| Summary: | 碩士 === 國立臺灣大學 === 農業化學系 === 81 === A chitinase-producing bacterium strain NTU-FC-1 was isolated
from soil at 45 ℃.It was identified as Bacillus coagulans
accor- ding to the basic morphological and biochemical
characteristics. This strain grew in a very simple medium .
The optimum culture medium was composed of1%soymeal peptone,0.2
% colloidal chitin, 0.03 % KHPO and 10 % wheat bran extract (5g
wheat bran/100 mL HO), with the initial pH adjusted to7.0.It
was cultured at 40℃ with a shaking rate of150 rpm.After24 hour
incubation,the enzyme productivity reached the maximum value
of 0.45 unit of chitinase activity/mL of culture broth.
Chitinase was purified by UF concen- tration,fractionation
with acetone ,adsorption with colloidal chitin,digestion at
50 ℃,and seperation by Fractogel TSK DEAE 650(M)
chromatography. After these steps , the refined chitinase was
obtained. The specific activity of the refined chitinase
creased by 146 folds and the activity recovery was 23.4 %.
The refined chitinase was very stable at 50 ℃ and the
activity was completely maintained for at least 5 days.Three
chitinases were found in the refined enzyme. By preparative
elec- trophoresis,they were seperated as E1, E2 and E3. Their
optimum temperatures were 60 , 70 and 60 ℃ , and optimum pH
were 5, 55∼6 , respectively. The major hydrolysis pro- ducts
of colloidal chitin by these chitinases were all N,N''''-di-
acetyl-chitobiose .The molecular weight for E1 , E2 and E3 were
77,000, 69,200 and 59,000 ,respectively.By Schiff''''s reagent
staining, E1 was found to be a glycoprotein with sugar
content of 14.3 % (w/w). The glycan chain was mainly composed
of galac- turonic acid ( aboout 43.3% ) and glucose ( about
10.5% ). By using ultra-filtration reactor, the optimum
condition for producing N,N''''-diacetylchitobiose and N,N'''',N"-
triacetyl- chitotriose was: enzyme/substrate ratio : 6 unit/100
mL 1.6 % colloidal chitin;flow rate : 7.6 mL/min;and substrate
concen-
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