Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Geotrichum candidum 4013: Extracellular lipase versus cell-bound lipase from the single strain

Two types of lipases (extracellular and cell-bound) were produced by Geotrichum candidum 4013 in liquid medium and were used as biocatalysts in blackcurrant oil hydrolysis. Reaction products were analysed for the degree of conversion from which enzyme activity was evaluated, and the composition of free fatty acids was compared to the composition of oil substrate. The enzyme activity was measured also before and after the reaction in SC-CO₂. The fatty acid composition of the acids liberated from oil by hydrolysis suggests a specificity of the cell-bound and extracellular enzymes from Geotrichum candidum 4013. The extracellular lipase displays low selectivity to the polyunsaturated fatty acids, and the cell-bound lipase possesses selectivity to the saturated fatty acids. Enantioselectivity of the tested processes achieved with both induced enzymes was high (from 43 to 242). The activity of all enzymes has markedly increased after their exposure to SC-CO₂. The treatment of enzymes by SC-CO₂ could be easy-to-use approaches to improve the efficiency of enzymatic reactions.     

Immunocytochemical identification and localization of lipase in cells of the mycelium of Penicillium cyclopium variety

The localization of lipase in cells of the fungus Penicillium cyclopium was investigated. It was shown by differential centrifugation of a homogenate of mycelial cells that the activity of the enzyme is associated with the cell wall. A study of ultrathin sections of mycelium fixed using the method of Zvyagintseva in an electron microscope showed that the final products of lipolytic activity of the enzyme is localized on the cell wall. Antibodies were raised against the purified A and B lipases from P. cyclopium and their specificity was assessed by enzyme-linked immunosorbent assay. The antibody preparation was used in cytochemical investigation by immunogold labelling. This study permits the localization of cell-bound lipase mainly in the cell wall and in the periplasmic space. The identity of the cell-bound lipase with one of the two extracellular lipases is also demonstrated.     

Analysis of Bacillus megaterium lipolytic system and cloning of LipA,a novel subfamily I.4 bacterial lipase

The lipolytic system of Bacillus megaterium 370 was investigated, showing the existence of at least two secreted lipases and a cell-bound esterase. A gene coding for an extracellular lipase was isolated and cloned in Escherichia coli. The cloned enzyme displayed high activity on short to medium chain length (C(4)-C(8)) substrates, and poor activity on C(18) substrates. On the basis of amino acid sequence homology, the cloned lipase was classified into subfamily I.4 of bacterial lipases.     

Carbon source impact on Yarrowia lipolytica KKP 379 lipase production

The production of lipases by microorganisms is strongly influenced by the culture conditions. The optimum culture conditions for enzyme production are strain- and species-dependent. The aim of this study was to evaluate the impact of the carbon source used in the culture medium on the profile of lipases produced by Yarrowia lipolytica KKP 379. We observed a different pattern of extracellular and cell-bound lipase production, which was the highest in the early exponential phase. The extracellular lipase activity increased in the late exponential phase due to the lower accumulation of lipase molecules in cell walls. The best carbon source for extracellular lipase production by Y. lipolytica KKP 379 was olive oil. Glucose, dodecane and olive oil had a positive effect on biomass yield. Dodecane and/or glycerol utilization in microbiological lipase production was possible, but this process could not proceed without the addition of some activators such as olive oil in the cultivation medium.     

Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica

AIMS: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors. METHODS AND RESULTS: The level of lipase production and LIP2 induction, measured using an LIP2-LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations. The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase. SIGNIFICANCE AND IMPACT OF THE STUDY: Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y. lipolytica mutant. Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium. Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration. We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media. CONCLUSIONS: This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y. lipolytica.     

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.     

Cell-Bound Thiaminase I of Bacillus thiaminolyticus

The distribution of the extracellular enzyme, thiaminase I, was determined for logarithmically growing cultures of Bacillus thiaminolyticus. About 60% of the enzyme is associated with the cells throughout the growth cycle. The remainder of the enzyme is in the culture medium. The release of the cell-bound thiaminase I is examined under a variety of conditions. The rate and extent of release is dependent on the pH and the nature of the incubation solution. The release process appears to be relatively independent of de novo protein synthesis, energy derived from oxidative phosphorylation, or divalent metal ions. The absence of carbon or nitrogen sources has little effect on the release of the enzyme. Cell-bound thiaminase I probably is the immediate precursor for extracellular thiaminase I found in the culture medium. Washed cells continue to release thiaminase I at the expense of cell-bound enzyme. In addition, purified cell-bound thiaminase I is indistinguishable from purified extracellular thiaminase I by a number of physical and kinetic criteria.     

Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.     

Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.     

Lipase and esterase formation by psychrophilic and mesophilic Acinetobacter species.

