• Journal of Life Science
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• v.17 no.1 s.81
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• pp.6-11
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• 2007

[ $\beta$ ]-Fructofuranosidases, a family 32 of glycoside hydrolases (GH32), share three conserved domains including the W(L/M)(C/N)DP(Q/N), FRDPK, and ECP(D/G) motifs. The functional role of the conserved acidic residues within three domains of levan fructotransferase, one of the $\beta-fructofuranosidases$, from Microbacterium sp. AL-210 was studied by site-directed mutagenesis. Each mutant was overexpressed in E. coli BL21(DE3) and purified by using Hi-Trap chelating affinity chromatography and fast performance liquid chromatography. Substitution of Asp-63 by Ala, Asp-195 by Asn, and Glu-245 by Ala and Asp decreased the enzyme activity by approximately 100-fold compared to the wild-type enzyme. This result indicates that three acidic residues Asp-63, Asp-195, and Glu-245 play a major role in catalysis. Since the three acidic residues are present in a conserved position in inulinase, levanase, levanfructotransferase, and invertase, they are likely to have a common functional role as nucleophile, transition state stabilizer, and general acid in $\beta-fructofuranosidases$.

• Microbiology and Biotechnology Letters
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• v.19 no.2
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• pp.158-162
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• 1991

Endoinulinase was purified from a commercial inulin preparation produced by Aspergillus ficuum using ion exchange chromatography on CM-Sephadex C-50 and DEAE-Sepharose 6B, HPLC gel filtration on a Protein Pak 125 Colum and HPLC ion exchange chromatography on a TSK DEAE-5pw Column. The endoinulinase had a molecular weight of 72,000${\pm}$1,000 and was glycoprotein with 23 to 25% w/w sugar content. The enzyme was much more active on inulin with random cleavage mode than on sucrose and on palatinose: The ration of activity on inulin and sucrose (I/S ratio) was 10~14.

• Microbiology and Biotechnology Letters
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• v.25 no.5
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• pp.448-453
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• 1997

Enzyme activities, production of killer toxin and some functionality of forty seven yeasts isolated from traditional Meju were investigated in culture broth and cell free extracts. Activities of $\alpha$-galactosidase, invertase and inulinase were detected in cell free extracts of 38 strains, 43 strains and 45 strains, respectively and acidic and neutral protease activities also were detected in culture broth of all the strains, $\beta$-Galactosidase activity was detected in cell free extracts of OE-20 and S-14 strains. Killer toxins were produced by OE-12, S-8 (Candida spp.), OE-19 (Zygosaccharomyces spp.) and S-3 (Saccharomyces spp.). Culture broth of OE-23 and S-9 showed 61.3% and 59.2% of antioxidant activity to $\alpha$, $\alpha$-diphenyl-$\beta$-picrylhydrazyl(DPPH), but nitrite-scavenging ability as well as inhibition of tyrosinase and polyphenol oxidase were not appeared in all the strains.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.344-349
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• 2004

A gene encoding inulin-degrading enzyme of Bacillus sp. snu-7 with ORF of 1536 nucleotides was cloned. And it was overexpressed as His-tagged protein in E. coli BL21(DE3) pLysS using pRSET B vector containing mature enzyme sequence. Maximum enzyme production was achieved by IPTG (0.1 mM) induction at $OD_{600}$ 1.2 and $30^{\circ}C$ followed by 6 h incubation. The expressed protein purified through immobilized metal affinity chromatography showed molecular mass of 60 kDa on SDS-PAGE. Results of thin-layer chromatography using inulin as a substrate showed the enzyme to be an exotype inulinase capable of producing only monomeric fructose as a product. $K_m$ and $k_{cat}$, for the hydrolyses of inulin and sucrose were $2.28\pm0.08$ mM and 358.05$\pm$20.38 $min^{-l}$, and 22.02$\pm$0.41 mM and 4619.11$\pm$215.12 $$min^{-1}, respectively. Optimal activity of the exoinulinase occurred at pH 7.0 and $50^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.21 no.1
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• pp.18-22
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• 1993

An assay system by using antibody was adopted to screen the endoinulinase producingfungi due to its high specificity toward endoinulinase, To determine whether the affinity-purified rabbit serum, which were generated against the purified endoinulinase, can react only with the endoinulinase, rocket immunoelectrophoresis was performed. The results showed that the serum specifically reacts with endoinulinase but not with exoinulinase and other proteins in the culture media. Using this polyclonal antibody, a strain from 62 fungal colonies was selected and it secreted an endoinulinase in the culture media to the amount comparable to that of Aspergillus ficuum A Tee 16882 known as a high endoinulinase producer.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.733-738
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• 2007

An extracellular exoinulinase($2,1-\beta-D$ fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable(100%) for 3 h at the optimum temperature of $50^{\circ}C$. $Mn^{2+}\;and\;Ca^{2+}$ produced a 2A-fold and 1.2-fold enhancement in enzyme activity, whereas $Hg^{2+}\;and\;Ag^{2+}$ completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6mg/ml and 41.3mg/ml, respectively.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.101-109
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• 2010

