Catalyzed Ester Synthesis UsingCandida rugosaLipase Entrapped by Poly(N-isopropylacrylamide-co-itaconic Acid) Hydrogel

This study reports the synthesis of polymeric matrices based onN-isopropylacrylamide and itaconic acid and its application for immobilization of lipase fromCandida rugosa. The lipase was immobilized by entrapment method. Free and immobilized lipase activities, pH and temperature optima, and storage stability were investigated. The optimum temperature for free and entrapped lipase was found to be 40 and 45°C, while the optimum pH was observed at pH 7 and 8, respectively. Both hydrolytic activity in an aqueous medium and esterolytic activity in an organic medium have been evaluated. Maximum reaction rate (Vmax) and Michaelis-Menten constants (Km) were also determined for immobilized lipase. Storage stability of lipase was increased as a result of immobilization process. Furthermore, the operational stability and reusability of the immobilized lipase in esterification reaction have been studied, and it was observed that after 10 cycles, the residual activity for entrapped lipase was as high as 50%, implying that the developed hydrogel and immobilized system could provide a promising solution for the flavor ester synthesis at the industrial scale.

Download Full-text

Improved Catalytic Performance of Carrier-Free Immobilized Lipase by Advanced Cross-Linked Enzyme Aggregates Technology

10.21203/rs.3.rs-505612/v1 ◽

2021 ◽

Author(s):

Xia Jiaojiao ◽

Yan Yan ◽

Bin Zou ◽

Adesanya Idowu Onyinye

Keyword(s):

Low Cost ◽

Immobilized Lipase ◽

Candida Rugosa ◽

Residual Activity ◽

Candida Rugosa Lipase ◽

Catalytic Performance ◽

Functional Surface ◽

Carrier Free ◽

Effective Synthesis ◽

Repeated Use

Abstract The cross-linked enzyme aggregates (CLEAs) are one of the technologies that quickly immobilize the enzyme without a carrier. This carrier-free immobilization method has the advantages of simple operation, high reusability and low cost. In this study, ionic liquid with amino group (1-aminopropyl-3-methylimidazole bromide，IL) was used as the novel functional surface molecule to modify industrialized lipase (Candida rugosa lipase, CRL). The enzymatic properties of the prepared CRL-FIL-CLEAs were investigated. The activity of CRL-FIL-CLEAs (5.51 U/mg protein) was 1.9 times higher than that of CRL-CLEAs without surface modification (2.86 U/mg protein). After incubation at 60℃ for 50 min, CRL-FIL-CLEAs still maintained 61% of its initial activity, while the value for CRL-CLEAs was only 22%. After repeated use for five times, compared with the 22% residual activity of CRL-CLEAs, the value of CRL-FIL-CLEAs was 51%. Further kinetic analysis indicated that the Km values for CRL-FIL-CLEAs and CRL-CLEAs were 4.80 mM and 8.06 mM, respectively, which was inferred that the affinity to substrate was increased after modification. Based on the above results, it was indicated that this method provided a new idea for the effective synthesis of immobilized enzyme.

Download Full-text

The influence of composition of poly(n-isopropylacrylamide-co-itaconic acid) hydrogel on immobilized Candida rugosa lipase activity

Hemijska industrija ◽

10.2298/hemind0806339m ◽

2008 ◽

Vol 62 (6) ◽

pp. 339-344

Author(s):

Nikola Milasinovic ◽

Melina Kalagasidis-Krusic ◽

Zorica Knezevic-Jugovic ◽

Jovanka Filipovic

Keyword(s):

