| Summary: | 碩士 === 國立中興大學 === 食品科學系 === 86 === AbstractThe importance of the enzyme saccharification of raw
starch has been well recognized from the viewpoints of energy
saving and effective biomass utilization. Previously, we
isolated a bacterium - Cytophaga sp. from soil, that had a great
potential for production of raw starch digesting amylase ( RSDA
). In this study, the rsda gene of Cytophaga sp. was cloned in
phage particles using Lambda ZAP II. A kb DNA fragment of
Cytophaga sp. chromsomal DNA digested with EcoR I was hybridized
with the DIG-labeled PCR probe derived from the N- and an
internal N- terminal amino acid sequence of RSDA. Through a
series of subcloning for locating the rsda-coding region, the
DNA insert was reduced to 3.2 kb and was ligated with pAcUW21.
We designated the recombinant plasmid pARMH10. The expressed
RSDA with a molecular weight of approximately 60 kDa, had the
property to hydrolyze raw starch as does the RSDA purified from
Cytophaga sp.. The sequence of 3.2 kb inserted DNA from
subclone was obtained upon ABI BigDye Terminator Cycle
Sequencing. The rsda gene consists of 1,455 bp encoding a
protein of 485 amino acid residues, which have a characteristic
signal peptide of 19 amino acid residues upstream of its N-
terminus. A typical promoter sequence and Shine-Dalgarno ( S.
D. ) region were located upstream of the initiation codon ATG.
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