Immobilization of Inulinase on Concanavalin A-Attached Super Macroporous Cryogel for Production of High-Fructose Syrup

In this study, concanavalin A (Con A)-attached poly(ethylene glycol dimethacrylate) [poly(EGDMA)] cryogels were used for immobilization of Aspergillus niger inulinase. For this purposes, the monolithic cryogel column was prepared by radical cryocopolymerization of EGDMA as a monomer and N,N′-methylene bisacrylamide as a crosslinker. Then, Con A was attached by covalent binding onto amino-activated poly(EGDMA) cryogel via glutaraldehyde activation. Characterization of cryogels was performed by FTIR, EDX, and SEM studies. Poly(EGDMA) cryogels were highly porous and pore size was found to be approximately 50–100 μm. Con A-attached poly(EGDMA) cryogels was used in the adsorption of inulinase from aqueous solutions. Adsorption of inulinase on the Con A-attached poly(EGDMA) cryogel was performed in continuous system and the effects of pH, inulinase concentration, and flow rate on adsorption were investigated. The maximum amount of inulinase adsorption was calculated to be 27.85 mg/g cryogel at 1.0 mg/mL inulinase concentration and in acetate buffer at pH 4.0. Immobilized inulinase was effectively used in continuous preparation of high-fructose syrup. Inulin was converted to fructose in a continuous system and released fructose concentration was found to be 0.23 mg/mL at the end of 5 min of hydrolysis. High-fructose content of the syrup was demonstrated by thin layer chromatography.

[1]  Bo Mattiasson,et al.  Affinity cryogel monoliths for screening for optimal separation conditions and chromatographic separation of cells. , 2006, Journal of chromatography. A.

[2]  Jing Li,et al.  Inulinase production by a marine yeast Pichia guilliermondii and inulin hydrolysis by the crude inulinase , 2007, Journal of Industrial Microbiology & Biotechnology.

[3]  R. Romero,et al.  Preparation and Characterization of CaO Nanoparticles/NaX Zeolite Catalysts for the Transesterification of Sunflower Oil , 2011 .

[4]  S. Jain,et al.  Production and Properties of Microbial Inulinases: Recent Advances , 2011 .

[5]  Q. D. Nguyen,et al.  Continuous production of oligofructose syrup from Jerusalem artichoke juice by immobilized endo-inulinase , 2011 .

[6]  J. Cabral,et al.  Stability evaluation of an immobilized enzyme system for inulin hydrolysis , 2007 .

[7]  J. Carlsson,et al.  Preparation of high-density Concanavalin A adsorbent and its use for rapid, high-yield purification of peroxidase from horseradish roots. , 2004, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[8]  A. Denizli,et al.  Biofunctionalization of magnetic poly(glycidyl methacrylate) microspheres with protein A: Characterization and cellular interactions , 2009 .

[9]  U. Reichl,et al.  Lectin-affinity chromatography for downstream processing of MDCK cell culture derived human influenza A viruses. , 2007, Vaccine.

[10]  J. Cabral,et al.  Immobilization of inulinase for sucrose hydrolysis , 2005 .

[11]  Prabhjeet Singh,et al.  Proizvodnja inulinaze: najnovija dostignuca , 2006 .

[12]  A. Tuncel,et al.  Synthesis of uniform, fluorescent poly(glycidyl methacrylate) based particles and their characterization by confocal laser scanning microscopy , 2005 .

[13]  John S. White,et al.  Straight talk about high-fructose corn syrup: what it is and what it ain't. , 2008, The American journal of clinical nutrition.

[14]  L. Fonseca,et al.  From Inulin to Fructose Syrups Using Sol–Gel Immobilized Inulinase , 2011, Applied biochemistry and biotechnology.

[15]  E. Danial,et al.  Novel Carrier of Grafted Alginate for Covalent Immobilization of Inulinase , 2009 .

[16]  A. Place,et al.  Genetic variation and relative catalytic efficiencies: lactate dehydrogenase B allozymes of Fundulus heteroclitus. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Mazutti,et al.  Immobilization of inulinase obtained by solid-state fermentation using spray-drying technology , 2012 .

[18]  A. Denizli,et al.  Concanavalin A immobilized poly(ethylene glycol dimethacrylate) based affinity cryogel matrix and usability of invertase immobilization. , 2012, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[19]  A. Vaidya,et al.  Immobilization of inulinase from Aspergillus niger NCIM 945 on chitosan and its application in continuous inulin hydrolysis , 2013 .

[20]  M. Puri,et al.  Production of high fructose syrup from Asparagus inulin using immobilized exoinulinase from Kluyveromyces marxianus YS-1 , 2007, Journal of Industrial Microbiology & Biotechnology.

[21]  Adsorption of inulinases in ion-exchange columns , 2000, Applied biochemistry and biotechnology.

[22]  P. Singh,et al.  Hydrolysis of inulin by immobilized thermostable extracellular exoinulinase from Aspergillus fumigatus , 2006 .

[23]  P. Bernfeld,et al.  [17] Amylases, α and β , 1955 .

[24]  H. Treichel,et al.  Evaluation of enzymatic activity of commercial inulinase from Aspergillus niger immobilized in polyurethane foam , 2013 .

[25]  J. Kong,et al.  Fourier transform infrared spectroscopic analysis of protein secondary structures. , 2007, Acta biochimica et biophysica Sinica.

[26]  Guanglei Liu,et al.  Inulinase-expressing microorganisms and applications of inulinases , 2009, Applied Microbiology and Biotechnology.

[27]  R. Monti,et al.  Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus , 2008 .

[28]  B. Mattiasson,et al.  Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate-containing fluids and cell culture applications. , 2007, Journal of separation science.

[29]  R. Monti,et al.  Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus , 2000, Journal of Industrial Microbiology and Biotechnology.

[30]  M. Holyavka,et al.  Inulinase Immobilization on Macroporous Anion-Exchange Resins by Different Methods , 2009, Bulletin of Experimental Biology and Medicine.

[31]  A. Denizli,et al.  Binding of antibodies to concanavalin A-modified monolithic cryogel , 2006 .

[32]  Y. Chisti,et al.  Production of fructose from inulin using mixed inulinases from Aspergillus niger and Candida guilliermondii , 2007 .

[33]  Débora Oliveira,et al.  Immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571 using modified sodium alginate beads , 2012, Bioprocess and Biosystems Engineering.