Pengoptimuman, penulen dan pencirian enzim mannanase daripada aspergillus niger USM F4 melalui pemfermentasian substret pepejal (SSF) dengan menggunakan isirong kelapa sawit sebagai substrat.
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Date
2009-05
Authors
Ab. Rashid, Syarifah
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Abstract
From the 9 isolates of fungi that were screened, the isolate USM F4 was found capable to
produce higher mannanase activity than the other 8 of fungal isolates. The colony and
microscopic morphologies identification showed that this fungal was very identical to
Aspergillus niger. The production of mannanase by A. niger USM F4 was futher
optimised based on the cultural conditions and fermentation medium in two main
systems; flask and tray systems. The optimisation of cultural conditions and fermentation
medium consisted physical and chemical parameters. The conditions in a 250 ml flask
were 80% (v/w) water ~ontent, particle size of substrate ::::; 0.5 mm, inoculum size of 1 x
10 7 spore/ml, 1 Og of substrate quantity, incubated at 30±2°C, without mixing effect, with
the addition of 2% (w/w) molases and 4% (w/w) ammonium nitrate and with the
cultivation time of 5 days. Under the optimum conditions, the maximum production of
mannanase activity was 433.84 ± 0.40 U/g substrate. There was an increment of about
54% after optimisation compared with before optimisation (281.97 U/g substrate). For
tray system, two sizes were tested those were 27x 19x6 em and 4 Sx40x7 em. For the tray
size of 27x19x6 em, almost the same conditions for a flask system were requested; 0.4
em substrate thickness (100 g substrate quantity), 80% (v/w) water content, particle size
of substrate :S 0.5 mm, inoculum size of 1 x 107 spore/ml, incubated at 30±2°C, without
mixing effect, with the addition of 2% (w/w) molases and 4% (w/w) ammonium nitrate
and with the cultivation time of 5 days. For the other tray size (4Sx40x7 em), the
optimised cultural conditions and fermentation medium were 0.5 em substrate thickness
(400 g substrate quantity), 80% (v/w) water content, particle size of substrate:::; 0.5 mm,
inoculum size of 1 x 107 spore/ml, incubated at 30±2°C, need to mix for every 24 hours,
with the addition of 2% (w/w) molases and 4% (w/w) ammonium nitrate and with the
cultivation time of5 days. A total mannanase production was 701.53 ± 0.82 U/g substrate
(for the tray size of 27x19x6 em) and 918.68 ± 1.59 U/g substrate (for the size of
45x40x7 em). Compared to a flask system, the increment was about 62% (for the tray
size of 27x19x6 em) and 112% (for the tray size of 4Sx40x7 em). A crude mannanase
was purified by ultrafiltration, Superdex 75 gel filtration chromatography and QSepharose
Fast Flow anion exchange chromatography. The purified mannanase was
homogenous on SDS-PAGE. The purified mannanase had a specific activity of 196.42
U/mg, with 0.55% recovery and overall purification of 4-fold. The molecular mass of
purified mannanase estimated by SDS-PAGE was about 47.4 kD. The optimal pH and
temperature for purified and crude mannanase activity was 4.0 and 60°C, respectively.
Both enzymes were stable at pH 4.0 and temperature of 60°C until 30 minutes. Chemicals
and anions such asS~+, Zn2+, Fe3+, Cu2+, Ca2+, Mn2+, Al3+, Na+, K+, M!f+, EDTA, 2-
mercaptoethanol and SDS at the concentration of 1.0 mM inhibited the purified
mannanase activity. Overall, sodium dodecyl sulfate (SDS) became the most inhibitor
component compared to other metal anions and chemicals. Mannanase activity was
highly specific towards locust bean gum (LBG) at the concentration of0.5% (w/v).
Description
Keywords
Pencirian enzim mannanase , Asper illus niger USM F4 , Pemfermentasian pepejal (SSF) , Isirong kelapa sawit