PURIFICATION AND SOME PROPERTIES OF LINAMARASE FROM CASSAVA (Manihot esculenta) CORTEX

Abstract

The enzyme linamarase (E. C. 3.2.1.21) has been purijied from cassava cortex (Manihot caculenta) by techniques involving acetone precipitation, ammonium sulfate fractionation, gel jiltration and ion-exchange chromatography.Two peaks of linamarase activity (A & B ) were eluted by linear gradient on DEAE-Sephadex column. Both forms of the enzyme were found to catalyze similar reactions, however, at different rates. Double reciprocal plots from initial velocity data gave K,,, of I .47 mM & I .44 mM for linamarase A & B, respectively, with linamarin as substrate. The K,,, values with the artijicial substrate paranitrophenyl glucoside (PNP-G) were 0.48 mM. Both enzyme forms exhibited maximal activity at pH 6.0 and 30°C.Arrhenius plots gave activation energy (E,) as 49 for linamarase A & B , respectively. Analysis of cyanide levels in cassava and its products with the crude enzyme showed the leaves to contain high levels of the toxic factor.

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

“…The value for GELIN 0 was not (p< 0.05) higher than 2.42mg/100g of commercial native linamarese (CNLIN). The presence of two active isoenzymes in commercial native linamarse (CNLIN) was earlier reported by Nok and Ikediobi (1999). Later reports by Wither et al (2002) revealled that a good number of isozymes are responsible for the wide spectrum of substrates specificity The molecular weight(Dalton units) of 22,000-26,000 , for GELIN 3 -GELIN0 were not highly significant (p<0.05) different form the molecular weight of carbonic anlydrase of 30,000 daltons but however, significantly different (p> 0.05) from the 63,000 of phosphorylase (b) 94,000 serum albumin 67,000, ovalbumin 43,000 The values for the GELIN were however, higher than trypsin inhibitor (20,000) lacalbumin 14,000 and fragements of myoglobin 17,200, 14 600 , 8,200, 6,380 and 2,5600) making the GELIN 0 -GELIN 3 and CNLIN to be classified as medium molecular weight enzyme fractions.…”

“…The performance of GELIN 3 were significantly (P< 0.05) higher than CNLIN. Table II Nok and Ikediobi (1999) showing that the total activity was in the range of 9-14micromole HCN released per minute.…”

“…The insoluble proteins were estimated as described by Nok & Ikediobi(1999) An automated refrigerated centrifuge with Lowrys based principle was applied in the estimation of insoluble protein impurity.…”

“…Activity kinetic profiles parameters applied for the characterization of the enzyme samples include; total activity, specific activity, purity fold, yield and purification efficiency. Calculation of data from total activities and impurity of enzyme samples are shown mathematically in equations (1-5) as described by Nok and Ikediobi (1999) and Onyike et al, 2001.…”

“…Linamarase (β-glucosidases) is economically very important because of its role as a detoxification agent for cyanogenic glucosides thereby improving food safety. Even though it can be produced from cassava (Manihot esculenta Crantz) tubers, the yields of native linamarase from this source is usually too low (Nok and Ikediobi, 1999). Linamarase from cassava is expensive and it is economically unwise to produce the enzyme from edible tubers.…”