Enzymatic Synthesis of Oleyl Oleate, a Liquid Wax Ester, in a Stirred Tank Reactor

High performance enzymatic synthesis of oleyl oleate, a liquid wax ester was successfully synthesized via enzymatic esterification reaction of oleic acid and oley alcohol. Immobilised Candida antartica lipase B (Novozym 435) was used as biocatalyst. The study was divided into four parts which are the optimisation of reaction synthesis at different scales, the reactor study in term of mixing efficiency, the stability of immobilised enzyme and the analysis and characterization of the product of the reaction. Preliminary synthesis of oleyl oleate was carried out in a small scale reaction with a total volume of 3.5 mL using screw-capped vials. Optimisation reaction study via conventional method of varying one parameter at-a-time approach was carried out. A high percentage conversion yield of >90% was achieved at optimum reaction time of 5 min, reaction temperature of 40-6O0C1 molar ratio of substrates (oleyl alcoholloleic acid) of 2:1, amount of enzyme of 0.4 g and organic solvents of Log P23.5 at fixed agitation speed of 150 rpm. Investigation in larger scale production of oleyl oleate was performed using 2 L stirred tank reactor (STR). The reaction was scaled-up to 300X with a total volume of 1.05 L. A high percentage conversion of oleyl oleate was achieved of >95% by conventional experiment method at reaction time of 30 min, agitation speed of 400 rpm, reaction temperature of 45-50°C, molar ratio of substrate (oleyl alcoholloleic acid) of 2:l and amount of enzyme of 90 g. The reaction synthesis was further optimised by response surface method (RSM) based on five-level, three-variable central composite rotatable design (CCRD) to evaluate the interactive effects of important parameters in larger scale processing. Generally, simultaneously increasing amount of enzyme, agitation speed and reaction temperature would improved the yields. A high percentage conversion of 97.4% was achieved under the optimum condition, which compared well with the maximum predicted value of 97.7%. In order to improve the production and productivity of oleyl oleate to the highest amount that can be produced in a 2 L STR, the reaction was synthesized in a solvent-free system. Maximum scaling-up of substrate concentration that can be achieved in the reactor vessel was 900X as compared to 300X previously. The production and productivity of oleyl oleate were successfully improved from 295.39 g/L/h to 705.76 g1Uh and 31 0.16 glh to 952.78 glh, respectively. Reactor study on the performance of 2 L STR as a mixing device was evaluated to improve the mixing efficiency. The rheological property of the reaction mixture exhibited Newtonian behaviour. Rushton turbine impeller showed better performance in degree of mixing, as compared to AL Hydrofoil impeller whereby a high Reynolds number of >lo4 was achieved at 400 rpm, which exhibit a turbulent flow pattern. There was significant effect to the mixing improvement on the enzyme particles distribution by using a 2 impellers system with spacing of 30 mm. The enzyme showed high stability against heat as shown by the high percentage conversion of wax ester. Novozym 435 retained its synthetic activity up to 9 uses and 4 uses in screw-capped vials and STR, respectively. The effect of shear forces due to the mechanical agitation speed on the enzyme morphology was determined by scanning electron microscope (SEM). Although small rupture on the surface of enzyme was observed when increasing the agitation speed, the enzyme activity was very high even at high agitation speed.Analysis of product was evaluated by spectroscopy method of Fourier transform-infrared spectroscopy (FT-IR) and gas chromatography-mass spectroscopy (GC-MS) to identify the product obtained. Characteristics of oleyl oleate were also examined, which include iodine value, saponification value, acid value and ester value. Solubility of oleyl oleate in methanol and ethanol was comparatively lower as compared to the solubility at higher chain length of alcohols. This compound seems compatible in most of oils and stable even after heating up to 90°C and overnight storage at room temperature

Study on anti-quorum sensing potentials and phytochemical constituents of Euphorbia hirta

