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1.
The effect of various physico-chemical factors on production of intra- and extracellular phospholipase A1 bySalmonella newport was investigated. Maximum intracellular enzyme levels were observed when cells were grown in brain heart infusion broth,
after 12 h of incubation at 37°C. Highest level of extracellular phospholipase A1, however, was seen in synthetic medium (pH 7.0) after 24 h of incubation at 37°C. Agitation during incubation had no effect
on the intracellular enzyme synthesis but enhanced extracellular enzyme levels. Addition of surfactants to the growth media
significantly decreased both intra- and extracellular phospholipase A1 production. 相似文献
2.
O. A. Stepnaya L. P. Ryazanova V. I. Krupyanko I. S. Kulaev 《Biochemistry. Biokhimii?a》2001,66(6):662-666
Interactions of a negatively charged exopolysaccharide of Xanthomonas campestris IBPM 124 with its extracellular enzymes (muramidase, endopeptidase, and neutral phosphatase) and also with egg lysozyme, lysostaphin, muramidase of Streptomyces globisporus, and a bacteriolytic enzyme complex of Streptomyces albus were studied. All these enzymes were positively charged under the conditions of their maximal activity. It was shown that interaction of the acidic exopolysaccharide from X. campestris with these enzymes changed their kinetic parameters. The change was either positive (increase in reaction rate) or negative (decrease in reaction rate) and depended on the enzyme and type of substrate cleaved. Due to such interactions, the acidic exopolysaccharide secreted by X. campestris into the environment not only retained and transported positively charged exoenzymes into the near-cellular space, but also regulated their activity. 相似文献
3.
Ann Oaks Ineke Stulen Karen Jones Michael J. Winspear Santosh Misra Ingrid L. Boesel 《Planta》1980,147(5):477-484
The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH4)2SO4 gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed.
New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33 相似文献
4.
Cellular levels of protein and two acid hydrolases, acid phosphatase (EC 3.1.3.2) and acid proteinase, were followed during cyst differentiation, arbitrarily divided into five stages, in the ciliate Histriculus muscorum Kahl. Extracellular enzyme activities were also measured. Protein content decreased gradually during cyst differentiation. In mature cysts the protein content was ca. 60% that of stationary phase organisms. The activities of both acid hydrolases remained unchanged during stage 1 and then decreased gradually; acid proteinase decreased more rapidly. Both enzymes remained slightly active in the mature cysts. The acid proteinase activity of stage 1 was reduced by cycloheximide treatment at time zero, whereas the enzyme was no longer sensitive to the inhibitor when treated at 1.5 h (late stage 1) after the first wash with encysting medium. Acid phosphatase activity was insensitive to the inhibitor. Extracellular release of acid phosphatase increased linearly at least until stage 5, although the extracellular release of acid proteinase was not detected. Cycloheximide blocked the extracellular release of acid phosphatase after stage 1. These results suggest that de novo synthesis of acid proteinase occurs during stage 1 and that lysosomes may play an important role during early stages of cyst differentiation. 相似文献
5.
6.
Mona A. Esawy Yannick Combet-Blanc 《World journal of microbiology & biotechnology》2006,22(3):197-200
Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The
immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated
5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that
of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature
for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting
enzyme. The activation energy (E
A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E
A = 1.59 and 1.99 Kcal mol−1 respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0
for both free and immobilized enzyme. The Michaelis constant K
m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme. 相似文献
7.
The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH4Cl 600 mg l–1) to Regime III (NH4CI 1200 mg l–1), whereas production of protease was highest in Regime II (600 mg l–1 < NH4Cl <1200 mg l–1). 相似文献
8.
