Fungal Lipase Research Articles

Role of an electrostatic network of residues in the enzymatic action of the Rhizomucor miehei lipase family.

We have used continuum electrostatic methods to investigate the role of electrostatic interactions in the structure, function, and pH-dependent stability of the fungal Rhizomucor miehei lipase (RmL) family. We identify a functionally important electrostatic network which includes residues S144, D203, H257, Y260, H143, Y28, R80, and D91 (residue numbering is from RmL). This network consists of residues belonging to the catalytic triad (S144, D203, H257), residues located in proximity to the active site (Y260), residues stabilizing the geometry of the active site (Y28, H143), and residues located in the lid (D91) or close to the first hinge (R80). The lid and the first hinge are associated with the interfacial activation of lipases, where an alpha-helical lid opens up by rotating around two hinge regions. All network residues are well conserved in a set of 12 lipase homologues, and 6 of the network residues are located in sequence motifs. We observe that the effects of modeled mutations R86L, D91N, and H257F on the pH-dependent electrostatic free energies differ significantly in the closed and open conformations of RmL. Mutation R86L is especially interesting since it stabilizes the closed conformation but destabilizes the open one. Site-site electrostatic interaction energies reveal that interactions between R86 and D61, D113, and E117 stabilize the open conformation.

Purification and properties of a lipase fromPenicillium chrysogenum isolated from industrial wastes

A fungal lipase was isolated from the wastes of industrial cultures of Penicillium chrysogenum employed in antibiotics production. Lipase activity was only detected in solid aggregates present in the protein concentrates. To extract the lipase, Triton X-100 was the most suitable surfactant. The lipase was further purified by hydrophobic interaction and anion exchange chromatography. A molecular mass of approx 40 kDa and a pI of approx 3.8 were estimated. Optimum pH and temperature were 7.9–8.1 and 45 °C respectively. Although its activity in the hydrolysis of lipids and esters is not very high, the lipase from P chrysogenum proved to be notably efficient in several synthetic reactions in non-aqueous media. © 2000 Society of Chemical Industry

The Conformational Changes of the Fungal Lipase from Humicola lanuginosa Induced by the Formation of Bile Salt and Mixed Micelles

The effect of sodium taurodeoxycholate and a mixture of sodium taurodeoxycholate/phosphatidyl-choline on the activation of fungal lipase from Humicola lanuginosa (HLL) was investigated by monitoring the specific activity and the changes in intrinsic protein fluorescence. A large increase in the inactivation rate was observed at about the critical micellar concentration of bile salt. On the contrary, a high activation of lipase was achieved by the presence of mixed micelles. Steady-state fluorescence quenching measurements were performed to resolve the fluorescence contribution of W89 residue in emission of four-tryptophan-containing HLL lipase. The W89 residue is located in the “lid” helix which participates in interfacial activation of the enzyme. The assignment of W89 residue was confirmed by use of the W89F mutant and inhibited form of lipase. The FQRS (fluorescence quenching resolved spectra) method was used to decompose the total emission spectrum of HLL lipase. The FQRS results show that the fluorescence of the W89 residue is similar in inhibited and inactivated HLL lipase and exhibits a maximum of emission at about 345 ± 1 nm (λex = 295 nm). In the mixed micelle solution the fluorescence of the W89 residue may be resolved into two components, with fluorescence maxima at 337 and 347 nm, respectively (λex = 295 nm). It is concluded that HLL lipase undergoes a conformational transition upon specific interactions with both anionic and mixed micelles, resulting in a change in the microenvironment of the W89 residue.

ChemInform Abstract: Chemoselective Hydrolysis of Esters by Fungal Lipase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Chemoselective Hydrolysis of Esters by Fungal Lipase

Lipase, isolated from Aspergillus niger, exhibits chemoselectivity towards hydrolysis of fatty acid esters and does not hydrolyse carbamates; this has been demonstrated with substrates such as 5 ( N-methoxycarbonyl- N-ethoxycarbonylmethylamino)-2-aryl-2,4-dihydro-3 H-1,2,4-triazol-3-ones.

