Enzyme and Microbial Technology (ENZYME MICROB TECH)

Publications in this journal

• Article: Highly enantioselective esterification of racemic ibuprofen in a packed bed reactor using immobilised Rhizomucor miehei lipase

  Antoni Sánchez, Francisco Valero, Javier Lafuente, Carles Solà
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  ABSTRACT: A systematic study of the enantioselective resolution of ibuprofen by commercial Rhizomucor miehei lipase (Lipozyme® IM20) has been carried out using isooctane as solvent and butanol as esterificating agent. The main variables controlling the process (temperature, ibuprofen concentration, ratio butanol:ibuprofen) have been studied using an orthogonal full factorial experimental design, in which the selected objective function was enantioselectivity. This strategy has resulted in a polynomial function that describes the process. By optimizing this function, optimal conditions for carrying out the esterification of racemic ibuprofen have been determined. Under these conditions, enantiomeric excess and total conversion values were 93.8% and 49.9%, respectively, and the enantioselectivity was 113 after 112 h of reaction. These conditions have been considered in the design of a continuous reactor to scale up the process. The esterification of ibuprofen was properly described by pseudo first-order kinetics. Thus, a packed bed reactor operating as a plug-flow reactor (PFR) is the most appropriate in terms of minimizing the residence time compared with a continuous stirred tank reactor (CSTR) to achieve the same final conversion. This reactor shows a similar behavior in terms of enantioselectivity, enantiomeric excess, and conversion when compared with batch reactors. A residence-time distribution (RTD) shows that the flow model is essentially a plug flow with a slight nonsymmetrical axial dispersion (Peclet number = 43), which was also corroborated by the model of CSTR in series. The stability of the system (up to 100 h) and the possibility of reutilization of the enzyme (up to four times) lead to consider this reactor as a suitable configuration for scale up of the process.
  Enzyme and Microbial Technology 05/2013;
• Article: A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

  P. Raghu, T. Madhusudana Reddy∗, K. Reddaiah, L.R. Jaidev, G. Narasimha
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  ABSTRACT: A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 �M to 30 × 10−5M and 1–150 �M. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 �M, 20 �M for PG and 0.57 �M, 1.92 �M for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.
  Enzyme and Microbial Technology 03/2013;
• Article: Laccase-poly(lactic-co-glycolic acid) (PLGA) nanofiber: highly stable, reusable, and efficacious for the transformation of diclofenac.

  Palanivel Sathishkumar, Jong-Chan Chae, Afeesh R Unnithan, Thayumanavan Palvannan, Hak Yong Kim, Kui-Jae Lee, Min Cho, Seralathan Kamala-Kannan, Byung-Taek Oh
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  ABSTRACT: Nanobiocatalysis has received growing attention for use in commercial applications. We investigated the efficiency, stability, and reusability of laccase-poly(lactic-co-glycolic acid) (PLGA) nanofiber for diclofenac transformation. NH stretching vibrations (3400-3500 cm(-1) and 1560 cm(-1)) in FT-IR spectra confirmed immobilization of laccase on PLGA nanofibers. The relative activity of immobilized laccase was 82% that of free laccase. Immobilized laccase had better storage, pH, and thermal stability than free laccase. The immobilized laccase produced complete diclofenac transformation in three reuse cycles, which was extended to 6 cycles in the presence of syringaldehyde. Results suggest that laccase-PLGA nanofiber may be useful for removing diclofenac from aqueous sources and has potential for other commercial applications.
  Enzyme and Microbial Technology 07/2012; 51(2):113-8.
• Article: High-yield secretion of multiple client proteins in Aspergillus

  Segato F, Damasio ARL, Gonçalves TA, de Lucas RC, SquinaFM, Decker SR, Prade RA
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  ABSTRACT: Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50–100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.
  Enzyme and Microbial Technology 01/2012;
• Article: Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu.

  Thuy T Tran, Gashaw Mamo, Laura Búxo, Nhi N Le, Yasser Gaber, Bo Mattiasson, Rajni Hatti-Kaul
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  ABSTRACT: Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.
  Enzyme and Microbial Technology 07/2011; 49(2):177-82.
• Article: Biologically synthesized fluorescent CdSNPs encapsulated by PHB

  Pandian SRK, Venkataraman Deepak, Kalishwaralal Kalimuthu, Sangiliyandi Gurunathan
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  ABSTRACT: Here an attempt was made to biologically synthesize fluorescent cadmium sulfide nanoparticles and to immobilize the synthesized nanoparticles in PHB nanoparticles. The present study uses Brevibacterium casei SRKP2 as a potential producer for the green synthesis of CdS nanoparticles. Biologically synthesized nanoparticles were characterized and confirmed using electron microscopy and XRD. The size distribution of the nanoparticles was found to be 10–30 nm followed by which the consequence of time, growth of the organism, pH, concentration of CdCl2 and Na2S on the synthesis of nanoparticles were checked. Enhanced synthesis and fluorescence emission of CdS nanoparticles were achieved at pH 9. The synthesized CdS NPs were immobilized with PHB and were characterized. The fluorescent intensity of the CdS nanoparticles remained unaffected even after immobilization within PHB nanoparticles.
  Enzyme and Microbial Technology 04/2011; 48 319–325(4-5):319–325.
• Article: Corrigendum to "Biocatalysts in microbial fuel cells" [Enzyme Microb. Technol. 47 (2010) 179-188].

