1. Candida rugosa and Porcine Pancreatic Lipase Immobilization into
Calcium Alginate and Using for Aroma Esters
Gül Özyılmaz, Esra Gezer
Mustafa Kemal Üniversitesi, Fen-Edebiyat Fakültesi, Kimya Bölümü HATAY
ABSTRACT
Candida rugosa lipase (CRL) and Porcin Pancreatic lipase (PPL) were immobilized into calcium alginate (Ca-Alg) beads via entrapment and used to produce of three industrially important flavors: isoamyl acetate (banana flavour), ethyl valerate (green apple flavour) and butyl acetate (pineapple flavour) in hexane. İmmobilization conditions were optimized in terms of sodium alginate and CaCl2 concentrations by determination of enzyme coupling, activity and enzyme leaking in hexane medium. The best results were obtained as 2.5 % sodium alginate for both CRL and PPL, whereas as 2 M and 2.5 M CaCl2 for CRL and PPL, respectively. Different reaction parameters such as enzyme concentration at immobilization, the amount of immobilized lipase in esterification medium, temperature, substrate concentration and reaction time for enhancing ester formation were investigated. The best and worst ester yield was obtained for isoamyl acetate and ethyl valerate when PPL was used as catalyst.
INTRODUCTION
The increasing demand for fragrance and flavour esters using in the food, cosmetic and pharmaceutical industries make it necessary to find alternative way instead of exracting from their natural materials which are too scarce or expensive for commercial use. The industrial ester synthesis using chemical reactions are tedious, non-selective and consume a large amount of energy. Using enzymes for these synthesis processes allows products which have better odor and color to be attained [1]
Lipases (triacylglycerol hydrolase, EC ) are a family of enzymes that in their natural environment catalyze the hydrolysis of fats. However, under appropriate operation conditions, lipases have shown to be very active catalysts in esterfication reactions [2]. There are several studies about flavour ester synthesis in non-aqueous medium using lipase by by esterification [3-5]
The aim of this work is to produce three commercially important flavour esters: isoamyl acetate (banana flavour), ethyl valerate (green apple flavour) and butyl acetate (pineapple flavour) by using Candida rugosa lipase (CRL) and Porcine Pancreatic lipase (PPL) immobilized into calcium alginate gel (Ca-Alg). The effect of sodium alginate and CaCl2 concentration on immobilization was investigated. In esterification reactions firstly flavour ester synthesis was carried out at solvent-free system and in hexane medium to compare the effect of non-polar solvent. It was also investigated the effects of various esterification parameters such as enzyme concentration at immobilization, the amount of immobilized enzyme, temperature, concentration of substrates and reaction time.
MATERIAL AND METHODS
Material
Candida rugosa lipase, porcine pancreatic lipase, isoamyl alcohol (IAA), buthyl alcohol (BA), ethyl alcohol (EA), glacial acetic acid (AA), valeric acid (VA), sodium alginate (Na-Alg), and all other chemicals were purchased from Sigma Chemical Co., St. Louis, MO, USA.
Lipase immobilization into calcium alginate gel
A hundred milliliters of Na-Alg solution containing CRL (or PPL) was dropped by syringe into 200 ml of CaCl2 solution at 4C. As soon as the mixed solution was dripped into CaCl2 solution, calcium alginate (Ca-Alg) gels were formed by cross-linking. After 2 h hardening at 4C, Ca-Alg beads were separated from CaCl2 solution by vacuum filtration, washed twice with 25 ml of distilled water.
Optimization of immobilization parameters
Immobilization was carried out by using Na-Alg and CaCl2 solutions at different concentrations. Na-Alg: % (CaCl2 and enzyme concentration constant at 2 M and 2 mg ml-1, respectively.)
CaCl2 : M (Na-Alg constant at the best value)
Three parameters were investigated for each sample: The amount of bound enzyme, Esterification activity by using 4-nitrophenol and acetic acid [6] and Leaking the entrapped enzyme to hexane medium during 180 min
Flavor ester synthesis
Flavor esters were firstly synthesized at solvent-free medium and hexane medium to determine proper medium.
Flavor ester synthesis was carried out in screw-capped bottle using 0.1 g of Ca-Alg beads and 10 ml substrate mixture of 25 mM acid (AA or VA) and 25 mM alcohol (IAA, BA or EA) in hexane with shaking at 200 rpm. At the end of the 1 h of reaction time, unconsumed acid was extracted twice by 10 ml of 25 mM NaOH and then by water. NaOH content of extraction solutions were titrated using 25 mM HCl solution. Difference of HCl volumes between lipase containing Ca-Alg and enzyme-free Ca-Alg were used to calculate the amount of acid substrate participated in the esterification reactions.
