• Title, Summary, Keyword: Phospholipase B

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Molecular Cloning of a Putative Gene Encoding Phospholipase B (plbA) from Aspergillus nidulans (사상설 진균 Aspergillus nidulans의 Phospholipase B 유전자(plb A)의 클로링)

  • Hong, Sa-Hyun;Cho, Eun-Min;Song, Seung-Eun;Eom, Chi-Yong
    • Microbiology and Biotechnology Letters
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    • v.36 no.3
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    • pp.189-194
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    • 2008
  • The phospholipase B (PLB) families are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. In this study, we report the putative gene encoding phospholipase B (plbA) containing lipase motifs was cloned for the first time from the filamentous fungus, Aspergillus nidulans. plbA was isolated from A. nidulans genomic DNA library using a PCR-amplified probe, which is designed on the basis of sequence information derived from the conserved lipase regions of various PLBs. The deduced product of plbA is of 626 amino acids. From the assigned sequence, PlbA showed 72% identity with Penicillium notatum PLB but have low similarity with phospholipase A of other organisms.

Molecular Cloning and Characterization of Alkaliphilic Phospholipase B (VFP58) from Vibrio fluvialis

  • AHN SUN HEE;JEONG SEUNG HA;KIM JIN MAN;KIM YOUNG OK;LEE SANG JUN;KONG IN SOO
    • Journal of Microbiology and Biotechnology
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    • v.15 no.2
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    • pp.354-361
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    • 2005
  • Vibrio fluvialis, an enteropathogenic bacterium, produces a phospholipase which is thought to be an important factor in the pathogenesis of disease. In this study, the phospholipase gene (vfp) was identified from V fluvialis (KCTC 2473) and its sequence was determined. The entire open reading frame was composed of 1,689 nuc1eotides and 563 amino acids. The phospholipase gene (vfp) was overexpressed in Escherichia coli as a his-tag fused protein. This recombinant protein (rVFP58) was solubilized with 6 M urea and purified by Ni-NTA affinity chromatography. The action mode of rVFP58 was determined by TLC and GC-MS, and it showed phospholipase B activity, which had both phospholipase A and lysophospholipase activities. The rVFP58 showed a maximum activity at pH around 9- 10 and temperature of about 40OC, and it was stable under alkaline condition over pH 9. The cytotoxicity of rVFP58 was evaluated, using a fish cell line, CHSE-2l4, and was found to cause significant cell death after 14 h of exposure to 250 $\mu$g of the protein.

Bacillus cereus에 의한 Phospholipase C (PLC) 생산

  • Seo, Guk-Hwa;Lee, Jong-Il;Bornscheuer, Uwe T.
    • 한국생물공학회:학술대회논문집
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    • pp.232-234
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    • 2002
  • Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. This study focuses on the production of PLC by B. cereus and recombinant E. coli with fusion protein gene (plc::gfp). Fermentation processes have been monitored by a 2-dimensional fluorescence sensor.

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Screenin of Phospholipase D Producing Actinomycetes (방선균으로부터 Phospholipase D 생산균주의 탐색)

  • 손동화;심재용;윤석후
    • Microbiology and Biotechnology Letters
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    • v.22 no.4
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    • pp.333-339
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    • 1994
  • In order to screen microorganisms producing phospholipase D (PLD) [EC 3.1.4.4], culture broths of about 900 strains of soil bacteria were subjected to examine for the PLD activity. When the hydrolytic activity of PLD (H-activity) in the supernatant was determined, 64 strains produced PLD more than 0.3 unit/ml and all of them were actinomycetes. Among 26 culture broths tested, 6 ones had transphosphatidylation activity (T-activity) of 30~68%. When the strains except one were cultivated on 3 different media at 30$\circ$C for 3 days under aerobic condition, strain # 1090 on medium B (yeast extract 1%, peptone 1%, glucose 1.5%, glycerol 1%, CaCO$_{3}$ 0.4%, and pH 7.2) produced PLD with much higher H- and T-activity, which were 8.3 units/ml and 76.3%, respectively. Subsequently, time course of PLD production of the strain # 1090 during cultivation with aeration of 1 v/v/m and agitation of 400 rpm at 30$\circ$C for 5 days on medium B in jar fermentor was investigated. H-activty of PLD reached almost maximum (about 9 units/ml) after 32 hours and maximal T-activity was found to be about 80%.

