Applied Biochemistry and Biotechnology (v.160, #7)

Monoglyceride and Diglyceride Production Through Lipase-Catalyzed Glycerolysis and Molecular Distillation by Patricia Bogalhos Lucente Fregolente; Gláucia Maria F. Pinto; Maria Regina Wolf-Maciel; Rubens Maciel Filho (1879-1887).
Distilled glycerides are obtained through distillation of the system mono-diglycerides which is produced from the esterification reaction between a triglyceride with glycerol. In this work, monoglycerides (MG) and diglycerides (DG) are produced through lipase-catalyzed glycerolysis of soybean oil using Candida antarctica B in a solvent-free system. To separate the products of the reaction in order to obtain essentially MG and an oil of DG, it is necessary to use a suitable process in order to preserve the stability of the components and to keep the products free of inappropriate solvents. So, after 24 h of enzymatic reaction, the mixture of acylglycerols and fatty acids was distilled into a centrifugal molecular distiller, since it provides a free solvent and lower temperature environment to increase the desired product concentration. Starting from a material with 25.06% of triglycerides (TG), 46.63% of DG, 21.72% of MG, 5.38% of free fatty acids (FFA), and 1.21% of glycerol, the MG purity in the distillate stream was 80% at evaporator temperature (T E) equal to 250 °C and feed flow rate (Q) equal to 10.0 mL/min. At these conditions, the MG recovery was 35%. The material collected in the residue stream presented DG-enriched oil with TG unhydrolyzed, residual MG, and low acidity (29.83% of TG, 53.20% of DG, 15.64% of MG, and 1.33% of FFA), which is suitable to replace TG oil in the human diet.
Keywords: Molecular distillation; Distilled monoglycerides; Diglycerides; Lipase-catalyzed glycerolysis

Nickel-Impregnated Silica Nanoparticle Synthesis and Their Evaluation for Biocatalyst Immobilization by Reddy Shetty Prakasham; G. Sarala Devi; Chaganti Subba Rao; V. S. S. Sivakumar; T. Sathish; P. N. Sarma (1888-1895).
In the present investigation, impact of nickel-impregnated silica paramagnetic particles (NSP) as biocatalyst immobilization matrices was investigated. These nanoparticles were synthesized by sol–gel route using a nonionic surfactant block co polymer [poly (ethylene glycol)-block-poly-(propylene glycol)-block-poly (ethylene glycol)]. Diastase enzyme was immobilized on these particles (enzyme-impregnated NSP) as model enzyme and characterized using Fourier-transform infrared spectroscopy and X-ray crystallography. Analysis of enzyme-binding nature with these nanoparticles at different physiological conditions revealed that binding pattern and activity profile varied with the pH of the reaction mixture. The immobilized enzyme was further characterized for its biocatalytic activity with respect to kinetic properties such as Km and Vmax and compared with free enzyme. Paramagnetic nanoparticle-immobilized enzyme showed more affinity for substrate compared to free one. The nature of silica and nickel varied from amorphous to crystalline nature and vice versa upon immobilization of enzyme. To the best of our knowledge, this is the first report of its kind for change of nature from one form to other under normal temperatures upon diastase interaction with NSP.
Keywords: Diastase; Nickel–silica nanoparticles; Immobilization/impregnation; Enzyme kinetics

The Effect of Trifluoroethanol on Tyrosinase Activity and Conformation: Inhibition Kinetics and Computational Simulations by Zhi-Rong Lü; Long Shi; Jun Wang; Daeui Park; Jong Bhak; Jun-Mo Yang; Yong-Doo Park; Hong-Wei Zhou; Fei Zou (1896-1908).
We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of tyrosinase. TFE increased the degree of secondary structure of tyrosinase, which directly resulted in enzyme inactivation. A reciprocal study showed that TFE inhibited tyrosinase in a slope-parabolic mixed-type inhibition manner (K I = 0.5 ± 0.096 M). Time-interval kinetic studies showed that the inhibition was best described as first order with biphasic processes. Intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescences were also measured to gain more insight into the supposed structural changes; these showed that TFE induced a conspicuous tertiary structural change in tyrosinase by exposing hydrophobic surfaces. We also predicted the tertiary structure of tyrosinase and simulated its docking with TFE. The docking simulation was successful with significant scores (binding energy for Autodock4 = −4.75 kcal/mol; for Dock6 = −23.07 kcal/mol) and suggested that the TRP173 residue was mainly responsible for the interaction with TFE. Our results provide insight into the structure of tyrosinase and allow us to describe a new inhibition strategy that works by inducing conformational changes rather than targeting the active site of the protein.
Keywords: Tyrosinase; Trifluoroethanol; Inhibition kinetics; Secondary structure; Docking simulation

