Mixed-solid substrate fermentation. A novel process for enhanced lipase production by Candida rugosa

Candida rugosa was cultivated in a mixed-solid substrate containing coconut oil cake (COC) and fine and coarse wheat bran (1:1:1) with an initial water activity (a_w) of 0.92. The substrate was modified by adding a mineral solution (5%), corn steep liquor (6%), maltose (2%), peptone (3%), olive oil (10%), gum arabic (0.4%), different fatty acids (0.3%) and Tweens (0.5%). Fermentation in a column fermenter significantly improved the lipase yield to 118.2 Units per gram of dry fermented substrate [U/gds] at 72 h. This result was obtained 24 hours earlier than in our former studies (87.76 U/gds at 96 h) in COC, and the yield showed a 38% increase. Growth was measured indirectly by determining the glucosamine content in the cell wall of the yeast contained in the fermented matter, after its hydrolysis.     

Effect of different carbon sources on lipase production by Candida rugosa

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

Effects of different fatty acids in lipase production by Candida rugosa

Summary Oleic acid has been reported as a good inducer of lipase production by Candida rugosa. In order to know if this enzyme is induced by oleic acid itself or by a metabolite, different short chain fatty acids were tested. Butyric acid was the best carbon source to growth microorganism but it did not induce lipase production. Although caprylic and capric acid were the best inducers of lipase production, at concentrations up 1 g/l they have toxic effect in Candida rugosa growth. Thus, from the point of view of industrial production oleic acid could be considered as the best substrate tested.     

Strategies for the production of lipase by Candida rugosa; neural estimation of biomass and lipase activity

Summary Various strategies for the production of lipase by Candida rugosa Diddens and Lodder (ATCC 14830) in stirred tank reactors were investigated. The strain was first screened for lipase production with agar plate assay based on the fluorescent dye Rhodamine B, followed by adaptation to the production medium used. The highest lipase activity of 7.6 U/ml was obtained on a medium containing 40 g olive oil/l and 1 g glucose/l, with supplying pure oxygen. Neural networks were used in the estimation of biomass and lipase activity.     

Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies

Simulation studies have predicted that maximum lipase activity is reached with fed-batch operation strategies. In this work, two different fed-batch operational strategies have been studied: constant substrate feeding rate and specific growth rate control. A constant substrate feeding rate strategy showed that maximum aqueous lipolytic activity (55 U/mL) was reached at low substrate feeding rates, whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. In the second fed-batch strategy studied, a feedback control strategy has been developed based on the estimation of state variables (X and mu) from the measurement of indirect variables such as CER by means of mass spectrometry techniques. An on-off controller was then used to maintain the specific growth rate at the desired value by adjusting the substrate feeding rate. A constant specific growth rate strategy gave higher final levels of aqueous lipolytic activity (117 U/mL) at low specific growth rates. At higher specific growth rates the enzyme remained accumulated inside the cell, as was observed with a constant feeding fed-batch strategy. With a constant specific growth rate strategy, lipase production by Candida rugosa was enhanced 10-fold compared to a batch operation. Purification studies have demonstrated that lipolytic and esterasic specific activity ratios of Candida rugosa isoenzymes can be modified by using different operational conditions. These studies have also showed that the isoenzymes obtained in a controlled growth rate strategy are around three- to four-fold more active than those obtained in a constant feeding rate strategy.     

Optimization of medium composition for lipase production by Candida rugosa NCIM 3462 using response surface methodology

A sequential optimization approach using statistical design of experiments was employed to enhance the lipase production by Candida rugosa in submerged batch fermentation. Twelve medium components were evaluated initially using the Plackett-Burman 2-level factorial design. The significant variables affecting lipase production were found to be glucose, olive oil, peptone, (NH4)2SO4, and FeCl3.6H2O. Various vegetable oils were tested in the second step, and among them, groundnut oil was found to be the best inducer for lipase production by C. rugosa. The third step was to identify the optimal values of the significant medium components with groundnut oil as the inducer using response surface methodology. The regression equation obtained from the experimental data designed using a central composite design was solved, and analyzing the response surface contour plots, the optimal concentrations of the significant variables were determined. A maximum lipase activity of 5.95 U.mL-1, which is 1.64 times the maximum activity obtained in the Plackett-Burman experimental trials, was observed. The optimum combination of medium constituents contained 19.604 g.L-1 glucose, 13.065 mL.L-1 groundnut oil, 7.473 g.L-1 peptone, 0.962 g.L-1 (NH4)2SO4, 0.0019 g.L-1 FeCl3.6H2O, and other insignificant components at the fixed level. A predictive model of the combined effects of the independent variables using response surface methodology and an artificial neural network was proposed. The unstructured kinetic models, logistic model, and Luedeking-Piret model were used to describe cell mass and lipase production. The parameters of the models were evaluated and the lipase production by C. rugosa was found to be growth associated.     

High lipase production by Candida rugosa is associated with G1 cells. A flow cytometry study

Growth of C. rugosa on three different culture media was analysed by laser flow cytometry to evaluate physiological growth conditions allowing effective lipase production. The highest productivity was associated with an increased proportion of cells in the G1 phase and was independent of the effect of the medium on lipase formation.     

