Optimal immobilization of beta-galactosidase from Pea (PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications.

Response surface methodology (RSM) and centre composite design (CCD) were used to optimize immobilization of beta-galactosidase (BGAL) from Pisum sativum onto two matrices: Sephadex G-75 and chitosan beads. The immobilization efficiency of 75.66% and 75.19% were achieved with Sephadex G-75 and chitosan, respectively. There was broad divergence in physico-chemical properties of Sephadex-PsBGAL and chitosan-PsBGAL. Chitosan-PsBGAL was better suited for industrial application based on its broad pH and temperature optima, higher temperature stability, reusability etc. Sephadex-PsBGAL and chitosan-PsBGAL showed much variation in their catalytic properties with respect to soluble enzyme. About 50% loss in activity of Sephadex-PsBGAL and chitosan-PsBGAL were observed after 12 and 46 days at 4 degrees C, respectively. Chitosan-PsBGAL showed higher rate of lactose hydrolysis present in milk and whey at room temperature and 4 degrees C than Sephadex-PsBGAL. In both cases, lactose of milk whey was hydrolyzed at higher rate than that of milk.

[1]  A. Karshikoff,et al.  The optimization of protein‐solvent interactions: Thermostability and the role of hydrophobic and electrostatic interactions , 1995, Protein science : a publication of the Protein Society.

[2]  Ron Usami,et al.  Development of the enzyme reactor system with an amperometric detection and application to estimation of the incipient stage of spoilage of chicken , 2000 .

[3]  Armin Fiechter,et al.  Advances in Biochemical Engineering , 1971 .

[4]  B. Krajewska.  Chitin and its derivative as supports for immobilization of enzymes , 1991 .

[5]  Perry D. Haaland,et al.  Experimental design in biotechnology , 1989 .

[6]  A. M. Kayastha,et al.  Improved stability of urease upon coupling to alkylamine and arylamine glass and its analytical use , 2006 .

[7]  K. Kalishwaralal,et al.  Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology. , 2008, Bioresource technology.

[8]  Roger A Sheldon,et al.  A new, mild cross‐linking methodology to prepare cross‐linked enzyme aggregates , 2004, Biotechnology and bioengineering.

[9]  G. Junter,et al.  Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics. , 1994, FEMS microbiology letters.

[10]  A. Ault,et al.  An introduction to enzyme kinetics. , 1974, Journal of chemical education.

[11]  Ipsita Roy,et al.  Lactose hydrolysis by Lactozym™ immobilized on cellulose beads in batch and fluidized bed modes , 2003 .

[12]  C. Choi,et al.  A graphical method of determining the Michaelis-Menten constant free of external mass transfer resistance for immobilized enzymes in a packed bed reactor , 1981, Biotechnology Letters.

[13]  B. Krajewska,et al.  Urease immobilized on chitosan membrane: preparation and properties. , 2007, Journal of chemical technology and biotechnology.

[14]  N. C. Price,et al.  Fundamentals of Enzymology: The Cell and Molecular Biology of Catalytic Proteins , 1999 .

[15]  N. Kretchmer.  Lactose and lactase. , 1972, Scientific American.

[16]  Xuemei Li,et al.  Pilot-scale lactose hydrolysis using β-galactosidase immobilized on cotton fabric , 2007 .

[17]  D. Keilin,et al.  Specificity of glucose oxidase (notatin). , 1952, The Biochemical journal.

[18]  P. De Luca,et al.  In vitro studies of the influence of ELF electromagnetic fields on the activity of soluble and insoluble peroxidase , 2003, Bioelectromagnetics.

[19]  A. Tanriseven,et al.  A novel method for the immobilization of β-galactosidase , 2002 .

[20]  Darrell W. Donahue,et al.  Application of chitosan‐entrapped β‐galactosidase in a packed‐bed reactor system , 2004 .

[21]  S. Khare,et al.  Entrapment of proteins by aggregation within sephadex beads , 1991, Applied biochemistry and biotechnology.

[22]  R. Juang,et al.  Stability and catalytic kinetics of acid phosphatase immobilized on composite beads of chitosan and activated clay , 2004 .

[23]  Ju-fang Wang,et al.  Optimization of fermentation media for nitrite oxidizing bacteria using sequential statistical design. , 2008, Bioresource technology.

[24]  Ivar Storrø,et al.  Alginate as immobilization material: III. Diffusional properties , 1992, Biotechnology and bioengineering.

[25]  L. McBean,et al.  Nutritive Value of Dairy Foods , 1988 .

[26]  Byron Horton Webb,et al.  Fundamentals of dairy chemistry , 1974 .

[27]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[28]  E. del Campillo,et al.  Biochemistry of the multiple forms of glycosidases in plants. , 1984, Advances in enzymology and related areas of molecular biology.