An extremely alkaliphilic bacterial strain, Bacillus sp. PPKS-2, was isolated from rice mill effluents and screened for the production of extracellular keratinase. The maximum production of keratinase occurred after 48 h in shaking culture at pH 11.0 and 37 °C in a medium containing 0.5% soybean flour. The strain grew and produced alkaline keratinase using chicken feather and horn meal as the sole source of carbon and nitrogen. An addition of 0.1% soybean flour or feather hydrolysate and sodium sulfite to feather medium increased the production and complete solubilization of feather took place within 5 days under solid-state fermentation conditions. The partially purified enzyme displayed maximum activity at pH 11.0 and 60 °C in a broad range of NaCl, 0–16%, and was not inhibited by sodium dodecyl sulfate (10%), ethylenediaminetetraacetic acid (10 mM), H2O2 (15%), and other commercial detergents. Immobilization of the whole cells proved to be useful for continuous production of keratinase and feather degradation. The enzyme was effectively used to remove hair from goat hide. The strain PPKS-2 can be effectively used for solid waste management of poultry feather in submerged as well as solid-state fermentation.
Keywords: Bacillus halodurans PPKS-2; Keratinase; Feather degradation; Dehairing; Immobilization; Solid-state fermentation

Acid-induced unfolding of a Tetracoccosporium sp. polygalacturonase enzyme (PG) was studied by a comprehensive series of biophysical and biochemical techniques. At pH 1.0, PG acquires partially folded state, which reveals characteristics of molten globule (MG) state, i.e., reduction of defined tertiary structure with minimal changes in the secondary structure. In this study PG unfolds exposing its hydrophobic surface to a greater extent than the native form at acidic pH with more tryptophan residues exposed to the solvent. Collectively, our data imply the presence of MG state of PG at low pH, suggesting the phenomenon of hydrophobic collapse model for folding and integration into cell membrane.
Keywords: Aggregation; Molten globule; Polygalacturonase; Refolding; Tetracoccosporium sp.

Expression of a Chitinase Gene from Metarhizium anisopliae in Tobacco Plants Confers Resistance against Rhizoctonia solani by Marcelo Fernando Kern; Simone de Faria Maraschin; Débora Vom Endt; Augusto Schrank; Marilene Henning Vainstein; Giancarlo Pasquali (1933-1946).
The chit1 gene from the entomopathogenic fungus Metarhizium anisopliae, encoding the endochitinase CHIT42, was placed under the control of the CaMV 35S promoter, and the resulting construct was transferred to tobacco. Seventeen kanamycin-resistant transgenic lines were recovered, and the presence of the transgene was confirmed by polymerase chain reactions and Southern blot hybridization. The number of chit1 copies was determined to be varying from one to four. Copy number had observable effects neither on plant growth nor development. Substantial heterogeneity concerning production of the recombinant chitinase, and both general and specific chitinolytic activities were detected in leaf extracts from primary transformants. The highest chitinase activities were found in plants harboring two copies of chit1 inserts at different loci. Progeny derived from self-pollination of the primary transgenics revealed a stable inheritance pattern, with transgene segregation following a mendelian dihybrid ratio. Two selected plants expressing high levels of CHIT42 were consistently resistant to the soilborne pathogen Rhizoctonia solani, suggesting a direct relationship between enzyme activity and reduction of foliar area affected by fungal lesions. To date, this is the first report of resistance to fungal attack in plants mediated by a recombinant chitinase from an entomopathogenic and acaricide fungus.
Keywords: Chitinase; Metarhizium anisopliae ; Disease resistance; Rhizoctonia solani ; Genetic transformation; Nicotiana tabacum