Ferulic acid esterase-III fromAspergillus niger does not exhibit lipase activity

The nine closest matches of the deduced primary sequence of ferulic acid esterase (FAE-III/ FAEA) from Aspergillus niger to any other proteins in a redundant database were with fungal lipases (32-26% identity). In this paper we show that FAE-III does not function significantly as a lipase; it exhibits no detectable activity on triglycerides, and has 10 5 -fold to 10 6 -fold lower activity than Rhizopus niveus lipase on diglycerides. Conversely, lipases exhibit ∼ 2.5 x 10 6 -fold lower ferulic acid esterase activity on methyl ferulate compared to FAE-III. Further, lipases possess no detectable activity on O-[5-O-(trans-feruloyl) -α-9-arabinofuranosyl]-(1 → 3)-O-β-3-xylopyranosyl-(1 → 4)-3-xylopyranose (FAXX), which is the substrate with the highest catalytic efficiency so far reported for FAE-III. These results show that FAE-III exhibits no significant lipase activity, and lipases exhibit no significant ferulic acid esterase activity.

Ferulic acid esterase‐III from Aspergillus niger does not exhibit lipase activity

The nine closest matches of the deduced primary sequence of ferulic acid esterase (FAE-III/FAEA) from Aspergillus niger to any other proteins in a redundant database were with fungal lipases (32–26% identity). In this paper we show that FAE-III does not function significantly as a lipase; it exhibits no detectable activity on triglycerides, and has 105-fold to 106-fold lower activity than Rhizopus niveus lipase on diglycerides. Conversely, lipases exhibit ∽2.5×106-fold lower ferulic acid esterase activity on methyl ferulate compared to FAE-III. Further, lipases possess no detectable activity on O–[5–O-(trans-feruloyl)-α-L-arabinofuranosyl]-(1→3)–O–β-D-xylopyranosyl-(1→4)-D-xylopyranose (FAXX), which is the substrate with the highest catalytic efficiency so far reported for FAE-III. These results show that FAE-III exhibits no significant lipase activity, and lipases exhibit no significant ferulic acid esterase activity. © 1999 Society of Chemical Industry

Effect of Acetate and pH on Sunflower Oil Assimilation by Mucor circinelloides f. circinelloides

Summary Mucor circinelloides f. circinelloides CBS 108.16 exhibited poor cell growth and substrate assimilation (32.5% of extracellular lipids) when grown with sunflower oil as sole carbon source. By contrast, in the presence of both sunflower oil (30 g/l) and sodium acetate (10 g/l), nearly complete utilization of both substrates (97.5% of sunflower oil, 100% of sodium acetate) occured and biomass production was increased about five-fold to 23 g dry weight/l after 72 h. A significantly higher content of γ-linolenic acid in the fungal neutral lipid fraction (5.2%) was also observed after 168 h of growth. These different patterns were attributed to the change in pH of the medium during cell growth in the presence and absence of acetate. In the absence of sodium acetate the pH decreased to 2.2, whereas in its presence it increased to about pH 8.0. During metabolism of sunflower oil in the presence of sodium acetate, the percentage of saturated fatty acids in the medium increased, suggesting a higher specificity of the fungal lipase for unsaturated fatty acids. For growth experiments in a small fermenter in the absence of sodium acetate, the gradual pH increase of sodium acetate containing medium was mimicked by an identical programme-controlled pH increase. Similar results as in the presence of sodium acetate were obtained. This observation indicated that the pH increase alone during cultivation was responsible for the increased sunflower oil utilization, biomass and GLA production.

Chemoselective Hydrolysis of Esters by Fungal Lipase

Lipase, isolated from Aspergillus niger, exhibits chemoselectivity towards hydrolysis of fatty acid esters and does not hydrolyse carbamates; this has been demonstrated with substrates such as 5 (N-methoxycarbonyl-N-ethoxycarbonylmethylamino)-2-aryl-2,4-dihydro-3H-1,2,4-triazol-3-ones.

Pancreatic enzymes: secretion and luminal nutrient digestion in health and disease.

Severe pancreatic exocrine insufficiency leading to malabsorption of nutrients is one of the most important late features of chronic pancreatitis. In contrast to other key enzymes, pancreatic synthesis and secretion of lipase is impaired more rapidly, its intraluminal survival is shorter due to its higher susceptibility against acidic and proteolytic denaturation, and its luminal digestive action is hardly compensated by nonpancreatic mechanisms. As a consequence, steatorrhea is in general more severe and occurs several years before clinical malabsorption of protein or starch. Apart from the detrimental effects of nutrient deficiency, profound alterations of upper gastrointestinal secretory and motor functions may be an additional and hitherto underestimated consequence of increased nutrient delivery to distal intestinal sites. Effective reduction of nutrient malabsorption in pancreatic insufficiency requires delivery of sufficient enzymatic activity into the duodenal lumen simultaneously with meal nutrients. Modern enteric-coated pancreatin microsphere preparations attempt to achieve this by optimizing the size of individual microspheres and chemical properties of the coating. However, lipid digestion cannot be completely normalized in most patients by current standard therapy. In the future, acid and protease stable bacterial and fungal lipases with additional pH optima in the acidic milieu or animal or bioengineered human gastric lipase preparations may offer superior therapeutic alternatives. This review first summarizes current knowledge about secretion and luminal fate of pancreatic enzymes and their effects on nutrient digestion in health and chronic pancreatitis. Second, rationale, current standards, options, and future aspects of enzyme replacement therapy are discussed.

