An evaluation of potential interferences in a fluorimetric assay for the rapid detection of thermotolerant coliforms in sewage

C.M. DAVIES and S.C. APTE.2000. A 1-h fluorimetric assay of β- d -galactosidase activity was evaluated for determining thermotolerant coliforms (TTC) in sewage samples. Above TTC concentrations of 2·3 × 10³ colony-forming units (cfu) 100 ml⁻¹, the assay response was related to TTC concentration. However, below this concentration, a large background signal was observed which was independent of TTC concentration. A separation scheme involving various filtration treatments and additions of a β- d -galactosidase inhibitor was devised and used to quantify the sources of this anomalous assay response. The interferences encountered were largely due to the presence in sewage of non-specific cell-free enzymes or other cell-free substances that were capable of hydrolysing the fluorogenic substrate. Despite this apparent limitation, the fluorimetric enzyme assay has potential as an 'early warning' indicator of treatment process failure and gross sewage contamination and leakage in situations where TTC concentrations exceed 2·3 × 10³ cfu 100 ml⁻¹     

A Sensitive Radiometric Assay for Tryptophan Hydroxylase Applicable to Crude Extracts

Abstract: We describe here a simple and convenient method for assay of tryptophan 5-monooxygenase (hydroxylase), applicable to enzyme in all states of purification. It is based on the enzyme-catalysed formation of 5-hydroxy-[4-³H]tryptophanfrom [5-³H]tryptophan, and the subsequent acid-dependent quantitative release of ³H as ³H₂O; unreacted substrate is removed with activated charcoal. The assay is linear with respect to both protein concentration and time, and gives results similar to those in a standard fluorimetric assay.     

Separation and characterization of 61- and 57-kDa lipases from Geotrichum candidum ATCC 66592.

Two lipolytic proteins (61 and 57 kDa) present in a Sephadex G-100 fraction of extracellular lipase from Geotrichum candidum ATCC 66592 were separated using high-performance liquid chromatography. Crossed electrofocusing immunoelectrophoresis was used to demonstrate that the 61-kDa lipase fraction contained two forms of lipase with pI 4.5 and 4.7. However, when deglycosylated with endoglycosidase H, the two forms gained an identical pI, 4.6. The 57-kDa lipase fraction contained one form of lipase with pI close to 4.5. Although the 61- and 57-kDa lipases were immunologically identical, the substrate specificity differed. Thus, the 61-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was 60% of the initial velocity of hydrolysis of oleic acid methyl ester, whereas the 57-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was only 7% of the initial velocity of hydrolysis of oleic acid methyl ester.     

Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.     

cDNA cloning and characterization of Geotrichum candidum lipase II

Geotrichum candidum produces two extracellular lipases, I and II. A lipase II cDNA clone was isolated from a cDNA library by colony hybridization using the 32P-labeled fragment of lipase I cDNA isolated previously. The nucleotide sequence of lipase II cDNA determined by the dideoxy chain terminating method includes the N- and C-terminal amino acid sequences of lipase II, and the overall amino acid composition deduced from the cDNA coincides with that deduced on amino acid analysis of this protein. The cloned lipase II cDNA codes a protein of 544 amino acids and a part of the signal sequence of 13 amino acids. The peptide chain lengths of lipases I and II are the same, their overall identity being 84%. Furthermore, four Cys residues are completely conserved, which may participate in the formation of disulfide bridges. A homology search indicated that the G. candidum lipases and Candida cyclindracea lipase are homologous enzymes and that they are members of the cholinesterase family.     

单因子-响应面法优化白地霉Y162产脂肪酶条件

对白地霉Y162液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适碳源为橄榄油,氮源为黄豆粉和NH4Cl,无机盐为BaCl2和MgCl2。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的橄榄油、BaCl2和NH4Cl三个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,得出橄榄油、BaCl2和NH4Cl最佳浓度分别为2.35％,0.36％,1.35％。优化后液体发酵液中脂肪酶活力提高到31.85 U/mL,比初始酶活力14.16 U/mL提高了2.25倍,表明单因子-响应面结合法可显著优化白地霉Y162液体发酵产脂肪酶条件。     

Fluorimetric Assay of the Activity of Extracellular Lipases of Pseudomonas fluorescens and Serratia marcescens

