• Journal of Microbiology and Biotechnology
• /
• v.27 no.4
• /
• pp.791-807
• /
• 2017

The type II secretion system (T2SS), which transports selected periplasmic proteins across the outer membrane, has rarely been studied in nonpathogens or in organisms classified as Betaproteobacteria. Therefore, we studied Cupriavidus metallidurans (Cme), a facultative chemilithoautotroph. Gel analysis of extracellular proteins revealed no remarkable differences between the wild type and the T2SS mutants. However, enzyme assays revealed that native extracellular alkaline phosphatase is a T2SS substrate, because activity was 10-fold greater for the wild type than a T2SS mutant. In Cme engineered to produce three Ralstonia solanacearum (Rso) exoenzymes, at least 95% of their total activities were extracellular, but unexpectedly high percentages of these exoenzymes remained extracellular in T2SS mutants cultured in rich broth. These conditions appear to permit an alternative secretion process, because neither cell lysis nor periplasmic leakage was observed when Cme produced a Pectobacterium carotovorum exoenzyme, and wild-type Cme cultured in minimal medium secreted 98% of Rso polygalacturonase, but 92% of this exoenzyme remained intracellular in T2SS mutants. We concluded that Cme has a functional T2SS despite lacking any abundant native T2SS substrates. The efficient secretion of three foreign exoenzymes by Cme is remarkable, but so too is the indication of an alternative secretion process in rich culture conditions. When not transiting the T2SS, we suggest that Rso exoenzymes are probably selectively packaged into outer membrane vesicles. Phylogenetic analysis of T2SS proteins supports the existence of at least three T2SS subfamilies, and we propose that Cme, as a representative of the Betaproteobacteria, could become a new useful model system for studying T2SS substrate specificity.

• YAKHAK HOEJI
• /
• v.41 no.2
• /
• pp.212-219
• /
• 1997

Multiple forms of extracellular phospholipase $A_2$ have been detected in human amniotic fluid (HAF). When HAF was subjected to heparin-Sepharose column chromatography, phospholipase $A_2$ activity was detected in both heparin-non binding and binding fraction. The activity of heparin-non binding fraction was further purified by sequential uses of column chromatographies on butyl-Toy-opearl 650M and DEAE-Sephacel. DEAE-Sephacel fraction contained three different phospholipase $A_2$ activities (Peak I, II, III). The molecular weight of DEAE-Sephacel fraction phospholipase $A_2$ determined by SDS-PAGE were about 52KDa (Peak I). Peak II, III required micromolar $Ca^{2+}$ ion for its maximum activity, but Peak I enzyme showed calcium independent phospholipase $A_2$ activity and showed broad range of pH (6.0~10.0) optimum. All these enzymes were not recognized by a monoclonal antibody raised against phospholipase $A_2$ from human synovial fluid. These results suggest that HAF might contain multiple forms of extracellular phospholipase $A_2$, which may neither belong to the 14KDa group II phospholipase $A_2$ family nor cytosolic phospholipase $A_2$.

• Microbiology and Biotechnology Letters
• /
• v.16 no.2
• /
• pp.119-125
• /
• 1988

$\beta$-Glucosidase of Cellulomonas sp. CS1-1 in cellular compartment was localized with cell-bound form while Avicelase and carboxymethylcellulase (CMCase) were appeared with extracellular enzyme. Cell growth on cellulose or CMC minimal broth was increased by glucose addition. $\beta$-Glucosidase production on cellobiose or CMC minimal broth was repressed by the addition of glucose. However, on CMC minimal broth, the enzyme production was specially stimulated by cellobiose addition. $\beta$-Glucosidase production was also induced by CMC, starcth and maltose compared with glycerol, arabinose, xylose and trehalose. From the above results, it was concluded that glucose effect on $\beta$-glucosidase biosynthesis showed catabolite repression, but enzyme production was induced by cellobiose, CMC, and starch, indicating that $\beta$-glucosidase is inducible enzyme. Yeast extract stimulated $\beta$-glucosidase production more than peptone and ammonium sulfate. $\beta$-Glucosidase activity was increased with 50mM MgCl$_2$in 10mM potassium phosphate buffer (pH 7.0). Optimum conditions for enzyme activities were pH 6.0 and 42$^{\circ}C$, Km value of $\beta$-glucosidase for p-nitrophenyl-$\beta$-D-glucosidase was 0.256mM and Ki for $\beta$-D(+)-glucose was 9.0mM.

