Continuous hydrolysis of olive oil by immobilized lipase in organic solvent

Lipase (EC 3.1.1.3) from Candida rugosa was immobilized with DEAE-Sephadex A50, Sephadex G50, Sephadex LH-20, Amberlite IRA94, and Amberlite XAD-7. The enzye immobilized with DEAE-Sephadex A50 was found to be most effective for continuous hydrolysis of olive oil in isooctane. For the continuous reaction, 0.2 g of dry immobilized enzyme was swollen with predetermined amount of water, and packed in a glass column reactor. When the organic solvent (Isooctane) containing olive oil substrate was cocurrently fed with aqueous buffer, the two phases were evenly distributed throughout the packed bed without surfactant supplement or prior mixing of the two phases. A small amount of the surfactant (AOT) was used only in packing procedure, and no additional surfactant was necessary thereafter. Effects of initial water content of the swollen gel, buffer types, and strength were examined in the continuous reaction. Our results suggest that the operational half-life was affected by desorption of the bound enzyme. Under the conditions of 20% olive oil in isooctane and 25 mM triethanolamine buffer (pH 7.0), operational half life was 220 h at 30 degrees C. The reactor was also operable with n-hexane, but the operational stability of the immobilized enzyme in n-hexane was only half of that in isooctane. Our results indicate that various enzyme carrier having hydrophilic or amphiphilic properties could be used for two-phase continuous reaction in packed-bed column, reactor without any surfactant supply or prior dispersion of the two immiscible phases. (c) 1992 John Wiley & Sons, Inc.     

The hydrolysis of triglycerides by immobilized lipase in a hydrophilic membrane reactor

In the present article a method is described to immobilize lipase from Candida rugosa on a hollow fiber membrane, and the use of such a system for the hydrolysis of lipids is reported. The membranes were ENKA hydro philic Cuprophan((R))-type hollow fibers, having a large specific surface area. The immobilized lipase exhibited a high stability: the half-life time was 43 days at a temperature of 30 degrees C. Furthermore, it is proved that kinetic studies can be carried out with this system, operated in a batch or continuous mode. The relation between conversion rate and degree of hydrolysis was determined. On this basis, a dynamic model of the process was developed that describes the relation between reaction conditions and the conversion rate.     

Continuous immobilized cell reactor for amide hydrolysis

Summary This article deals with continuous hydrolysis of acrylamide into acrylic acid using the wild-typeBrevibacterium sp. R312 which can hydrolyze all water-soluble amides into their corresponding acids. Biotransformation has been carried out in a fluidized bed reactor specially designed to obtain good contact conditions between cells entrapped into small calcium alginate beads (2–3 mm) and low-concentration acrylamide solutions (10–40g·l^–1). Different flow rates, biocatalyst loads and substrate concentrations have been investigated. Kinetic constants for the immobilized enzyme have been identified. It appears that the Michaelis constant does not change with operating conditions and remains roughly equal to the value obtained for free cells. In contrast, the maximum rate of hydrolysis is considerably decreased, as if only cells on the outskirts of beads were involved in the transformation. On the whole it is proved that corynebacteria cells could be usefully used for the bioconversion of amides in a continuous immobilized cell reactor; the higher the solid hold-up and/or the smaller the beads, the more efficient the biological transformation.     

Continuous hydrolysis of tallow with immobilized lipase in a microporous membrane

Thermostable lipase from Thermomyces lanuginosus was immobilized in untreated microporous membranes. Melted tallow pumped through the membrane did not wash the enzyme out. From 0.4 to 0.9% of the soluble activity remained after immobilization with half-lives of 1-2 months or more at 50 degrees C. Membranes can be acid/base washed and reloaded with enzyme with no adverse effects. Buffer was required for a long half-life, and recycling the buffer improved the mass transfer of glycerol out of the immobilized lipase reactor. Immobilized activity was unaffected when the pH of the aqueous product changed from 5.5 to 6.5.     

