Fecal microflora of 15 healthy elderly persons with a median age of 84 years in a rural area whose inhabitants tend to be long-lived (Yuzurihara-area, Uenohara, Yamanashi Prefecture) was compared with the microflora of individuals with a median age of 68 years in an urban area (Tokyo). The diet of the elderly persons in the Yuzurihara area is characterized by a high intake of dietary fiber. Total numbers of anaerobic bacteria were significantly smaller in the elderly persons in the Yuzurihara area than those in the Tokyo area. A significantly large number of bifidobacteria, but not of lecithinase-negative clostridia, was observed in the elderly persons in the Yuzurihara area. Large numbers and high incidences of bacilli and lecithinase-positive clostridia (mainly Clostridium perfiringens) were found in the elderly persons in the Tokyo area. Twenty-five genera and over 81 species were isolated from the elderly persons in the Yuzurihara area, and 25 genera and over 92 species were isolated from the elderly persons in the Tokyo area. Furthermore, significantly larger numbers of Bijdobacterium adokscentis and Fusobacterium mortiferum strains were found in the Yuzurihara group, but significant reductions in the Bacteroides buccae-oris group, B. thetaiotaomicron, Bacteroides spp., C. coccoides, C. paraputrificum, and Clostridium spp. were observed in the same group. A significantly higher isolation rate of Bacillus subtilis was observed in the elderly persons in the Tokyo area. The difference in the fecal microflora between elderly persons in Yuzurihara and those in the Tokyo area might be due to a difference in the intake of dietary fiber.

The effects of four-week raffinose intake (15 g/day) on the fecal microflora and fecal properties were studied in healthy human volunteers. The significantly increasing numbers of Bifidobacterium spp. were observed during the raffinose intake, whereas the numbers of lecithinase-negative Clostridium spp. and bacteroidaceae during the intake were significantly lower than those before and after the intake. The percentage of Bifidobacterium spp. was increased from 11.6-15.5% of the total to 58.2-80.1% of the total during the intake. The fecal pH values during the feeding were lower than those before and after the intake.

Alkaliphilic Nocardiopsis sp. strain F96 produced three beta-1,3-glucanase isozymes of different molecular masses (BglF1, BglF2 and BglF3). The N-terminal amino acid sequences of BglFs indicated that these isozymes were the products of a single gene. The beta-1,3-glucanase gene (bglF) was cloned from the chromosomal DNA of strain F96. The bglF gene encoded a polypeptide of 270 amino acids including a signal sequence. The deduced amino acid sequence of mature BglF exhibited the highest homology to those of glycoside hydrolase (GH) family 16 beta-1,3-glucanases, suggesting that the enzyme belonged to the GH family 16. The mature region of bglF gene was functionally expressed in Escherichia coli. The optimum pH and temperature of purified recombinant BglF were pH 9.0 and 70 degrees C, respectively. This enzyme efficiently hydrolyzed insoluble beta-1,3-glucans and showed the highest activity toward a beta-1,3-1,4-glucan rather than beta-1,3-glucans. These results suggested that BglF would be a novel beta-1,3-glucanse. Mutational analysis revealed that Glu123 and Glu128 should be the catalytic residues of BglF.

The effects of brown rice, containing fourfold as much dietary fiber as polished rice, on the human fecal microflora were determined. Significantly increased numbers of Bifidobacterium adolescentis and Enterococcus faecalis were observed during the brown rice intake, whereas the total counts and the numbers of bacteroides, Eubacterium aerofaciens, and Escherichia coli during the intake were lower than those before and after the intake. Lower numbers of clostridia and low incidences of Clostridium paraputrificum and C. perfringens were observed during the brown rice intake.

Most attempts to modify the properties of enzymes by effective in producing the electrostatic field at an activesite of amino acid substitution around the active sites have resulted in the enzyme, the 143rd glutamate, judging from the electrostatic suppression of the biological activity, suggesting that the structure of natural enzymes should be almost optimized evolutionally calculation, indicating that the change of electric charge at this to show the highest activity. In contrast, we found an interesting site may affect the activity [6]. Secondly, the 119th glutaminesite of a well-known metalloendopeptidase, thermolysin (EC.was estimated to play a role in stabilizing the structureby form-3.4.24.4), where almost all the amino acid replacement causes a ing a hydrogen bond with the hydroxyl group of the 103rd remarkable increase in the hydrolytic activity. Negative correlaserine, as already proposed for the neutral protease from Baciltion between the activity and the thermal stability was observed, lus stearothermophilus [7]. The breakage of the hydrogen bond The flexibility around the substrate binding site is suggested to may affect the activity and the thermal stability with changing be a key to the correlation. Nature may have selected the amino the flexibility around the substrate binding site. acid at this site, which suppresses the flexibility of the molecule, It should be noted that the cDNA sequencing [8] and our to get the highest thermal stability at the expense of the activity, re-examination of direct amino acid sequencing of the enzyme Key words: Enzyme stability; Activity; Design principle; [9] have corrected the known sequence by two residues, and Amino acid mutation that the l l9th happens to correspond to one of them. The corrected sequence is identical with that of a neutral protease from Bacillus stearothermophilus [7].

Effects of a high cholesterol (HC) diet and HC diet supplemented with polydextrose on fecal flora, fecal bacterial enzyme activity and fecal putrefactive products in eight healthy volunteers were studied. They were fed a low cholesterol (LC) diet, HC diet, HC diet supplemented with polydextrose (15 g/day) (HCP) at 2-week intervals consecutively for 6 weeks. Polydextrose supplementation increased fecal weight and decreased fecal pH and the stool concentration of indole, p-cresol, iso-valeric acid and iso-butyric acid. A significant reduction of Clostridium in the feces was also observed during the HCP diet period. The frequency of occurrence and number of Clostridium perfringens in the feces were lower for the HCP diet than for the HC diet. Epidemiologic and experimental studies indicate that geographic, urbanrural, and socioeconomic differences exist in the incidence of colon and rectal cancer (4,8,18) and that dietary factors, particularly a high fat intake and a relatively low intake of certain types of dietary fiber, play important roles in their etiology (2,26,31,32). Carcinogens or cocarcinogens are produced in the gut by bacterial action on dietary components or on secretions produced in response to the diet (14). Studies comparing feces of subjects from Scotland, England and the United States (high-risk area for large bowel cancer) to subjects from India, Japan, 53

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