Characterization of chitosanase of bacillus licheniformis mb-2 isolated from manado hot spring water

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EKOWATI CHASANAH. Characterization of Chitosanase of Bacillus
licheni,formis MB-2Isolated from Manado Hot Spring Water. Under direction of
MAGGY THENAWIDJAYA SUHARTONO, PURWIYATNO HARIYADI, and
ARIEF BUD1 WITARTO
Chitosanase (EC 3.2.1.132) is an important enzyme in the production of
chitosan oligomers. This research dealt with chitosanase of Indonesian origin,
which was produced by chitinolytic bacteria isolated from Manado hot spring
water, referred to as B. licheniformis MB-2.Specifically, the aims of the research
were: (1) to produce and purify chitosanase from the bacteria, (2) analyze

biochemical characteristics of the enzyme, (3) classify the enzyme based on
biochemicd and preliminary bioinfmatics molecular study. The result showed
that the optimum media for producing the enzyme were 0.24% chitosan, 0.25%
casiton, 1% MgS04, 1.4% K2HP04, 0.02% CaC12.2H20,0.002% FeS04.7H20.
Enzyme harvested at the third day had an activity of 0.7 UlmL, while that
harvested at the seventh day showed an activity of 0.8 UlmL. Purification through
the HIC system (Butyl Sepharose 4 FF) resulted in 2 major active fractions, i.e.,
FI and F2. The SDS-PAGE and zymogram analysis applied on these fractions
showed that Fi fraction had 2 chitosanases with molscular weight of 70.7 and 55.9
kDa while F2 showed a single band with approximate molecular weight of 75
kDa.Further purification of F1using Gel Filtration system with Sephadex G-75
matrix failed to separate the isozymes. Cl~aracterizationperformed following the
purification showed that Fz chitosanase functioned optimally at 70°C and pH 6-7.
When incubated at 70 O C , 80 O C and 90°C, the tin values of this enzyme were
26.56 rnin., 18.44 min., and 16.74 min., respectively and the k values were
0.026/min., 0.037/min, and 0.04/min. Deactivation energy was found to be 5720.5
kallmol°K. Stability tests showed that the enzyme's activity was increased by the
addition of 1 m M ~ n at~high
' temperature incubation (80 OC and 90°C). The
chitosanase activity decreased insignificantly by the presence of 10% etanol and

reduced substantially by 10% propanol. It was also found that the enzyme's
stability was unaffected by the presence of 1 M NaCl, 6M urea but decreased by
2M Guanidine Hydrochloride. This enzyme does not degrade colloidal and glycol
chitins, but hydrolyzes glycol chitosan up to 0.8% and colloidal chitosan up to
11%. It was found that 85% deacetylated (DDA) soluble chitosan was most
susceptible to this enzyme, followed by 90% and 100% DDA soluble chitosan.
The K, values of the loo%, 90%, and 85% DDA soluble chitosans were found as
0.58, 0.24, and 0.23 mg/ml, while the,V
values were 261, 668 and 843 Ulmg,
respectively. The hydrolysis products of F2chitomase at 24 h incubation (70°C)
were pentamer and hexamer, indicating that F2chitosanase has a good prospect for
producing biofunctional chitooligosacc~des. PCR cloning based on
bioinfomatics- molecular study, in addition to biochemical characteristics,
showed that the enzyme might not of the Family 8 and 46 of Glycosyl Hydrolase.

Keywordr :thermophile, chitosunme, characterization