A fluorimetric assay was carried out on the activity of extracellular lipase concentrations obtained from Serratia marcescens and Pseudomonas fluorescens using as substrates fatty acyl esters of 4-methylumbelliferone (4-methylumbelliferone elaidate, 4-methylumbelliferone nonanoate, 4-methylumbelliferone butyrate, 4-methylumbelliferone palmitate and 4-methylumbelliferone oleate) at pH 4.0, 6.0, 8.0 and 10.0. The Ser. marcescens and Ps. fluorescens were cultured in Pope and Skerman's basal medium (Skerman 1957) supplemented with 0.5% (w/v) of a commercial medium. The extracellular lipases were isolated and purified by ammonium sulphate precipitation. The assay was carried out by relating the fluorescent intensity emitted by two lipase concentrations on five substrates against four standard curves. These standard curves were prepared by estimating the intensity of fluorescence given by varying dilutions of 4-methylumbelliferone at the four pH levels. The results indicated that the oleic ester of 4-methylumbelliferone was a suitable substrate at pH 8.0 and pH 10.0. These pH values were also optimum for fluorescent intensity on substrates of 4-methylumbelliferone elaidate, 4-methylumbelliferone butyrate and 4-methylumbelliferone palmitate. However, on substrate 4-methylumbelliferone nonanoate, the optimum pH was 4.0.     

Extracellular enzyme activities associated with epiphytic microbiota on submerged stems of the reed Phragmites australis

Abstract Fluorogenic 4-methylumbelliferyl (MUF) compounds were used as analogue substrates for assay of extracellular enzyme activities associated with epiphytic microbiota at submerged Phragmites australis stem surfaces. Incubations at a range of MUF substrate concentrations indicated that saturation of enzyme activity was achieved at a MUF substrate concentration of about 200 μmol 1⁻¹. Later determinations at a single, saturation, concentration of MUF substrate were, therefore, carried out at about 200 μmol l⁻¹. Such determinations were undertaken using P. australis stems from eight gravel-pit ponds. The rate of enzymatic hydrolysis of MUF phosphate (analogue substrate for phosphatase activity) was > MUF β- d -glucopyranoside (β- d -glucosidase) > MUF β- d -galactopyranoside (β- d -galactosidase) > MUF sulphate (sulphatase) and MUF palmitate (lipase) on stems from all eight ponds. Thus the relative magnitude of the various components of total epiphyton extracellular enzyme activity might be a conservative feature.     

Study on immunocapture-chemiluminescence assay of lipase activity in a biological sample.

A new approach for the determination of lipase (triacylglycerol lipase, EC.3.1.1.3) activity in a biological sample was investigated by combining an immunocapture technique with a chemiluminescence (CL) assay method in order to eliminate interference with CL detection. The proposed method consists of an immunocapture step to trap lipase and a subsequent step for CL detection of the activity of the captured lipase. The CL detection is based on the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) reaction and utilizes a proenhancer substrate [a lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI)] which liberates an active enhancer, HDI, by enzymatic hydrolysis. A polyclonal antibody prepared with porcine pancreas lipase was used for the immunocapture. The proposed immunocapture-CL method effectively eliminated the interference with the CL reaction from biological components and enabled the determination of spiked porcine pancreas lipase activity in serum samples in the range 0.41-1.1 U(HDI) (1 U(HDI) corresponds to the amount which liberates 1 pmol HDI/min at 37 degrees C from the substrate). The method was further applied to the assay of the activity for human pancreas lipase in serum and the results showed good correlation (r = 0.871) with those by the conventional colorimetric method.     

Correlation between high-performance liquid chromatography and automated fluorimetric methods for the determination of dopamine, 3, 4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid in nervous tissue and cerebrospinal fluid

The correlation between automated fluorimetric methods and high-performance liquid chromatography is described for the determination of homovanillic acid and 5-hydroxyindoleacetic acid in cerebrospinal fluid, and for dopamine, 3, 4-dihydroxyphenylacetic acid and homovanillic acid in striata of rat brain. The automated fluorimetric methods for 3, 4-dihydroxyphenylacetic acid and homovanillic acid showed a good correlation with the high-performance liquid chromatographic methodology. The fluorimetric determination for dopamine was somewhat less reliable than the high-performance liquid chromatographic assay. The fluorimetric assay for 5-hydroxyindoleacetic acid correlated poorly with the chromatographic method.     

