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     

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.     

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.     

Continuous synthesis of esters by cell-bound fungal lipases in an organic solvent

Summary Strains of Penicillium cyclopium and Rhizopus arrhizus secrete two extracellular lipases and contain an intracellular lipase. As these intracellular enzymes exhibited good synthetic activities in organic solvent, we designed a loop fixed-bed reactor for the continuous synthesis of esters. In a preliminary study we optimized the yield of ester synthesis using response surface methodology. With dodecanoic dodecyl ester as a model compound, the yields of ester synthesis were higher than 90%. It has been demonstrated that the reactor designed for this study is more efficient than a stirred batch reactor and more efficient than a fixed-bed reactor without a loop current or without a second catalytic column. In application, we have shown that lipases from R. arrhizus and P. cyclopium do not esterify tertiary alcohols like many lipases. Correspondence to: L. C. Comeau     

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.     

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.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.     

Surface-Bound Nuclease of Staphylococcus aureus: Localization of the Enzyme

The cellular localization of staphylococcus nuclease, previously known as an exoenzyme, was investigated, and the following results were obtained. (i) When Staphylococcus aureus cells were converted to protoplasts by cell wall lytic enzyme L-11 (a bacteriolytic enzyme purified from Flavobacterium sp. which specifically hydrolyzes amide and peptide linkages of murein layers), over 80% of the cell-bound nuclease was released into the surrounding sucrose medium. (ii) The cell-bound nuclease was associated with the cell-wall membrane fraction of mechanically disrupted cells. (iii) The nuclease activity of cell-wall membrane fractions from cells during early and late stages of protoplast formation were compared. Less activity was found in the late stage. These results suggest that nuclease may be located at or near the surface of the cells. The distribution of cell-bound nuclease in the cell-wall membrane fraction varied with the growth conditions of S. aureus. The activity of alkaline phosphatase, another surface enzyme, was also investigated. Less of this enzyme than nuclease was released when the cells were converted to protoplasts.     

In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus

Many surface proteins of Gram-positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C-terminal sorting signal. To achieve cell-wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell-wall-spanning region and cell-wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell-wall anchoring of enzymatically active lipase. The cell-wall-immobilized lipase (approximately 10000 molecules per cell) retained more than 80% of the specific activity, compared to the C-terminally unmodified S. hyicus lipase secreted by S. carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the influence of the distance between the cell-wall sorting signal and the C-terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the pep-tidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coliβ-lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S, carnosus. These results demonstrate that it is possible to immobilize normally soluble enzymes on the cell wall of S. carnosus - without radically altering their catalytic activity - by fusing them to a cell-wall-immobilization unit, consisting of a suitable cellwall-spanning region and a standard cell-wall sorting signal.     

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.     

Synthetic activity enhancement of membrane-bound lipase from <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> by pretreatment with isooctane

The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase™ firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4°C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.     

Cell surface and cellular debris-associated heat-stable lipolytic enzyme activities of the marine alga Nannochloropsis oceanica

Microbial surface display of lipases can be effectively employed for the development of whole-cell biocatalysts for industrial bioconversions. In the present work, we report for the first time the presence of thermostable lipolytic enzyme activities against p-nitrophenyl laurate, both on the cell surface and the cellular debris fraction of the marine microalga Nannochloropsis oceanica (strain CCMP1779). Whole cell-associated lipolytic activity (WCLA) shows a 2.5-fold stimulation after heat treatment at 100?°C for 60?min, while the activity of the respective cell debris is retained for 15?min. In contrast, heat treatment renders the soluble fraction of the disrupted cells inactive. The progress curve of cellular debris-associated lipase activity is biphasic and levels off very fast. Treatment with the surfactants SDS, Triton X-100 and CHAPS, which are known to inhibit lipase activity in various degrees, results in a loss of both cell bound and cell debris lipolytic activities (CDLA). The highest whole cell lipase catalytic efficiency was observed against p-nitrophenyl butyrate and the optimum pH for hydrolysis was determined at pH 7.0. Both unheated and heated undisrupted whole cell biocatalysts are also catalytically active against olive oil. High-salt concentrations (1M NaCl) lead to about 50% whole cell enzyme inhibition whereas the activity of heated cells increases. These findings offer novel insight into the biocatalytic properties and the biotechnological applicability of microalgal lipases from N. oceanica.     

