The lipolytic activity of Thermomyces lanuginosus strains isolated from different natural sources

The ability of 144 Thermomyces lanuginosus wild strains isolated from biohumus, mushroom and garden composts, decayed leaves, hazelnuts, and raw coffee beans to hydrolyze synthetic (tributyrin, Tween 20, Tween 40, Tween 60, and Tween 80) and natural fatty substrates (sunflower, soybean, rapeseed and corn oil) was evaluated, and whether the lipolytic activity depended on the isolation source determined. All strains incubated at 55 °C on solid media containing 1% synthetic and 15% natural fatty substrates hydrolyzed both types of substrate. Mean lipolytic activity on natural substrates was significantly higher than on synthetic substrates. The highest mean activity index was noted after growth on sunflower oil, followed by soybean oil and tributyrin; indices on other fatty substrates were low. Strains isolated from raw coffee beans showed the highest mean index, followed by those from biohumus and garden compost; the lowest index being for strains isolated from hazelnuts. Thus, the lipolytic activity index depended on the specific fatty substrate and the source of the isolates.     

Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases

Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P < 0.05), while the growth rates on the different media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein-1). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.     

Lipase Production and Activity as a Function of Incubation Time, pH and Temperature of Four Lipolytic Micro-organisms

S ummary . The lipolytic activity in supernatant fractions of cultures of Saccharomycopsis lipolytica, Micrococcus caseolyticus, Bacillus licheniformis , and a Staphylococcus sp. was studied. Nutrient broth with and without emulsified olive oil was used as substrate. Optimal pH values and temperatures for the lipase produced by the 4 different micro-organisms were determined. The lipolytic activity generally reached a maximum after incubation for 2–6 days. The subsequent decrease in the lipolytic activity was associated with a high proteolytic activity only for Micrococcus caseolyticus . The lipolytic activity was decreased by the presence of olive oil in the medium. Determination of the lipolytic activity after a certain time of incubation, the maximal lipolytic activity and a time-integrated lipolytic activity are compared as estimators for the potential hydrolytic capacity of micro-organisms.     

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by the US Department of Agriculture.     

Lipolytic activity of suspended and membrane immobilized lipase originating from indigenous Burkholderia sp. C20

In this work, a simple, inexpensive, and efficient method of preparing immobilized lipase is presented. The lipase originating from a newly isolated indigenous strain Burkholderia sp. C20 was immobilized onto cellulose nitrate (CN) membrane via filtration. The CN-immobilized lipase was able to retain 60% of its original activity after repeated uses for nine times. The thermal stability of the lipase was also slightly improved after immobilization. The optimal reaction conditions of CN-lipase were pH 9.0 and 55 degrees C, which are similar to those for the suspended lipase. Both suspended and immobilized lipase could hydrolyze the six oil substrates examined, while immobilized lipase displayed less specificity over the oil substrates. Kinetic analysis shows that the dependence of lipolytic activity of both suspended and immobilized lipase on oil substrate concentration can be described by Michaelis-Menten model with good agreement. The estimated kinetic constants for suspended lipase (v(max)=243.9 U/mg, K(m)=0.024 mM) and immobilized lipase (v(max)=32.8 U/mg, K(m)=5.61 mM) were quite different. Employment of immobilization seemed to result in a decrease in v(max) and an increase in K(m), most likely due to the mass transfer resistance arising from formation of micelles during the lipase immobilization process.     

Lipolytic activity of Antarctic cold-adapted marine bacteria (Terra Nova Bay, Ross Sea)

