Naji Arafat Mahat

A facile enzymatic synthesis of geranyl propionate by physically adsorbed Candida rugosa lipase onto multi-walled carbon nanotubes

In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6h at 55°C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate.

Influence of rain and malathion on the oviposition and development of blowflies (Diptera: Calliphoridae) infesting rabbit carcasses in Kelantan, Malaysia.

The influence of rain and malathion on the initial oviposition as well as development of blowfly species infesting rabbit carcasses decomposing in sunlit and shaded habitats were studied over a period of 1 year in Kelantan, Malaysia. Chrysomya megacephala (Fabricius) was the most dominant species that infested the carcasses, followed by Chrysomya rufifacies (Macquart). In general, rain, depending on its intensity, delayed initial oviposition by 1-2 days and prolonged the pupation period by 1-3 days. The presence of malathion in the carcasses delayed initial oviposition by 1-3 days and prolonged the pupation period by 2-3 days. These findings deserve consideration while estimating postmortem interval since rain is a commonplace occurrence in Malaysia and malathion is one of the common poisons as an agent for choice to commit suicide.

Modelling and optimization of Candida rugosa nanobioconjugates catalysed synthesis of methyl oleate by response surface methodology

Esters, such as methyl oleate, are functionally important compounds in many industrial sectors, mainly as components for manufacturing of emulsifiers, detergents, intermediate stabilizers and wetting agents. Acid functionalization of multi-walled carbon nanotubes (F-MWCNTs) by using a mixture of HNO3 and H2SO4 (1:3, v:v) was employed as the matrix for the adsorption of Candida rugosa lipase (CRL) as nanobioconjugates (CRL-MWCNTs) for the production of methyl oleate. Structural information of the developed CRL-MWCNTs was confirmed using Fourier Transform Infrared spectroscopy, thermogravimetric analysis and transmission electron microscopy, which revealed a successful attachment of CRL onto the F-MWCNTs. Process parameters (reaction time, temperature and alcohol:acid molar ratio) were optimized for high percent conversion of methyl oleate. Structural analysis established that CRL was successfully attached to the surface of the F-MWCNTs. Under the optimized conditions, which were 13.87 h, 51 °C, molar ratio oleic acid:methanol (1:3.80), a high ester yield of 90.90% was attained. Also, under conditions of the shortest reaction time of 6 h, 47.07 °C and acid:methanol ratio of 1:2.54, the CRL-MWCNTs catalysed a 74.51% ester yield. Hence, we established that response surface methodology (RSM) can be a practical technique for the prediction of the conditions that favour the high yield production of methyl oleate. Under optimized conditions, the CRL-MWCNTs nanobioconjugates are potentially good and economical biocatalysts for the production of methyl oleate, as well as other types of commercially important fatty-acid methyl esters.

Response surface methodological approach for optimizing production of geranyl propionate catalysed by carbon nanotubes nanobioconjugates

Terpene esters of short-chain fatty acids are essential oils that have big importance in food, cosmetic and pharmaceutical industries as flavours and fragrances. Geraniol and citronellol are the most important substances. Considering the ever-increasing demand for such products, their enzymatic production from natural raw materials by using environmentally friendly and economically attractive processes may prove advantageous. In this contribution, we would like to present an alternative option for the production of geranyl propionate using nanobioconjugates consisting of Candida rugosa lipase adsorbed onto multi-walled carbon nanotubes (CRL-MWCNTs). We investigated the effects of incubation time, temperature, solvent log P and substrate molar ratio, and determined the optimum conditions. The yield of geranyl propionate catalysed by CRL-MWCNTs nanobioconjugates was significantly influenced by two factors, namely, temperature and time of the reaction. Under the optimum reaction conditions of 55 °C, solvent n-heptane (log P = 4.0), geraniol to propionic acid molar ratio of 5:1 and reaction time of 6 h, the use of CRL-MWCNTs resulted in 51.3% production of geranyl propionate. Therefore, the investigation revealed that geranyl propionate was successfully synthesized under mild conditions with reasonably high yield within a short period of time. The CRL-MWCNTs nanobioconjugates demonstrated a potential as economical and environmentally smarter biocatalysts for the production of geranyl propionate.

Synthesis of geranyl propionate in a solvent-free medium using Rhizomucor miehei lipase covalently immobilized on chitosan–graphene oxide beads

ABSTRACT The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan–graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box–Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76 ± 0.02% of recovered protein had yielded 7.2 ± 0.04 mg g−1 and 211 ± 0.3% U g−1 of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20 hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70 rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.

