PURIFICATION AND QUALITY EVALUATION OF LINAMARASE

(Β-GLUCOSIDASE) GENETICALLY ENGINEERED FROM
SACCHAROMYCES CEREVISIAE

• 1Ikya, J.K. 1Ariahu, C.C. 2Ayatse, J.O.I
  1Department of Food Science and Technology, University of Agriculture, Makurdi
  2Federal University Dutsin-Ma
• Email: aveyina2012@gmail.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-ethylsephadex
  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 (Mwt), 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 (pHopt), temperature (Topt), total activity, specific
  activity and enzyme efficiency. 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 genetically engineered linamarase (β-glucosidase) group acted
  linamarin, lotaustralin, para-nitrophenylglucoside (PNPG), dhurrin, amygdalin, prunasin
  and taxiphyllin at a wide range of pH 1-14 and 25-35 oC each exhibiting highest activity
  at optimum pHopt and Topt of 6.8 and 35 oC The wide pH tolerance at low temperatures
  and specific activity towards cyanogenic glucosides degradation suggest a possible use of
  the genetically engineered linamarase from S. cerevisiae in detoxification capable of
  providing food security from increased production and exportation of plant-based food
  products.