A model for lipase production by Candida rugosa

A model adequately describing the lipase production by Candida rugosa has been developed, calibrated and validated using new experimental data. Process modelling has been done using CAMBIO software (Computer Aided Modelling of BIOprocesses), allowing to easy and interactively test various hypothesis and reaction schemes.Olive oil, oleic acid and glycerol has been used as substrates. The model satisfactorily describes the time evolution of biomass growth as well as lipase production in all cases. In particular diauxic behavior is successfully characterized.Model development process has helped in obtaining a 3-fold increase in lipase production when using oleic acid as substrate instead of the original olive oil used.List of Symbols Oil g/l Oil concentration - Fa g/l Fatty acids concentration - Gly g/l Glycerol concentration - Cr g/l Biomass (dry weight) - Lp U/ml Lipase - _p Oil hydrolysis rate - _gly Uptake rate on glycerol - _fa Uptake rate on fatty acids - _lp Increase rate of lipase - Y _ca Biomass/Fatty acids yield - Y _cg Biomass/Glycerol yield - Y _la Lipase/Fatty acids yield - k _l Specific growth rate on fatty acids - K _c Saturation constant - K _I Inhibition constant for lipase - k11 Specific growth rate on glycerol - k ₃ Oil hydrolysis parameter     

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.     

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.     

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.     

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.     

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.     

Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes

The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

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.     

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.     

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.     

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     

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.     

Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.     

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。     

Structured modeling and state estimation in a fermentation process: Lipase production by Candida rugosa

A simple structured mathematical model coupled with a methodology of state and parameter estimation is developed for lipase production by Candida rugosa in batch fermentation. The model describes the system according to the following qualitative observations and hypothesis: Lipase production is induced by extracellular oleic acid present in the medium. The acid is transported into the cell where it is consumed, transformed, and stored. Lipase is excreted to the medium where it is distributed between the available oil-water interphase and aqueous phase. Cell growth is modulated by the intracellular substrate concentration. Model parameters have been determined and the whole model validated against experiments not used in their determination. The estimation problem consists in the estimation of three state variables (biomass, intra- and extracellular substrate) and two kinetic parameters by using only the on-line measurement provided by exhaust gas analysis. The presented estimation strategy divides the complex problem into three subproblems that can be solved by stable algorithms. The estimation of biomass (X) and the specific growth rate (mu), is achieved by a recursive prediction error algorithm using the on-line measurement of the carbon dioxide evolution rate. mu is then used to perform an estimation of intracellular substrate and the other kinetic parameter related to substrate transport (A) by an adaptive observer. Extracellular substrate is then evaluated by means of the estimated values of intracellular substrate and biomass through the material balance of the reactor. Simulation and experimental tests showed good performance of the developed estimator, which appears suitable to be used for process control and monitoring. (c) 1995 John Wiley & Sons, Inc.     

Enhanced activity and enantioselectivity of Candida rugosa lipase immobilized on macroporous adsorptive resins for ibuprofen resolution

The lipase from Candida rugosa was immobilized on three commercially available macroporous adsorptive resins for kinetic resolution of ibuprofen. One resin, CRB02, increased the enzyme activity by 50% to 0.027 g g(-1) min(-1). The deactivation constant (0.19 h(-1)) of the immobilized enzyme was half of that of the native enzyme and the enantioselectivity (E = 29.2) of the immobilized lipase was 2.2 times as much as that of the native lipase for the kinetic resolution of ibuprofen with 1-propanol in isooctane at 30 degrees C.