Characterization of Genetically Engineered Linamarase (β-glucosidase) from Saccharomyces cerevisiae

Abstract

The characterization parameters of genetically engineered linamarase (β-glucosidase) from Saccharomyces cerevisiae due to action of the enzyme on linamarin as influenced by degree of purification, pH and temperature were investigated. Commercial native linamarase (CNLIN) was used as control. Linamarase genes (chromosomal DNA) and plasmids (circular DNA) isolated from bitter cassava and yeast respectively were restricted and ligated to produce recombinant genes (r-DNA). The r-DNA were introduced into the nucleus of CaCl2 induced competent Saccharomyces cerevisiae cells which transformed into strains capable of producing genetically engineered linamarase (GELIN). Recombinant S. cerevisiae cells at the stationary phase of growth were recovered, 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. The physical characterization parameters of the enzyme extracts such as impurity levels, molecular weights (Mwt), number of isoenzyme, sulphur amino acids (methionine and cysteine) 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 using linamarin (cassava) as substrates for characterization of activity kinetic profiles such as optimum pH (pHopt), temperature (Topt), total activity, specific activity, purity fold, yield and efficiency ratio. The results indicated that the genetically engineered linamarase(β-glucosidase) consisted of 3 isoenzyme forms. Purification conferred different ionic charges of zero to GELIN0, unit positive charge GELIN1, and unit negative charge to GELIN2 and GELIN3 respectively. Ranges for other parameters were Mwt (22,000-26,000 Daltons), insoluble protein impurity (0.4 -3.5 mg/100g sample) and purity fold (11.5 -1.0) for GELIN3 - GELIN0). Methionine and cystiene varied from 2.0 to 2.6% and 3.0 to 20% respectively (CNLIN - GELIN3). The native commercial enzyme (CNLIN) acted only at pH 6.8 on linamarin with pHopt and Topt of 6.8 and 35 oC respectively. The wide pH tolerance and specific activity towards linamarin degradation suggest a possible use of the genetically engineered linamarase from S. cerevisiae in detoxification of cassava for increased production exportation of cassava-based food products.