Mechanical Properties ◽

Itaconic Acid ◽

Lipase Activity ◽

Acid Content ◽

Immobilized Enzyme ◽

In Situ Polymerization ◽

Immobilized Lipase ◽

Candida Rugosa ◽

Candida Rugosa Lipase

The application of lipases as catalysts in chemical reactions has been deterred by the high cost of isolation and purification of enzymes, the instability of their structure when they are isolated from their natural environment, contamination of products with residual protein, their sensitivity to process conditions, etc. These problems could be overcome using immobilized lipases. Immobilization is achieved by fixing enzymes to or within solid supports and as a result a heterogeneous system is obtained. The present paper reports on the immobilization of Candida rugosa lipase in hydrogels based on N-isopropylacrylamide and itaconic acid. Immobilization of lipase is carried out by two different methods. In the first method, lipase is added to the reaction mixture before polymerization and crosslinking (in situ polymerization), while in the second method the synthetized hydrogels are immersed in lipase solution and left to rich the equilibrium swelling. The specific activities of the immobilized lipase were determined in both cases and compared. The amount of the immobilized lipase is higher if the immobilization is carried out by immersing hydrogel in lipase solution. It was observed that in both cases lipase activity increases with an increase of the itaconic acid content up to 10 wt% and thereafter decreases. From the measurements of shear storage moduli (G') it was concluded that the increase of the itaconic acid content decreases the mechanical properties of the hydrogels. SEM analysis confirmed the highly porous structure of hydrogels. It was found that greater pores were achieved when the enzyme was immobilized by in situ polymerization. When the enzyme was immobilized by in situ polymerization the itaconic acid content had not great effect on the mass of the immobilized enzyme, except for the 100/0 sample. On the contrary, for the samples where the enzyme was immobilized by swelling, the increase of the itaconic acid content increases the mass of the immobilized enzyme. Concerning the activity of the immobilized lipase, the swelling degree and mechanical properties of the investigated hydrogels, the best results were performed by the 95/5 hydrogel sample.

Download Full-text

Immobilization of lipase from Candida rugosa into copolymer hydrogels of poly(N-isopropylacrylamide-co-itaconic acid) synthesized in the presence of surfactants

Hemijska industrija ◽

10.2298/hemind110915063m ◽

2011 ◽

Vol 65 (6) ◽

pp. 667-673

Author(s):

Nikola Milasinovic ◽

Nedeljko Milosavljevic ◽

Jovanka Filipovic ◽

Zorica Knezevic-Jugovic ◽

Melina Kalagasidis-Krusic

Keyword(s):

Mechanical Properties ◽

Itaconic Acid ◽

Nonionic Surfactants ◽

Enzyme Stability ◽

Binding Capacity ◽

Candida Rugosa ◽

Reference Sample ◽

Hydrolytic Activity ◽

Dynamic Mechanical Properties ◽

Lipase From Candida Rugosa

To overcome the problems of free enzyme application as catalysts in chemical reactions, i.e. high costs of isolation and purification processes, high sensitivity to process conditions, insufficient enzyme stability etc., a different immobilization techniques are to be used. Immobilization to/within solid support improves enzyme stability decreasing its denaturation. This paper deals with hydrogels of N-isopropylacrylamide and itaconic acid with incorporated nonionic surfactants (Triton X-100, Brij 30 and Tween 80) synthesized in distilled water at room temperature by free radical polymerization. These hydrogels were used as supports for immobilization of enzyme, lipase from Candida rugosa by post-entrapment method. The aim was to investigate the effect of the nonionic surfactants on the lipase binding capacity, as well as on its hydrolytic activity. In order to characterize the obtained hydrogels FT-IR analysis has been performed. Further, the swelling behaviour of these samples in buffer solution of pH 6.80 has been investigated. The dynamic - mechanical properties of hydrogels and detailed have been studied, too. The immobilized lipase showed somewhat reduced hydrolytic activity, as compared to the activity of free lipase as well as in comparison to the lipase immobilized to the reference sample (sample synthesized under the same polymerization conditions, but in the absence of surfactants). It was concluded that the addition of surfactants increased the hydrogel mesh size. The surfactant addition did not affect the dynamic - mechanical properties of the investigated hydrogels. The largest percentage of specific activity and yield of activity were presented by the reference sample, too. It is obvious that the absence of surfactants charged groups has no influence on the lipase binding capacity, and the obtained activity yields are to be expected.

