Interconversion of Two Lipases from Rhizopus delemar

Rhizopus (Rh.) delemar were cultivated under the various conditions and the productivity of three lipases (A, B, and C) were examined. There seemed to be no remarkable change of A-lipase production so far as examined, however, the productions of B- and C-lipases were changed with correlation depending on the composition of the medium. B-lipase was increasingly produced as much as C-lipase production decreased by the presence of phospholipid in a culture medium. The property of C-lipase was so changed by the incubation with phospholipid as to agree with that of B-lipase. Besides the above, the property of B-lipase was changed by its purification to that of C-lipase.

From these results, it seems that B-lipase protein is the same as that of C-lipase and a phospholipid is related to the intercoversion from C-lipase to B-lipase.     

The Purification and the Properties of Three Kinds of Lipases from Rhizopus delemer

The study was undertaken to clarify whether three kinds of lipases (EC 3.1.1.3) secreted from Rhizopus delemar are originally different or identical with each other. First of all, the purification of those lipases was carried out and their enzymatic properties were examined. Their properties including the stability on heat and pH, precipitabilities at a certain pH, the behaviours on a SE-Sephadex C50 column and on a Sephadex G200 column and so on were compared.

From the results, A-lipase is clearly different from the other two lipases. On the other hand, it seems that B- and C-lipases are originally identical.     

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     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Lipases of Fonsecaea pedrosoi and Phialophora verrucosa

Lipase activity was demonstrable titrimetrically in the culture filtrates of Fonsecaea pedrosoi and Phialophora verrucosa on the 6th day of incubation reaching a peak on the 15th and 12th days respectively for the two fungi. Purified lipases of F. pedrosoi and P. verrucosa, with specific activities of 36.0 and 39.4 fold increase respectively, were obtained by cold acetone extraction, gel filtration followed by ion exchange chromatography. The lipases had the same optimum pH (5.5) and temperature (35° C). The molecular weights of the lipases of F. pedrosoi and P. verrucosa, estimated by gel filtration on Sephadex G-100, were 25000 and 20000, respectively and the enzymes showed broad susbstrate specificity. The possible role of lipase in the pathogenesis of infection caused by the fungi is discussed.     

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Biochemical and Structural Characterization of Two Site-Directed Mutants of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> Lipase

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all enzymes retained their initial activities.     

Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90°C and between pH 6.5 and 10; the half-life of the enzymes at 75°C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53–2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.     

Purification and properties of the lipases from Rhodotorula pilimanae Hedrick and Burke

Summary The Rhodotorula pilimanae CBS 5804 strain secretes into the culture medium two lipases: their pH optima are 4 and 7. The two lipases were purified by precipitation with acetone followed by chromatography on SP-Sephadex C50 and Sephadex G200. The purification factors achieved in comparison with the supernatant culture were x74 for lipase I and x90 for lipase II. The molecular weights were estimated at 172,800 and 21,400 for lipase I and lipase II, respectively. Their activities are optimal between 45°C and 55°C. The activation energies were 5.9 kcal·mole^-1 for lipase I and 12.4 kcal·mole^-1 for lipase II. The inactivation energies were about 21.9 and 17.7 kcal·mole^-1 for lipase I and lipase II, respectively. The enzymes are slightly inhibited by Cu²⁺, Co²⁺, Hg²⁺, Mn²⁺, N-acetylacetone, acetic acid and sodium lauryl sulphate. EDTA did not affect their enzymatic activity. These two lipases are secreted in the culture media in the absence of inducer; their biosynthesis is not inhibited by glucose. These lipases hydrolyse primarily the 1-(or 3-)position of all triglycerides tested.     

Purification and partial characterization of two new cold-adapted lipases from mesophilic Geotrichum sp. SYBC WU-3

Two cold-adapted lipases (Lipase-A and Lipase-B in the paper) of mesophilic Geotrichum sp. SYBC WU-3 were purified by using (NH₄)₂SO₄ fractionation, chromatography separation on a DEAE-cellulose-32 column and a Sephadex G100 column. The molecular mass of Lipase-A and Lipase-B were determined to be approximately 41.1 and 35.8 kDa, respectively by SDS-PAGE. The optimum temperature for the activity of Lipase-A was found to be 20 °C, and that of Lipase-B was 15 °C. Lipase-A and Lipase-B had good stability when temperature was below 40 °C. Both the optimum pH for the activity of the lipases was 9.5. Lipase-A retained about 80% of its activity when pH was between 3 and 6 and Lipase-B maintained over 80% activity in the pH range of 3–8. The two lipases showed hydrolysis efficiency to various p-nitrophenyl esters, but they were more active with shorter p-nitrophenyl esters (C2 and C4).     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