New Potential Biocatalysts by Laccase Immobilization in PVA Cryogel Type Carrier by Michaela Dina Stanescu; Magdalena Fogorasi; Boris L. Shaskolskiy; Simona Gavrilas; Vladimir I. Lozinsky (1947-1954).
Laccases are enzymes belonging to the Oxidoreductases class. These enzymes may be good biocatalysts for different processes, at laboratory and industrial levels. A successful use at industrial scale demands a higher stability of the enzyme. As an easy way to obtain longer life biocatalysts, the immobilization process is recommended. Thus, the paper presents different ways of obtaining new biocatalysts by a laccase covalent immobilization on a macroporous carrier based on poly(vinyl alcohol) cryogel. Different procedures of covalent immobilization are described, the newly obtained biocatalysts being characterized. According to the experimental data, the stability of the immobilized enzyme increased and the pH profile changed, compared with those of the free enzyme.
Keywords: Laccase covalent immobilization; Poly(vinyl alcohol) cryogel

The action modes of an oligosaccharide-producing multifunctional amylase (OPMA) were investigated using glucose and some oligosaccharides as its substrates. OPMA did not cause the hydrolysis of maltose or isomaltose, but it catalyzed the α-1,6-transglycosylation of maltose to produce isomaltose or did the self-condensation of isomaltose to form isomaltotetraose and 4-O-α-isomaltosyl isomaltose. OPMA exhibited strong α-1,6-transglycosylation activity in addition to its α-1,4-hydrolytic activity on higher oligosaccharides substrates rather than bisaccharides. OPMA displayed high acceptor specificity in its transglycosylation or condensation reaction. OPMA seemed to only take glucose or isomaltose as the acceptor molecule in its transglycosylation or condensation reaction, which made glucose or isomaltose form higher products, and as a result, glucose or isomaltose were absent in the final products. In view of the simultaneously formation of several transglycosylation or condensation products, it was predicted that there might be separate donor and acceptor sites in OPMA’s active center and the fact that the catalytically active form of this enzyme included its homodimer or homotrimer supported this prediction. Accordingly, a special pathway, isomaltose pathway, for OPMA catalysis was proposed to emphasize the central or important signification of isomaltose in OPMA catalysis.
Keywords: Amylase; Action mode; Condensation; Hydrolysis; Transglycosylation

The mechanism of action of the anti-apoptotic oncogene Bcl-2 and Ca+2 regulation in ischemia–reperfusion injury is still obscure. In this present study, we investigated mitochondrial Ca+2 overloads and mechanism of action of Bcl-2. Eighteen Wistar rats were divided into sham-operated control group (I) (n = 6), ischemia and reperfusion group (II) (n = 6), and amlodipine-treated group (1 mg kg−1 body weight/daily by oral route for 7 days before inducing ischemia–reperfusion maneuver) (III) (n = 6). Rats were subjected to 1 h of hepatic ischemia followed by 3-h reperfusion. Mitochondrial Ca2+ content was determined and damage was confirmed by transmission electron microscopy. Decrease of mitochondrial Ca+2 level is related to reduction of apoptosis and cellular changes, viz. increased Bcl-2 expression followed by reduction in secondary endoplasmic reticulum, whereas ischemia/reperfusion group shows overloading Ca+2 ions and decrease in Bcl-2 expression as compared to sham-operated rats. Thus, Bcl-2-dependent reduction of Ca+2 is an important component of the anti-apoptotic program in ischemia–reperfusion injury.
Keywords: Ca+2 ; Bcl-2; Apoptosis; Ischemia–reperfusion

E. coli, P. aeruginosa, and B. cereus Bacteria Sterilization Using Afterglow of Non-Thermal Plasma at Atmospheric Pressure by Farshad Sohbatzadeh; Abasalt Hosseinzadeh Colagar; Saeed Mirzanejhad; Somayeh Mahmodi (1978-1984).
We developed and employed a new geometrical structure of dielectric barrier discharge in atmospheric pressure for bacterial broad spectrum sterilization. We utilized a plasma source having an AC power supply at 50 HZ and 5,400 V (rms value). We prepared suspensions of the Gram-negative bacteria species (Escherichia coli, Pseudomonas aeruginosa) and a Gram-positive of Bacillus cereus with Luria–Bertani broth media up to OD600 nm = 0.25 of McFarland standard. Afterglow of non-thermal atmospheric pressure plasma treated these suspensions. The influence of the atmospheric plasma afterglow on the species was assayed in different time durations 5, 10, and 15 min. The spectroscopic results of this investigation indicated that the survival reduction of the species can reach to 100% for P. aeruginosa in an exposure time of 10 min, E. coli and B. cereus in an exposure time of 15 min.
Keywords: Dielectric barrier discharge; Non-thermal atmospheric plasma; Sterilization; Escherichia coli ; Pseudomonas aeruginosa ; Bacillus cereus