Hydrolysis of menhaden oil by a Candida cylindracea lipase immobilized in a hollow-fiber reactor

A lipase from Candida cylindracea immobilized by adsorption on microporous polypropylene fibers was used to selectively hydrolyze the saturated and monounsaturated fatty acid residues of menhaden oil at 40 degrees C and pH 7.0. At a space time of 3.5 h, the shell and tube reactor containing these hollow fibers gives a fractional release of each of the saturated and monounsaturated fatty acid residues (i.e., C14, C16, C16:1, C18:1) of ca. 88% of the corresponding possible asymptotic value. The corresponding coproduct glycerides retained over 90% of the initial residues of both eicosapentaenoic (EPA; C20:5) and docosahexaenoic (DHA; C22:6) acids. The half-life of the immobilized lipase was 170 h when the reactor was operated at the indicated (optimum) conditions. Rate expressions associated with a generic ping-pong bi-bi mechanism were used to fit the experimental data for the lipase catalyzed reaction. Both uni- and multiresponse nonlinear regression methods were employed to determine the kinetic parameters associated with these rate expressions. The best statistical fit of the uniresponse data was obtained for a rate expression, which is formally equivalent to a general Michaelis-Menten mechanism. After reparameterization, this rate expression reduced to a pseudo-first-order model. For the multiresponse analysis, a model that employed a normal distribution of the ratio of Vmax/Km with respect to the chain length of the fatty acid residues provided the best statistical fit of the experimental data.     

Hydrolysis of rice bran oil using immobilized lipase in a stirred batch reactor

Candida cylindracea lipase was immobilized by adsorption on acid washed glass beads. It was observed that protein loading of the support depends on the size of the particle, with smaller particle containing higher amount of protein per unit weight. Initial reaction rate linearly varied up to enzyme concentration of 17.25 U/mL. Amount of free fatty acids produced was linearly proportional up to the enzyme loading of 1650 μg/g of bead. Achievement of chemical equilibrium took longer time in the case of less protein loading. Degree of hydrolysis was found to decrease in second and third consecutive batch operations on repeated use of immobilized lipase.     

Inulin hydrolysis by an immobilized yeast-cell reactor

Whole cells of Pichia polymorpha have been shown to possess inulinase (2, 1-β-d-fructan fructanohydrolase, EC 3.2.1.7) activity. This activity was slightly different from that of the purified enzyme: optimum pH was modified, optimum temperature was higher and thermal stability was improved. Whole cell immobilization by adsorption on beech wood-shavings was straightforward. A reactor of this type permits the bioconversion of inulin into d-fructose (and d-glucose) with sufficient cell growth to ensure the stability of the system. A chicory extract was hydrolysed completely to a high fructose syrup during an experiment lasting 75 days.     

Effect of water on hydrolysis of olive oil by immobilized lipase in reverse phase system

Summary Candida rugosa lipase immobilized by adsorption on the swollen Sephadex LH-20 and Sephadex LH-60 could effectively hydrolyze olive oil at high concentration in a reverse phase system. Initial water content was found to be the most important factor that determines both the hydrolysis rate and the degree of hydrolysis; approximately 54% of water in the gels was readily utilized.     

Effect of solvents on hydrolysis of olive oil by immobilized lipase in reverse phase system

Summary Lipase fromCandida rugosa was immobilized by adsorption on three supports which could contain water available for the hydrolysis of olive oil in a reverse phase system. To select the most suitable solvent for this system, the effect of organic solvents on the stability and catalytic activity of immobilized lipase for the hydrolysis reaction has been examined. The results revealed that isooctane was superior to any other solvents tested in this study for enzymatic fat splitting in a reverse phase system. Also the effect of the solvent polarity on the hydrolysis of olive oil has been examined in detail using various organic solvents mixed with an equivolume of isooctane. It was found that the hydrolysis of olive oil by immobilized lipase was markedly affected by the polarity of reaction solvents.     

Maltodextrin hydrolysis in a fluidized-bed immobilized enzyme reactor

The present work deals with maltodextrin hydrolysis by glucoamylase immobilized onto corn stover in a fluidized bed reactor. An industrial enzyme preparation was covalently grafted onto corn stover, yielding an activity of up to 372 U/g and 1700 U/g for support particle sizes of 0.8 and 0.2 mm, respectively. A detailed kinetic study, using a differential reactor, allowed the characterization of the influence of mass transfer resistance on the reaction catalyzed by immobilized glucoamylase. A simple and general mathematical model was then developed to describe the experimental conversion data and found to be valid.     

