroz JH, de Rezende ST (2010) Characterization of an exoinulinase produced by Aspergillus terreus CCT 4083 grown on sugar cane bagasse. J Agric Food Chem 58:8386–8391CrossRefGoogle Scholar
  15. Kim KY, Rhee S, Kim SI (2005) Role of the N-terminal domain of endoinulinase from Arthrobacter sp. S37 in regulation of enzyme catalysis. J Biochem Tokyo 138:27–33CrossRefGoogle Scholar
  16. Kurtzman CP (1998) Discussion of teleomorphic and anamorphic ascomycetous yeasts and a key to genera. In: Kurtzman CP, Fell JW (eds) The yeasts, a taxonomic study, 4th edn. Elsevier, Amsterdam, pp 111–121CrossRefGoogle Scholar
  17. Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 73:331–371CrossRefGoogle Scholar
  18. Kushi RT, Monti R, Contiero J (2000) Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus. J Ind Microbiol Biotechnol 25:63–69CrossRefGoogle Scholar
  19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  20. Lim SH, Ryu JM, Lee H, Jeon JH, Sok DE, Choi ES (2011) Ethanol fermentation from Jerusalem artichoke powder using Saccharomyces cerevisiae KCCM50549 without pretreatment for inulin hydrolysis. Bioresour Technol 102:2109–2111CrossRefGoogle Scholar
  21. Lu HZ, Ca YM, Wu ZW, Jia JH, Bai FY (2004) Kazachstania aerobia sp nov., an ascomycetous yeast species from aerobically deteriorating corn silage. Large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol 54:2431–2435CrossRefGoogle Scholar
  22. Makimura K, Murayama SY, Yamaguchi H (1994) Detection of a wide-range of medically important fungi by the polymerase chain-reaction. J Med Microbiol 40:358–364CrossRefGoogle Scholar
  23. Meyer SA, Payne RW, Yarrow D (1998) Candida Berkhout. In: Kurtzman CP, Fell JW (eds) The yeasts, a taxonomic study, 4th edn. Elsevier, Amsterdam, pp 454–573CrossRefGoogle Scholar
  24. Naidoo K, Ayyachamy M, Permaul K, Singh S (2009) Enhanced fructooligosaccharides and inulinase production by a Xanthomonas campestris pv. phaseoli KM 24 mutant. Bioproc Biosystems Eng 32:689–695CrossRefGoogle Scholar
  25. Nakamura T, Kurokawa T, Nakatsu S, Ueda S (1978) Crystallization and general properties of an extracellular inulinase from Aspergillus sp. Nippon Nogeikagaku Kaishi 52:159–166CrossRefGoogle Scholar
  26. Nakase T, Suzuki M (1986) Bullera megalospora, a new species of yeast forming large ballistospores isolated from dead leaves of Oryza sativa. Miscanthus sinensis and Sasa sp. in Japan. J Gen Appl Microbiol 32:225–240CrossRefGoogle Scholar
  27. Nakase T, Jindamorakot S, Imanishi Y, Am-In S, Ninomiya S, Kawasaki H, Limtong S (2010) Candida potacharoeniae sp. nov. and Candida spenceri sp. nov., two novel galactose-containing ascomycetous anamorphic yeast species isolated in Thailand. J Gen Appl Microbiol 56:287–295CrossRefGoogle Scholar
  28. Nguyen QD, Sujto NM, Bujna E, Rezessy-Szabo JM (2010) Production of extracellular inulinase by Thermomyces lanuginosus: optimisation of media compositions and environmental conditions. J Biotechnol 150:S321–S322CrossRefGoogle Scholar
  29. Ohta K, Akimoto H, Matsuda S, Toshimitsu D, Nakamura T (1998) Molecular cloning and sequence analysis of two endoinulinase genes from Aspergillus niger. Biosci Biotech Biochem 62:1731–1738CrossRefGoogle Scholar
  30. Pandey A, Soccol CR, Selvakumar P, Soccol VT, Krieger N, Fontana JD (1999) Recent developments in microbial inulinases—its production, properties, and industrial applications. Appl Biochem Biotechnol 81:35–52CrossRefGoogle Scholar
  31. Pons T, Olmea O, Chinea G, Beldarrain A, Marquez G, Acosta N, Rodriguez L, Valencia A (1998) Structural model for family 32 of glycosyl-hydrolase enzymes. Proteins 33:383–395CrossRefGoogle Scholar