Lipase - Catalyzed glycerolysis of sunflower oil to produce partial glycerides.

Partial glycerides were prepared by glycerolysis of sunflower oil in presence of lipase enzyme as catalyst. Six lipases of different origins were used and compared for their catalytic activity. These include Chromobacterium lipase, pancreatic lipase, Rhizopus arrhizus lipase, lyophilized lipase (plant lipase) in addition to two lipase preparations derived from Rhizopus japonicas ; Lilipase A-10 and Lilipase B-2. Chromobacterium lipase was found to be the most active as glycerolysis catalyst whereas lyophilized lipase; a plant preparation from wheat germ was the least active. The results have also shown that the lipase type affects also the product polarity and hence its field of application as a food emulsifier. Less polar products can be obtained using Chromobacterium lipase whereas the more polar ones using a fungal lipase preparation «Lipase A-10». The product polarity is also influenced by the process temperature but the mode of its effect is different for different lipases.

Fluorescence study of fungal lipase from Humicola lanuginosa

Time-resolved and steady-state fluorescence quenching measurements have been performed to study two different conformations of the fungal lipase from Humicola lanuginosa. The intrinsic fluorescence of tryptophan Trp89 residue, located in the ‘lid’ region, has been used as a probe for the dynamics of protein. The native (‘closed-lid’) form of the enzyme has been found to decay as a triple exponential with time constants and relative contributions of 5.4 ns (74.3%), 2.2 ns (20.4%) and 0.4 ns (5.3%). A comparison of recovered decay parameters obtained for native and mutated H. lanuginosa lipase shows that Trp89 contributes about 61% to the class of emitting species with the lifetime of 5.4 ns. The fluorescence quenching data show that three out of four tryptophans (i.e., 117, 221 and 260 residues) with H. lanuginosa lipase are totally quenchable by acrylamide while completely inaccessible to iodide. On the contrary, the Trp89 residue is available for both quenchers. Using steady-state iodide fluorescence quenching data and the fluorescence-quenching-resolved-spectra (FQRS) method, the total emission spectrum of the native lipase has been decomposed into two spectral components. One of them, unquenchable by iodide, has a maximum of fluorescence emission at 330 nm and the second one, exposed to the solvent, emits at 338 nm. The resolved spectrum of the redder component corresponds to the Trp89 residue, which participates in about 65% of the total H. lanuginosa emission. The dynamic Stern—Volmer quenching constants calculated for both native (‘closed-lid’) and inhibited (‘open-lid’) lipase are 2.71 and 4.49 M −1, respectively. The values obtained indicate that Trp89 is not deeply buried in the protein matrix. Our results suggest that distinct configurations of fungal lipase can be monitored using the fluorescence of the Trp89 residue located in the ‘lid’-helix which participates in an interfacial activation of the enzyme.

Insights into the molecular basis for fatty acyl specificities of lipases from Geotrichum candidum and Candida rugosa

Despite immense progress in our comprehension of lipase structure and function during the past decade, the basis for lipase acyl specificities has remained poorly understood. This review summarizes some recent advances in the understanding at the molecular-level of substrate acyl recognition by two members in a group of large ( M w∼60 kDa) microbial lipases. Two aspects of acyl specificity will be focused upon. (i) The unique preference of a fungal Geotrichum candidum lipase for long-chain cis (Δ-9) unsaturated fatty acid moieties in the substrate. Mutational analysis of this lipase identified residues essential for its anomalous acyl preference. This information highlighted for the first time parts in the lipase molecule involved in substrate acyl differentiation. These results are discussed in the context of the 3D-structure of a G. candidum lipase isoenzyme and structures of the related Candida rugosa lipase in complex with inhibitors. (ii) The mechanism by which the yeast C. rugosa lipase discriminates between enantiomers of a substrate with a chiral acyl moiety. Molecular modeling in combination with substrate engineering and kinetic analyses, identified two alternative substrate binding modes. This allowed for the proposal of a molecular mechanism explaining how long-chain alcohols can act as enantioselective inhibitors of this enzyme. A picture is thus beginning to emerge of the interplay between lipase structure and fatty acyl specificity.