Download Full-text

Activated Carbon as Support for Lipase Immobilization

Eurasian Chemico-Technological Journal ◽

10.18321/ectj298 ◽

2007 ◽

Vol 5 (2) ◽

pp. 115

Author(s):

Mohd Basyaruddin Abdul Rahman ◽

Mahiran Basri, Mohd Zobir Hussein ◽

Raja Nor Zaliha Raja Abdul Rahman ◽

Yau Kim Yan ◽

Abu Bakar Salleh

Keyword(s):

Activated Carbon ◽

Room Temperature ◽

Storage Stability ◽

Physical Adsorption ◽

Immobilized Lipase ◽

Candida Rugosa ◽

Lipase Immobilization ◽

Immobilized Lipases ◽

Different Types ◽

Native Lipase

Lipase from Candida rugosa was immobilized onto four different types of activated carbon; KI/2030, KI/3040, KI/5060 and KI/6070. The immobilized lipase was used in the esterification of oleic acid and 1-butanol in hexane. The effects of difference pore sizes, surface area, reaction temperature, thermostability of the immobilized lipases, storage stability in organic solvent and leaching studies were investigated. Among the four samples, KI/6070 gave the highest activities and stability in all the parameters investigated. Immobilized lipases generally exhibit activities higher than the native lipase for the parameters studied, with optimum temperature of 40°C. Immobilized lipases are more stable than native lipase in hexane at room temperature up to 12 days. Leaching study proved that the immobilization of lipase using physical adsorption is cheap and easy. This method was found to be suitable for the attachment of enzyme on the support.

Download Full-text

Surface Modification of Macroporous Matrix for Immobilization of Lipase for Fructose Oleic Ester Synthesis

BULLETIN OF CHEMICAL REACTION ENGINEERING AND CATALYSIS ◽

10.9767/bcrec.11.3.573.339-345 ◽

2016 ◽

Vol 11 (3) ◽

pp. 339 ◽

Cited By ~ 2

Author(s):

Hani Hilmanto ◽

Chusnul Hidayat ◽

Pudji Hastuti

Keyword(s):

Surface Modification ◽

Immobilized Lipase ◽

Wave Number ◽

Esterification Reaction ◽

Protein G ◽

Ester Synthesis ◽

Bending Vibrations ◽

Hydrophobic Matrix ◽

The Matrix ◽

Stretching Vibration

The objective of this research was to modify the matrix surfaces to obtain both hydrophobic matrix (HM) and hydrophilic-hydrophobic matrix (HHM) for enzymatic synthesis of fructose oleic ester (FOE). The modification was performed by the attachment of 2-phenylpropionaldehyde (PPA) and PPA followed by polyethyleneimine (PEI) for HM and HHM, respectively. The results from FT-IR analysis showed that the peak of stretching vibration of NH2 bond decreased and it was followed by an increase of the peak vibration of –C=N– bond at wave number 1667 cm-1. The peak of bending vibrations of the C=C bond also increased. It indicated that PPA was successfully attached on matrix. For HHM, an increase in the peak area of NH2 bond indicated that PEI was also successfully attached on the matrix. The optimum conditions of lipase adsorption were obtained at buffer pH 7 containing 0.5 M NaCl (9.27 mg protein/g matrix) and without NaCl (9.23 mg protein/g matrix) for HM and HHM, respectively. For FOE synthesis, the best immobilized lipase concentration was about 8% and 6% for HM and HHM, respectively. The optimum time of esterification was 24 h and 18 h for HM and HHM, respectively, in which the yields were 75.96% and 85.29%, respectively. The immobilized lipase could be used up to 3 cycles of esterification reaction. Copyright © 2016 BCREC GROUP. All rights reservedReceived: 21st December 2015; Revised: 23rd February 2016; Accepted: 14th April 2016How to Cite: Hilmanto, H., Hidayat, C., Hastuti, P. (2016). Surface Modification of Macroporous Matrix for Immobilization of Lipase for Fructose Oleic Ester Synthesis. Bulletin of Chemical Reaction Engineering & Catalysis, 11 (3): 339-345 (doi:10.9767/bcrec.11.3.573.339-345)Permalink/DOI: <a href="http://doi.org/10.9767/bcrec.11.3.573.339-345">http://doi.org/10.9767/bcrec.11.3.573.339-345</a>

