Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates.

A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.     

A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes

Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases.     

Activation and inhibition of human cancer cell hyaluronidase by proteins

Results regarding hyaluronidase activity in tumor extracts or cell lines are subject to variations according to the method used for the assay and, sometimes, within an assay. Hyaluronidase was assayed at pH 3.8 in the culture medium of the human cancer-derived cell lines SA87 and H460M by several techniques: HPLC, Reissig technique, ELSA technique, and zymographic analysis. The optimal pH was between 3.3 and 4 in solutions at constant 150 mM sodium concentration. The enzyme was reversibly inhibited by sodium concentration over 200 mM. The activity of purified hyaluronidase increased in the presence of low concentrations of the specific HA-binding glycoprotein hyaluronectin, or of bovine serum albumin or immunoglobulins, or of human albumin, transferrin, or hemoglobin, showing that proteins cooperate in enzyme activity. The ELSA technique showed that optimal pH was slightly lower in the presence of HN than that with BSA. The optimal BSA concentration was determined with the ELSA technique at 0.1 g/liter, and excess of either protein inhibited hyaluronidase. When measured with the Reissig technique, the activity of purified enzyme in the presence of 0.1 g/liter BSA was up to fourfold that without BSA. The cooperative effect of BSA was visualized by zymography. We conclude that the total protein content of hyaluronidase solutions must be considered to correctly interpret quantitation of the enzyme in sera or tissue extracts because protein concentrations above 200 microg/liter lead to underestimation of the enzyme.     

Regulation of proteolytic enzymes in axenically grown myxamoebae of Physarum polycephalum

The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.Abbreviations BSA bovine serum albumin - SD semi-defined (medium with low protein content; Table 1) The investigation formed a part of the Ph.D. thesis of A. Haars, Göttingen, 1976     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Diffusion through a Double-Sided Plate: Development of a Method to Study Alga-Bacterium Interactions

Bacteria and algae isolated from a wastewater oxidation pond were inoculated onto opposing surfaces of double-layer agar plates (Lutri plates) to determine the usefulness of such plates for studying microbial interactions. The altered growth characteristics of various algae depending on the species of bacteria on the adjacent medium surface indicated that there was diffusion of extracellular products through the agar, suggesting that this simple assay can be used for screening potential interactions of actively growing organisms.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Lipoprotein lipase of cultured mesenchymal rat heart cells. IV. Modulation of enzyme activity by VLDL added to the culture medium.

Lipoprotein lipase activity was studied in rat heart cell cultures grown in the presence of 20% fetal calf and horse serum and a medium concentration of triacylglycerol of 0.03 mg/ml. After 6--8 days, when the enzyme activity had reached high levels, the cells were incubated for 24 h in a medium containing 20% serum derived from fasted or fed rats. No change in enzyme activity occurred in the presence of fasted rat serum, but a 50% fall was observed with fed rat serium. When the complete culture medium was supplemented with rat plasma VLDL (0.075--0.75 mg triacylglycerol) a pronounced decrease in lipoprotein lipase activity occurred after 3--5 h of incubation. Similar extent of enzyme fall was observed also in the presence of triacylglycerol-rich lipoproteins isolated from rat plasma after feeding of safflower oil or lard, even though the fatty acid composition of the triacylgylcerol varied markedly. As the addition of VLDL to the culture medium resulted in a lesser fall of heparin releasable than residual activity it seems that there was no direct inhibition of surface bound enzyme activity and that the transport of the enzyme to the cell surface was not affected. These data indicate that addition of VLDL to the culture medium resulted in a fall in enzyme synthesis, while total protein synthesis as determined by incorporation of [3H]leucine, remained unchanged. This inhibition could be reproduced by increasing free fatty acid concentration of the medium, however addition of excess albumin to VLDL-containing medium did not prevent the fall in enzyme activity. The present results obtained with cultured rat hearts cells suggest that in vivo plasma levels of triacylglycerol-rich lipoproteins could modulate the lipoproteins could modulate the lipoprotein lipase activity of the heart.     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli

To develop a rapid reporter system for the screening of stationary-phase promoters in Escherichia coli, the expression pattern of the green fluorescent protein (GFP) during bacterial cultivation was compared with that of the commonly used β-galactosidase. Using GFP with enhanced fluorescence, the expression pattern of both reporter systems GFP and β-galactosidase were similar and showed a typical induction of gene activity of the reporter genes, i.e. increase of expression at the transition from exponential to stationary phase. The expression was affected by the culture medium, i.e. in contrast to the complex medium (LB medium), the stationary-phase specific induction was only observed in synthetic medium (M9) when amino acids were added, whereas there was generally no induction in MOPS medium. To develop a rapid screening method on agar plates for stationary-phase promoters, a photographic approach was used, continued with computational image treatment. A screening method is presented which enables an on-line monitoring of gene activity.     

Prescreening bacterial colonies for bioactive molecules with Janus plates, a SBS standard double-faced microbial culturing system

Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method’s utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells.     