AIMS: The aim of this study was to investigate the lipolytic activity of cold-adapted Antarctic marine bacteria and, furthermore, the combined effect of some environmental factors on this enzymatic process. METHODS AND RESULTS: Strains were assayed for lipolytic activity on a basal medium amended with seven individual fatty acid esters. A significant activity was observed for 148 isolates (95.5% of the total screened). The interactive effect of pH, temperature and NaCl concentration on the substrates was tested for six representative isolates, identified as Pseudoalteromonas, Psychrobacter and Vibrio. Differences between strains according to NaCl and pH tolerances were observed. Only one strain degraded the substrate more efficiently at 4 degrees C than at 15 degrees C. CONCLUSIONS: Our findings demonstrate that the lipolytic activity of Antarctic marine bacteria is rather variable, depending on culture conditions, and occurs in a wide range of salt concentration and pH. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolation and characterization of bacteria that are able to efficiently remove lipids at low temperatures will provide insight into the possibility to use cold-adapted bacteria as a source of exploitable enzymes. Moreover, research on the interactive effects of salt concentration, pH and temperature will be useful to understand the true enzyme potentialities for industrial applications.     

Identification of a carboxylesterase-producing Rhodococcus soil isolate

Subtropical soil microbial isolates were screened for carbohydrate, tributyrin, or olive oil hydrolysis using agar plates supplemented with the corresponding substrates. A heterotrophic, aerobic, Gram-positive strain displaying activity on tributyrin was selected and further characterized. Analysis of the morphological and physiological traits of the strain placed it as a member of the genus Rhodococcus. Further 16S rDNA sequencing revealed a 99% identity to Rhodococcus erythropolis. The strain displayed lipolytic activity on fatty-acid-derivative substrates of short chain length, with cell extract fractions having highest activity, as confirmed by the presence, after zymogram analysis, of a ca. 60-kDa intracellular protein band with activity on 4-methylumbelliferone-butyrate substrate. The presence of such a lipolytic enzyme, similar to those found in other Gram-positive bacteria, indicates that the strain could be of interest for certain biotechnological applications, like the synthesis of pharmaceuticals or biocide detoxification.     

Exploring the specific features of interfacial enzymology based on lipase studies

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.     

Collagenolytic Activity of Some Marine Bacteria

Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources.     

The Isolation and Characterization of Strains of Lipolytic Bacteria from the Ovine Rumen

The first anaerobic lipolytic bacterium isolated from the rumen was Anaerovibrio lipolytica . In this study strains of anaerobic lipolytic bacteria were isolated from a sheep rumen. All the new isolates were Gram negative curved rods with flagella. The bacteria, which produced propionic acid as a major fermentation product, could ferment only a small range of substrates. The new isolates are thought to belong to the same genus as Anaerovibrio lipolytica .     

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K _m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.     

Ecological and physiological studies on fungi associated with human hair

Thirty-seven species attributed to 19 genera of keratinophilic fungi were recovered from 100 human hair samples collected from the Assiut governorate. The genera Aspergillus followed by Penicillium and Chrysosporium were frequently isolated from 65, 43 and 30% of the samples respectively. Fifteen species and 13 genera of thermophilic and thermotolerant fungi (recovered at 45 degrees C) were identified. The thermotolerant Aspergillus fumigatus was frequently encountered and emerged from 82% of the samples. Thirteen isolates of keratinophilic and 20 isolates of thermophilic fungi were tested for lipolytic and proteolytic activities. All the keratinophilic fungi showed lipolytic and proteolytic activities while 100 and 85% of the thermophilic fungi showed lipolytic and proteolytic activities. Using the paper-disc plate method, 12 types of shampoos and oils were tested for their antifungal activities on 42 strains of keratinophilic and thermophilic or thermotolerant fungi. Three out of four types of shampoo proved to be highly effective against all the test fungi.     

Biochemical properties of Streptococcus macedonicus strains isolated from Greek Kasseri cheese

A total of 32 Streptococcus macedonicus strains, isolated from Greek Kasseri cheese, were screened for biochemical properties of technological importance in milk fermentation processing, such as acid production, proteolytic and lipolytic activity, citrate metabolism, exopolysaccharide production, antimicrobial activity and biogenic amines production. All strains were found to be moderate acidifiers in milk. Only four strains could hydrolyse milk casein, while 11 strains showed lipolytic activity against tributyrin. Using amino acid derivatives of 4-nitroaniline as substrates, the highest peptidase activities were determined against phenylalanine- and glycine-proline-4-nitroanilide. Using fatty acid derivatives of 4-nitrophenol, it was shown that all strains exhibited esterase activities up to caprylate, with highest values against butyrate and caproate. Only one showed activity up to palmitate; this was also the most active strain against tributyrin. Five of the 32 strains could metabolize citrate but none of them produced exopolysaccharides. Nine strains displayed antimicrobial activity towards Clostridium tyrobutyricum, while no antimicrobial activity was detected against Listeria innocua and Propionibacterium freudenreichii subsp. shermanii. Finally, none was able to decarboxylize ornithine, histidine or lysine, and only four strains produced tyramine from tyrosine.     

Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits

During cold storage after milk collection, psychrotrophic bacterial populations dominate the microflora, and their extracellular enzymes, mainly proteases and lipases, contribute to the spoilage of dairy products. The diversity, dynamics, and enzymatic traits of culturable psychrotrophs in raw milk from four farms were investigated over a 10-month period. About 20% of the isolates were found to be novel species, indicating that there is still much to be learned about culturable psychrotrophs in raw milk. The psychrotrophic isolates were identified and classified in seven classes. Three classes were predominant, with high species richness (18 to 21 species per class) in different seasons of the year: Gammaproteobacteria in spring and winter, Bacilli in summer, and Actinobacteria in autumn. The four minor classes were Alphaproteobacteria, Betaproteobacteria, Flavobacteria, and Sphingobacteria. The dominant classes were found in all four dairies, although every dairy had its own unique "bacterial profile." Most but not all bacterial isolates had either lipolytic or both lipolytic and proteolytic activities. Only a few isolates showed proteolytic activity alone. The dominant genera, Pseudomonas and Acinetobacter (Gammaproteobacteria), showed mainly lipolytic activity, Microbacterium (Actinobacteria) was highly lipolytic and proteolytic, and the lactic acid bacteria (Lactococcus and Leuconostoc) displayed very minor enzymatic ability. Hence, the composition of psychrotrophic bacterial flora in raw milk has an important role in the determination of milk quality. Monitoring the dominant psychrotrophic species responsible for the production of heat-stable proteolytic and lipolytic enzymes offers a sensitive and efficient tool for maintaining better milk quality in the milk industry.     

The effect of dietary menhaden,olive, and coconut oil fed with three levels of vitamin E on plasma and liver lipids and plasma fatty acid composition in rats

The effect of dietary fats with varying degrees of unsaturation in the presence of different concentrations of vitamin E on tissue lipid levels was studied in rats. Rats were fed either menhaden oil, olive oil or coconut oil at 15% levels with either 0.1, 0.3 or 0.6 mg/g of vitamin E as alpha-tocopherol for four weeks. Rat serum and liver were analyzed for total cholesterol, HDL-cholesterol, triacylglycerol and phospholipids. In addition, fatty acid composition of serum lipids was also analyzed. Serum total cholesterol and triacylglycerol were significantly lower in rats fed menhaden oil than in those fed olive or coconut oil, while the HDL-cholesterol was significantly higher in serum of rats fed menhaden and olive oil than in those fed coconut oil. Levels of vitamin E in the diet had only a significant effect on serum cholesterol and liver phospholipids. The Pearson correlation coefficient showed a significant positive relationship between serum triacylglycerol and total cholesterol, and a negative correlation between triacylglycerol and HDL-cholesterol, and between total and HDL-cholesterol.In the liver, total cholesterol was significantly higher in rats fed coconut oil than in rats fed menhaden oil. Total liver phospholipids were lower in rats fed either coconut oil or olive oil compared to those fed menhaden oil, especially with higher levels of vitamin E intake. Higher levels of vitamin E in the diet appear to increase triacylglycerol and phospholipids in livers of rats fed menhaden oil. In the liver a significant negative correlation was observed between phospholipids and cholesterol. The type and degree of unsaturation (polyunsaturated fatty acids in menhaden oil, monounsaturated fatty acids in olive oil and saturated fatty acids in coconut oil) significantly affected plasma and tissue lipids.     

