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Facters affecting the production of raw-starch-digeting amylase
of Cytophaga sp.. A Cytophaga sp. isolated from soil had a great
potential for the production of raw starch digesting amylase.
The effects of growth conditions and medium composition on
enzyme production were investigated. The basal medium contained
1 % polypeptone, 0.2 % yeast extract, 0.5 % beef extract and 0.5
% raw corn starch. Enzyme production was increased when the
contents of raw starch was reduced. Enzyme production was also
increased when polypeptone was replaced by polypeptone-S or
defatted soybean. The optimal culture medium consists of 0.5 %
polypeptone-S, 0.2 % yeast extract and 0.2 % raw corn starch at
pH 6.5. The amylase activity in the supernatant of the culture
with raw starch as the one of the carbon source reached maximal
level ( 220 U/ml ) after 24 h incubation at 35℃. Cloning and
sequencing of S-layer protein gene from Cytophaga sp.Protein
compositions of the total cell of Cytophaga sp. were analyzed by
two-dimensional gel electrophoresis. One major protein
constituted about 16 % of the protein contents of the cells as
revealed by Bioimage analysis. It had a molecule mass of 120
kDa determined by SDS-PAGE. The 120 kDa protein was removed from
the cell after treating with 4 M guanidine hydrochloride ( pH
7.4 ) at 37℃ for 5 min. There was also a leaching out of this
protein from the cell during growing. By fractionation the 120
kDa protein was shown to be located in the cell envelope. The
cell surface location of the protein was also confirmed by IgG
colloidal gold labelling of whole cells preadsorbed with 120 kDa
protein antiserum. From above results, the 120 kDa protein was
considered as an S-layer protein of Cytophaga sp..AbstractS-
layer protein is the major protein of Cytophaga sp.. Polyclonal
antibodies against purified S-layer protein were used to screen
aλZAPII library containing chromosomal Cytophaga sp. DNA.
Several phages capable of expressing this S-layer protein were
isolated from Escherichia coli. The nucleotide sequences of
positive clones were determined. The internal N-terminal amino
acid sequence of S-layer protein was consistent with the
sequence from Met-499 to Ala-513 amino acid residues (
MKDGSRKSFPITVTA ) translated from nucleotide sequence of
positive clone. The amino acid sequence of this clone does not
contain N-terminal amino acid sequence of S-layer protein. It
is suggested that the clone is a part of the S-layer protein
gene and lacked the 5'' end sequence. By the inverse PCR and PCR
sequence techniques, the complete nucleotide sequence of S-layer
protein gene was determined. It encoded a protein of 1047 amino
acids. The first 32 amino acids resembled a putative secretion
signal, giving rise to a mature S-layer protein of 1015 amino
acids. The N-terminal amino acid sequence (
ASDATGIYKDAVNYLIEKGI ) of S-layer protein was consistent with
the amino acid sequences translated from the nucleotide
sequences. Amino acid sequence alignment analysis revealed a
level simiarity of ranging from 29 % to 43 % with the S-layer
protein from other bacteria.
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