  Vinay Sharma, P P Kundu
  Enzyme and Microbial Technology 02/2011; 48(2):193.
• Article: Potential of the waste from beer fermentation broth for bio-ethanol production using the simultaneous saccharification and fermentation process without any additional enzyme, microbial cells and carbohydrates

  Jung Hwan Ha, Nasrullah Shah, Mazhar Ul-Islam, Joong Kon Park
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  ABSTRACT: The potential of the waste from beer fermentation broth (WBFB) for the production of bio-ethanol using simultaneous saccharification and fermentation process without any extra additions of saccharification enzymes, microbial cells or carbohydrate was tested. The major microbial cells in WBFB were isolated and identified. The variations in compositions of WBFB with stock time were investigated. There was residual activity of starch hydrolyzing enzymes in WBFB. The effects of reaction modes e.g. static and shaking on bio-ethanol production were studied. After days of cultivation using the supernatant of WBFB at 30 the ethanol concentration reached 103.8 g/in shaking culture and 91.5 g/in static culture. Agitation experiments conducted at temperature-profile process in which temperature was increased from 25 to 67 ◦shortened the simultaneous process time. The original WBFB was more useful than the supernatant of WBFB in getting the higher concentration of ethanol and reducing the fermentation time. From this whole study it was found that WBFB is cheap and suitable source for bio-ethanol production. © 2011 Elsevier Inc. All rights reserved.
  Enzyme and Microbial Technology 01/2011; 49:298-304.
• Article: Biodegradation of fuel oxygenates by sol–gel immobilized bacteria Aquincolatertiaricarbonis L108

  A. Pannier, C. Oehm, A. R. Fischer, P. Werner, U. Soltmann, H. Böttcher
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  ABSTRACT: The development of long-term storable biofilters containing bacteria which are capable to degrade recalcitrant environmental compounds like the fuel oxygenates methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) is of special interest for the treatment of contaminated water. Fuel oxygenates have been proven to be more persistent to biodegradation than other gasoline components. Only a few microorganisms, e.g. Aquincola tertiaricarbonis L108, are capable to biodegrade these substances.In order to develop highly efficient and long-term stable biocomposite materials for bioremediation, the applicability of sol–gel matrices for the immobilization of A. tertiaricarbonis was tested. Two different sol–gel immobilization techniques were used: (A) the immobilization within ceramic-like shapes by using the freeze-gelation technique and (B) sol–gel coatings on an inert, porous material. For comparison, the widely used alginate-bead immobilization technique has been applied. The embedding of A. tertiaricarbonis within freeze-gelation composites (method A) proved to be not applicable because this strain was too sensitive regarding the applied freezing and drying conditions even though cell-protecting additives were added. In contrast, A. tertiaricarbonis immobilized on porous expanded clay pellets (method B) could be stored under humid atmosphere at least for a time of 8 months without a significant reduction of their metabolic activity.
  Enzyme and Microbial Technology 11/2010; 47(6):291–296.
• Article: Evaluation of the incidence of diffusional restrictions on the enzymatic reactions of hydrolysis of penicillin G and synthesis of cephalexin

  Pedro Valencia, Lorena Wilson, Carolina Aguirre, A. Illanes
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  ABSTRACT: The impact of mass transfer limitations on penicillin G acylase immobilized in glyoxyl-agarose particles of different sizes and enzyme loads was evaluated for the reactions of hydrolysis of penicillin G and synthesis of cephalexin under the hypothesis that the impact of internal diffusional restrictions on the catalytic potential of the enzyme will be of a greater magnitude for a fast reaction of hydrolysis than a slower reaction of synthesis. Experimental evidences were obtained from batch reactor operation where increase in enzyme load and particle size has a much stronger impact on the former reaction. Additional evidences were obtained by the impact of the above biocatalyst properties on the apparent Michaelis constants for substrates. Michaelis constants of penicillin G acylase catalysts of different enzyme load and particle sizes varied between 0.73 and 4.55 mM and between 3.0 and 11.1 mM for the hydrolysis of penicillin G and the synthesis of cephalexin respectively. Michaelis constants for penicillin G acylase biocatalysts subjected to progressive size reduction were reduced from 5.0 to 0.46 mM and from 7.2 to 2.1 mM for hydrolysis of penicillin G and synthesis of cephalexin respectively. Higher fluctuation between these values in hydrolysis of penicillin G reflects higher impact of diffusional restrictions on this reaction. Thièle modulus for the substrate was much higher for penicillin G hydrolysis than for cephalexin synthesis in a wide range of substrates concentrations. Ratio between moduli for hydrolysis of penicillin G and synthesis of cephalexin was 15 at saturating concentration of phenylglycine methyl ester and increased at lower concentrations of such substrate. Results highlight the importance of designing the biocatalyst according to the reaction in which it will be used, being particularly important in the case of penicillin G acylase that is currently being used both in reactions of hydrolysis and synthesis.
  Enzyme and Microbial Technology 07/2010;