Gene Cloning, Expression, and Characterization of Recombinant Aerolysin from Aeromonas hydrophila by Vijai Singh; Pallavi Somvanshi; Gaurav Rathore; D. Kapoor; B. N. Mishra (1985-1991).
Aerolysin is a significant virulent toxin protein secreted by Aeromonas hydrophila; it produces deep wound infections and hemorrhagic septicemia. The complete aerolysin gene (1,482 bp) was amplified from A. hydrophila. Furthermore, it was cloned and expressed into Escherichia coli BL21(DE3) codon plus RP cells using 0.5 mM IPTG for induction. The protein size was 54 kDa as estimated by SDS-PAGE, and it was purified by Ni–NTA affinity chromatography. Anti-His antibodies were used to characterize the expressed aerolysin by Western blotting and showed hemolytic activity with fish red blood cells. Aerolysin may be used as immunoassays for earlier control of A. hydrophila and is also compatible for vaccination.
Keywords: Aeromonas hydrophila ; Aerolysin; Expression; RBCs; Vaccine

First Evidence of Aerobic Biodegradation of BTEX Compounds by Pure Cultures of Marinobacter by Sabrina Berlendis; Jean-Luc Cayol; Frédéric Verhé; Sophie Laveau; Jean-Luc Tholozan; Bernard Ollivier; Richard Auria (1992-1999).
Marinobacter vinifirmus was shown to degrade toluene as sole carbon and energy source under aerobiosis and at NaCl concentrations in the range 30–150 g/L. Maximum toluene consumption rate, total CO2, and biomass productions were measured in the presence of 60 g/L of NaCl. Under these conditions, 90% of the carbon from toluene was recovered as CO2 and biomass. Maximum specific toluene consumption rate was about 0.12 mgC toluene mgC biomass−1 h−1 at NaCl concentrations between 30 and 60 g/L. It decreased to 0.03 mgC toluene mgC biomass−1 h−1 at 150 g/L. Besides toluene, M. vinifirmus degraded benzene, ethylbenzene, and p-xylene. Benzene and toluene were utilized to a lesser extent by another Marinobacter sp., Marinobacter hydrocarbonoclasticus.
Keywords: Halophilic bacterium; Toluene biodegradation; Aerobic; Marinobacter hydrocarbonoclasticus ; Marinobacter vinifirmus

Sonoassisted Microbial Reduction of Chromium by Mathur Nadarajan Kathiravan; Ramalingam Karthick; Naggapan Muthu; Karuppan Muthukumar; Manickam Velan (2000-2013).
This study presents sonoassisted microbial reduction of hexavalent chromium (Cr(VI)) using Bacillus sp. isolated from tannery effluent contaminated site. The experiments were carried out with free cells in the presence and absence of ultrasound. The optimum pH and temperature for the reduction of Cr(VI) by Bacillus sp. were found to be 7.0 and 37 °C, respectively. The Cr(VI) reduction was significantly influenced by the electron donors and among the various electron donors studied, glucose offered maximum reduction. The ultrasound-irradiated reduction of Cr(VI) with Bacillus sp. showed efficient Cr(VI) reduction. The percent reduction was found to increase with an increase in biomass concentration and decrease with an increase in initial concentration. The changes in the functional groups of Bacillus sp., before and after chromium reduction were observed with FTIR spectra. Microbial growth was described with Monod and Andrews model and best fit was observed with Andrews model.
Keywords: Bacillus sp.; Sonolysis; Chromium reduction; Electron donors; Growth kinetics