Continuous glycerolysis of olive oil by Chromobacterium viscosum lipase immobilized in liposome in reversed micelles

Chromobacterium viscosum lipase which has adsorbed on liposome and solubilized in microemulsion droplets of glycerol containing a little amount of water could catalyze the glycerolysis of olive oil. Studies on the continuous glycerolysis of olive oil by the immobilized enzyme was done at 37 degrees C in continuous stirred vessel bioreactor with polysulfone membrane. The effect of the flow rate of substrate (olive oil) in isooctane on the conversion and composition of the outlet was investigated using high-performance liquid chromatography (HPLC). The conversion increased with decrease in the flow rate. And we studied the effect of water content in the glycerol-water-lipase solution on the glycerolysis reaction. The conversion to desirable products, mono- and di-olein, was improved without a substantial production of oleic acid at lower water concentrations, i.e., below 8.0% (w/v) which corresponds to a w(o) value of 0.97. At water concentration higher than 8.0% (w/v), the amount of free fatty acid was dramatically increased. Higher operational stability of the enzyme reactor, and the half-line of the enzyme continuous reaction was about 7 weeks.     

Continuous ethanol production by immobilized yeast in a fluidized reactor

Summary In order to minimize the adverse effect of CO₂ gas in a packed bed immobilized yeast reactor, a fluidized bed reactor was used for the continuous production of ethanol from glucose. Immobilized yeast was prepared by entrapping whole cells of Saccharomyces cerevisiae within a Caalginate matrix. It was found that the efficiency of the ethanol production in a fluidized bed reactor was 100% better than that for a packed bed reactor system. The alcohol productivity obtained was 21 g/l/hr in a fluidized bed reactor at 94% of conversion level.     

Continuous ethanol production by immobilized yeast reactor

Summary Saccharomyces cerevisiae yeast immobilized in calcium alginate gel beads was employed in packed-bed column reactors for continuous ethanol production from glucose or cane molasses, and for beer fermentation from barley malt wort. With properly balanced nutrient content or periodical regeneration of cells by nutrient addition and aeration, ethanol production could be maintained for several months. About 7 percent (w/v) ethanol content could be easily maintained with cane molasses diluted to about 17.5 percent (w/v) of total reducing sugars at about 4 to 5 h residence time. Beer of up to 4.5 percent (wv) of ethanol could be produced from barley wort at about 2 h residence time without any addition of nutrients.     

Continuous penicillin G hydrolysis in an electro-membrane reactor with immobilized penicillin G acylase

Penicillin G (2%, w/v in phosphate buffer, pH 8) was hydrolysed in a flow-through, miniature electro-membrane reactor with the penicillin G acylase immobilized in 5% (w/v) polyacrylamide (diam. 10 mm, thickness 2.6 mm, enzyme activity 24 U ml^–1). The conversion of penicillin G increased from 0.15 to almost 0.5 when the electric current applied to the reactor was changed from –600 to +600 A/m² with a substrate residency of 1 h. Symbols and abbreviations c _j ^p & concentration of component j in product stream (M) c _j ^s & concentration of component j in substrate stream (M) c _s ^o & substrate concentration at reactor inlet (M) C _j ^p=c _j ^p/c _S ⁰ & scaled concentration of component j in product stream C _j ^s=c _j ^s/c _S ⁰ & scaled concentration of component j in substrate stream i & electric current density (A/m²) j & reaction component, j P, Q or S P & main reaction product (6-aminopenicillanic acid) PGA & penicillin G acylase Q & side reaction product (phenylacetic acid) S & substrate (penicillin G) Y _s=C _P ^s+C _P ^p & substrate conversion & mean residence time of substrate and product streams in reactor (h) =C _Q ^s+C _Q ^p+C _S ^s+C _S ^s & check-sum of scaled concentrations =C _P ^p/(C _P ^s+C _P ^p) & separation factor of 6-aminopenicillanic acid (0 1)     

Lactose hydrolysis by immobilized beta-galactosidase in capillary bed reactor

The hydrolysis of lactose by immobilized beta-galactosidase was studied in a continuous-flow capillary bed reactor operating at 30 degrees C. Solutions containing 50, 100, and 150 g lactose and 0.5 g sodium acetate/L were fed to the reactor. Lactose conversions ranging from 24% to greater than 99% were achieved at reactor space times ranging from 0.06 to 6.3 min. These conversion data were successfully modeled in terms of a plug flow reactor model and a form of Michaelis-Menten kinetics which included competitive inhibition by both the alpha and beta forms of galactose.     