Ji-Liang Tang Clare L. Gough Michael J. Daniels 《Molecular & general genetics : MGG》1990,222(1):157-160
Summary A recombinant plasmid pIJ3079 contains DNA sequences from Xanthomonas campestris pv campestris involved in coordinate negative regulation of production of the extracellular enzymes protease, endoglucanase, amylase and polygalacturonate lyase, and extracellular polysaccharide (EPS). Wild-type bacteria harbouring pIJ3079 and therefore carrying extra copies of the gene(s) therein showed reduced enzyme and EPS production and reduced aggresiveness to plants. Localised Tn5 mutagenesis of the corresponding region of the genome gave mutants producing higher levels of enzymes and EPS than the wild type, suggesting that the gene(s) may negatively regulate production in the normal cell. Enzyme and EPS production in the mutants was still dependent on previously characterised positive regulatory genes. 相似文献
9.
Linda Thorne Khoosheh K. Gosink Thomas J. Pollock 《Journal of industrial microbiology & biotechnology》1989,4(2):135-144
Summary Mutants ofXanthomonas campestris B 1459 were isolated that are defective in secretion of both cellulase and amylase. Both enzymes accumulated in the periplasmic space. The defects in secretion of cellulase or amylase were partly overcome by introducing into the mutants specific multiple copies of DNA cloned fromX. campestris, and presumed to code for cellulase or amylase enzymes. The mutant strains also showed reduced amounts of extracellular pectinase and protease activities, as if the mutants were generally defective for secretion of extracellular enzymes. The mutants showed reduced pathogenesis for turnip seedlings. The secretion-defective mutants may allow production of xanthan gum with reduced cellulose, pectin, protein and starch-degrading enzyme activities, thereby allowing more widespread mixing of microbially produced xanthan gum with these commercially important water-soluble polymers. 相似文献
10.
Rate of photosynthesis and activities of photosynthetic carbon reduction cycle enzymes were determined in pods (siliqua),
whereas rate of dark CO2 fixation, oil content and activities of enzymes involved in dark CO2 metabolism were measured in seeds ofBrassica campestris L. cv. Toria at different stages of pod/seed development. The period between 14 and 35 days after anthesis corresponded to
active phase of seed development during which period, seed dry weight and oil content increased sharply. Rate of pod photosynthesis
and activities of photosynthetic carbon reduction cycle enzymes were maximum in younger pods but sufficiently high levels
were retained up to 40 days after anthesis. The rate of dark14CO2 fixation in seeds increased up to 21 days after anthesis and declined thereafter but maintaining sufficiently high rates
till 35 days after anthesis. Similarly various enzymes viz., phosphoenolpyruvate carboxylase, NAD+-malate dehydrogenase and NADP+-malic enzyme, involved in dark CO2 metabolism retained sufficient activities during the above period. These enzyme activities were more than adequate to maintain
the desired supply of malate which mainly arises from dark CO2 fixation in seeds and further translocated to leucoplasts for onward synthesis of fatty acids. Enzyme localization experiments
revealed phosphoenolpyruvate carboxylase and enzymes of sucrose metabolism to be present only in cytosol, whereas enzymes
of glycolysis were present both in cytosolic and leucoplastic fractions. These results indicated that oil synthesis in developingBrassica seeds is supported by pod photosynthesis and dark CO2 fixation in seeds as the former serves as the source of sucrose and the latter as a source of malate 相似文献
11.
Kameshnee Naidoo Manimaran Ayyachamy Kugen Permaul Suren Singh 《Bioprocess and biosystems engineering》2009,32(5):689-695
Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL−1) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL−1 after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using
sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L−1 h−1) and specific (119,025 U g−1 h−1) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L−1 h−1 and specific productivity of 72 g g−1 h−1 FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone.
The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase.
This mutant has potential for large-scale production of inulinase and fructooligosaccharides. 相似文献
12.
Qing-peng Sun Yong-kun Yu Shan-xia Wan Fu-kuan Zhao Yu-lan Hao 《Plant Growth Regulation》2009,57(1):7-13
The changes in cytosolic Ca2+ levels play an important role in the jasmonic acid (JA) signal transduction pathway. We demonstrate that an increase in cytosolic
free Ca2+ concentration ([Ca2+]cyt) of Arabidopsis leaf cells was affected by pretreatment with heparin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8).