Download Full-text

Free Candida Rugosa Lipase–Catalyzed Synthesis Of Citronellyl Butyrate In n–Hexane By Direct Esterification: Effect Of Reaction Parameters

Jurnal Teknologi ◽

10.11113/jt.v50.173 ◽

2012 ◽

Author(s):

Irvan Dahlan ◽

Azlina Harun @ Kamaruddin ◽

Ghasem D. Najafpour

Keyword(s):

Butyric Acid ◽

Active Sites ◽

Immobilized Lipase ◽

Candida Rugosa ◽

Candida Rugosa Lipase ◽

Molar Ratio ◽

Esterification Reaction ◽

Reaction Parameters ◽

Direct Esterification ◽

Competitive Nature

Sintesis sitronelil butirat melalui pengesteran langsung telah dikaji di dalam n–hexane sebagai pelarut organik yang dimangkinkan oleh lipase bebas dan lipase tersekatgerak daripada Candida rugosa. Kajian telah dijalankan untuk mengesahkan pengaruh pelbagai parameter pada pembentukan sitronelil butirat oleh lipase bebas daripada Candida rugosa, iaitu kesan kepekatan lipase, nisbah molar substrat, suhu, tiga jenis bahan sokongan untuk lipase tersekatgerak dan nisbah sekatgerak. Peningkatan kepekatan lipase bebas menyebabkan peningkatan kepada penukaran asid. Sifat peningkatan tidak berubah dari tindak balas pengesteran dapat diperhatikan pada kepekatan lipase tinggi yang memberikan kepekatan optimum lipase pada 3.33 g/l dengan penukaran asid sebanyak 92%. Kemungkinan ini disebabkan oleh tapak aktif lipase yang berlebihan yang berada di dalam zarah lipase pukal, yang tidak memberi sumbangan bererti kepada tindak balas. Aktiviti lipase didapati terencat dengan bertambahnya kepekatan asid butirik (pada kepekatan sitronelol tetap) dan sitronelol (pada kepekatan asid butirik tetap). Ini disebabkan adanya persaingan semulajadi pengikatan alkohol dan asid. Penukaran asid optimum diperolehi pada suhu 40°C selepas 24 jam pengeraman. Bagaimanapun, atas dari suhu ini, aktiviti pengesteran yang dimangkinkan oleh lipase mula menurun kerana penyahaslian protein. Daripada tiga jenis sokongan yang digunakan untuk lipase tersekatgerak, Amberlite MB–1 menunjukkan penukaran asid tertinggi berbanding dengan Amberlite XAD–1180 dan Celite 545. Penukaran asid optimum diperolehi pada nisbah sekatgerak 10 mg lipase/g penyokong. Pada nisbah sekatgerak ini, lipase mengoptimumkan sentuhan dengan permukaan penyokong dengan mengekalkan konformasi yang aktif pada tahap optimum. Kata kunci: Sitronelil butirat; lipase Candida rugosa; pengesteran langsung; lipase tersekatgerak; pelarut organik Free and immobilized Candida rugosa lipases were investigated for the synthesis of citronellyl butyrate by direct esterification reaction in n–hexane as organic solvent. A set of experiments was carried out to verify the influence of various parameters on the formation of citronellyl butyrate by free Candida rugosa lipase, such as lipase loading, substrate molar ratio, temperature, three kinds of support for immobilization, and ratio of immobilization. The conversion was increased with increasing lipase loading. The behavior of leveling–off in esterification was observed at higher lipase loading which gave the optimal amount of lipase loading at 3.33 g/l with 92% conversion. This might be due to the excess of lipase active sites, which remained inside the bulk of lipase particles, was not contributing significantly to the reaction. Increasing butyric acid and citronellol concentrations (at fixed citronellol and butyric acid concentrations, respectively) inhibited the lipase activity due to competitive nature of alcohol and acid binding. Optimal acid conversion was obtained at 40°C after 24–h incubation time. Above this temperature, however, the activity of lipase–catalyzed esterification begins to decrease due to denaturation of protein. From the three kinds of supports for immobilized lipase, Amberlite MB–1 showed the highest conversion compared to Amberlite XAD–1180 and Celite 545. The optimal acid conversion was obtained at lipase loading of 10 mg lipase/g support. At this loading, lipase attempts to optimize its contact with the surface of the support whereby optimum active conformation was retained. Key words: Citronellyl butyrate; Candida rugosa lipase; direct esterification; immobilized lipase; organic media