多拷贝毕赤酵母重组菌表达GCL摇瓶优化和高密度发酵

目的:构建高效表达白地霉脂肪酶的毕赤酵母重组菌株,并对筛选得到的菌株进行摇瓶发酵条件优化和分批补料高密度发酵工艺研究。方法:将诱导型表达载体pPIC9K-gcl电转化至毕赤酵母GS115。通过橄榄油-罗丹明B平板和摇瓶发酵筛选高脂肪酶活力的重组菌株,运用基于TaqMan探针的实时荧光定量PCR 法确定其拷贝数,并对菌株进行摇瓶发酵条件优化。在此基础上,研究重组菌在3L 发酵罐中的高密度发酵工艺。结果:筛选得到一株具有3 个白地霉脂肪酶基因拷贝的菌株GS115/pPIC9K-gcl 78#,初始酶活力为220 U/ml。当摇瓶发酵条件为甲醇诱导96 h,每24 h甲醇添加量1 %,接种量2 %,培养基初始pH 7.0,500 ml摇瓶装液量50 ml,甲醇诱导温度25℃ 时酶活力达735 U/ml。3L 发酵罐高密度发酵176.5 h,酶活力达到3360 U/ml,总蛋白含量达到4.30 g/L,且发酵过程中细胞活性一直保持在96 % 以上。结论:基因拷贝数与重组菌株的产酶水平呈正相关,摇瓶优化可显著提高重组菌株的产酶能力,为白地霉脂肪酶的工业化生产奠定了技术基础。     

Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes

A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g . cm-3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19-1.22 g . cm-3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.     

Simple gas chromatography method for lipase assay

A gas chromatography method for lipase assay using tributyrin as substrate is presented. Tributyrin is hydrolyzed by lipase to produce free butyric acid that is directly quantified by gas chromatography. The estimation of lipase activity takes only 6 min after enzyme reaction. The technique needs a small enzyme sample and is useful for analysis of large number of lipase samples. © Rapid Science Ltd. 1998     

Use of specific polyclonal antibodies to detect heterogeneous lipases from Geotrichum candidum.

Geotrichum candidum CMICC 335426 was previously shown to produce two lipases termed lipase A and lipase B, lipase B being highly specific for hydrolysis of esters of cis-delta 9 fatty acids. We now describe the isolation of polyclonal antibodies specific for lipase A and lipase B. These antibodies were used in Western blotting techniques to detect the appearance of the lipases during the course of the fermentation of G. candidum CMICC 335426. A and B were found to be produced simultaneously in the extracellular medium at the start of the growth phase. The two lipases were always present at similar levels in the medium. The specific antibodies were then used to detect the presence of A- and B-like lipases in crude lipase samples from other strains of G. candidum. The lipases were found at different levels in all these samples, and the specificities of the crude lipases varied significantly from one strain to another. Differences in specificity could therefore be explained by different levels of specific (B-type) and non-specific (A-type) lipases in the medium. This was verified by purifying A- and B-type lipases from the G. candidum strain ATCC 34614.     

A fluorimetric method for measuring the activity of soil enzymes

Summary A fluorimetric method is described for the measurement of the activity of a range of soil enzymes. The method is based on the measurement of 4-methylumbelliferone (MUB), a fluorescent product liberated on hydrolysis of the enzyme substrate. The main advantage of the method over colorimetric techniques is that separation of MUB from the soil is unnecessary and the method is therefore suitable for routine, automated analyses. The method was used to measure the activity of β-cellobiase, β-galactosaminidase, β-glucosidase and β-xylosidase over a wide range of substrate concentration and in a range of soils. Kinetic parameters are reported for these enzymes. The method was also shown to be suitable for the assay of arylsulphatase and acid and alkaline phosphatase in soil. The technique should be applicable to a wide range of soil hydrolases, using the same assay methods.     