• Korean Journal of Microbiology
• /
• v.36 no.2
• /
• pp.130-135
• /
• 2000

Decomposition rate of organic matiter in the mud flat of Sunchon Bay was estimated. Physicochemical parameters, cellulose degradation rate. distribution of heterotrophic bacteria, and extracellular enzymatic activities were measured from August 1997 to July 1998. Soil temperatures, water contents, concentration of $PO_4$-P and organic matter were -1-~$30^{\circ}C$, 42.1-53.1%, 0.0779-0.1961 mgig and 1.99-7.64%, respectively. Decomposition rate of cellulose film ranged from 7.7 to 100%imonth, high in summer and low in winter. The number of heterotrophic bacteria ranged from $0.87{\times}10^6 to 3.6{\times}10^7 $CUFsIg dq soil. Enzymatic activities of phosphatase, $\alpha$-D-gluEosidase, $\beta$-D-glucosidase and cellobiohydrolase, which were measured as decomposition rate of methylumbelliferyl(MLiF)-substrate, were 152.23-1779.80 nMIhr, 2.67-202.18 nM/hr, 5.03-258.26 M h r and 3.42-63.07 nM/hr, respectively Cellulose degradaaon rate and extracellular extracellular enzymatic activities were conelated with each other, and showed high correlation coefticiency with soil temperature.

• Journal of Wetlands Research
• /
• v.11 no.1
• /
• pp.123-130
• /
• 2009

Hyporheic zone is an area where hydraulic exchanges occur between surface water and ground water. Such transient area is anticipated to facilitate diverse biogeochemical reactions by providing habitats for various microorganism. However, only a few data are available about microbial properties in hyporheic zone, which would be important in better understanding of biogeochemical reactions in whole streams. The study site is Naesung stream, located in the north Kyoung-Sang Province, of which sediment is sandy with little anthropogenic impacts. Soil samples were collected from a transect placed perpendicular to stream flow. The transect includes upland fringe area dominated by Phragmites japonica, bare soil, and soil adjacent to water. In addition, soil samples were also collected from downwelling and upwelling areas in hyporheic zone within the main channel. Soils were collected from 3 depth in each area, and water content, pH, and DOC were measured. Various microbial properties including extracellular enzyme activities ($\beta$-glucosidase, N-acetylglucosaminidase, phosphatase and arylsulfatase), and microbial community structure using T-RFLP were also determined. The results exhibited a positive correlation between water content and DOC, and between extracellular enzyme activities and DOC. Distinctive patterns were observed in soils adjacent to water and hyporheic zone compared with other soils. Overall results of study provided basic information about microbial properties of hyporheic zone, which appeared to be discernable from other locations in the stream corridor.

• Journal of Microbiology and Biotechnology
• /
• v.8 no.4
• /
• pp.353-360
• /
• 1998

A bacterial strain PN33 producing large amounts of extracellular pectin lyase (PNL, EC 4.2.2.10) was isolated from soil. The isolated bacterium was identified as a strain of Bacillus sp. Production of PNL by the strain was induced only by pectins, with a higher degree of esterification, which had been added to the culture medium as a sole carbon source. The optimal medium for PNL production was determined to consist of 10 g pectin, 2 g yeast extract, 4 g $K_2HPO_4{\cdot}3H_2O$, 0.6 g $MgSO_4$, and 0.11 g $CaCl_2$ per liter (pH 7.0). The PNL activity in the culture supernatant reached the highest level of 132 mU/ml after 32 h cultivation at $37^{\circ}C$ in the optimal medium. The PNL produced was purified to homogeneity by ammonium sulfate fractionation (50~80%), and cation exchange and size exclusion chromatographies. The molecular mass of the enzyme was estimated to be approximately 52 kDa by SDS-PAGE. Almost the same mass was determined by nondenaturing PAGE, indicating that the functional enzyme had a monomeric structure. As expected, the PNL exhibited higher activities on the highly esterified pectins whereas it gave no detectable activity on polygalacturonic acid. The enzyme showed the highest activity at the acidic pH of 6.0, exceptional for a bacterial PNL. Maximum activity was measured at $40^{\circ}C$, although the stability f the purified enzyme was poor at this temperature. alcium (1 mM) was found to activate the PNL activity by $50\%$, and also remarkably increased the thermal stability f the enzyme. Phenylmethylsulfonylfluoride (PMSF) and iethylpyrocarbonate (DEPC) inhibited the PNL activity lmost completely at the concentration of 5 mM. This result ndicates that some serine and histidine residues of the nzyme may play an essential role for catalytic function of he enzyme.

• Mycobiology
• /
• v.41 no.2
• /
• pp.67-72
• /
• 2013

Pathogenic microbes secrete various enzymes with lipolytic activities to facilitate their survival within the host. Lipolytic enzymes include extracellular lipases and phospholipases, and several lines of evidence have suggested that these enzymes contribute to the virulence of pathogenic fungi. Candida albicans and Cryptococcus neoformans are the most commonly isolated human fungal pathogens, and several biochemical and molecular approaches have identified their extracellular lipolytic enzymes. The role of lipases and phospholipases in the virulence of C. albicans has been extensively studied, and these enzymes have been shown to contribute to C. albicans morphological transition, colonization, cytotoxicity, and penetration to the host. While not much is known about the lipases in C. neoformans, the roles of phospholipases in the dissemination of fungal cells in the host and in signaling pathways have been described. Lipolytic enzymes may also influence the survival of the lipophilic cutaneous pathogenic yeast Malassezia species within the host, and an unusually high number of lipase-coding genes may complement the lipid dependency of this fungus. This review briefly describes the current understanding of the lipolytic enzymes in major human fungal pathogens, namely C. albicans, C. neoformans, and Malassezia spp.

