Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.     

Mass production of lipase by fed-batch culture of Pseudomonas fluorescens

Summary High concentration production of an extracellular enzyme, lipase, was achieved by a fed-batch culture of Pseudomonas fluorescens. During the cultivation, temperature, pH and dissolved oxygen concentration wwre maintained at 23°C, 6.5 and 2–5 ppm, respectively. Olive oil was used as a carbon source for microbial growth. To produce lipase effectively the specific feed rate of olive oil had to be maintained in a range of 0.04–0.06 (g oil) · (g dry cell)^-1 · h^-1. The CO₂ evolution rate was monitored to estimate the requirement of olive oil. The ratio of feed rate of olive oil to the CO₂ evolution rate was varied in the range of 20–60 g oil/mol CO₂. The higher value of the ratio accelerated microbial growth, but did not favour lipase production. Once the high cell concentration of 60 g/l had been achieved, the ratio was changed from 50 to 30 g oil/mol CO₂ to accelerate the lipase production. By this CO₂-dependent method a very high activity of lipase, 1980 units/ml, was obtained. Both the productivity and yield of lipase were prominently increased compared with a conventional batch culture.     

Effective production of Pseudomonas fluorescens lipase by semi-batch culture with turbidity-dependent automatic feeding of both olive oil and iron ion

Summary An automatic feeding system to supply olive oil in semi-batch culture was established by monitoring cell concentration with a laser turbidimeter combined with a microcomputer and a pulse motor. In this automatic feeding system, specific olive oil supply rate (g olive oil) · (g dry cell)^-1 · h^-1, q ₀, was changed in an appropriate range. Attempts were made to produce lipase by a turbidity-dependent automatic fed-batch culture of Pseudomonas fluorescens. It was found from the semi-batch cultures with turbidity-dependent feeding of olive oil and with varied initial Fe ion concentrations that excess Fe ion was inhibitory to formation of the lipase. Turbidity-dependent automatic simultaneous feeding of olive oil and Fe ion was performed to obtain semi-deficiencies of both the oily substrate in the culture liquid and Fe content of the cells. Using this semi-batch culture, high lipase activity, 5600 units/ml, was attained at an optimal value of q ₀.     

Production and Regulation of Lipase Activity from <Emphasis Type="Italic">Penicillium restrictum</Emphasis> in Submerged and Solid-State Fermentations

Different carbon (C) sources, mainly carbohydrates and lipids, have been screened for their capacity to support growth and lipase production by Penicillium restrictum in submerged fermentation (SmF) and in solid-state fermentation (SSF). Completely different physiological behaviors were observed after the addition of easily (oleic acid and glucose) and complex (olive oil and starch) assimilable C sources to the liquid and solid media. Maximal lipolytic activities (12.1 U/mL and 17.4 U/g) by P. restrictum were obtained with olive oil in SmF and in SSF, respectively. Biomass levels in SmF (12.2–14.1 mg/mL) and SSF (7.0–8.0 mg/g) did not varied greatly with the distinct C sources used. High lipase production (12.3 U/g) using glucose was only attained in SSF, perhaps due to the ability of this fermentation process to minimize catabolite repression.     

Lipase production by Trichosporon fermentans WU-C12, a newly isolated yeast

From the soil samples of various locations, 245 strains of microorganisms were isolated by the enrichment culture method using olive oil as a carbon source. Of these microorganisms one deuteromycotinous yeast was the best producer of extracellular lipase, and the strain WU-C12 was identified as Trichosporon fermentans from the morphological and taxonomical properties. When cultivated at 30°C for 4 d in the medium containing 8% (w/v) corn steep and 3% (v/v) olive oil as sources of nitrogen and carbon, T. fermentans WU-C12 produced 126 U/ml of extracellular lipase. When 3% (v/v) tung oil was used instead of 3% (v/v) olive oil, 146 U/ml of the lipase was produced. Although lipase production decreased to 40 U/ml by the addition of 2% (w/v) glucose to the corn steep-olive oil medium, the strain WU-C12 produced 34 U/ml of lipase in the medium containing 2% (w/v) glucose instead of 3% (v/v) olive oil. On the other hand, T. fermentans WU-C12 could grow and produce lipase in the medium containing n-paraffin as a carbon source.     

