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

JuliusKwagh-harIkya¹, CharlesChukwumaAriahu¹ and JamesOrteseIorzuaAyatse²

¹Department of Food Science and Technology, University of Agriculture, Makurdi, Nigeria
²Federal University Dutsin-Ma, Katsina State, Nigeria.

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 (M_wt), 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 (pH_opt), temperature (T_opt), 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 M_wt (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 pH_opt and T_opt 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.

genetically engineered;linamarase (β-glucosidase);accharomyces cerevisiae;cassava

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