Identification and characterization of Lactobacillus species isolated from fillets of vacuum-packed smoked and salted herring (Clupea harengus)

Seventy-eight strains were isolated from fillets of vacuum-packed smoked and salted herring ( Clupea harengus ) on APT glucose medium at 5°C or 20°C . All the isolates belonged to the genus Lactobacillus . CO₂ production and arginine degradation were characteristic of the isolates. A dichotomous key is proposed, using only four characters (growth on Rogosa broth, fermentation of ribose, presence of meso -diaminopimelic acid, isomer of lactate produced) for presumptive identification of lactobacilli from fish. The isolates could be allocated to three groups related to Lactobacillus groups II or III of Kandler and Weiss. Most of the strains possess important technological capacities : tolerance of high levels of NaCl, liquid smoke and bile salts. No lipolytic activity was found but some proteolytic activity was found on casein. Inhibitory activity against pathogenic or spoilage organisms was frequent and probably due to the production of hydrogen peroxide, or organic acids or both. No Carnobacterium strains were isolated despite the use of suitable temperature and isolation medium, probably because of the smoking of the fish.     

Substrate Utilization Profiles of Bacterial Strains in Plankton from the River Warnow, a Humic and Eutrophic River in North Germany

Bacteria are very important degraders of organic substances in aquatic environments. Despite their influential role in the carbon (and many other element) cycle(s), the specific genetic identity of active bacteria is mostly unknown, although contributing phylogenetic groups had been investigated. Moreover, the degree to which phenotypic potential (i.e., utilization of environmentally relevant carbon substrates) is related to the genomic identity of bacteria or bacterial groups is unclear. The present study compared the genomic fingerprints of 27 bacterial isolates from the humic River Warnow with their ability to utilize 14 environmentally relevant substrates. Acetate was the only substrate utilized by all bacterial strains. Only 60% of the strains respired glucose, but this substrate always stimulated the highest bacterial activity (respiration and growth). Two isolates, both closely related to the same Pseudomonas sp., also had very similar substrate utilization patterns. However, similar substrate utilization profiles commonly belonged to genetically different strains (e.g., the substrate profile of Janthinobacterium lividum OW6/RT-3 and Flavobacterium sp. OW3/15-5 differed by only three substrates). Substrate consumption was sometimes totally different for genetically related isolates. Thus, the genomic profiles of bacterial strains were not congruent with their different substrate utilization profiles. Additionally, changes in pre-incubation conditions strongly influenced substrate utilization. Therefore, it is problematic to infer substrate utilization and especially microbial dissolved organic matter transformation in aquatic systems from bacterial molecular taxonomy.     

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.     

Screening Botryosphaeria species for lipases: Production of lipase by Botryosphaeria ribis EC-01 grown on soybean oil and other carbon sources

Nine isolates of Botryosphaeria spp. were screened for lipases when cultivated on eight different plant seed oils and glycerol, and all produced lipases. Botryosphaeria ribis EC-01 produced highest lipase titres on soybean oil and glycerol, while eight isolates of Botryosphaeria rhodina produced significantly lower enzyme titres. B. ribis EC-01 produced lipase when grown on different fatty acids, surfactants, carbohydrates and triacylglycerols, with highest enzyme titres produced on Triton X-100-emulsified stearic (316.7 U/mL), palmitic (283.5 U/mL) and oleic (247.4 U/mg) acids, and soybean oil (105.6 U/mL), as well as castor oil (191.2 U/mg); an enhancement of 9-fold over soybean oil-grown cultures. Glycerol was also a good substrate for lipase production. The crude lipase extract was optimally active at pH 8.0 and 55 °C, stable between 30 and 55 °C and pH 1–10, and tolerant to 50% (v/v) glycerol, methanol and ethanol. The crude lipase showed affinity for substrates of short, average and long-chain fatty acids (different esters of p-nitrophenol and triacylglycerols). Zymograms developed with 4-methylumbelliferyl-butyrate showed two bands of lipolytic activity at 45 and 15 kDa. This is the first report on the production of lipases by B. ribis grown on these different carbon sources.