Lipase production by immobilized Candida rugosa cells

Summary Immobilization of Candida rugosa cells on a solid support for extracellular lipase production has been explored. The use of Ca-alginate beads and of mixed matrix of polyurethane foam/Ca-alginate beads enabled us to operate a batch and a continuous four-phase fluidized bed bioreactor. Cells co-entrapped together with polyurethane into Ca-alginate did not show higher lipase production levels than the cells entrapped in Ca-alginate gels. The addition of gum arabic to the medium greatly enhanced lipase production without affecting the hydrodynamic operating conditions significantly. This fact demonstrates that the reactor system is limited in terms of organic substrate dispersion and direct contact with cells. Correspondence to: C. Solà     

A structural basis for enantioselective inhibition of Candida rugosa lipase by long-chain aliphatic alcohols.

Molecular modeling showed that the enantiomers of heptyl 2-methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast-reacting S-enantiomer may well occupy the previously identified acyl-binding tunnel in the active site of the lipase. By contrast, the slow-reacting R-enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long-chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2-methyldecanoic acid. Long-chain aliphatic alcohols may coordinate to the acyl-binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast-reacting S-enantiomer, thus resulting in a lowered enantioselectivity in the resolution.     

Structure and conformational flexibility of Candida rugosa lipase

Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat enzyme with a hexahistidine tag in a baculovirus-insect cell expression system, and purified the enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 micromol/min/mg protein. The purified recombinant enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the purified enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4x10(-3) (37 degrees C, pH 9.0). These experimental results with a purified enzyme preparation quantitatively confirmed the reversibility of the enzyme reaction previously observed with crude enzyme preparations.     

Two conformational states of Candida rugosa lipase.

The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole do not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap.     

Stability studies and effect of the initial oleic acid concentration on lipase production by Candida rugosa

The production of lipase by Candida rugosa in batch cultures was studied. The initial concentration of the carbon source employed, oleic acid, had an important effect on the final lipolytic activity levels. The maximum lipase/substrate yield and specific productivity obtained correspond to an initial oleic acid concentration of 2 g/l. At higher concentrations, up to 8 g/l oleic acid, specific productivity decreased. Lipase production was not observed below 1 g/l oleic acid. Lipase inactivation in culture broth due to surface forces and shear stress at the gas/liquid interface was not observed. There was no shear stress denaturation at stirring rates of 250, 500 and 750 rpm. No temperature inactivation was detected up to 50° C. Two different lipases with a similar molecular weight of 60kDa were purified from culture broth.     

New differences between isoenzymes A and B from Candida rugosa lipase

Water adsorption isotherms of pure lipases A and B from Candida rugosa are different and can be used to distinguish between the isoenzymes. The maximum esterification yield (50%, 20h) can be achieved at initial 0.9<1.0. Lipase B is more stereoselective (49% yield, 98% enantiomeri excess) than lipase A (47% yield, 72% enantiomerci excess) but both isoenzymes mainly esterify the (S) 2(4-isobutylphenyl)propionic acid (Ibuprofen).     

Enhanced activity of Candida rugosa lipase modified by polyethylene glycol derivatives

The derivatives of polyethylene glycol (PEG) were prepared by reacting PEG with propylene oxide to enhance its hydrophobicity and introduce a branched structure. The PEG derivatives were activated with cyanuric chloride and used to modify the lipase fromCandida rugosa. The maximum specific activity of lipase modified with the PEG derivatives was about 2-fold of that modified with PEG for the esterification of oleic acid and lauryl alcohol in hexane.     

Coconut cake – a potent substrate for the production of lipase by Candida rugosa in solid-state fermentation

Investigations were conducted with the aim of producing extracellular lipase from Candida rugosa by solid-state fermentation (SSF), using coconut oil cake (COC) as a solid substrate. To optimize production, various modifications were made to enrich the substrate by supplementing it with mineral solution, different carbon sources and several inorganic as well as organic nitrogen sources. Among them, urea (1%), peptone (3%) and maltose (5%) were found to be most suitable. Addition of olive oil (10%) encouraged lipase synthesis. The maximum lipase activity in the enriched substrate was 87.76 units per gram of dry fermented substrate [U/gds] compared to 25.81 U/gds in the raw cake at 96 h of fermentation, and growth was as high as 14.44 mg/gds of glucosamine. This was reached at 72 h in the enriched substrate. C. rugosa growth was calculated indirectly by estimating the glucosamine content in the cell wall after its hydrolysis. The enzyme yield was far better than any values reported as yet.     

Rapid purification of two lipase isoenzymes from Candida rugosa

Summary We describe a two-step method for the purification of two lipases (lipases A and B) from C. rugosa. The purification procedure includes Phenyl-Sepharose and Sephacryl HR 100 chromatographies. The enzymes obtained were pure according to criteria of specific activity and neutral sugar content.     

海藻糖对脂肪酶的保护机理及酶失活动力学

采用自制的磁性固定化酶(MIE),考察了高温下二糖类对酶的保护作用。结果显示:海藻糖对悬浮于水溶液中的MIE没有保护作用;而在高温干燥后,对酶的保护作用效果依次为:海藻糖>乳糖>蔗糖,支持‘玻璃态学说’;此外,采用两步失活动力学模型能够较好的拟合酶的失活过程,并且得到酶的失活速率常数k和半衰期t1/2,加入海藻糖和乳糖之后,MIE的半衰期分别增长了31和23倍。