Inhibition of proliferation of retrovirus-immortalized macrophages by LPS and IFN-γ: Possible autocrine down-regulation of cell growth by induction of IL1 and TNF

The GG₂EE macrophage tumor cell line was previously established by immortalization of C3H/HeJ mouse bone marrow cells with the J2 retrovirus which contains the v-myc and v-raf oncogenes. Studies on the control of GGZEE cell proliferationin vitro have recently been performed. We observed that the combination of 5–25 U/ml recombinant mouse interferon- (rmIFN-) plus 0.03 – 0.3 µg/ml lipopolysaccharide (LPS) markedly inhibited the proliferation of GG₂EE cells (by >95%)in vitro, while either agent alone inhibited only by <40% and 0–10%, respectively. Subsequent studies established that biologically active ILI-like (2–4 U/ml) and TNF-like (50–100 U/ml) activities were released into the supernatants of LPS-treated GG₂EE cells. The combination of IFN- + LPS induced more (6–8 U/ml) ILI release. These results suggested that the inhibition of proliferation of GG₂EE cells by IFN- + LPS could have been mediated in part by cytokines produced by the cells themselves. rhIL1 at a concentration of 10 U/ml inhibited GG₂EE proliferation by 25–30%, while rmIFN- (25 U/ml) + rhIL1 (10 U/ml) inhibited proliferation by 98%. Thus, 10 U/ml rhIL1 could completely replace LPS in the LPS + rmIFN- combination. Further, the combination of low doses of rhIL1 (0.1 to 1 U/ml) plus rmTNF (250 U/ml), which together inhibited proliferation by <20% synergized with doses of 5 to 25 U/ml rmIFN- to inhibit proliferation of GG₂EE cells by 98–99%. These results suggest that cytokines produced by the cells themselves can synergize with rmIFN- to inhibit the oncogene-driven proliferation of GG₂EE cells.     

Production and potential applications of a xylanase from a new strain of Streptomyces cuspidosporus

Enzyme production by a new mesophilic Streptomyces isolate was investigated which grew optimally on 1% (w/v) xylan and 10% (w/v) wheat bran at pH 7 and 37 °C. Xylan induced only CMCase (0.29 U/ml) besides xylanase (22–35 U/ml, 40–49 U/mg protein). Wheat bran induced xylanase (105 U/ml, 17.5 U/mg protein), CMCase (0.74 U/ml), -xylosidase (0.009 U/ml), -glucosidase (0.026 U/ml), -L-arabinofuranosidase (0.049 U/ml), amylase (1.6 U/ml) and phytase (0.432 U/ml). The isolate was amenable to solid state cultivation and produced increased levels of xylanase (146 U/ml, 28 U/mg protein). The pH and temperature optima of the crude xylanase activity were 5.5 and 65 °C respectively. The pI was 6.0 as determined by PEG precipitation. The crude enzyme was applied in treatment of paper pulp and predigestion of poultry feed and was found to be effective in releasing sugars from both and soluble phosphorus from the latter.     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

Characterization of <Emphasis Type="Italic">Mucor miehei</Emphasis> lipase immobilized on polysiloxane-polyvinyl alcohol magnetic particles

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding. The resulting immobilized biocatalyst was recycled by seven assays, with a retained activity around 10% of its initial activity. K_m and V_max were respectively 228.3 M and 36.1 U mg of protein^–1 for immobilized enzyme. Whereas the optimum temperature remained the same for both soluble and immobilized lipase (45 °C), there was a shift in pH profiles after immobilization. Optimum pH for the immobilized lipase was 8.0. Immobilized enzyme showed to be more resistant than soluble lipase when assays were performed out of the optimum temperature or pH.     