Euphorbia hirta is an annual broad-leaved herb and widely used as traditional medicine to treat various ailments. This herb was tested on the anti quorum sensing (anti-QS) potentials in fresh (edible or macerated) forms and acetone extracts via biomonitor strain Chromabacterium violaceum (ATCC 12472). The biomonitor strain has an ability to produce purple pigment (violacein) under QS-control. The different parts of E. hirta extracts were then subjected to preliminary phytochemical screening using standard procedures and finally analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). Preliminary screening on fresh parts of this herb revealed that leaves exhibited the highest anti-QS activities towards C. violaceum. The results also exhibited the wide variation in the anti-QS activities on whole plants, flowers, stems, leaves and roots of E. hirta from acetone extraction. The highest anti-QS activities were recorded by leaves and flowers extracts as the lowest of minimum QS inhibition concentration values (1.8906 mg/ml) were indicated by both extracts respectively. Phytochemical screening of E. hirta extracts revealed the presence of carbohydrates, lipids, protein, flavonoids, alkaloids, saponins, resins, steroids, acidic compounds, tannins, glycosides, phenols and terpenoids. The quantitave phytochemical assays via GC-MS indicated that this herb rich with fatty acids, terpenoids and phenolic compounds. Keywords: Euphorbia hirta, Chromabacterium violaceum, anti-quorum sensing, phytochemical assays

Isolation and identification of amylolytic bacteria from ragi

Ragi is a traditional dry starter culture frequently used in many fermented food products in Asian countries. Ragi contains starch that can be degraded by amylases provided by bacteria and other microorganisms during fermentation in producing fermented foods. This study was carried out to isolate bacteria from ragi and to screen for their ability to hydrolyse starch. Seven bacterial colonies were isolated from ragi, with three samples showing starch hydrolysis activity. Two of the starchdegrading bacterial isolates were identified using partial 16S rRNA gene sequencing as Bacillus licheniformis with 97% and 98% similarity, and one sample identified as a lactic acid bacteria, Enterococcus faecium (98%). Isolation and identification of these bacteria from ragi can provide a promising source of amylase that can be further studied and manipulated for the development of starter culture and to improve the quality of traditional ragi-based fermented food products

Optimization of lipase-catalyzed synthesis of palm amino acid surfactant using response surface methodology (RSM)

Amino acid surfactants are high-value surfactants which have excellent emulsifying characteristics and minimal toxicity to the living body. Enzymatic synthesis of palm kernel amino acid surfactant was optimized by response surface methodology (RSM) using palm kernel olein (PKO) and l(+)-lysine catalyzed by Lipozyme RM IM. The reaction was performed in batch mode stirred tank reactor (STR) with one multi-bladed impeller. A central composite rotatable design (CCRD) was employed to evaluate the interactive effects of various parameters. The parameters were temperature (A): (40.00–70.00 °C), impeller speed (B): (100.00–400.00 rpm), substrates ratio (C): (1.00–4.00 mmol) and enzyme amount (D): (5.00–8.00 g). The optimum condition derived via RSM at fixed reaction time of 24 h was temperature; 47.50 °C, impeller speed; 323.96 rpm, substrates ratio; 3.25 mmol and enzyme amount; 7.25 g. The experimental yield was 89.03% under the optimum condition, which compared well with the maximum predicted value of 93.77%

Green synthesis of lauryl palmitate via lipase-catalyzed reaction

Enzyme catalysis is most attractive for the synthesis of fine organic compounds, which are difficult to prepare and to handle by conventional means. In this work, green synthesis of lauryl palmitate, a wax ester was successfully carried out by lipase-catalyzed esterification of palmitic acid and lauryl alcohol. In this study, commercial immobilized lipase from Candida antartica (Novozym 435) was used as biocatalyst. The effect of various reaction parameters were optimized to obtain a high yield of wax esters. The optimum condition to produce lauryl palmitate was at reaction time (RT); 10 min, temperature (T); 40°C, amount of enzyme (E); 0.4 g, molar ratio of substrate (N); 2:1 and organic solvents of log P>3.5. The product was then subjected to characterize using Fourier-transform infrared spectroscopy (FT-IR) and Gas chromatography spectroscopy (GC) to ensure the purity of product obtained. Analysis of yield showed that at optimum condition, lauryl palmitate was produced in short time with high purity, >90%

Identification of bacteria associated with Holothuria (Mertensiothuria) leucospilota from Pangkor Island

Holothuria (Mertensiothuria) leucospilota is the most abundant sea cucumber species in Malaysia. This study aimed to identify bacteria isolated from the external and internal body parts of H. leucospilota collected from the coastal water of Pangkor Island, Perak, Malaysia. A total of 26 bacterial samples were isolated using streak plate method from eight body parts of two fresh H. leucospilota specimens and from the surrounding surface sediments and seawater. Identification of the bacterial isolates was based on microscopic examination, 16S rDNA amplification and phylogenetic analysis using the neighbour-joining method. Three genera of bacteria were identified namely Vibrio, Bacillus, and Acinetobacter. The genus Vibrio was found to be the main bacterial group associated with the H. leucospilota specimens from Pangkor Island