Download Full-text

Preparation of Immobilized Lipase Based on Hollow Mesoporous Silica Spheres and Its Application in Ester Synthesis

Molecules ◽

10.3390/molecules24030395 ◽

2019 ◽

Vol 24 (3) ◽

pp. 395 ◽

Cited By ~ 7

Author(s):

Zhe Dong ◽

Meng-Ying Jiang ◽

Jie Shi ◽

Ming-Ming Zheng ◽

Feng-Hong Huang

Keyword(s):

Reaction Time ◽

Mesoporous Silica ◽

Immobilized Lipase ◽

Lipid Production ◽

Candida Rugosa ◽

Molar Ratio ◽

Esterification Reaction ◽

Enzymatic Esterification ◽

Free System ◽

Hollow Mesoporous Silica

In this study, Candida rugosa lipase (CRL) was immobilized into modified hollow mesoporous silica (HMSS) materials with different hydrophobicity. Among propyl-(C3), phenyl-(C6), octyl-(C8), and octadecyl-(C18) modified HMSS as well as native HMSS, taking advantage of more hydrophobic microenvironment, the HMSS-C18-CRL showed exceptional performance in enzymatic esterification reaction. Using the novel HMSS-C18 with immobilized CRL (HMSS-C18-CRL), we investigated the esterification of phytosterols with polyunsaturated fat acid (PUFA) in a solvent-free system for the production of phytosterols esters. Response surface methodology (RSM) was applied to model and optimize the reaction conditions, namely, the enzyme load (5–25%), reaction time (10–110 min), molar ratio of α-linolenic acid (ALA)/phytosterols (1:1–7:1) and represented by the letters E, T, and M respectively. Best-fitting models were successfully established by multiple regressions with backward elimination. The optimum production was achieved at 70 min for reaction time, 20% based on the weight of substrate for enzyme loading, and 5.6:1 for ALA/phytosterols molar ratio. Under optimized conditions, a conversion of about 90 ± 2% was achieved. These results indicated that HMSS-C18-CRL demonstrates to be a promising catalyst and can be potentially applied in the functional lipid production.

Download Full-text

Improved production of biolubricants from soybean oil and different polyols via esterification reaction catalyzed by immobilized lipase from Candida rugosa

Fuel ◽

10.1016/j.fuel.2017.11.119 ◽

2018 ◽

Vol 215 ◽

pp. 705-713 ◽

Cited By ~ 49

Author(s):

Elisa D.C. Cavalcanti ◽

Érika C.G. Aguieiras ◽

Priscila R. da Silva ◽

Jaqueline G. Duarte ◽

Eliane P. Cipolatti ◽

...