Acinetobacter O16, a psychrophilic species, produced extracellular lipase (measured by hydrolysis of olive oil, tributyrin, or beta-naphthyl laurate) when grown on a complex medium (peptone plus yeast extract). Most lipase was produced during the logarithmic phase of growth. Very little cell-bound lipase was formed. These cells also produced an esterase (measured by the hydrolysis of beta-naphthyl acetate). At first, all esterase was cell bound; significant amounts appeared in the external medium late in growth. Breaking the cells did not increase cell-bound lipase activity. After breaking of the cells, most of the cell-bound lipase and esterase activity was solubilized, even after very high speed centrifugation. No appreciable amounts of these enzymes were released by osmotic shock. Lipase formation was greatly affected by nutrient conditions. Lowering either the yeast extract of the peptone content of the normal complex medium lowered or abolished lipase formation. Esterase activity was lowered to a lesser extent. Cells growing in synthetic amino acid plus vitamin medium or in acid-hydrolyzed casein produced substantial amounts of esterase but no cell-free or cell-bound lipase. However, if sodium taurocholate was added to these media, lipase was produced. Greatest production occurred if a mixture of di- and poly-peptides was also present. Taurocholate also stimulated lipase production in the normal complex medium. Adding Tween 80 or ethanol to the normal complex medium inhibited lipase production. Sodium acetate, oleic acid, olive oil, or Tween 20 added to synthetic media did not affect lipase production. The psychrophile grew more quickly at 30 degrees C than at 15 or 20 degrees C but produced more lipase at the lower temperatures. Esterase production was about the same at 20 and 30 degrees C. A mesophilic Acinetobacter species produced the same amount of lipase and esterase at 20 and 30 degrees C. The best production of lipase by the psychrophile occurred in standing cultures.     

Genetic and Biochemical Studies on Cell-Bound α-Amylase in Bacillus subtilis Marburg

A small but significant amount of alpha-amylase activity was detected in the cells of Bacillus subtilis Marburg. The cell-associated activity was almost constant regardless of the level of extracellular alpha-amylase activity. The cell-bound amylase activity could be separated into three components, upon Sephadex G-75 chromatography, referred to as components A, B, and C. Component C showed the same properties as the extracellular alpha-amylases so far examined. Component A had a molecular weight greater than 70,000, as judged from the elution position on Sephadex G-75, and became smaller upon treatment with trypsin but was still larger than that of component C. An alpha-amylase mutant that lacked extracellular alpha-amylase completely because of a mutation within the structural gene of the enzyme was found to lose all three cell-bound amylase components simultaneously. These data suggest strongly that the cell-bound amylase components are precursors of the extracellular alpha-amylase and that the alpha-amylase of this organism is produced under the direction of the same gene whether the enzyme is within or outside the cell.     

Purification and characterization of the extracellular and cell-bound lipases from a Penicillium cyclopium variety

Summary A new strain of Penicillium cyclopium was found on mouldy Grenoble walnuts. From the culture filtrate, two new extracellular lipases (A- and B-lipase) were isolated and characterized. The mycelium contained one of the two forms of lipase (B-lipase). The specificity of the cell-bound lipase linked to the mycelium was then studied. The enzyme was specific to shortchain homogeneous triacylglycerols; it preferred to attack the o-position rather than the p-position, and showed stereospecificity on the sn-1 position. Correspondence to: L.-C. Comeau     

Temperature regulation of lipase secretion by Pseudomonas fluorescens strain MFO

The psychrotrophic bacterium Pseudomonas fluorescens is a milk contaminant known to secrete a lipase that is a nuisance for the dairy industry but may have a biotechnological interest. Strain MFO secretes this enzyme upon induction under various conditions. Regardless of the inducer and growth temperature, a single enzyme is produced. However, optimal production occurs when the culture is grown at 17.5° C. Other exported proteins (an extracellular protease and two periplasmic phosphatases) have previously been shown to display exactly the same optimal temperature of production. In contrast, constitutive cell-bound esterase and cytochrome oxidase are produced at a roughly constant rate regardless of the growth temperature. The relevance of these results are discussed in terms of multifunctional regulation and interest for the dairy industry. Correspondence to: N. Orange     

Effect of lipids and detergents on the production and solubilization of extracellular lipase inYarrowia lipolytica

Triglycerides, oleic acid but not fatty acid-containing, nonionic detergents (Spans, Tweens) were able to stimulate the synthesis of cell-bound and soluble lipase ofYarrowia lipolytica grown in a complex medium containing citrate and urea. The optimal concentration of olive oil for induction was 0.5% (W/V). The combined effect of a high ionic strength (0.75 mol/L KCl) and of digitonin (2 mmol/L) at pH 7.6 resulted in solubilization of 80% of the cell-bound lipase and a significant activation of the enzyme. Comparison of twoY. lipolytica strains showed the effects of Mg²⁺ and Fe³⁺ concentrations in the medium on the synthesis of the enzyme to be strongly strain-dependent.     

Substrate specificity,regioselectivity and hydrolytic activity of lipases activated from Geotrichum sp.