白地霉Y162脂肪酶基因克隆及其在毕赤酵母中的高效表达

借助生物信息学,对已克隆的地霉属脂肪酶全长基因序列进行同源比对,根据保守序列设计引物,在基因组DNA和cDNA水平上,于国内首次克隆了Geotrichum candidum Y162脂肪酶基因.Gcandidum Y162脂肪酶基因全长1692bp,不含内含子,编码包括19个氨基酸信号肽在内的563个氨基酸.与NCBI GenBank中已报道的地霉属脂肪酶氨基酸序列有86%的一致性.将该基因克隆到pPIC9K表达载体上,转化毕赤酵母GS115,摇瓶发酵96h后毕赤酵母分泌表达55 U/mL重组脂肪酶,实现了脂肪酶的高效表达.酶学性质研究表明,该重组脂肪酶对C9位顺式双键的甘油酯具有明显的底物特异性;对甲醇、甘油等有机溶剂呈现耐受性;最适温度和最适pH分别为50℃和8.0,在pH6.0～10.0及60℃以下能保持60%以上的酶活力.底物特异性、有机溶剂、温度及pH耐受性赋予该重组酶良好工业应用潜力.     

Synthesis of various kinds of esters by four microbial lipases

Ester synthesis by microbial lipases, using homogeneous enzyme preparations, were investigated. The amount of synthesized ester was estimated by alkalimetry, and products were identified by thin-layer chromatography and infrared spectroscopy. Lipases from Aspergillus niger, Rhizopus delemar, Geotrichum candidum and Penicillium cyclopium synthesized esters from oleic acid and various primary alcohols. Only Geotrichum candidum lipase synthesized esters of secondary alcohols. Esters of tertiary alcohols, phenols or sugar alcohols were not synthesized by any lipase. Rather high concentrations of alcohol were required to synthesize the esters of ethylene glycol, propylene glycol or trimethylene glycol. Lipases from Aspergillus niger and Rhizopus delemar synthesized oleyl esters of various fatty acids and some dibasic acids. In contrast, lipases from Geotrichum candidum and Penicillium cyclopium synthesized oleyl esters only from medium or long chain fatty acids.     

Fluorimetric assay of tobacco leaf dehydrogenases with resazurin.

A versatile fluorimetric assay based on the reduction of resazurin to resorufin demonstrated high specific activities for a number of important pyridine nucleotide-linked dehydrogenases in tobacco leaves. The Michaelis constant for the important photosynthetic enzyme, D-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (EC 1.2.1.13), determined by the fluorimetric method, was considerably lower than constants determined by conventional extraction and assay methods reported for the enzyme from other plants. The sensitivity of the fluorimetric method enabled the use of dilute enzyme preparations with resultant low background and high substrate specificity. Inclusion of the anti-oxidant diethyldithiocarbamate in the extraction medium preserved the enzymes during extraction. Primary amines inhibited competitively, and phenazine methosulfate non-competitively each of the eight dehydrogenases tested with the fluorimetric assay. The Mn2+ dependence of NADP-linked dehydrogenases specific for isocitrate and malate was confirmed. The method is rapid, requires a simple combination of ingredients and should be useful for surveying dehydrogenase activity in leaves.     

Purification and characterization of an extracellular lipase of Galactomyces geotrichum

Summary Galactomyces geotrichum secretes an extracellular lipase with a relative molecular weight of 57±2 kDa in the unglycosylated state and 62±2 kDa when glycosylated. The enzyme has optimal activity at pH 7.75 and 30°C and has a pI of 5.2. The specificity of the lipase is similar to lipase A of Geotrichum candidum, but the amino acid composition of the lipases are different.     

A stable, radioactive substrate emulsion for assay of lipoprotein lipase.

A method is described for the assay of lipoprotein lipase, using a stable, radioactive substrate emulsion. Fatty acid-labeled trioleoylglycerol was emulsified by homogenization in glycerol with lecithin as detergent. This anhydrous emulsion was stable for at least six weeks. Substrate solutions for enzyme assay were prepared by diluting the emulsion with buffer containing serum and albumin. The fatty acid produced on hydrolysis was isolated in a one-step liquid-liquid partition system. Incubations with extracts of acetone powders from adipose tissue displayed characteristics of lipoprotein lipase activity, i.e., serum dependence and inhibition by NaCl and protamine. The method is rapid (less than 1 hour), sensitive and reproducible, and suitable for routine use.