PURIFICATION AND QUALITY EVALUATION OF LINAMARASE

(Β-GLUCOSIDASE) GENETICALLY

¹Ikya, J.K,  ¹Ariahu, C.C.   ²Ayatse,  J.O.I

¹ Department of Food Science and Technology, University of Agriculture, Makurdi

²Federal university Dutsin-Ma

Email: aveyina@yahoo.com

ABSTRACT

Linamarase  (β-glucosidase)  was genetically engineered from genes (chromosomal DNA) and plasmids (circular DNA) isolated from bitter cassava and yeast respectively. Both genes  were restricted and ligated to produce recombinant gene (r-DNA) which was introduced into the nucleus of CaCl₂ induced competent Saccharomyces cerevisiae cells which transformed into strains capable of producing genetically engineered linamarase (GELIN). Recombinant otherwise genetically modified yeast ( S. cerevisiae) cells at the stationary phase of growth were harvested, homogenized and centrifuged to obtain crude extracts designated as GELIN₀. Carboxy methyl cellulose, diethyl amino-ethyl-sephadex and diethyl amino-ethyl-cellulose were used to purify the crude extracts resulting in GELIN₁, GELIN₂ and GELIN₃, respectively and stored under refrigerated conditions before further study and commercial native linamarase (CNLIN) was used as control.  The physico-chemical characteristics of genetically engineered linamarase  from Saccharomyces cerevisiae as influenced severally by degree of purification, pH and temperature were investigated. The parameters on physico-chemical characteristics of the enzyme extracts such as impurity levels, molecular weights (M_wt), number of isoenzyme, sulphur amino acids (methionine and cysteine), purity fold, yield and the electrical charges were evaluated using standard methods. The ability of the enzyme extracts and a commercial native linamarase (CNLIN) to hydrolyse cyanogenic glucosides was challenged to evaluate optimum pH (pH_opt), temperature (T_opt), total activity, specific activity and enzyme efficiency.