With pretreatment of heparin, an antagonist of inositol 1,4,5-trisphosphate (IP3) sensitive channels, the basal and JA induced fluorescence of [Ca2+]cyt were both decreased. Furthermore, heparin and TMB-8, another antagonist of IP3 sensitive channels, enhanced the JA-induced gene expression of JR1. These data suggest that there may be a fine tune control system between extracellular and intracellular Ca2+ mobilization in JA signaling pathway. 相似文献
13.
《Bioscience, biotechnology, and biochemistry》2013,77(9):2010-2013
Three endoglucanase genes (cel5A, cel5B, and cel61A) were cloned from an industrial fungus, Aspergillus kawachii. Yeasts transformed with these cDNAs showed endoglucanase activity in medium. Cel5A and Cel61A contained a type 1 cellulose-binding domain (CBD1) at the C-terminus of the enzyme. The putative catalytic regions of Cel5A and Cel5B showed homology with various endoglucanases belonging glycosyl hydrolase family 5 (GH5). Cel5B showed high homology with Cel5A in catalytic region, but it lacked CBD1 and linker. The cel5A contained four introns, whereas cel5B contained five introns. The putative catalytic region of Cel61A showed homology with enzymes belonging to GH61. The cel61A contained no introns. 相似文献
14.
A bacterium YT–25 which produces enzymes lytic against Pseudomonas aeruginosa was isolated from soil and it was identified as Bacillus subtilis.A1-enzyme, A2-enzyme, B-enzyme and NLF (Native Cell-Lytic Factor) which contribute the lysis of P. aeruginosa were purified from the culture filtrate of strain YT–25.Purified A1-enzyme, A2-enzyme and B-enzyme individually lysed the vegetative cells of P. aeruginosa in the presence of NLF.NLF is a low molecular basic peptide and seemed to alter the sufrace structure of P. aeruginosa.B-enzyme hydrolyzed the peptidoglycan purified from P. aeruginosa to release the reducing groups, but A1-enzyme and A2-enzyme released neither reducing groups nor free amino groups from the peptidoglycan. 相似文献
15.
Gadea Rico‐Pérez Alejandro Pezza M. Graciela Pucciarelli Miguel A. de Pedro Fernando C. Soncini Francisco García‐del Portillo 《Molecular microbiology》2016,99(3):546-556
Bacteria remodel peptidoglycan structure in response to environmental changes. Many enzymes are involved in peptidoglycan metabolism; however, little is known about their responsiveness in a defined environment or the modes they assist bacteria to adapt to new niches. Here, we focused in peptidoglycan enzymes that intracellular bacterial pathogens use inside eukaryotic cells. We identified a peptidoglycan enzyme induced by Salmonella enterica serovar Typhimurium in fibroblasts and epithelial cells. This enzyme, which shows γ‐D‐glutamyl‐meso‐diaminopimelic acid D,L‐endopeptidase activity, is also produced by the pathogen in media with limited nutrients and in resting conditions. The enzyme, termed EcgA for e ndopeptidase responding to c essation of g rowth’, is encoded in a S. Typhimurium genomic island absent in Escherichia coli. EcgA production is strictly dependent on the virulence regulator PhoP in extra‐ and intracellular environments. Consistent to this regulation, a mutant lacking EcgA is attenuated in the mouse typhoid model. These findings suggest that specialised peptidoglycan enzymes, such as EcgA, might facilitate Salmonella adaptation to the intracellular lifestyle. Moreover, they indicate that readjustment of peptidoglycan metabolism inside the eukaryotic cell is essential for host colonisation. 相似文献
16.
Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH1 and NADP-IDH2 (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH1 while NADP-IDH2 represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH2 was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH1, and as a consequence NADP-IDH2 does not result from a post-translational modification of NADP-IDH1. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH1 is restricted to the cytosol and NADP-IDH2 to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH2 was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA
bovine serum albumin
- DEAE
diethylaminoethyl
- NADP-IDH
NADP-isocitrate dehydrogenase
- NADP-IDH1
cytosolic NADP-IDH
- NADP-IDH2
chloroplastic NADP-IDH 相似文献
17.
【背景】野油菜黄单胞菌(Xanthomonas campestris pv. campestris, Xcc)引起十字花科植物黑腐病,在全球范围内造成经济损失,亟须深入研究其致病机理,开发新的黑腐病防控措施。细菌脂肪酸合成系统不仅为细胞膜合成提供原料,其中间代谢产物还是许多生物活性分子合成的底物,具有重要的生理功能,也是抗菌药物筛选的重要靶标。【目的】研究XccfabZ对扩散信号分子(diffusible signal factor, DSF)类信号产量、致病力、胞外酶、胞外多糖和运动性等方面的影响。【方法】利用报告菌株检测法分析了不同替换突变株的DSF类群体感应信号产量。利用同源重组原理,在DSF类信号高产菌株中获得替换突变株,利用高效液相色谱(highperformanceliquid chromatography, HPLC)法测定DSF类信号产量。利用剪叶法检测替换突变株对寄主植物甘蓝的致病力,并分析了不同菌株的胞外多糖、胞外酶和运动性差异。【结果】报告菌株检测法和HPLC法都证明大肠杆菌fabZ替换突变株(XccΔfabZ/pSRK-EcfabZ)中DSF类信号产量显著下降。... 相似文献
18.
J.F. Evans P. Dupuis A.W. Ford-Hutchinson 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,840(1):43-50
Leukotriene A4 hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A4 to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B4 was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent Km for leukotriene A4 between 2 · 10?5 and 3 · 10?5 M. The purified leukotriene A4 hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A4 hydrolase from previously purified epoxide hydrolases. 相似文献
19.
M. R. Sharipova E. V. Shakirov N. P. Balaban L. A. Gabdrakhmanova M. A. Shilova G. N. Rudenskaya I. B. Leshchinskaya 《Microbiology》2000,69(5):552-558
The activities of proteinases in the culture fluid and cellular fractions ofBacillus intermedius 3–19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total
pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during
active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in
the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium.
The production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2-to 4-fold
on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound
proteins. The addition of C0Cl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release
of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm.
The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted. 相似文献
20.
Li XL Skory CD Ximenes EA Jordan DB Dien BS Hughes SR Cotta MA 《Applied microbiology and biotechnology》2007,74(6):1264-1275
The catalytic domain encoded by an adenine–thymine (AT)-rich xylanase gene (xynA) of the anaerobic fungus Orpinomyces was expressed in Hypocrea jecorina under the control of the cel7A promoter and terminator. No XynA protein was detected in H. jecorina culture supernatants when the original sequence was fused to the H. jecorina cel5A region coding for its signal peptide, carbohydrate-binding module, and hinge. Replacing the xynA (56% AT content) with a synthetic sequence containing lower AT content (39%) supported the extracellular production (150 mg
l−1) of the fusion xylanase by H. jecorina. Northern analysis revealed that successful production after the decrease in AT content was related to higher levels of the
xylanase-specific mRNA. Another construct with an RDKR-coding sequence inserted between the cel5A linker and the xynA catalytic domain allowed production of the fully processed active xylanase catalytic domain. Both the fusion (40 kDa) and
the fully processed (28 kDa) forms displayed enzymatic properties of family 11 xylanases. Both the R and the Kex2-like KR
sites were recognized during secretion, resulting in a mixture of two amino termini for the 28-kDa xylanase. The work demonstrated
for the first time that glycoside hydrolases derived from anaerobic fungi can be produced by H. jecorina.
The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture
over other firms or similar products not mentioned. 相似文献