Anatomy of lipase binding sites: the scissile fatty acid binding site

Shape and physico-chemical properties of the scissile fatty acid binding sites of six lipases and two serine esterases were analyzed and compared in order to understand the molecular basis of substrate specificity. All eight serine esterases and lipases have similar architecture and catalytic mechanism of ester hydrolysis, but different substrate specificities for the acyl moiety. Lipases and esterases differ in the geometry of their binding sites, lipases have a large, hydrophobic scissile fatty acid binding site, esterases like acetylcholinesterase and bromoperoxidase have a small acyl binding pocket, which fits exactly to their favorite substrates. The lipases were subdivided into three sub-groups: (1) lipases with a hydrophobic, crevice-like binding site located near the protein surface (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). The length of the scissile fatty acid binding site varies considerably among the lipases between 7.8 Å in cutinase and 22 Å in Candida rugosa and Rhizomucor miehei lipase. Location and properties of the scissile fatty acid binding sites of all lipases of known structure were characterized. Our model also identifies the residues which mediate chain length specificity and thus may guide protein engineering of lipases for changed chain length specificity. The model was supported by published experimental data on the chain length specificity profile of various lipases and on mutants of fungal lipases with changed fatty acid chain length specificity.

The decay of the fluorescence anisotropy of tryptophan residues in fungal lipase fromHumicola lanuginosa

Pulse nanosecond fluorescence anisotropy decay has been used to study the mobility of tryptophan residues within fungal lipase fromHumicola lanuginosa. The decay of emission anisotropy of protein in native, inhibited and mutated form has been investigated in buffered water and 50% v/v glycerol solutions. The rotational motions of the lipase were analyzed in terms of two different kinetic models. It was found that the fluorescence emission anisotropy decay can best be desribed with two rotational correlation times: 0.63 and 5.45 ns in water and 0.98 and 10.70 ns and in 50% v/v glycerol solution. Using the same experimental conditions the decay of inhibited and mutatedH. lanuginosa lipase showed a similar biexponential character. These results are interpreted in terms of local or segmental motion arising from a mass of about 1083 daltons which corresponds to the ‘lid’-helix fragment of the enzyme.

Separation and Quantitation of Low Amounts of Enzymatic Lipolysis Products Obtained in the Presence of High Triglyceride Concentrations

A method has been developed for the qualitative and quantitative determination of enzymatic lipolysis products in the presence of high triglyceride concentrations, using aminopropyl bonded silica cartridges and capillary gas chromatography. Two reactions are catalyzed in the presence of the Rhizopus javanicus lipase: hydrolysis of triglycerides to diglycerides, and diglycerides to monoglycerides. Lipids were extracted from the hydrolyzed olive oil emulsion and separated into fractions with aminopropyl bonded silica cartridges by means of sequential elution. The elution procedure was well characterized and optimized to obtain a maximum recovery and purity for each lipid fraction. Further analysis was done with capillary gas chromatography. The method is suitable to study the properties of lipases in vitro in the presence of high triglyceride concentrations and different amounts of bile salts. The expected inhibition of the fungal lipase by higher concentrations of bile salts, under physiological conditions, was limited.

Pancreatic dysfunction and treatment options.

Pancreatic steatorrhea and pancreatic diabetes are the dominant symptoms of patients in the decompensated stage of chronic pancreatitis (CP). In this stage, the nutritional state is greatly disturbed and hypoglycemia and labile infection are involved. Pancreatic enzyme replacement therapy is the principal treatment method for pancreatic steatorrhea. Before initiating this therapy, dietary fat intake must be determined and pancreatic lipase and bicarbonate secretion function must be evaluated. Upper small intestinal pH is regulated by gastric acid secretion, and abnormal gastric emptying changes lipolysis. In addition, precipitation of bile acids in the upper small intestine and ileal brakes due to undigested fats and carbohydrates must be considered. Porcine pancreatin, bacterial lipase, and acid-resistant fungal lipase are used as enzymes for replacement therapy. Conventional, entero-coating, and enteric-coated microsphere preparations of porcine pancreatin are available for treatment and are formulated to protect against gastric acids, to dissolve enzymes at optimum pH, and to be emptied simultaneously with food from the stomach. Gastric acid secretion suppressants, such as H2 blockers or a proton pump inhibitor, can also be used concomitantly with pancreatin preparations. In consideration of both strengths and weaknesses of these preparations, types and dosages of enzyme replacement therapy should be carefully prescribed, and fecal fats should be examined repeatedly by a simple and rapid method during treatment. Attention should also be paid to changes in body weight and nutritional indices (e.g., nutritional parameters, fat-soluble vitamins). The relationship between carbohydrate maldigestion/malabsorption in CP patients and treatment of pancreatic diabetes are topics for future research.

