Pengoptimuman penghasaan dan penulenanenzdd keratinase daripada microsporum gypseum s(f)23 pencilan temp a tan menggunakan fermentasi kultur tenggelam
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Date
2009-07
Authors
Zainul Kamal, Syazni
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Abstract
Chicken feathers which consist 90% keratin are produce as a waste by poultry processing
industries in huge quantities every year. So, this research was developed to produce
keratinase that are able to degrade keratin into protein and amino acid to be use in poultry
dietary. The isolate that was used in the production of keratinase was identified as
Microsporum gypseum. The isolate was isolated using a baiting technique from soil
sample from a horse stable located in Georgetown, Pulau Pinang. Microsporum gypseum
S(F)23 achieved maximum extracellular keratinase activity after 6 days of incubation.
Optimization of cultural conditions and medium compositions showed an increment of
287.6% or 0.903 U/ml compared with the activity before optimization which was 0.233
U/ml. The optimum cultural conditions were agitation speed of 150 rpm, initial pH
medium of 8.0, incubation temperature of 30°C and inoculum size 15% (v/v) l.Oxl05
spores per ml. Meanwhile, optimal medium composition were (w/v) ground chicken
feathers, 1 %; NaCl, 0.05%; KH2P04, 0.04%; K2HP04, 0.03% and peptone, 0.5%.
Keratinase is an inducible enzyme and only produced by Microsporum gypseum S(F)23
culture in the presence of keratin substrate, which are chicken feathers. The presence of
glucose in the culture medium at low concentration inhibits keratinase activity.
Keratinase was purified from the culture broth by ultrafiltration (Amicon Centricon 3
kDa system) and Sephadex G-75 gel filtration chromatography, twice. Peak was purified
about 7.411 fold with a yield of 0.831% and specific activity of 0. 704 U/mg protein. Its
molecular weight was estimated to be around 27 000 Dalton by SDS-P AGE. Some of the
biochemical characteristics were identified. The purified keratinase had the optimum pH
of8.0 and stable at pH range of8.0 to 9.0 and retained 100% activity after 2 hours. While
optimum temperature for purified keratinase was 40°C and stable at temperature range
from 37°C to 40°C and retained more than 80% activity after 2 hours. Furthermore, 1
mM of Ca2+ and Na2+ were found to activate keratinase activity while 1 mM of Mi+,
Zn2+, Hg+, Ba2+, Co2+, K+, Pb2+, Cd2+ and EDTA inhibited keratinase activity. Keratinase
had broad substrate specification and active towards soluble proteins and insoluble
proteins.
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Keywords
Penulenan enzim keratinase , Microsporum gypseum S(F)23 , Menggunakan fermentasi kultur tenggelam