pH-optima in lipase-catalysed esterification

Though lipases are frequently applied in ester synthesis, fundamental information on optimal pH or substrate concentration, can almost only be found for the reverse reaction – hydrolysis. This study demonstrates that the pH-optima of lipase-catalysed esterifications differ significantly from the optima of the hydrolysis reaction. In the esterification of n-butanol and propionic acid with lipases of Candida rugosa (CRL) and Thermomyces lanuginosa (TLL) pH-optima of 3.5 and 4.25, respectively, were found. This is about 3–4 units (CRL) and 7 units (TLL) in pH lower than optimum for hydrolysis. Enzyme activity increased with increasing concentrations of protonated acid indicating that the protonated acid rather than the deprotonated form is substrate for esterification. The rate of esterification can be drastically increased by ensuring acid concentrations up to 1000 mmol L^?1 for CRL and 600 mmol L^?1 for TLL in the reaction system.     

Enzyme production by the nematode-trapping fungus,Duddingtonia flagrans

Duddingtonia flagrans degrades peptides, proteins, starch, pectin, lipase, lecithin and oils when grown on agar medium. Serine proteases with optimal activity at pH 8.5 to 10.5 were produced when it was grown in submerged culture. It also produced phospholipase C with optimal activity at pH 8.5, lipases with high activity at pH 3.5 and at 7.5 to 8.5 and pectin-degrading enzymes with pH optima of 3 and 8. The pH of the culture medium affected the types of lipases and pectin degrading enzymes produced but not the proteases or phospholipase C.     

Synthesis of calix[n]arene-based silica polymers for lipase immobilization

The objective of this study was to prepare new calix[n]arene-based silica polymers for immobilization of Candida rugosa lipase. The amino functionalized calix[4]arene (C4P), calix[6]arene (C6P) and calix[8]arene (C8P)-based silica polymers were used for the covalent attachment of C. rugosa lipase using glutaraldehyde as a coupling agent. The characterization of synthesized CnP polymers and immobilized lipases were made by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. The hydrolytic activities of immobilized lipases (CnP-L) were evaluated and compared with the free enzyme. The activity recovery of immobilized CRL (C. rugosa lipase) based on the carrier C4P, C6P and C8P reaches 74.6%, 68.5% and 51.4%, respectively. The optimal pH and temperature region of the immobilized lipases for the hydrolysis of p-NPP were 7.0 and 50 °C. Nevertheless, the immobilized lipase has good stability, adaptability and reusability in comparison with the free enzyme.     

Proteolytic Activity in Commercial Triacylglycerol Hydrolase Preparations

The proteolytic activity of 34 commercial lipase preparations (CLP) was determined using a labeled casein substrate. Only three CLP were free from proteolytic activity. Porcine pancreatic lipases exhibited levels of proteolytic activity comparable to or greater than that of a reference porcine trypsin. Bacterial lipases contained up to 10% of the proteolytic activity of commercial trypsin. Proteolytic activities in lipases from fungal species were present at low levels (<1% of the activity in trypsin). Among preparations of fungal origin, lipases from Aspergillus niger and Mucor javanicus were highest in proteolytic activity; Aspergillus oryzae and Pseudomonas cepacia lipases were lowest. Proteins in CLP were separated by non-denaturing PAGE; between 4 and 17 protein bands in the range &#104 6.5- &#83 200 kDa were observed. With the exception of a single pair of Rhizomucor miehei lipases, the distribution of apparent molecular weights (AMW) was unique to each preparation. Bands of caseinolytic activity in commercial lipases were visualized by applying a zymographic technique. CLP contained between 0 (P. cepacia lipases) and 6 (porcine pancreas lipase and Rhizopus oryzae lipase) discrete proteolytic bands. Common themes of proteolytic AMW emerged, including 21-23 kDa and 30-35 kDa bands.     

Characterization of two novel lipase genes isolated directly from environmental sample

Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35°C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.     