Substrate specificity (typoselectivity), regioselectivity and hydrolytic activity of induced lipases from three strains (4012, 4013, 4166) of Geotrichum candidum and that of Geotrichum ludwigii (48) were investigated. The lipases were induced in two types of culture media, of which the medium containing peptone as nitrogen source was proved to give better results. Olive oil was employed as inductor for the lipase activity. Activated lipases represented mostly extracelullar lipases, which penetrated through cellular membrane into medium. The activity of cell-bound lipase was also determined. Most of lipases belong to the group of specific lipases able to hydrolyse ester bonds in the positions sn-1 and sn-3 ester of triacylglycerols (1,3-selective lipases) and display specificity to saturated fatty acids. All activated lipases from Geotrichum sp., extracellular and cell-bound, were used as biocatalyst in the blackcurrant oil hydrolysis.     

Effect of different carbon sources on lipase production by Candida rugosa

Different carbon sources affecting growth and lipase production in Candida rugosa were studied by using batch cultures on defined medium. Carbohydrates and acids non-related to fats did not induce lipase production. The highest yields of enzyme were obtained with lipids or fatty acids as carbon sources. Tween 80 stimulated lipase biosynthesis and secretion outside the cell. Combinations of two types of substrates, carbohydrates and fatty acids, did not improve lipase production, and in some cases, their consumption was produced in a sequential pattern. Glucose presented a repressing effect on lipase production. Moreover, glucose was found to be effective in stimulating lipase secretion by cells with a high level of cell-bound lipase activity because of their previous growth in oleic acid.     

Capacity of N- and C-domains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> flo1p cell wall protein homologue to expose lip2 lipase on cell surface of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeast

The closest homologue of the Saccharomyces cerevisiae Flo1p cell wall protein was detected in Yarrowia lipolytica yeast (YALI0C09031p) by the method of genomic analysis, and capacity of its N- and C-domains to expose the Lip2 lipase on the cell surface was studied. The efficient fixation of the enzyme on the Y. lipolytica cell wall surface was demonstrated. The activity of the cell-bound lipase was 9170 and 3200 units per 1 g of dry solid matter when using N- and C-domains of the cell wall protein, respectively. At the same time, in the case of immobilization using the N-domain, approximately 30% of the total lipase activity was detected in the culture medium, whereas when using C-domain of the cell wall protein YALI0C09031p, practically all lipase was in the immobilized state. Obtained values of the level of the cell-bound lipase activity considerably exceed previously published data opening a prospect for new technological solutions which meet industrial needs.     

Chromosomal mapping and cloning of the lipase gene of Pseudomonas aeruginosa

Various mutants (lip) of Pseudomonas aeruginosa PAO 2302 that lacked extracellular lipase activity were isolated. They were selected on a calcium-triolein agar. The phenotypic characteristics of two of these mutants suggested that they were defective in the gene coding for lipase: both lip mutants produced no lipase in liquid- and on solid medium. They were nonpleiotropic with regard to various other exoproducts. None of the mutants released any putatively cell-bound lipase after treatment of cells with Triton X-100 or alginate. The electrophoretic protein- and LPS-profiles of outer membranes derived from lip mutants and the parental strain were identical. The lip locus was mapped on the chromosome of P. aeruginosa PAO 1 by FP5- and R68. 45-mediated crossings and by transduction with phage G101. The lip locus was cotransduced with pyrF only (60%) indicating a map position at about 57 min. The lipase gene was cloned on a 3.1 kb SalI fragment using vector pKT248. The newly constructed plasmid was able to complement the lipase deficiency of the two lip mutants of P. aeruginosa.     

Helicobacter pylori EstV: identification, cloning, and characterization of the first lipase isolated from an epsilon-proteobacterium

Bacterial lipases are attracting an enormous amount of attention due to their wide biotechnological applications and due to their roles as virulence factors in some bacteria. Helicobacter pylori is a significant and widespread pathogen which produces a lipase(s) and phospholipases that seem to play a role in mucus degradation and the release of proinflammatory and cytotoxic compounds. However, no H. pylori lipase(s) has been isolated and described previously. Therefore, a search for putative lipase-encoding genes was performed by comparing the amino acid sequences of 53 known lipolytic enzymes with the deduced proteome of H. pylori. As a result, we isolated, cloned, purified, and characterized EstV, a novel lipolytic enzyme encoded by open reading frame HP0739 of H. pylori 26695, and classified it in family V of the bacterial lipases. This enzyme has the properties of a small, cell-bound carboxylesterase (EC 3.1.1.1) that is active mostly with short-chain substrates and does not exhibit interfacial activation. EstV is stable and does not require additional cofactors, and the maximum activity occurs at 50 degrees C and pH 10. This unique enzyme is the first lipase isolated from H. pylori that has been described, and it might contribute to ulcer development, as inhibition by two antiulcer substances (beta-aescin and glycyrrhizic acid) suggests. EstV is also the first lipase from an epsilon-proteobacterium to be described. Furthermore, this enzyme is a new member of family V, probably the least-known family of bacterial lipases, and the first lipase of this family for which kinetic behavior, inhibition by natural substances, and other key biochemical features are reported.