• Journal of Ecology and Environment
• /
• v.33 no.3
• /
• pp.261-270
• /
• 2010

Increasing atmospheric $CO_2$ affects the soil carbon cycle by influencing microbial activity and the carbon pool. In this study, the effects of elevated $CO_2$ on extracellular enzyme activities (EEA; ${\beta}$-glucosidase, N-acetylglucosaminidase, aminopeptidase) in salt marsh sediment vegetated with Suaeda japonica were assessed under ambient atmospheric $CO_2$ concentration (380 ppm) or elevated $CO_2$ concentration (760 ppm) conditions. Additionally, the community structure of sulfate-reducing bacteria (SRB) was analyzed via terminal restriction fragments length polymorphism (T-RFLP). Sediment with S. japonica samples were collected from the Hwangsando intertidal flat in May 2005, and placed in small pots (diameter 6 cm, height 10 cm). The pots were incubated for 60 days in a growth chamber under two different $CO_2$ concentration conditions. Sediment samples for all measurements were subdivided into two parts: surface (0-2 cm) and rhizome (4-6 cm) soils. No significant differences were detected in EEA with different $CO_2$ treatments in the surface and rhizome soils. However, the ratio of ${\beta}$-glucosidase activity to N-acetylglucosaminidase activity in rhizome soil was significantly lower (P < 0.01) at 760 ppm $CO_2$ than at 380 ppm $CO_2$, thereby suggesting that the contribution of fungi to the decomposition of soil organic matter might in some cases prove larger than that of bacteria. Community structures of SRB were separated according to different $CO_2$ treatments, suggesting that elevated $CO_2$ may affect the carbon and sulfur cycle in salt marshes.

• Journal of Animal Science and Technology
• /
• v.48 no.1
• /
• pp.77-90
• /
• 2006

The purpose of this study was to isolate cellulolytic and hemicellulolytic anaerobic bacteria inhabiting from gut of ruminants and investigate their hydrolytic enzyme activities. Extracellular CMCase activities of H-strains isolated from the rumen of a Holstein dairy cow were higher than those of D- and DC- strains from the rumen and large intestine of Korean spotted deer. Most isolated bacteria utilized more efficiently Dehority's artificial medium containing starch, glucose and cellobiose (DAS) than those in Dehority's artificial medium containing cellulose only (DAC). The results of biochemical reactions and sugar fermentation indicated that the isolated bacteria belong to one of bacterial strains of Peptostreptococcus spp., Bifidobacterium spp., Prevotela ruminicola/buccae, Clostridium beijer/butyricum and Streptococcus intermedis which are not highly cellulolytic. Activities of Avicelase, xylanase, β-D-glucosidase, α-L-arabinofuranosidase and β-xylosidase of the isolated anaerobic bacteria in DAS were higher than those in DAC. In conclusion, the results indicated the higher enzyme activities of the isolated strains cultured in DAS medium were mainly caused by their specific carbohydrate utilization for enzyme production and growth rate. The highly cellulolytic bacteria were not isolated in the present experiment. Thus further research is required to investigate characteristics of gut bacteria from Korean spotted deer.

• Journal of Life Science
• /
• v.29 no.2
• /
• pp.158-163
• /
• 2019

The purpose of this study was to isolate a new marine agarase-producing bacterium. Agarase can hydrolyze agar and agarose to produce agarooligosaccharides or neoagarooligosaccharides, which possess many physiological functions. Strain DH-3 was isolated from seawater collected from the coast of Yeosu at Jeollanam province, Korea. A 16S rDNA sequence analysis showed this strain to be Persicobacter sp. DH-3. Extracellular agarase was prepared from culture media of Persicobacter sp. DH-3 and used for characterization. Relative activities at 20, 30, 40, 50, 60, and $70^{\circ}C$ were 50, 55, 70, 100, 90, and 50%, respectively. Relative activities at pH 5, 6, 7, and 8 were 75, 100, 90, and 75%, respectively. The enzyme showed maximum activity at $50^{\circ}C$ in a 20 mM Tris-HCl buffer at pH 6. This enzyme could be useful, as agar is in liquid state at $50^{\circ}C$. Agarase activities were maintained at 80% or more for 2 hr at 20, 30, and $40^{\circ}C$. Thin layer chromatography analysis suggested that Persicobacter sp. DH-3 produced extracellular ${\beta}$-agarases as it hydrolyzed agarose to produce neoagarohexaose and neoagarotetraose. In addition, zymogram analysis confirmed that Persicobacter sp. DH-3 produces at least three agar-degrading enzymes with molecular weights of 45, 70, and 140 kDa. Therefore, it is expected that agarases from Persicobacter sp. DH-3 could be used to produce functional neoagarooligosaccharides.