Protein produced by Bacillus subtilis ATCC21332 in the presence of Cymbopogon flexuosus essential oil

Proteins levels produced by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antimicrobial agents or antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics or natural compounds in nature as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was focusing on the effect of essential oil from Cymbopogon flexuosus in regulating proteins production by Bacillus subtilis ATCC21332. The Minimum Inhibition Concentration (MIC) of the C. flexuosus essential oil on B. subtilis was determined by using microdilution assay, resulting 1.76mg/ml. The bacteria cells were further exposed to the C. flexuosus essential oil at concentration of 0.01 MIC for 72 h. The proteins were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile showed that a band with approximate size of 30 kDa was appeared for the treated bacteria with C. flexuosus essential oil. Thus, B. subtilis ATCC21332 in stressful condition with the presence of C. flexuosus essential oils at low concentration could induce the protein production. The isolated protein also showed antimicrobial activity against selected Gram-positive and Gram-negative bacteria

Large scale production of liquid wax ester by immobilized lipase

Oleyl oleate, a liquid wax ester was synthesized by an immobilized Candida antartica lipase B (Novozym 435) as biocatalyst using oleic acid and oleyl alcohol. The effects of various reaction parameters were optimized to obtain a high yield of liquid wax esters. Investigation in large scale production was performed in batch mode of stirred tank reactor (STR) with one multi-bladed impeller. The optimum condition to produce liquid wax ester was, reaction time (RT); 30 min, temperature (T); 50 °C, amount of enzyme (E); 90 g (900,000 PLU), agitation speed (A) of 400 rpm, number of impeller tip (N) of 2 and molar ratio of oleyl alcohol to oleic acid (M); 2:1. Analysis of the yield showed that at optimum condition, >90% liquid wax esters were produced. The stability of Novozym 435 showed at high percentage (>80%) up to 4 cycles

Optimization of lipase catalyzed synthesis of Ethyl Valerate, a green apple flavor using Response Surface Methodology (RSM).

Ethyl valerate, a green apple flavor is short-chain ester with fruity notes is widely used in food, cosmetic and pharmaceutical industries. Traditionally, flavor esters are produced by chemical method or extracted by natural sources. However, with the steadily growing demand for natural flavor compounds, the biosynthesis of such esters by lipase under mild conditions has been receiving much attention for producing these valuable products. In this study, enzymatic synthesis of ethyl valerate in solvent free system, was successfully optimized via response surface method (RSM) based on 5-level, 4-variable of central composite rotatable design (CCRD). The parameters were reaction time (30-60 min), reaction temperature (30-60 ℃), amount of enzyme (10-25%, w/w) and shaking speed (50-150 rpm). The optimum condition derived via RSM for the reaction was reaction time of 48 min, reaction temperature of 30 ℃, enzyme amount of 25%, (w/w) and shaking speed of 51 rpm. The actual experimental yield was 84.28 % under the optimum condition, which compared well with the maximum predicted value of 84.61%. Comparison of predicted and experimental values reveals good correspondence between them, implying that empirical models derived from RSM can be used to adequately describe the relationship between the factors and response in the synthesis of ethyl valerate

Process improvement in the production of oleyl oleate, a liquid wax ester in stirred tank reactor

Lipase-catalyzed production of oleyl oleate, a liquid wax ester by esterification of oleic acid with oleyl alcohol was performed in 2 L stirred tank reactor (STR). Process improvement study in reactor operation was carried out in batch mode STR with single impeller mounted on the centrally located shaft. Rushton turbine impeller showed better performance in degree of mixing, as compared to AL Hydrofoil impeller, whereby a high Reynolds number of >104 was achieved at 400 rpm, which exhibit a turbulent flow pattern [1]. Homogenous enzyme particles suspension was obtained at lower impeller speed (≤200 rpm) by using AL Hydrofoil impeller. However, there was significant effect to the mixing improvement on the enzyme particles distribution by using 2 impellers system with spacing of 30 mm even at lower agitation speed (≤200 rpm). High stability of Novozym 435 was observed even in the presence of shear forces on the enzyme particles due to the mechanical agitation speed. Wax ester reaction mixture in STR follows Newtons’ law due to the linear relation between the shear stress (τ) and shear rate (dυ/dy) [2]. The process was successfully carried out to the highest amount of wax ester that can be produced in 2 L STR whereby, the production and productivity of oleyl oleate were improved from 295.39 g/L/h to 705.76 g/L/h and 310.16 g/h to 952.78 g/h, respectively