Enhancement of enzymatic hydrolysis rate of olive oil in water by dimethyl β-cyclodextrin

Summary The hydrolysis rate of olive oil byCandida cylindracea lipase in an aqueous solution without surfactants can be increased up to 6-fold by the addition of up to 60 mg/ml of dimethyl -cyclodextrin (DMCD).     

Immobilisation ofCandida cylindracea lipase on a new range of ceramic supports

Summary Lipase fromC. cylindracea was covalently immobilised to a number of surface-treated ceramic supports (3–10 mg. (g dry wt support)^–1). At room temperature, the immobilised lipase could convert R,S-citronellol and butyric acid to citronellyl butyrate at rates in the range 7–51 mol. (mg lipase.min)^–1. The lipase maintained 90–100% of its initial activity over a period of 150 days.     

Solvent-free glycerolysis of palm and palm kernel oils catalyzed by commercial 1,3-specific lipase from Humicola lanuginosa and composition of glycerolysis products

Glycerolysis of palm and palm kernel oils were conducted using a commercial 1,3-specific lipase from Humicola lanuginosa (trade name: SP 398) as catalyst (500 units lipase g^–1 oil) at 40°C and oil:glycerol (1:2 mol mol^–1) in a solvent-free system. After 24 h, the glycerolysis products of palm and palm kernel oils consisted of 23% triacylglycerols, 18% monoacylglycerols, 38% diacylglycerols and 18% triacylglycerols, 31% monoacylglycerols, 42% diacylglycerols, respectively. The monoacylglycerol fraction of the glycerolysis product of palm oil was enriched in oleic acid. Palmitic acid content of the monoacylglycerol fraction of the same product was less than that of the original oil. Under the same conditions, monacylglycerol fraction of the palm kernel oil glycerolysis product was enriched in palmitic, stearic and oleic acids.     

Scanning electron microscopic studies of lipase-catalysed esterification catalysis for the synthesis of stearoyl lactate and p-cresyl laurate

The state of three lipases, two from Rhizomucor miehei and one from porcine pancreas, employed in the esterification reactions leading to the preparation of food additive esters were investigated by scanning electron microscopy (SEM). The lipases employed in the synthesis of stearoyl lactic acid and p-cresyl laurate in 10 ml solvent at 40–60 °C in shake-flask experiments and 150 ml in non-polar solvents at 50–60 °C in bench-scale level experiments were compared. All three lipases, which were subjected to high temperatures and non-polar solvents for a prolonged period of incubation of 72–120 h, showed decrease in the compactness when compared to unused lipase. The presence of buffer preserved the activity and compactness and the absence of the same reduced the amount of enzyme per unit area on the support. R. miehei lipase samples subjected to reaction in presence of 0.0004 ml of 0.1 M buffer/mg enzyme preparation at different pH values (4.0–9.0) showed a decrease in compactness of the enzyme on the surface which correlated to an increase in esterification activity. An increase in volume of buffer (0.0002–0.003 ml/mg enzyme preparation) in the reaction mixture at pH 7.0 showed a decrease in compactness and also a reduction in activity. The studies indicate that a compromise between pH and volume of buffer can lead to variation in the extent of adsorption, distribution and activity, enabling the achievement of maximum conversions in the esterification reactions.     

Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris

Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization. In this study, we utilized PCR to remove an unnecessary linker of pGAPZC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 in the Pichia expression system. Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6–38.4°C), glucose concentration (0.3–3.7%), yeast extract (0.16–1.84%), and pH (5.3–8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris. Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1°C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6. The predicted value of lipolytic activity was 246.9±39.7 U/ml, and the actual value was 253.3±18.8 U/ml. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.     