海南近海水域产碱性蛋白酶菌株的分离筛选及发酵条件优化

【背景】微生物蛋白酶在工业生物技术上具有广阔的应用前景。在微生物蛋白酶中,碱性蛋白酶占全球酶总产量的50%以上,获取产碱性蛋白酶的新微生物资源意义重要。【目的】在海南近海贝类养殖基地海泥中筛选获得高产碱性蛋白酶的菌株,对其生长特性进行探究并优化菌株产酶条件,获得新的蛋白酶生产资源。【方法】以酪素培养基为筛选培养基,采用形态学结合系统发育分析鉴定菌株,通过响应面实验优化菌株的产酶条件。【结果】筛选获得一株高产碱性蛋白酶的菌株F3,鉴定为粘质沙雷氏菌（Serratia marcescens）。菌株在最优产酶条件下发酵酶活达到（339.36±4.30） U/mL。【结论】筛选获得的菌株粘质沙雷氏菌F3有较好的产碱性蛋白酶的能力。     

一株羽毛降解菌的分离与鉴定

【目的】从土壤中分离并鉴定羽毛降解菌,测定其生长最适温度及起始pH,并观察酶活动态。【方法】采用系列稀释法和选择培养基法筛选目的菌株,基于16S rRNA基因序列及Biolog方法鉴定其分类地位,利用全自动生长曲线分析仪监测菌株的最适生长条件,并通过测定蛋白水解活性观察其酶活动态。【结果】从混合羽毛的土壤样品中筛选到一株羽毛降解菌,命名为菌株GIMN1.015,初步判定该菌株属于芽孢八叠球菌属(Sporosarcina)。最适生长pH为9.0,温度为30°C。蛋白水解活性最高值出现在培养后96 h。【结论】菌株GIMN1.015在利用羽毛角蛋白资源中具有潜在的应用价值。这是芽孢八叠球菌在羽毛降解方面的首次报道。     

A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.     

The effect of the iron content in the culture medium on the nature of the protein spectrum in Neisseria meningitidis

The protein spectra of Neisseria meningitidis, strain A 208, in the process of its cultivation on solid culture medium based on peptone agar under the conditions of the surplus or deficiency of ions of trivalent iron in the medium. The creation of iron deficiency by the introduction of two iron-binding admixtures, desferol and diethylenetriaminepentaacetic acid (DTPA), was found to lead to the production of two additional proteins with molecular weights of 72 kD and 36-37 kD by meningococcal cells. Of these two admixtures, DTPA more readily stimulated the production of low-molecular protein with a molecular weight of 36-37 kD. This protein was found to be noticeably labile, while protein with a molecular weight of 72-73 kD showed no such lability. As the result of the cultivation of meningococci in iron-deficient medium, the content of protein in microbial residue was 2- to 3-fold greater than that obtained by the cultivation of meningococci in culture medium with the surplus of iron in the form of ferric nitrate.     

Properties of secreted protease from vegetative Trichoderma atroviride mycelia cultivated with protein inducer reveal a complex protein-recognition mechanism

In this study, the submerged cultivation of Trichoderma atroviride with protein substrate (bovine serum albumin, casein, ovalbumin, etc.) led to the secretion of protease activity (PA) into the medium. The secretion started within 30 h and rose to the maximum after 72-h cultivation. It continued upon the prolonged cultivation (up to 8 days) with lower secreted PA. Zymography of the secreted protease (SDS-PAGE with 0.2% gelatine), revealing high molecular weight (m.w.) protease(s) (~200 kDa) with high autolytic activity as the only secretory product. Enzymological characteristics of high m.w. proteases elicited by different inducer proteins were surprisingly different, suggesting that mycelia can recognize not only the presence of inducer proteins but also some qualitative characteristics. Low m.w. protease(s), as found from other studies was(were) seen after prolonged cultivation only, as a band with m.w. about 36 kDa. Expression of known Trichoderma spp. genes encoding secreted proteases, prb1 and proA, showed that only prb1 was expressed after 3–4 days of cultivation, i.e., after the early-secretion phase. The secretory activity of the earlier phase was impaired by tunicamycin and brefeldin A and was significantly stimulated by uncoupler. The existence of a biphasic fungal secretory response and the protein quality recognition represent previously unrecognized processes.     

In vitro cultivation and behavior of aortic endothelium cells in a low serum culture medium

Endothelial cells isolated from calf aorta were used in the subculture No. 4 to 10 for experiments to establish standardized and well reproducible conditions of cultivation. The cells can be cultivated in the commercial medium Eagle-MEM with following supplements: 0.1 g L-glutamine, 5.0 g peptone, 0.5 g serum albumin, and 10 ml (= 1%) bovine serum per 1000 ml medium (MEMPAS). With the aid of immunofluorescence technique the cell type specific marker Factor VIII antigen was shown to be localized especially in the perinuclear region of the cells. The cells were characterized with regard to their growth behaviour. Both, the MEMPAS and the Eagle-MEM with 10 per cent serum increases the cell number in the first 4 days of the exponential growth to the same values. The use of MEMPAS in connection with a strict cultivation regime from the deep frozen cell suspension to culture in scintillation vials guaranteed well reproducible conditions of cultivation. In 14 non-selected experiments distributed over a longer period of time it was found that with regard to the values of the cell number on the respective day the cultures can be divided into two groups, which differ with statistical significance. In further experiments it was possible to confirm this result. Medium, conditioned by endothelial cells (K-MEMPAS) increases the cell number and the growth rate. From these results it was concluded that endothelial cells of vessels are able to produce growth factors with self-stimulating effects. At this time the endothelial cell line is stored in deep frozen state up to the 25th subculture. The endothelial cells cultivated in the described standardized conditions are useful for screening of cell type specific factors with angiogenic activity.