Kinetics of Amycolatopsis mediterranei DSM 43304 lipase-mediated ...

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MATERIALS & METHODS Biocatalyst A. mediterranei DSM 43304 having high lipolytic activity was screened and optimized for lipase production as described by Dheeman et al. [13]. The extracellular lipase from A. mediterranei DSM 43304 was immobilized onto Celite matrix and the hydrolytic activity of the Celite-immobilized lipase was measured titrimetrically using an olive oil emulsion in 50 mM Tris-HCl buffer (pH 8) at 37 °C [14]. With this assay, the activity of Celite-immobilized lipase was 120 IU g -1 . The water content of the immobilized enzyme particles, by comparison of weight before and after heating at 105 °C for 12 h, was determined to be 2.2% (w/w). The same initial batch of immobilized lipase was used throughout the realization of this work. Esterification reaction Unless stated otherwise, synthesis of isoamyl acetate was carried out in a mechanically agitated glass reactor with 50 mL capacity. The reactor containing 300 mM of isoamyl alcohol and 750 mg Celite-immobilized A. mediterranei DSM 43304 lipase as a catalyst in n-hexane was placed in a thermostatic water bath providing a constant temperature to within ±0.1 °C. When the reaction temperature reached the set value (37 °C), acetic acid was added, to a final concentration of 300 mM, to initiate the reaction (total reaction volume 10 mL). A reaction in the same conditions without enzyme was realized in parallel and was used as a control. All experiments were performed in duplicate and replicated at least twice. The samples were analyzed on a gas chromatograph (Perkin Elmer AutoSystem XL, MA, USA) connected to a DB-5 capillary column (30 m × 0.25 mm, d f 0.25 µm, Agilent JW Scientific, CA, USA) and a flame ionization detector (FID). Kinetic modeling For kinetic modeling a total of 20 batch experiments were performed providing a total of 180 duplicate data points. Initial reaction rates were obtained from the experimental concentration-time profiles by regression of the linear portion of the kinetic data. The experimental concentration profiles were fitted to the kinetic models using the Levenberg-Marquardt nonlinear regression algorithm available in the ODRPACK [15]. The differential equations resulting from the different ester synthesis mechanism simplifications used in the nonlinear regression was simulated using the ODEPACK library [16]. RESULTS & DISCUSSION Effect of reaction parameters The effect of varying the enzyme loading on the rate of reaction was investigated by gradually increasing the mass of enzyme from 0.25 to 1.50 g of enzyme (i.e., from 0.83 to 5.0 g of enzyme mol -1 of the limiting substrate) at a constant agitation of 120 rpm. The results showed that the reaction rate increased linearly with increased enzyme loading. For the enzymatic synthesis of other esters, similar behaviour was found in the literature [5, 17]. The curves overlapped for larger enzyme loadings (≥5.0 g mol -1 ), suggesting there was no free substrate to bind with the excess of enzyme or external mass transfer resistance was limiting the rate. Such a rate limitation was also reported for the enzymatic synthesis of ethyl palmitate, isoamyl acetate, and octyl acetate [5, 18, 19]. Since there was no significant increase in rate and conversion with increased enzyme loadings (>2.5 g mol -1 ), further experiments were performed using enzyme loadings of 2.5 g mol -1 (7.5%, w/v). In the present study, preliminary experiments were performed to ensure the absence of external and internal diffusion limitations in all experiments. The effect of speed of agitation on initial rate and conversion was studied over the range of 80 to 250 rpm. It was found that both the initial rate and conversion increased with agitation speeds from 80 to 200 rpm with no significant increase above 200 rpm, indicating the reaction rate and conversion were no longer limited by mass transfer limitations of immobilized enzyme at 200 rpm. However, above 200 rpm, there was no significant increase in reaction rate and conversion. In order to assess the influence of initial concentration of substrates on the reaction kinetics, with the aim ofoptimizing initial rate and conversion, experiments with different substrate molar ratios were examined. The enzyme loading (2.5 g mol -1 ) and the concentration of isoamyl alcohol were kept constant in this set of experiments. The initial rate of reaction and equilibrium conversion increased with increasing acetic acid concentration up to a critical value (600 mM); however, further increase of acetic acid concentration above 600 mM, i.e, for an acetic acid/isoamyl alcohol molar ratio >2, resulted in decreased initial rate and equilibrium conversion. The decreased initial rates and conversion may be due to the formation of ineffective enzyme-substrate complexes at high substrate concentrations [12, 17]. Moreover, polar substrates tend to accumulate in the micro-queous environment of the suspended enzyme and may reach concentration levels