Nucleation and growth of microbial lipase crystals from clarified concentrated fermentation broths.

Bulk crystallization is emerging as a new industrial operation for protein recovery. Characterization of bulk protein crystallization is more complex than protein crystallization for structural study where single crystals are grown in flow cells. This is because both nucleation and crystal growth processes are taking place while the supersaturation falls. An algorithm is presented to characterize crystallization using the rates of the two kinetic processes, nucleation and growth. The values of these rates allow ready comparison of the crystallization process under different operating conditions. The crystallization, via adjustment to the isoelectric pH of a fungal lipase from clarified fermentation broth, is described for a batch stirred reactor. A maximum nucleation rate of five to six crystals formed per microliter of suspension per second and a high power dependency ( approximately 11) on the degree of supersaturation were found. The suspended protein crystals were found to grow at a rate of up to 15-20 nm/s and also to exhibit a high power dependency ( approximately 6) of growth rate on the degree of supersaturation.

Cardiolipin hydrolysis by human phospholipases A 2: The multiple enzymatic activities of human cytosolic phospholipase A 2

The ability of mammalian phospholipases A 2 (PLA 2) to hydrolyse cardiolipin (diphosphatidylglycerol) was monitored with a fluorescent displacement assay which allows the use of natural phospholipid substrates. The mammalian enzymes used were porcine pancreatic (Group I) secretory PLA 2 (sPLA 2), human non-pancreatic (Group II) sPLA 2 and human cytosolic PLA 2 (cPLA 2). High activity was observed with porcine pancreas sPLA 2 whereas the human sPLA 2 demonstrated only minimal activity with this substrate. In comparison, sPLA 2 from Naja naja venom (Group I) also showed only modest activity with this substrate. Since many lipases possess PLA 1 activity, a representative enzyme from Rhizopus arrhizus was also assessed for its ability to hydrolyse cardiolipin which proved to be a good substrate for this fungal lipase. In all cases dilysocardiolipin was the major product while some monolyso intermediate was detected after chromatographic separation. Human cPLA 2 was unable to hydrolyse cardiolipin at a significant rate, however, both monolysocardiolipin and dilysocardiolipin, which are prepared by the PLA 2-catalysed hydrolysis of cardiolipin, were good substrates providing a further example of the extensive lysophospholipase activity of this enzyme. Moreover, cardiolipin that was initially hydrolysed in situ with either excess porcine pancreatic PLA 2 or R. arrhizus lipase (PLA 1) was subsequently hydrolysed by human cPLA 2. One explanation of this result is that human cPLA 2 is able to hydrolyse both 1-acyl and 2-acyl-lysophospholipids.

Sucrose diacrylate: A unique chemically and biologically degradable crosslinker for polymeric hydrogels

A series of degradable hydrogels based on different vinyl monomers such as acrylamide, sucrose-1′-acrylate, and acrylic acid were synthesized using sucrose-6,1′-diacrylate (SDA) as a crosslinking agent. SDA was prepared by enzymatic transesterification of vinyl acrylate with sucrose in pyridine. Base catalyzed hydrolysis of SDA in aqueous solution was studied as a function of pH. As expected, hydrolysis of SDA was faster at higher pHs such that poly(acrylamide), poly(sucrose 1′-acrylate), and poly(acrylic acid) hydrogels underwent substantial degradation at and above pH 7, 9, and 13, respectively. The degradation was characterized by changes in the swelling ratios of the hydrogels indicating breakage of the crosslinking agent. Degradation of the hydrogels at their chemically stable pHs was studied in presence of enzymes. Enzymes, including pepsin and a fungal Lipase, were able to degrade the poly(acrylamide) hydrogel at pH 4 and 5, respectively. Poly(acrylic acid) hydrogel was degraded in presence of a fungal protease at pH 7.8. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 2221–2229, 1997