BIOCHEMICAL AND MOLECULAR STUDIES ON β-GLYCOSIDASE FROM SORGHUM (Sorghum bicolor (L) Moench) AND CASSAVA (Manihot esculenta Crantz)
BIOCHEMICAL AND MOLECULAR STUDIES ON β-GLYCOSIDASE FROM SORGHUM (Sorghum bicolor (L) Moench) AND CASSAVA (Manihot esculenta Crantz)
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Date
2012-12
Authors
ADOBE, KWANASHIE
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Abstract
Genes encoding linamarase from cassava root cortex and dhurrinase from sorghum seeds
were amplified using the polymerase chain reaction. Crude extracts of linamarase and
dhurrinase containing most of the intracellular proteins were used to hydrolyze pnitrophenyl-β-D-glucopyranoside
(pNP) and linamarin extracted from cassava cortex. A
gene fragment corresponding to linamarase (1,504 bp) was amplified. The native enzyme
showed an optimum pH of 6.0 for pNP hydrolysis. The optimum temperature observed
from the experimental assay showed that cassava linamarase has maximum activity for
the hydrolysis of pNP at 65°C. The Q10, calculated between 40°C and 50°C was 1.17.
Activation energy (Ea) was found to be 17.32 KJ/mol (4.14 kcal mol-1). The KM for
linamarase was 5.75 x 10-3 µmol l-1 with a kcat value of 3.41 min-1 and Vmax of 1.96 x
10 -2 µmol l-1 min-1. A 1,695 bp fragment corresponding to dhurrinase gene was also
amplified. Dhurrinase enzyme activity was stable at room temperature (25oC) with
optimum temperature of 65oC. Temperature coefficient (Q10) calculated between 40oC
and 50oC gave a value of 2.53. Activation energy (Ea) of 51.06 KJ/mol was also
extrapolated. The pH activity curve put maximal activity for dhurrinase at pH 5.0. The
double reciprocal plot of dhurrinase showed a calculated KM of 1.39 x 10-2 µmol l-1,
Vmax of 1.01 x 10-1 µmol l-1 min-1 and kcat of 7.27 min-1. Initial velocity data clearly
showed that dhurrinase had an index of physiological efficiency (kcat) of 7.27 min-1. Half
life of enzyme activity (using linamarin as substrate) extrapolated from steady state data
favored dhurrinase (288.79 min), linamarase showing a half life of 117.47 min. Our
investigations have shown clearly that dhurrinase holds the capacity to totally detoxify
cassava and its products of cyanogenic principles more effectively than linamarase.
Description
A THESIS REPORT SUBMITTED TO THE POSTGRADUATE SCHOOL,
AHMADU BELLO UNIVERSITY, ZARIA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE MASTER
OF SCIENCE DEGREE IN BIOCHEMISTRY
(M Sc. BIOCHEMISTRY)
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF SCIENCE
AHMADU BELLO UNIVERSITY
ZARIA, NIGERIA.
DECEMBER, 2012
Keywords
BIOCHEMICAL,, MOLECULAR,, STUDIES,, β-GLYCOSIDASE,, SORGHUM,, Sorghum,, bicolor,, Moench,, CASSAVA,, Manihot,, esculenta,, Crantz,