Maryam Yousefi

Rapid and high-density covalent immobilization of Rhizomucor miehei lipase using a multi component reaction: application in biodiesel production

Aldehyde-functionalized silica and silica nanoparticles (SBA-15) were prepared as a matrix system for enzyme immobilization. Immobilization of Rhizomucor miehei lipase (RML) on these supports was performed via a multicomponent reaction under extremely mild conditions (25 °C, pH 7). Investigation on the mechanism of this reaction confirmed the Ugi four-component immobilization approach. The loading capacity of the supports and specific activity of the immobilized derivatives were interestingly improved. The results revealed very rapid immobilization of 10 and 60 mg of RML on 1 g of aldehyde-functionalized silica and SBA-15 after 10 and 30 minutes, respectively. Leaching experiments were performed by incubation of the immobilized derivatives in 1 M NaCl solution. The lack of the free lipase in the solution confirmed the covalent nature of the linkage. The thermal stability and co-solvent stability of the derivatives in the presence of three polar organic solvents (1-propanol, 2-propanol and dioxane) were greatly improved compared to the soluble enzyme. Both the derivatives were also used to catalyze the transesterification of colza oil with methanol to produce fatty acid methyl esters (FAMEs). In the case of RML immobilized on SBA-15 (SBA-RML), the presence of 40% of tert-butanol (v/v) as solvent in the reaction medium largely improved the conversion yield.

Covalent binding of hyper-activated Rhizomucor miehei lipase (RML) on hetero-functionalized siliceous supports

Physical adsorption onto hydrophobic supports has proven to be an effective way to improve the activity of lipases. Covalent binding, on the other hand, enhances the active lifetime of the immobilized biocatalysts. To combine the benefits of adsorption and covalent binding, immobilization of RML on new hetero-functional supports are reported. For this, chemical modification of silica and silica mesoporous nanoparticles was performed by the simultaneous use of two coupling linkers; Octyltriethoxysilane (OTES) for hydrophobic interaction and glycidoxypropyltrimethoxylsilane (GPTMS) for covalent linkage of RML. Altering the GPTMS/OTES ratio makes possible to have different amount of octyl and epoxy groups on the supports. The results showed that immobilization of RML on octyl-functionalized supports produces specific activity almost 1.5-2 folds greater than the specific activity of the free enzyme. The observed hyper-activation decreased with increasing epoxy groups on the supports confirming the enhancement of covalent nature of the attachment. Leaching experiment was also confirmed positive effect of the presence of epoxy groups on the supports. Regarding the specific activity of the immobilized preparations and desorption percentages of RML from each support, the most suitable carrier obtains from the functionalization of the supports in presence of GPTMS and OTES in the ratio of 1:1.

Synthesis of raloxifene-chitosan conjugate: A novel chitosan derivative as a potential targeting vehicle.

Chitosan is a biocompatible, non-toxic and biodegradable biopolymer. Due to the presence of functional groups on its surface, it can be modified and used as a carrier in targeted drug/gene delivery systems. In this study, raloxifene (a selective estrogen receptor ligand) was conjugated to chitosan using different methods. The conjugates were investigated by means of FTIR, TGA and physical properties assessments. Cell viability was evaluated by XTT assay. FTIR and TGA results confirmed that the conjugation between chitosan and raloxifene occurred more efficiently when trimethyl chitosan in the presence of triethylamine and excess amount of linker was used. XTT assay on MCF-7 cell line illustrated that more than 80% of cells were viable after 24h exposure to selected molecules. These findings confirm that the conjugation of raloxifene-chitosan can occur successfully using special synthesis condition and this novel chitosan derivative can be introduced as a potential drug/gene targeting agent.

A new abietane diterpenoid from Salvia xanthocheila Boiss.

From the chloroform extract of aerial part of Salvia xanthocheila Boiss. one new abietane diterpenoid, xantoquinone (5α,6α-dimethoxy-7,11,14-trioxoabieta-8,12-diene) and one known oleanene triterpenoid namely 1β,3β-dihydroxy-12-oleanene were isolated. The structure of the new terpenoid was elucidated by comprehensive spectroscopic analysis including electron ionisation-mass spectra, 1H NMR, 13C NMR, distortionless enchancement by polarisation transfer, H,H correlation spectroscopy, heteronuclear multiple-quantum coherence and heteronuclear multiple-bond correlation.

Immobilization of laccase on epoxy-functionalized silica and its application in biodegradation of phenolic compounds

A novel method of laccase immobilization on epoxy-functionalized silica particles was developed. Laccase from Myceliophthora thermophila was covalently immobilized onto epoxy-functionalized matrix by nucleophilic attack of amino groups of laccase to epoxy groups of the support. The enzyme loading on the support was about 30 mg/g under the optimum conditions (pH 4.5, 24 h). The effect of pH, temperature and organic solvent on immobilized enzyme activity was determined and compared with those of free enzyme. In general the immobilized enzyme was found to be stabilized compared to the free enzyme. Lineweaver-Burk plots were used to calculate kinetic parameters for ABTS oxidation. KM values were 24.0 and 25.3 μM while vmax values were 10.0 and 1.6 μM min-1 for free and immobilized laccase, respectively. The performance of the biocatalyst was evaluated by the degradation of phenolic compounds including phenol, p-chlorophenol and catechol. The removal efficiency of catechol by immobilized laccase was about 95% after 2 h.