The effect of esterification parameters
Ester synthesis was optimized in terms of by, Enzyme concentration used in immobilization (1-5 mg ml-1), The amount of Ca-Alg beads in flavor ester synthesis ( g), Reaction temperature (30-70 oC), Substrate (alcohol and acid) concentration ( mM) and Reaction time (1-48 h)
RESULTS AND DISCUSSION
Because cross-linking between alginate and Ca+2 ions leads to gelation, alginate and CaCl2 concentration are major parameters for enzyme gel entrapment [7]. The maximum activities were observed at 2.5 % Na-Alg concentration for both CRL and PPL. The highest activities were observed at 2 M CaCl2 and 2.5 M CaCl2 for PPL and CRL, respectively. Enzyme leaking was decreased by increase of Na-Alg and CaCl2 concentration.
Flavor esters were firstly synthesized at solvent-free medium and hexane medium and IAAc, EV and BAc yield in hexane medium was 34 %, 19% and 33 % more than those of solvent-free medium for CRL, respectively. The same tendency was observed for PPL synthesis: 14%, 42% and 6% more yield was obtained in hexane medium. Similar results related the increment the ester yields in organic medium were reported in literature earlier [8]. So, subsequent ester synthesis was carried out in hexane medium.
The effect of enzyme concentration at immobilization
The effect of the concentration of enzyme used during the immobilization step was studied and reported in Table given below: As seen, not only the amounts of the entrapped enzymes but also the esterification yield of Ca-Alg gels increased by increasing enzyme amount used in immobilization.
The effect of Ca-Alg amount
The production of IAAc and EV increased by increasing Ca-Alg amount up to 0.2 and 0.3 g, respectively, and then a slow decrease observed at higher enzyme amount (Fig 1).
In case of BA synthesis, ester yield was proportional by the amount of Ca-Alg used in hexane medium. 0.2 of Ca-Alg gel was used in esterification studies in subsequent experiments.
Figure 1 The effect of enzyme concentration using at immobilization
: Isoamyl acetate, :Ethyl valerate, ∆:Butyl acetate
The effect of temperature
As seen in Fig 2, The minimum effect of temperature on production was observed for EV especially at using CRL and the highest activity was observed at 50 and 45 C for CRL and PPL, respectively. In case of BA, maximum activities were observed at 45 and 50 C for CRL and PPL, respectively.
IAAc production was the highest at 40 C for both CRL and PPL, and after that temperature ester yield decreased dramatically.
Figure 2 The effect of temperature on ester yields.
: Isoamyl acetate, :Ethyl valerate, ∆:Butyl acetate
The effect of substrate concentration
As seen in Fig 3(a) and (b), the highest amount of BAc was achieved when 25 mM acid was used for both CRL and PPL. The BAc production was decreased sharply by increasing acid concentration after 25 mM. IAAc production was increased by increasing of AA concentration upto 75 and 50 mM for CRL and PPL, respectively. However, EV yield was increased by increasing VA concentration upto 100 mM. It was clearly concluded that AA concentration higher than 50 mM, a significant decrease in conversion yield was observed probably due to lipase denaturation by AA [5, 7, 8]. As can be seen in Fig 3(c) and (d), the most yields were obtained for IAAc at 25 and 50 mM of IAA concentration for CRL and PPL, respectively.
(b)
(d)
(a)
(c)
Figure 3 The effect of substrate concentrations on ester yields.
: Isoamyl acetate, :Ethyl valerate, ∆:Butyl acetate
While, the most amount of BAc was get at 50 mM of BA concentration for both CRL and PPL, this value was 75 mM EA for EV synthesis. The worst yield was observed for EV synthesis. It can be seen İn Fig 3 (c) and (d) that, PPL was more efficient than CRL to produce flavor esters at their optimal working conditions.
The effect of reaction time
As seen in Fig 4, ester production increased by time upto 10 h of reaction time and then slightly decreased while using CRL. Similar results were observed for PPL as production of EV and BAc, however, IAAc yield increased by increasing reaction time.
The best result was obtained for IAAc synthesis when PPL was used. However, the minimum yield was obtained for EV flavor when CRL was used as catalyst.
Figure 4 The effect of reaction time on ester yields.
: Isoamyl acetate, :Ethyl valerate, ∆:Butyl acetate,
Conclusion
Three industrial flavor esters were produced by using CRL and PPL immobilized into Ca-Alg gel. Immobilization conditions and production parameters were optimized. According to results, the best yield was obtained for IAAc when PPL was used, however the worst yield was get for EV while using CRL. PPL was more efficient for flavor ester synthesis than CRL.
Acknowledgements
The authors wish to thank the TUBITAK for supporting the this study (Project no: 107T919)
References
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(mg ml-1) CE
(mg g Ca-Alg-1)
Esterification yield of CRL
(mol ester h-1 g Ca-Alg-1)
Esterification yield of PPL
CRL
PPL
IAAc EV
BAc 1
6.3
4.8
100
200
150 83
175 2
12.3
6.2
165
290
250
108 3
20.9
8.9
190
375
300
168
325 4
29.9
12.9
275
196
450 5
31.6
13.2
308
475
360
210
400
Because the increment of ester production was lessened after 3 mg ml-1 enzyme concentration, immobilization of CRL and PPL were carried out using enzyme concentration at 3 mg ml-1 at following studies.