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Effects of Brazilin on the Phospholipase $A_2$ Activity and Changes on Intracellular Free Calcium Concentration in Rat Platelets

  • Hwang, Gwi-Seo;Kim, Ji-Young;Chang, Tong-Shin;Jeon, Sun-Duck;So, Dhong-Su;Moon, Chang-Kiu
    • Archives of Pharmacal Research
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    • v.21 no.6
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    • pp.774-778
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    • 1998
  • Brazilin [7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10(6H)-tetrol] inhibited thrombin-, collagen- and ADP-induced aggregation of washed rat platelets. T hrombin- and collagen-induced ATP release were also inhibited by brazilin in a concentration-dependent manner. Brazilin inhibited the formation of platelet thromboxane $A_2$ caused by thrombin, whereas it had no effect on the prostaglandin $D_2$ formation. Brazilin inhibited $^3H$-arachidonic acid liberation from membrane phospholipids of thrombin-stimulated platelets. Brazilin inhibited the rise of intracellular free calcium caused by thrombin. These results indicate that the inhibition of phospholipase ($PLA_2$) activity and [$[Ca^{2+}]_1$ elevation might be at least a part of antiplatelet mechanism of brazilin.

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Isolation and Characterization of MT2617-2B, a Phospholipase C Inhibitor Produced by an Actinomycetes Isolate (방선균 분리주가 생산하는 Phospholipase C 저해물질인 MT-2617-2B의 분리 및 특성)

  • Ko, Hack-Ryong;Lee, Hyun-Sun;Oh, Won-Keun;Ahn, Soon-Cheol;Kim, Bo-Yeon;Kang, Dae-Ook;Mheen, Tae-Ick;Ahn, Jong-Seog
    • Microbiology and Biotechnology Letters
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    • v.24 no.1
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    • pp.19-26
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    • 1996
  • A phospholipase C (PLC) inhibitor (MT267-2B) was isolated from the culture broth of actinomycetes isolate MT2617-2 by the extraction with n-butanol and column chromatographic techniques. The molecular weight of the inhibitor was 1057, by the spectroscopic analyses of IR, $^{13}C$-and $^{1}H$-NMR and ESI-MS. The chemical structure of MT2617-2B was found to be a macrolide compound consisted of a hemiketal ring, polyhydroxyl and polymethyl groups, which had a malonate and guanidine group as its side chain. MT2617-2B produced its two isomers having the same molecular weight by standing in methanol solution at room temperature. Therefore, MT2617-2B was identified as copiamycin and niphithricin A, macrolide antibiotics. The values of $IC_{50}$ against PLC-${\gamma}$1 and PLC-${\beta}$1 were 25 and 50${\mu}$g/ml, respectively. MT2617-2B had antimicrobial activities against Staphylococcus aureus and Candida albicans, but not against Escherichia coli.

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Production and Characterization of Extracellular Phospholipase D from Streptomyces sp. YU100

  • Lim, Si-Kyu;Choi, Jae-Woong;Chung, Min-Ho;Lee, Eun-Tae;Khang, Yong-Ho;Kim, Sang-Dal;Nam, Doo-Hyun
    • Journal of Microbiology and Biotechnology
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    • v.12 no.2
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    • pp.189-195
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    • 2002
  • Using Streptomyces sp. YU100 isolated from Korean soil, the fermentative production of phospholipase D was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, glucose and yeast extract were found to be the best. By varying the concentration of nutrients and calcium carbonate, the optimal culture medium was determined as 2.0% glucose, 1.5% yeast extract, 0.5% tryptone 0.3% calcium carbonate. During cultivation, the strain secreted most of the phospholipase D in the early stage of growth within 24 h. The phospholipase D produced in the culture broth exhibited hydrolytic activity as well as transphosphatidylation activity on lecithin (phosphatidylcholine). In particular, the culture broth showed 8.7 units/ml of hydrolytic activity when cultivated at $28^{\circ}C$ for 1.5 days. The phospholipase D was purified using 80% ammonium sulfate precipitation and DEAE-Sepharose CL-6B column chromatography, which produced a major band of 57 kDa on a 10% SDS-polyacrylamide gel with purity higher than 80%. The enzyme showed an optimal pH of 7 in hydrolytic reaction, and at pH 4 in a transphosphatidylation reaction. The enzyme activity increased until the reaction temperature was elevated to $60^{\circ}C$. The enzyme was relatively stable at high temperatures and neutral pH, but significantly unstable in the alkaline range. Among the detergents tested as emulsifiers of phospholipids, the highest enzyme activity was observed when 1.5% Triton X-100 was employed. However, no inhibitory effect by metal ions was detected. Under optimized reaction conditions, the purified enzyme not only completely decomposed PC to phosphatidic acid within 1 h, but also exhibited higher than 80% conversion rate of PC to PS by transphosphatidylation within 4 h.