Continuous production of monoacylglycerols from palm olein in packed-bed reactor with immobilized lipase PS

A packed-bed reactor (PBR) system using immobilized lipase PS as biocatalyst was developed for continuous monoacylglycerols (MAG) production. The condition for continuous MAG production using immobilized lipase PS (IM-PS) of 1.5 g (550 U) in PBR (0.68 cm i.d., 25 cm long) was optimized. The effect of molar ratio of glycerol to palm olein, water content in glycerol and residence time on MAG production was investigated. The optimal glycerol to palm olein molar ratio and water content in glycerol were 12:1 and 10% (w/w), respectively. The yield of MAG increased with increasing residence time. At a residence time of 7.5 h gave the highest yield of MAG of 60%. The long-term operation gave the highest yield of MAG 61.5% at 24 h of the operation time with the productivity of 1.61 g MAG/day. A half-life of the long-term process was 35 days of the operation time with the productivity of 0.81 g MAG/day. Furthermore, the large scale of MAG production was performed continuously with IM-PS of 15 g (5500 U) in PBR (1.5 cm i.d., 50 cm long). The highest yield of MAG in large-scale operation of 70.1% and the 11-fold increasing in productivity of 18.3 g MAG/day were obtained at 24 h of the operation time.     

Continuous interesterification of butteroil and conjugated linoleic acid in a tubular reactor packed with an immobilized lipase

Modified milkfats were produced via interesterification (acidolysis) reactions of butteroil and conjugated linoleic acid (CLA) in a packed bed reactor containing an immobilized lipase preparation from Candida antarctica. The rate expression for the interesterification reaction is of the generalized Michaelis–Menten form. Significant enrichment of butteroil in CLA residues was accomplished at reactor space times (fluid residence times) of 2–4 h at 40–60°C, but the optimum operating temperature was ca. 50°C. Approximately 80–90% of the free CLA fed to the reactor can be converted to its esterified form.     

Continuous hydrolysis of sucrose by invertase adsorbed in a tubular reactor

Studies were made of invertase adsorption on Amberlite ion exchange resins. Up to 4000 units of adsorbed enzymatic activity (aea) were obtainedper g of IRA 93 resin; for an aea of 1600 units, the maximum ratio of aea over units of soluble enzyme used for adsorption was close to 50%. Nodesorption occurred during extensive washing at 30°C with 0.01M sodiumacetate buffer at pH 5. Progressive desorption of aea from the invertase–IRA 93 complex occurred when buffer molarity and temperature were increased. Desorption differed only slightly when the buffer pH was 3 or 5. Theoptimum pH of aea was 3.2 with IRA 93 resin, and varied between 3.2 and 5.1with other resins, depending on their anionic or cationic nature. Batch hydrolysis of sucrose by IRA 93–adsorbed invertase followed 1st order kinetics with respect to the substrate concentration, as in the case of soluble invertase. Continuous sucrose hydrolysis with IRA 93–adsorbed invertase was performed in a tubular reactor, and the percent conversion was experimentally determined as a function of the flow rate. The reaction was experimentally determined 50% (w/v) sucrose solution, at pH4 and 30°C; at the selected flow rate, the ratio of sucrose hydrolysis remained constant and close to 76%. This shows that invertase was not desorbed from the tubular reactor. Some continuous hydrolyses were performed with an industrial sucrose solution: enzymatic activity seemed to be stable for anextended period for time (1 month) at 30°C and pH 3 or 4.     

固定化脂肪酶催化毛油合成生物柴油

本研究开发了一种用石油醚提取毛油的工艺,研究了以提取的毛油和甲醇为原料,用固定化Candida sp.99-125脂肪酶催化合成脂肪酸甲酯(FAMEs)的可行性。同时考察了磷脂对固定化酶活性、反应起始速率、固定化酶使用批次的影响以及毛油和精炼油对固定化酶使用批次等的影响。研究结果表明,用磷脂质量分数为1%的石油醚悬液浸泡过的脂肪酶比仅用石油醚浸泡过的脂肪酶初始转酯化速率显著下降。当大豆油中无磷脂时,15min时FAMEs的产率为26.2%;磷脂质量分数为5%时,FAMEs降为12.4%。当大豆油中磷脂质量分数小于1%时,固定化酶使用10个批次,FAMEs产率无明显变化。固定化脂肪酶催化石油醚浸提得到的大豆和小桐子毛油,经过10个批次反应FAMEs产率都保持在70%以上,该固定化酶直接催化毛油生产生物柴油具有良好的工业化前景。