Abstract
To develop a novel yeast whole-cell biocatalyst by yeast surface display technology that can hydrolyze chitin, the chitinaseC gene from Serratia marcescens AS1.1652 strain was cloned and subcloned into the yeast surface display plasmid pYD1, and the recombinant plasmid pYD1/SmchiC was electroporated into Saccharomyces cerevisiae EBY100 cell. Aga2p-SmChiC fusion protein was expressed and anchored on the yeast cell surface by induction with galactose, which was verified by indirect immunofluorescence and Western blotting. The chitinolytic activity of the yeast whole-cell biocatalyst or partially purified enzyme was detected by agar plate clear zone test, SDS-PAGE zymography and dinitrosalicylic acid method. The results showed that the chitinaseC gene from S. marcescens AS1.1652 strain was successfully cloned and expressed on the yeast cell surface, Aga2p-SmChiC fusion protein with molecular weight (67 kDa) was determined. Tests on the effect of temperature and pH on enzyme activity and stability revealed that the yeast whole-cell biocatalyst and partially purified enzyme possessed both thermal stability and activity, and even maintained some activity under acidic and weakly alkaline conditions. The optimum reaction temperature and pH value were set at 52 °C and 5.0, respectively. Yeast surface display technology succeeded in preparing a yeast whole-cell biocatalyst with chitinolytic activity, and the utilization of chitin could benefit from this process of enzyme preparation.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ahmed A, Taha RM, Mohajer S, Elaagib ME, Kim SK (2012) Preparation, properties and biological applications of water soluble chitin oligosaccharides from marine organisms. Rus J Mar Biol 38:351–358
Dahiya N, Tewari R, Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes: a review. Appl Microbiol Biotechnol 71:773–782
Dutta PK, Dutta J, Tripathi V (2004) Chitin and chitosan: chemistry, properties and applications. J Sci Ind Res 63:20–31
Fukuda T, Isogawa D, Takagi M, Kato-Murai M, Kimoto H, Kusaoke H, Ueda M, Suye S (2007) Yeast cell-surface expression of chitosanase from Paenibacillus fukuinensis. Biosci Biotechnol Biochem 71:2845–2847
Guo XF, Kikuchi K, Matahira Y, Sakai K, Ogawa K (2002) Water-soluble chitin of low degree of deacetylation. J Carbohydr Chem 21:149–161
Gutierrez-Roman MI, Dunn MF, Tinoco-Valencia R, Holguin-Melendez F, Huerta-Palacios G, Guillen-Navarro K (2014) Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP). World J Microbiol Biotechnol 30:33–42
Hackel BJ, Wittrup D (2009) Yeast surface display in protein engineering and analysis. In: Lutz S, Bornscheuer UT (eds) Protein engineering handbook, vol 1 & vol 2, 1st edn. Wiley, Weinheim, pp 621–648
Horn SJ, Sørlie M, Vaaje-Kolstad G, Norberg AL, Synstad B, Varum KM (2006) Comparative studies of chitinases A, B and C from Serratia marcescens. Biocatal Biotransform 24:39–53
Inaba C, Higuchi S, Morisaka H, Kuroda K, Ueda M (2010) Synthesis of functional dipeptide carnosine from nonprotected amino acids using carnosinase-displaying yeast cells. Appl Microbiol Biotechnol 86:1895–1902
Khor E (2001) The relevance of chitin. In: Khor E (ed) Chitin: fulfilling a biomaterials promise. Elsevier Science Ltd, Oxford, pp 1–8
Kuroda K, Ueda M (2011) Cell surface engineering of yeast for applications in white biotechnology. Biotechnol Lett 33:1–9
Liau CY, Lin CS (2008) A modified coomassie brilliant blue G 250 staining method for the detection of chitinase activity and molecular weight after polyacrylamide gel electrophoresis. J Biosci Bioeng 106:111–113
Littlechild JA, Guy J, Connelly S, Mallett L, Waddell S, Rye CA, Line K, Isupov M (2007) Natural methods of protein stabilization: thermostable biocatalysts. Biochem Soc Trans 35:1558–1563
Sezen K, Kati H, Nalcacioĝlu R, Muratoĝlu H, Demirbaĝ Z (2008) Identification and pathogenicity of bacteria from European shot-hole borer, Xyleborus dispar Fabricius (Coleoptera: Scolytidae). Ann Microbiol 58:173–179
Shigechi H, Koh J, Fujita Y, Matsumoto T, Bito Y, Ueda M, Satoh E, Fukuda H, Kondo A (2004) Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase. Appl Environ Microbiol 70:5037–5040
Shigemori T, Nagayama M, Yamada J, Miura N, Yongkiettrakul S, Kuroda K, Katsuragi T, Ueda M (2013) Construction of a convenient system for easily screening inhibitors of mutated influenza virus neuraminidases. FEBS Open Bio 3:484–489
Sitrit Y, Barak Z, Kapulnik Y, Oppenheim AB, Chet I (1993) Expression of Serratia marcescens chitinase gene in Rhizobium meliloti during symbiosis on alfalfa roots. Mol Plant Microbe Interact 6:293–298
Songsiriritthigul C, Lapboonrueng S, Pechsrichuang P, Pesatcha P, Yamabhai M (2010) Expression and characterization of Bacillus licheniformis chitinase (ChiA), suitable for bioconversion of chitin waste. Bioresour Technol 101:4096–4103
Sutrisno A, Ueda M, Abe Y, Nakazawa M, Miyatake K (2004) A chitinase with high activity toward partially N-acetylated chitosan from a new, moderately thermophilic, chitin-degrading bacterium, Ralstonia sp. A-471. Appl Microbiol Biotechnol 63:398–406
Suzuki K, Sugawara N, Suzuki M, Uchiyama T, Katouno F, Nikaidou N, Watanabe T (2002) Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation. Biosci Biotechnol Biochem 66:1075–1083
Synstad B, Vaaje-Kolstad G, Cederkvist FH, Saua SF, Horn SJ, Eijsink VG, Sorlie M (2008) Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method. Biosci Biotechnol Biochem 72:715–723
Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43:777–780
Tsai SL, Goyal G, Chen W (2010) Surface display of a functional minicellulosome by intracellular complementation using a synthetic yeast consortium and its application to cellulose hydrolysis and ethanol production. Appl Environ Microbiol 76:7514–7520
Tu S, Qiu X, Cao L, Han R, Zhang Y, Liu X (2010) Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite. J Invertebr Pathol 104:75–82
Wang Z, Qi Q, Wang PG (2006) Engineering of cyclodextrin glucanotransferase on the cell surface of Saccharomyces cerevisiae for improved cyclodextrin production. Appl Environ Microbiol 72:1873–1877
Acknowledgments
This work was supported by a cooperation project in industry, education and research of Guangdong province and the Ministry of Education of China (No. 2008B090500191).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, X., Jin, X., Lu, X. et al. Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display. World J Microbiol Biotechnol 30, 2577–2585 (2014). https://doi.org/10.1007/s11274-014-1681-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-014-1681-5