Keyword(s):

Soybean Oil ◽

Immobilized Lipase ◽

Candida Rugosa ◽

Esterification Reaction ◽

Lipase From Candida Rugosa

Download Full-text

Incorporation of Poly(Itaconic Acid) with Quaternized Thiazole Groups on Gelatin-Based Films for Antimicrobial-Active Food Packaging

Polymers ◽

10.3390/polym13020200 ◽

2021 ◽

Vol 13 (2) ◽

pp. 200

Author(s):

Celeste Cottet ◽

Andrés G. Salvay ◽

Mercedes A. Peltzer ◽

Marta Fernández-García

Keyword(s):

Antimicrobial Activity ◽

Itaconic Acid ◽

Food Packaging ◽

Crosslinking Agent ◽

Radical Scavenging ◽

Proton Nuclear Magnetic Resonance ◽

Esterification Reaction ◽

Alkylation Reaction ◽

Vapor Permeability ◽

Active Food Packaging

Poly(itaconic acid) (PIA) was synthesized via conventional radical polymerization. Then, functionalization of PIA was carried out by an esterification reaction with the heterocyclic groups of 1,3-thiazole and posterior quaternization by N-alkylation reaction with iodomethane. The modifications were confirmed by Fourier transform infrared (FTIR) and proton nuclear magnetic resonance (1H-NMR), as well as ζ-potential measurements. Their antimicrobial activity was tested against different Gram-negative and Gram-positive bacteria. After characterization, the resulting polymers were incorporated into gelatin with oxidized starch and glycerol as film adjuvants, and dopamine as crosslinking agent, to develop antimicrobial-active films. The addition of quaternized polymers not only improved the mechanical properties of gelatin formulations, but also decreased the solution absorption capacity during the swelling process. However, the incorporation of synthesized polymers increased the deformation at break values and the water vapor permeability of films. The antioxidant capacity of films was confirmed by radical scavenging ability and, additionally, those films exhibited antimicrobial activity. Therefore, these films can be considered as good candidates for active packaging, ensuring a constant concentration of the active compound on the surface of the food, increasing products’ shelf-life and reducing the environmental impact generated by plastics of petrochemical origin.

Download Full-text

Enzymatic Synthesis of Isopropyl Acetate by Immobilized Bacillus cereus Lipase in Organic Medium

Enzyme Research ◽

10.4061/2011/919386 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 21

Author(s):

Madan Lal Verma ◽

Wamik Azmi ◽

Shamsher Singh Kanwar

Keyword(s):

Acetic Acid ◽

Bacillus Cereus ◽

Water Activity ◽

Molecular Sieves ◽

Immobilized Lipase ◽

Molar Ratio ◽

Ester Synthesis ◽

Initial Water ◽

Isopropyl Acetate ◽

Mg Addition

Selective production of fragrance fatty acid ester from isopropanol and acetic acid has been achieved using silica-immobilized lipase of Bacillus cereus MTCC 8372. A purified thermoalkalophilic extracellular lipase was immobilized by adsorption onto the silica. The effects of various parameters like molar ratio of substrates (isopropanol and acetic acid; 25 to 100 mM), concentration of biocatalyst (25–125 mg/mL), reaction time, reaction temperature, organic solvents, molecular sieves, and initial water activity were studied for optimal ester synthesis. Under optimized conditions, 66.0 mM of isopropyl acetate was produced when isopropanol and acetic acid were used at 100 mM: 75 mM in 9 h at 55°C in n-heptane under continuous shaking (160 rpm) using bound lipase (25 mg). Addition of molecular sieves (3 Å × 1.5 mm) resulted in a marked increase in ester synthesis (73.0 mM). Ester synthesis was enhanced by water activity associated with pre-equilibrated saturated salt solution of LiCl. The immobilized lipase retained more than 50% of its activity after the 6th cycle of reuse.

Download Full-text