sufficient to cause denaturation of the enzyme molecule [20]. The present results are consistent with the reported inhibition of lipases by acetic acid during synthesis of esters [7, 21]. Water can have a dramatic influence on biocatalytic processes in non-aqueous media, depending upon the nature of the organic solvent used as a suspension medium [22, 23] and the form of enzyme protein employed [24]. Several mechanisms have been proposed for water-induced enzyme activation in nonaqueous milieu. It may act as a molecular lubricant, increasing internal flexibility of the enzyme [25] reducing unfavourable protein-protein interactions, responsible for structural distortions and aggregation or it may increase the active site polarity [23, 24] leading to enhanced enzyme activity in non-aqueous solvents. In the present study, the initial rate and equilibrium conversion were highest when 100 µL of water (1%, v/v) was added to the reaction mixture under otherwise similar conditions. Water accumulation during the esterification reaction possibly caused a decrease in the initial rate and equilibrium conversion, when the amount of initially added water was more than 100 µL. Thus, due to its effect on the thermodynamic balance of the reaction, an amount of water more than an optimum value (100 µL) resulted in decreased initial rates and equilibrium conversion indicating that hydrolysis outweighed synthesis (esterification). Reaction temperature has a profound effect on reaction rates, equilibrium conversion and enzyme stability [19, 26]. However, the effect of temperature on equilibrium conversion and initial rate is difficult to predict because it may influence the reaction efficiency depending on the source of the enzyme [26, 27], type of immobilization [26] and ionization state of the enzyme active site [28]. In accordance with transition state theory, increasing temperature would result in a positive effect on the rate constant; however, high temperatures may disrupt enzyme tertiary structure resulting in loss of catalytic activity [5]. Therefore, in view of the fact that at temperatures ≥60 °C the immobilized enzyme inactivates faster over the time period of the reaction (72 h) and the boiling point of the reaction mixture occurred at 69 °C, the influence of the temperature on the reaction kinetics was investigated in the temperature range of 30 to 50 °C. Since Celiteimmobilized A. mediterranei DSM 43304 lipase is thermostable, ester synthesis was observed at all temperatures from 30 to 50 °C and the initial rates and equilibrium conversions were found to increase with the temperature with a maximum rate and equilibrium conversion obtained at 50 °C. This suggests the predominance of kinetic effect in this temperature range [5, 17]. Esterification using optimal parameters A sequential strategy of experimental design proved to be useful in determining the conditions for maximizing the equilibrium conversion in n-hexane using Celite-immobilized A. mediterranei DSM 43304 lipase as a catalyst. Optimum conversion was obtained at an acetic acid/isoamyl alcohol molar ratio of 2, initial addition of 1% (v/v) of water and 7.5% (w/v) of enzyme (i.e. 2.5 g of enzyme mol -1 of alcohol) at 50 °C. Under these conditions, a 12 h reaction time was sufficient to reach the equilibrium molar conversion of 59%; however under non-optimized operational conditions the equilibrium molar conversion reached was 21% after 36 h of reaction time (Figure 1). Widely different conversion yields of isoamyl acetate in organic solvent systems have been reported in the literature. Hari Krishna et al. [29] used n- heptane for the synthesis of isoamyl acetate and obtained a conversion yield of >80% in 72 h using Rhizomucor miehei Lipozyme IM- 20 at 6.7 g mol -1 of substrate, whereas a maximum conversion yield of 100% was reported by Romero et al. [5] using Candida antarctica Novozyme 435 at 13.8 g mol -1 of substrate in n-hexane. Liaquat and Owusu Apenten [30] used n-hexane during esterification to obtain a conversion yield of 30% in 48 h with rapeseed seedling lipase at 200 g mol -1 of substrate. Furthermore, Larios et al. [31] reported a conversion yield of 74% for the synthesis of n-butyl acetate in n-hexane using C. antarctica lipase B at 50 g mol -1 of substrate. In the present investigation a non-commercial Celite-immobilized lipase at 2.5 g mol -1 of substrate was employed to achieve a conversion yield of 59% in 12 h, indicating a significant esterification at a lower enzyme concentration. Kinetic model based on time-course measurements Several studies have reported the kinetic modeling of lipase mediated esterification [10, 12, 32] and transesterification [17, 33, 34] reactions in organic media using commercially available immobilized lipases. However no attempt has been made to develop a kinetic model for the direct esterification using a noncommercial lipase. To the best of our knowledge the present study is the first report on the development of a kinetic model for direct esterification reaction in organic solvent catalyzed by a non-commercial actinomycete lipase. Esterification reactions of various organic acids with different alcohols by a variety of commercial lipases are often modeled using the so called Ping Pong Bi Bi mechanism, a well known and widely accepted mechanism for lipase-catalyzed reactions [9-11, 17]. In the present investigation, the Ping Pong Bi Bi mechanism coupled with a competitive inhibition by one of the substrates was assumed as a basis
Page 1: Kinetics of Amycolatopsis mediterra
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Kinetics of Amycolatopsis mediterranei DSM 43304 lipase-mediated ...

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