Efficient L-lactic acid production from Jerusalem artichoke tubers, by Lactobacillus casei G-02, using simultaneous saccharification and fermentation (SSF) in a fed-batch culture, is demonstrated. A kinetic analysis of the SSF revealed that the inulinase activity was subjected to product inhibition, whereas the fermentation activity of G-02 was subjected to substrate inhibition. It was also found that the intracellular NADH oxidase (NOX) activity was enhanced by the citrate metabolism, which dramatically increased the carbon flux of the Embden-Meyerhof-Parnas (EMP) pathway, along with the production of ATP. As a result, when the SSF was carried out at $40^{\circ}C$ after an initial hydrolysis of 1 h and included a sodium citrate supplement of 10 g/l, an L-lactic acid concentration of 141.5 g/l was obtained after 30 h, with a volumetric productivity of 4.7 g/l/h. The conversion efficiency and product yield were 93.6% of the theoretical lactic acid yield and 52.4 g lactic acid/l00 g Jerusalem artichoke flour, respectively. Such a high concentration of lactic acid with a high productivity from Jerusalem artichokes has not been reported previously, making G-02 a potential candidate for the economic production of L-lactic acid from Jerusalem artichokes on a commercial scale.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.196-200
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• 1999

A recombinant human $\alpha_{s1}$-casein was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Three different leader sequences derived from the mating factor $\alpha$l (MF$\alpha$l), inulinase, and human $\alpha_{s1}$-casein were used to direct the secretion of human $\alpha_{s1}$-casein into the extracellular medium. Among the three leader sequences tested, the native leader sequence of human $\alpha_{s1}$-casein was found to be the most efficient in the secretory expression of human $\alpha_{s1}$-casein, which implies that the native leader sequence of human $\alpha_{s1}$-casein might be used very efficiently for the secretory production of other heterologous proteins in yeast. The recombinant human $\alpha_{s1}$-casein was proteolytically cleaved as the culture proceeded. Therefore, an attempt was made to produce human $\alpha_{s1}$-casein using a S. cerevisiae mutant in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 72 h of culture, most of the human $\alpha_{s1}$-casein secreted by the wild type was cleaved, whereas more than 70% of the human $\alpha_{s1}$-casein secreted by yap3-disruptant remained intact. The results suggest that YAP3 might be involved in the internal cleavage of human $\alpha_{s1}$-casein expressed in yeast

• Journal of Life Science
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• v.9 no.2
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• pp.231-240
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• 1999

Regular exercise is effective in preventing coronary disease such as angina pectoris and infarction, inside it can lower the blood pressure and aids in weight control and release of stress. Risk factors of arteriosclerosis is hypertension, hyperlipidemia, diabetes, obesity, physical inactivity and excessive smoking. Arteriosclerosis begins at young age worsens with age, particulary in male. For people with risk factors of arteriosclerosis, it is important to prevent arteriosclerosis-related disease with dietary, living pattern and exercise prescription. Dietary fibers promote exercise of the digestive tract and shortens the time food remains inside the digestive tract. It can prevent obesity, hyperlipidemia, arteriosclerosis and colin cancer by blocking the absorption of cholesterol. Various vegetables and sea foods are lichen unsaturated fats and prevent the absorption of cholesterol inside the digestive tract. Essential fatty acids and unsaturated fats which are contained in vegetable oils, promotes metabolism while preventing absorption. In fruits, pectin water-soluble fiber, is present and lowers the level of cholesterol. By consuming foods that low in cholesterol and saturated fats, and rich in unsaturated fats, aliomentotherapy alone can reduce the plasma cholesterol by 10~l5$\%$. For ideal exercise, it should be aerobic with intensity of 60~80$\%$ HRmax, duration of 15~60min/day. The frequancy of 3~6/week is desirable the better exercise prescription is endurance aerobic exercise. To get more effect exercise, exercise consistency is very important.

• Microbiology and Biotechnology Letters
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• v.34 no.3
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• pp.278-287
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• 2006

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cycloinulooligosaccharides (cyclofructan, CF) of ${\beta}-(2{\to}1)$-linked D-fructofuranose as well as hydrolysis of cyclofructan. Sequences analysis indicated that CFTase was divided into five distinct regions containing three repeated sequences (R1, R3, and R4) at the N-terminus and C-terminus. Each domain function was investigated by comparison of wild type CFTase enzyme (CFT148) and deletion mutant proteins (CFT108: R1 and R3 deletion; CFT130: R4 deletion; and CFT88: R1, R3, and R4 deletion) of CFTase. The CFT108 mutant had both CFTase and CF hydrolyzing activity as CFT148 did. CFTase activities and CF hydrolysing activities were disappeared in CFT130 and CFT88 mutants. These results indicated that the C-terminal R4 region of P. polymyxa CFTase is necessary for cyclization and hydrolyzing activity.