The use of isocyanide-based multicomponent reaction for covalent immobilization of Rhizomucor miehei lipase on multiwall carbon nanotubes and graphene nanosheets

We describe here a novel and simple method for making bioconjugation and immobilization of Rhizomucor miehei lipase (RML) on carboxylated multiwall carbon nanotubes (MWCNT-COOH) and carboxylated graphene nanosheets (Gr-COOH) by using an isocyanide-based four-component reaction. In this approach, the enzyme supplies amino groups, the support supplies carboxylic acid groups, and the missing components (isocyanide and aldehyde) are added to the reaction medium. This coupling reaction was carried out in water at 25 °C, in which rapid and high enzyme loading were achieved. The maximum loading capacity of 530 mg and 680 mg was obtained for Gr-COOH and MWCNT-COOH, respectively. A variety of techniques including FTIR, Raman spectroscopy, XRD, SEM, and TGA were employed to characterize the immobilized derivatives of RML. The immobilized preparations showed significantly increased thermal stability and co-solvent stability as compared to the soluble enzyme. Kinetic parameters and optimum pH activity of RML and its immobilized preparations were also determined. The Km values of 0.44, 0.23, and 0.18 mM and the maximum reaction rates (Vmax) of 0.09, 0.1, and 0.08 mM min−1 were obtained for MWCNTs-RML, Gr-RML, and free RML, respectively. This approach may provide a general and efficient method to attach biomolecules on a variety of carboxylated solid surfaces at ambient conditions.

One pot three-component reaction for covalent immobilization of enzymes: application of immobilized lipases for kinetic resolution of rac-ibuprofen

This paper presents a novel strategy for the simple immobilization of biomolecules on epoxy-functionalized supports via a one-pot three component reaction. Lipase B from Candida antarctica (CALB) and Rhizomucor miehei lipase (RML) as model enzymes were immobilized on epoxy-functionalized silica and mesoporous silica nanoparticles (SBA-15). Investigation of the mechanism of this reaction confirmed the participation of three functional groups including carboxylic acid groups from the enzyme surface, epoxy groups from the support and isocyanide from the reaction medium. The results revealed very rapid immobilization of 10 and 40 mg of RML on 1 gr of the supports shortly after 30 minutes of incubation. The loading capacity of the supports was also dramatically improved with the maximum loading of 158 mg of CALB and 77 mg of RML on SBA-15 and silica, respectively. Silica-epoxy-RML showed an enantiomeric excess (ee) of 92% and an E-value of 29.9 in the enantioselective esterification of (R,S)-ibuprofen.

Chemical composition and antibacterial activity of the volatile oil from seeds of Artemisia annua L. from Iran

The composition of essential oil of the seeds of Artemisia annua L. was analysed by GC-MS. Overall, 16 volatile components were identified on the basis of their mass spectra characteristics and retention indices representing 95.5% of the total oil. Trans-3(10)-caren-4-ol (22.3%), artemisia ketone (18.6%), 1,8-cineole (14.9%), δ-selinene (13.0%) and α-pinene (8.2%) were the major compounds. Oxygenated monoterpenes were the main compounds with 51.6% followed by sesquiterpene hydrocarbons (13.3%), monoterpene hydrocarbons (9.9%) and other compounds (8.3%). The essential oil was highly active against Escherichia coli and Enterococcus faecalis.

Placenta-specific1 (PLAC1) is a potential target for antibody-drug conjugate-based prostate cancer immunotherapy

Our recent findings strongly support the idea of PLAC1 being as a potential immunotherapeutic target in prostate cancer (PCa). Here, we have generated and evaluated an anti-placenta-specific1 (PLAC1)-based antibody drug conjugate (ADC) for targeted immunotherapy of PCa. Prostate cancer cells express considerable levels of PLAC1. The Anti-PLAC1 clone, 2H12C12, showed high reactivity with recombinant PLAC1 and selectivity recognized PLAC1 in prostate cancer cells but not in LS180 cells, the negative control. PLAC1 binding induced rapid internalization of the antibody within a few minutes which reached to about 50% after 15 min and almost completed within an hour. After SN38 conjugation to antibody, a drug-antibody ratio (DAR) of about 5.5 was achieved without apparent negative effect on antibody affinity to cell surface antigen. The ADC retained intrinsic antibody activity and showed enhanced and selective cytotoxicity with an IC50 of 62 nM which was about 15-fold lower compared to free drug. Anti-PLAC1-ADC induced apoptosis in human primary prostate cancer cells and prostate cell lines. No apparent cytotoxic effect was observed in in vivo animal safety experiments. Our newly developed anti-PLAC1-based ADCs might pave the way for a reliable, efficient, and novel immunotherapeutic modality for patients with PCa.

Biotransformation of two furanocoumarins by the fungi species Aspergillus sp. PTCC 5266 and Aspergillus niger PTCC 5010

Abstract The microbial transformations of peucedanin and oreoselon by the fungi Aspergillus niger and Aspergillus sp. were investigated for the first time. Incubation of peucedanin with A. niger yielded a new hydroxylated metabolite with high yield (56%), which was characterized as 2-(1-hydroxypropan-2-yl)-3-methoxy-7H-furo[3,2-g]chromen-7-one. Oreoselon was converted to a new reduced metabolite methyl 3-(2,3-dihydro-6-hydroxy-2-isopropyl-3-oxobenzofuran-5-yl)propanoate in biotransformation by Aspergillus sp. The structures of the metabolites were determined by spectroscopic methods including IR, EI-MS, 1H NMR, 13C NMR, and elemental analysis.