The Comparison of the Properties of Two Lipases from Penicillium cyclopium Westring

Penicillium cyclopium Westring produced two kinds of lipases (EC 3.1.1.3) (A and B). At the begining of the cultivation, activity of B-lipase was detected more than that of A-lipase, though only A-lipase was accumulated at the last stage. The pH stability of B-lipase suggested that the eventual decrease of B-lipase activity was brought about by undesirable pH of culture fluid.

Two lipases were isolated respectively in homogeneous states and characterized. The pI’s of A- and B- lipases were 4.96 and 4.15 and their molecular weights were 27000 and 36000, respectively. In addition, the substrate specificities of two lipases were significantly different from each other.     

New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases

A Photobacterium strain, M37, showing lipolytic activity, was previously isolated from an intertidal flat of the Yellow Sea in Korea and identified as Photobacterium lipolyticum sp. nov. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,023 bp) corresponded to a protein of 340 amino acid residues with a molecular weight of 38,026. No sequence similarity was found with any known bacterial lipases/esterases; instead, the most similar enzymes were several filamentous fungal lipases. Although the similarity was very low (less than 16%), there were many conserved regions over the entire sequence and N-terminal oxyanion hole (RG) region, a signature sequence of filamentous fungal lipases. The novel protein M37 was produced in both a soluble and insoluble form when the Escherichia coli cells harboring the gene were cultured at 18°C. The soluble protein exhibited lipase activity in a pH-stat assay using an olive oil emulsion. The M37 lipase also displayed a maximum activity at 25°C and maintained its activity at a low temperature range (5–25°C) with an activation energy (E _a) of 2.07 kcal/mol. Accordingly, these results indicate that the M37 lipase from P. lipolyticum sp. nov. is a new cold-adapted enzyme.     

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.     

High yield <Emphasis Type="Italic">Rhizopus chinenisis</Emphasis> prolipase production in <Emphasis Type="Italic">Pichia pastoris</Emphasis>: Impact of methanol concentration

Rhizopus lipases have been successfully expressed in Pichia pastors and different fermentation strategies have been investigated. However, there is no sufficient study on the effects of methanol concentration on the production of Rhizopus lipases in P. pastors. In this study, the lipase from Rhizopus chinensis CCTCC M20102 was expressed under different fed-batch fermentation conditions at methanol concentrations ranging from 0.5 to 3.5 g/L. The lipase activity, stability, and productivities were analyzed. The optimum methanol concentration was 1 g/L, with the highest lipase activity of 2,130 U/mL, without degradation. Additional information was obtained from the analysis of methanol consumption and production rates. The results also suggested that the cell concentration at the end of the glycerol fed-batch phase was very important for cell viability and protease activity.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

Isolation of a novel lipase producing fungal isolate Aspergillus fumigatus and production optimization of enzyme

Abstract

Fungal lipases occupy a place of prominence among biocatalysts owing to their novel, multifold applications and resistance to high temperature and other operational conditions. In the present study, Aspergillus fumigatus isolated from oil-contaminated soil produced good amount of lipase activity with galactose (1%) as carbon source and peptone (0.1%) as nitrogen source after 72?h of incubation in the production medium at 45?°C and pH 10.0. The isolated enzyme was found to give its optimum reaction temperature at 40?°C and pH 9.0 with the substrate used as p-nitrophenyl benzoate. The activity of lipase was inhibited by the presence of metal ions. A 6.68-fold increase for lipase production was obtained by one variable at a time. Based on the findings of present study, lipase of A. fumigatus is a potential lipase and a candidate for industrial applications such as bioremediation, detergent, leather and pharmaceutical industries.     

Inhibition of Incorporation of l-Alanine into Uridine-diphospho N-acetylmuramic Acid by Glycine

Lipases from Aspergillus niger and Rhizopus delemar hydrolyzed triolein and produced l,2 (2,3)-diolein and 2-monoolein. These two lipases appears to have strong specificity towards the outer chains of the triglyceride. Comparing the proportions of fatty acids in position 1 (3) of cocoa butter with proportions of fatty acids liberated after limited hydrolysis of cocoa butter, it becomes clear that these two lipases do not hydrolyze the ester bond in position 2 of the triglyceride.

On the other hand, lipases from Geotrichum candidum Link and Penicillium cyclopium Westring attacked the fatty acid chains regardless of their positions. Geotrichum candidum lipase liberated oleic acid and palmitic acid in preference to stearic acid from cocoa butter.