  32. Ruivo CC, Lachance MA, Rosa CA, Bacci M Jr, Pagnocca FC (2005) Candida bromeliacearum sp. nov. and Candida ubatubensis sp. nov., two yeast species isolated from the water tanks of Canistropsis seidelii (Bromeliaceae). Int J Syst Evol Microbiol 55:2213–2217CrossRefGoogle Scholar
  33. Saber WIA, El-Naggar NE (2009) Optimization of fermentation conditions for the biosynthesis of inulinase by the new source; Aspergillus tamarii and hydrolysis of some inulin containing agro-wastes. Biotechnology 8:425–433CrossRefGoogle Scholar
  34. Sciarria TP, Romano S, Correnti A (2010) Use of mixed substrate composed of organic solid wastes and energetic crop plants (Jerusalem artichoke) for the combined production of hydrogen, ethanol and methane in a two-stage fermentation process. J Biotechnol 150:S141–S142CrossRefGoogle Scholar
  35. Sheng J, Chi ZM, Li J, Gao LM, Gong F (2007) Inulinase production by the marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase. Proc Biochem 42:805–811CrossRefGoogle Scholar
  36. Sheng J, Chi ZM, Gong F, Li J (2008) Purification and characterization of extracellular inulinase from a marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the purified inulinase. Appl Biochem Biotechnol 144:111–121CrossRefGoogle Scholar
  37. Singh RS, Sooch BS, Puri M (2007) Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1. Bioresour Technol 98:2518–2525CrossRefGoogle Scholar
  38. Skowronek M, Fiedurek J (2006) Purification and properties of extracellular endoinulinase from Aspergillus niger 20 OSM. Food Technol Biotechnol 44:53–58Google Scholar
  39. Spiro RG (1966) Analysis of sugars found in glycoproteins. Methods Enzymol 8:3–26CrossRefGoogle Scholar
  40. Thanonkeo P, Thanonkeo S, Yamada M (2010) Ethanol production from Jerusalem Artichoke (Helianthus tuberosus L.) by thermotolerant bacterium, Zymomonas mobilis. J Biotechnol 150:S152–S152CrossRefGoogle Scholar
  41. Vandamme EJ, Derycke DG (1983) Microbial inulinases-fermentation process, properties, and applications. Adv Appl Microbiol 29:139–176CrossRefGoogle Scholar
  42. Wang SA, Jia JH, Bai FY (2008) Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants. Antonie Van Leeuwenhoek 94:257–265CrossRefGoogle Scholar
  43. Wang SA, Li FL, Bai FY (2010) Candida laoshanensis sp. nov. and Candida qingdaonensis sp. nov., anamorphic, ascomycetous yeast species isolated from decayed wood. Int J Syst Evol Microbiol 60:1697–1701CrossRefGoogle Scholar
  44. Yarrow D (1998) Methods for the isolation, maintenance and identification of yeasts. In: Kurtzman CP, Fell JW (eds) The yeasts, a taxonomic study, 4th edn. Elsevier, Amsterdam, pp 77–100CrossRefGoogle Scholar
  45. Yuan WJ, Zhao XQ, Ge XM, Bai FW (2008) Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater. J Appl Microbiol 105:2076–2083CrossRefGoogle Scholar
  46. Yuan WJ, Chang BL, Ren JG, Liu JP, Bai FW, Li YY (2011) Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions. J Appl Microbiol 112:38–44CrossRefGoogle Scholar
  47. Zhang T, Gong F, Chi Z, Liu GL, Chi ZM, Sheng J, Li J, Wang XH (2009) Cloning and characterization of the inulinase gene from a marine yeast Pichia guilliermondii and its expression in Pichia pastoris. Antonie Van Leeuwenhoek 95:13–22CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Key Laboratory of Biofuels, Qingdao Institute of BioEnergy and Bioprocess TechnologyChinese Academy of SciencesQingdaoChina
  2. 2.College of Animal Science and TechnologyQingdao Agricultural UniversityQingdaoChina
  3. 3.Graduate School of the Chinese Academy of SciencesBeijingChina

Personalised recommendations