Interesterification of butter fat by partially purified extracellular lipases from Pseudomonas putida,Aspergillus niger and Rhizopus oryzae

Three extracellular lipases were produced by batch fermentation of Pseudomonas putida ATCC 795, Aspergillus niger CBS 131.52 and Rhizopus oryzae ATCC 34612 during the late phase of growth, at 72, 96 and 96 h, respectively. The lipases were partially purified by (NH₄)₂SO₄ fractionation. The lipase of P. putida was optimal at pH 8.0 whereas those from A. niger and R. oryzae were optimal at pH 7.5. The A. niger lipase had the lowest V _max value (0.51×10^-3 U/min) and R. oryzae the highest (1.86×10^-3 U/min). The K _m values for P. putida, A. niger and R. oryzae lipases were 1.18, 0.97, and 0.98 mg/ml, respectively. Native PAGE of the partially-purified lipase extracts showed two to four major bands. The interesterification of butter fat by A. niger lipase decreased the water activity as well as the hydrolytic activity. The A. niger lipase had the highest interesterification yield value (26%) and the R. oryzae lipase the lowest (4%). In addition, A. niger lipase exhibited the highest decrease (17%) in long-chain hypercholesterolemic fatty acids (C12:0, C14:0 and C16:0) at the sn-2-position; the P. putida lipase demonstrated the least favourable changes in specificity at the same position.F. Pabai and S. Kermasha are with the Department of Food Science and Agricultural Chemistry, McGill University, 21111 Lakeshore, Ste Anne de Bellevue, Québec, H9X 3V9, Canada; A. Morin is with the Food Research and Development Centre, Agricultural and Agri-Food Canada, 3600 Casavant Blvd West, Saint-Hyacinthe, Québec, J2S 8E3, Canada.     

Characterization of neutral lipases revealed the tissue-specific triacylglycerol hydrolytic activity in Nilaparvata lugens

The lipid metabolism plays an essential role in the development and reproduction of insects, and lipases are important enzymes in lipid metabolism. In Nilaparvata lugens, an important insect pest on rice, triacylglycerol hydrolytic activities were different among tissues, with high activity in integument, ovary, and fat body, but low activity in intestine. To figure out the tissue-specific triacylglycerol hydrolytic activity, we identified 43 lipases in N. lugens. Of these 43 lipases, 23 belonged to neutral lipases, so this group was selected to perform further experiments on triacylglycerol hydrolysis. The complete motifs of catalytic triads, β9 loop, and lid motif, are required for the triacylglycerol hydrolytic activity in neutral lipases, which were found in some neutral lipases with high gene expression levels in integument and ovary, but not in intestine. The recombinant proteins of 3 neutral lipases with or without 3 complete motifs were obtained, and the activity determination confirmed the importance of 3 motifs. Silencing XM_022331066.1, which is highly expressed in ovary and with 3 complete motifs, significantly decreased the egg production and hatchability of N. lugens, partially through decline of the lipid metabolism. In summary, at least one-third of important motifs were incomplete in all neutral lipases with high gene expression in intestine, which could partially explain why the lipase activity in intestine was much lower than that in other tissues. The low activity to hydrolyze triacylglycerol in N. lugens intestine might be associated with its food resource and nutrient components, and the ovary-specific neutral lipases were important for N. lugens reproduction.     

Expression and characterization of a novel heterologous moderately thermostable lipase derived from metagenomics in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Seven lipolytic genes were isolated and sequenced from a metagenomic library that was constructed following biomass enrichment in a fed-batch bioreactor submitted to high temperature (50–70°C) and alkaline pH (7–8.5). Among those sequences, lipIAF1-6 was chosen for further study and cloned in Streptomyces lividans 10–164. The G+C content within the sequence was 64.3%. The encoded protein, LipIAF1-6, was related to various putative lipases previously identified in different genome sequences. Homology of LipIAF-6 with the different lipases did not exceed 31%. The optimum pH (8.5) and temperature (60°C) of the purified enzyme were in agreement with the enrichment conditions. Furthermore, the enzyme was thermostable for as long as 30 min at 70°C. The maximum activity of the purified lipase was 4,287 IU/mg towards p-nitrophenyl (p-NP) butyrate (60°C; pH 8.5). LipIAF1-6 does not seem to need the presence of metal ions for its activity. The enzyme was slightly inhibited by 10 mM CoCl₂ (14%), HgCl₂ (12%), and dithiothreitol (DTT) (15%). The serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF) reduced activity by 39% and 71% when incubated at concentrations of 1 and 10 mM, respectively. Finally, LipIAF1-6 was stable in different organic solvents, and against several surfactants and oxidative agents commonly found in detergent formulations. These results are quite encouraging for further use of this enzyme in different industrial processes.