Structure and properties of oil palm-based nanocellulose reinforced chitosan nanocomposite for efficient synthesis of butyl butyrate

In this study, nanocellulose (NC) was successfully extracted from oil palm frond leaves (OPFL) using a combination of bleaching, alkaline treatment and acid hydrolysis. X-ray diffractogram revealed the extracted NC was crystalline with a crystallinity index of 70.2%. This indicates its suitability as nano-fillers for preparing the chitosan/nanocellulose (CS-NC) supports to immobilize Candida rugosa lipase (CRL) to produce the CRL/CS-NC biocatalysts. FTIR, FESEM and TGA characterizations of the CRL/CS-NC confirm the CRLs were successfully conjugated to the CS-NC supports. The air-dried CS-NC supports gave satisfactory immobilization of the CRLs (5.2mg/g) with the resultant CRL/CS-NCs catalysed conversions of ≥80% of butyl butyrate within 6h. Time course reaction profile revealed that 76.3% butyl butyrate conversion was achieved at 4h immobilization time using 3mg/mL of CRL/CS-NCs. NMR analyses on the purified butyl butyrate confirmed that the ester was successfully synthesized.

Provenance Establishment of Stingless Bee Honey Using Multi-element Analysis in Combination with Chemometrics Techniques†

As consumption of stingless bee honey has been gaining popularity in many countries including Malaysia, ability to identify accurately its geographical origin proves pertinent for investigating fraudulent activities for consumer protection. Because a chemical signature can be location‐specific, multi‐element distribution patterns may prove useful for provenancing such product. Using the inductively coupled‐plasma optical emission spectrometer as well as principal component analysis (PCA) and linear discriminant analysis (LDA), the distributions of multi‐elements in stingless bee honey collected at four different geographical locations (North, West, East, and South) in Johor, Malaysia, were investigated. While cross‐validation using PCA demonstrated 87.0% correct classification rate, the same was improved (96.2%) with the use of LDA, indicating that discrimination was possible for the different geographical regions. Therefore, utilization of multi‐element analysis coupled with chemometrics techniques for assigning the provenance of stingless bee honeys for forensic applications is supported.

Patterns of oviposition and development of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) on burned rabbit carcasses

Considering that crimes against animals such as illegal killing and cruelty have been alarmingly increasing and since burning is one of the common ways for disposing cadavers, ability to estimate minimum postmortem interval (PMI) using entomological data merits consideration. Chrysomya megacephala and Chrysomya rufifacies are common necrophagous species recovered from cadavers in many countries including Malaysia. Specific studies focusing on the oviposition and developmental patterns of both species on cadavers manifesting different levels of burn as described by the Crow-Glassman Scale (CGS) remain scarce. In four replicates, rabbit carcasses were burned to CGS levels #1, #2 and #3 by varying the amount of petrol used and duration of burning. Oviposition by C. megacephala and C. rufifacies was delayed by one day in the case of carcasses burned to the CGS level #3 (p<0.05) when compared with that of controls. Such delay in oviposition was not observed in the CGS level #1 and #2 carcasses. No significant differences (p>0.05) in the duration of development were found between control and burned carcasses. These findings deserve consideration while estimating minimum PMI since burning as a mean for disposing animal and human cadavers is gaining popularity.

A statistical approach for optimizing the protocol for overexpressing lipase KV1 in Escherichia coli: purification and characterization

ABSTRACT Lipase is one of the most important industrial enzymes, widely used in the preparation of food additives, cosmetics and pharmaceuticals. In order to obtain a large amount of lipase, in the present study, a gene encoding intracellular lipase was cloned from Acinetobacter haemolyticus. The recombinant lipase KV1 containing a His-tag was expressed in Esherichia coli BL21 (DE3) cells, using pET-30a as the expression vector. Using the central composite design, screening and optimization of induction conditions (cell density before induction, IPTG (isopropyl β-D-1-thiogalactopyranoside) concentration, post-induction temperature and post-induction time) were made. All parameters significantly (P < 0.05) influenced the expression of lipase KV1, rendering a 70% increase in enzyme production at optimum induction conditions (OD600 before induction: 0.6, IPTG concentration: 0.5 mmol/L, post-induction temperature: 40 °C, post-induction time: 16 h). The expressed recombinant lipase KV1 was purified using Ni-affinity chromatography, affording ∼3.1-fold of the enzyme with an estimated relative molecular mass of 39 kDa. The recombinant lipase KV1 exhibited its maximum activity at 40 °C and pH 8.0. Beneficially, the recombinant lipase KV1 retained its relative activities (>80%) even up to 24 h between pH 7−12; suggesting that the recombinant lipase KV1 may be suitable for a wide range of industrial applications.

Influence of Paraquat on Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) Infesting Minced‐beef Substrates in Kelantan, Malaysia

This study investigated the influence of paraquat, a prevalent poison used by suicides, on initial oviposition and development of Chrysomya megacephala (Fabricius) using minced‐beef substrates. Paraquat in lethal dose for human (40 mg/kg), two times the lethal dose (80 mg/kg) and five times the lethal dose (200 mg/kg) were mixed thoroughly with respective minced‐beef substrates (1 kg each) that were decomposed in a shaded habitat fully protected from rain. Results of four replications of the above experiment revealed that the presence of paraquat neither delayed initial oviposition nor prolonged the developmental stages of C. megacephala. Therefore, estimation of postmortem interval (PMI) based on empirical baseline data obtained using animal models devoid of any poisons would still be appropriate for estimating PMI in paraquat‐related deaths.