Tissue Type Expression of Phospholipase C β3 in Olive Flounder (Paralichthys olivaceus) Following Various Stimulation (다양한 자극에 의한 넙치의 Phospholipase C β3 조직별 발현 분석)

  • WOO, Soo-Ji;LEE, Hyung-Ho;CHUNG, Joon-Ki
    • Journal of Fisheries and Marine Sciences Education
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    • v.28 no.5
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    • pp.1266-1272
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    • 2016
  • Phospholipase C is a key enzyme of signaling pathways hydrolyzed phosphatidylinositol 4,5-bisphosphate to generate 2 second messengers. Among the PLC, $PLC-{\beta}$ subfamily consisted of 4 isoforms, $PLC-{\beta}$ 1~4. Here, we studied the tissue specific expression of $PLC-{\beta}3$ in olive flounder (Paralichthys olivaceus) following external stimulation like lipopolysaccharide (LPS), concanavalin A (ConA) and environmental stress compared with the inflammatory cytokines IL-1b. $PoPLC-{\beta}3$ gene transcripts has the effect in stimulated tissue compared to control. These results provide what we sure to be a important role for $PLC-{\beta}3$ activity in tissue and verify $PLC-{\beta}3$ as potential immune enzyme for signal transduction.

Glucosylsphingosine Activates Serotonin Receptor 2a and 2b: Implication of a Novel Itch Signaling Pathway

  • Afzal, Ramsha;Shim, Won-Sik
    • Biomolecules & Therapeutics
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    • v.25 no.5
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    • pp.497-503
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    • 2017
  • Recent reports claimed that glucosylsphingosine (GS) is highly accumulated and specifically evoking itch-scratch responses in the skins of atopic dermatitis (AD) patients. However, it was unclear how GS can trigger itch-scratch responses, since there were no known molecular singling pathways revealed yet. In the present study, it was verified for the first time that GS can activate mouse serotonin receptor 2a (mHtr2a) and 2b (mHtr2b), but not 2c (mHtr2c) that are expressed in HEK293T cells. Specifically, effects of GS on all mouse serotonin receptor 2 subfamily were evaluated by calcium imaging techniques. The GS-induced intracellular calcium increase was dose-dependent, and antagonists such as ketanserin (Htr2a antagonist) and RS-127445 (Htr2b antagonist) significantly blocked the GS-induced responses. Moreover, the proposed GS-induced responses appear to be mediated by phospholipase C (PLC), since pretreatment of a PLC inhibitor U-73122 abolished the GS-induced responses. Additionally, the GS-induced calcium influx is probably mediated by endogenous TRPC ion channels in HEK293T cells, since pretreatment of SKF-96365, an inhibitor for TRPC, significantly suppressed GS-induced response. In conclusion, the present study revealed for the first time that GS can stimulate mHtr2a and mHtr2b to induce calcium influx, by utilizing PLC-dependent pathway afterwards. Considering that GS is regarded as a pruritogen in AD, the present study implicates a novel GS-induced itch signaling pathway.

Study on recombinant expression of Phospholipase C gene (plc) in methylotrophic yeast Pichia pastoris and its properties

  • Seo, Kook-Hwa;Rhee, Jong-Il
    • 한국생물공학회:학술대회논문집
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    • pp.191-194
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    • 2003
  • The phospholipase C (PLC) hydrolyzes the polar head groups such as phosphocholine or phosphoethanolamine residues esterified at the sn-3 position of phospholipids. Pichia pastoris can utilize methanol as a carbon source and produce recombinant proteins under the control of the strong, tightly-regulated alcohol oxidase (AOX) promoter. In this study, we developed recombinant P. pastoris system for PLC expression and analyzed PLC activity.

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