Mechanism of Overproduction of Secreted Enzymes in the Mycelial Fungus Penicillium canescens

The fungus Penicillium canescens strain F178 (VKPM) and its niaD ^– mutant exhibited an increased capability of synthesizing extracellular enzymes -galactosidase (50–60 U/ml) and xylanase (100 U/ml). The synthesis was induced by arabinose and its catabolite, arabitol. A deficiency in arabitol dehydrogenase, leading to arabitol accumulation in the cell, was detected in the chain of reactions of arabinose catabolism. The increased synthesis of -galactosidase and xylanase in P. canescens is accounted for by (1) cellular accumulation of the inducer (arabitol) at low concentrations of arabinose in the medium and (2) prevalence of induction over repression.     

Lipase-catalyzed synthesis of capsaicin analogs by transacylation of capsaicin with natural oils or fatty acid derivatives in n-hexane

Transacylation of capsaicin with triolein using a commercial lipase gave olvanil in an 85% yield at 70°C for 144 h. When olive oil was employed, the major product was olvanil (62%). Safflower oil gave a mixture of olvanil (39%) and linoleoyl vanillylamide (32%). Perilla oil gave linolenoyl vanillylamide (13%). Myristic acid and its methyl ester could be used as an acyl donor, and myristoyl vanillylamide was obtained in 20–78% using several lipases.     

Enzyme formation by a yeast cell wall lytic Arthrobacter species: formation of amylase

The kinetics of amylolytic enzyme formation by a yeast cell wall lytic Arthrobacter species were studied. Cultivation on autoclaved cells of baker's yeast showed that amylase formation was closely related to trehalose and glycogen dissimilation. Growth on yeast glycogen (0.5%) proceeded quite rapidly ( = 0.31 h^–1) with extensive amylase formation during exponential cell multiplication and a further low increase in activity during the stationary phase. Beside amylolytic activity [450 units (U) l^–1] the formation of a relatively high level of -glucosidase (90 U l^–1) was detected, the latter almost exclusively bound to bacterial cells. Growth on 0.5% trehalose occurred at a reduced rate ( = 0.22 h^–1) with post-logarithmic enzyme synthesis in the stationary phase. Amylase activity attained a level of 1200 U l^–1, whereas -glucosidase was very low at 7.7 U l^–1. Continuous culture experiments in the chemostat showed maximal volumetric productivity of amylase (105 U l^–1 h^–1) at a dilution rate of 0.15 h^–1. Growth on various carbohydrates revealed low levels of amylolytic activity (<100 U l^–1), which were increased by a -1,4-glucans and oligosaccharides such as starch, dextrin, maltotriose and maltose. On 0.5% maltose, growth-associated enzyme synthesis (230 U l^–1) was detected at a reduced growth rate ( = 0.14 h^–1). Amylolytic enzyme preparations from the culture fluid showed an unusual cleavage pattern; acting on starch, the polymer was almost completely hydrolysed to maltotriose and maltose in a molar ratio of 3:1.Correspondence to: W. A. Hampel     

Continuous production of glucoamylase by immobilized growing cells ofAureobasidium pullulans

Summary Yeast-like cells ofAureobasidium pullulans were immobilized in Ca-alginate gel beads and employed for continuous production of glucoamylase in a fluidized-bed reactor (250 ml working volume). After an activation time of 48 h, to allow the in situ germination of the fungal blastospores, the reactor was operated continuously for over 150 h. A steady state enzyme concentration of 1.2–1.3 U ml^–1 of glucoamylase activity and an enzyme volumetric productivity of ca. 130 U ml^–1 h^–1 were obtained at a medium flow rate of 26 ml h^–1. Enzyme activity and volumetric productivity were influenced by fermentation conditions such as inoculum size and airflow rate.     

Enzymatic fractionation of evening primrose oil by rape lipase: Enrichment of gamma-linolenic acid

Summary Rape lipase discriminates strongly against -linolenic acid (allcis-6, 9, 12–183; GLA). GLA in the fatty acids from evening primrose oil was concentrated from 9.5% to 62% with a 95% yield during esterification of these fatty acids with butanol catalyzed by rape lipase. Hydrolysis of evening primrose oil catalyzed by the rape lipase raised the GLA content in unhydrolyzed acylglycerols to 28%.