Feedback inhibition existed in lovastatin biosynthesis from Aspergillus terreus. Exogenous lovastatin and other different polyketide antibiotics biosynthesized by polyketide synthase were supplemented to the cultures of A. terreus to investigate their influences on lovastatin production. Supplementing exogenous lovastatin of 100 mg l−1 at the early stage of fermentation and the fast stage of its biosynthesis resulted in decreases of 76.4% and 20% in final lovastatin production, respectively. However, the fungal cell growth was not affected; the growing cycle was only prolonged in the submerged cultivation. Separate supplementation of the five kinds of polyketide antibiotics such as tylosin, erythromycin, tetracycline, daunorobin, and rifamycin to the cultures resulted in increases of about 20~25% in the final lovastatin production. Especially, supplementing tylosin of 50 mg l−1 at the beginning of lovastatin biosynthesis led to the final lovastatin production of 952.7 ± 24.3 mg l−1, which was improved by 42% and 22% compared with that produced in the control and the original culture, respectively. These results are helpful to understand the regulations on lovastatin biosynthesis and improve the final desired metabolite contents in many antibiotics production.
Keywords: Aspergillus terreus ; Lovastatin; Production; Polyketide antibiotics; Tylosin

A cascade reaction combining the enzymatic hydrolysis of Penicillin G potassium salt (PGK) with the kinetically controlled enzymatic coupling of in situ formed 6-aminopenicillanic acid (6-APA) with p-hydroxyphenylglycine methyl ester (D-HPGM) to give amoxicillin as the final product by using a single enzyme has been demonstrated successfully. Ethylene glycol (EG) was employed as a component of reaction buffer to improve the synthesis yield. Reaction parameters, including different cosolvents, EG content, the loading of immobilized penicillin G acylase (IPA), and reaction temperature and time were studied to evaluate their effects on the reaction. The best result of 55.2% yield was obtained from the reaction which was carried out in the mixed media containing 40% sodium dihydrogen phosphate buffer (apparent pH 6.0) and 60% EG (v/v), with the initial concentration 150 mM and 450 mM of PGK and D-HPGM, respectively, catalyzed by 50 IU/mL IPA at 25 °C for 10 h. The IPA could be recycled for nine batches without obviously losing of catalytic activity. The important strategy will have potential application in the β-lactam antibiotics industry due to the advantages of saving the effort of isolating 6-APA, reducing usual enzymatic steps and the industrial cost of amoxicillin synthesis.
Keywords: Amoxicillin; Cascade; Enzymatic synthesis; Penicillin G acylase; Penicillin G potassium salt

Expression of a Glucose-tolerant β-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae by Bruno Benoliel; Marcio José Poças-Fonseca; Fernando Araripe Gonçalves Torres; Lidia Maria Pepe de Moraes (2036-2044).
A β-glucosidase gene (bgl4) from Humicola grisea var thermoidea was successfully expressed in Saccharomyces cerevisiae. The recombinant protein (BGL4 Sc ) was initially detected associated with yeast cells and later in the culture medium. BGL4 Sc showed optimal pH and temperature of 6.0 and 40 °C, respectively, and an apparent molecular mass of 57 kDa. The enzyme showed activity against cellobiose and synthetic substrates, and was inhibited more than 80% by Fe2+, Cu2+, Zn2+, and Al3+. Using p-nitrophenyl-β-d-glucopyranoside (pNPG) as substrate, BGL4 Sc presented a V max of 6.72 μmol min−1 mg total protein−1 and a K m of 0.16 mM under optimal conditions. Most important, BGL4 Sc is resistant to inhibition by glucose and the calculated K i value for this sugar is 70 mM. This feature prompts BLG4 Sc as an ideal enzyme to be used in the saccharification process of lignocellulosic materials for ethanol production.
Keywords: β-Glucosidase; Humicola grisea var. thermoidea ; Saccharomyces cerevisiae ; Glucose tolerance; Biomass conversion

A Process to Produce Penicillin G Acylase by Surface-Adhesion Fermentation Using Mucor griseocyanus to Obtain 6-Aminopenicillanic Acid by Penicillin G Hydrolysis by José Luis Martínez-Hernández; Marco Arnulfo Mata-Gómez; Cristóbal Noé Aguilar-González; Anna Ilyina (2045-2053).
The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V max, and the Michaelis constant, K M, were determined using the Michaelis–Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0–10 mM). K M and V max were determined to be 3.0 × 10−3 M and 4.4 × 10−3 mM/min, respectively.
Keywords: Penicillin G acylase (PGA); Surface-adhesion fermentation (SAF); Opuntia imbricata ; Mucor griseocyanus

Biochemical Properties of α-Amylase from Peel of Citrus sinensis cv. Abosora by Saleh Ahmed Mohamed; Ehab A. Drees; Mohamed O. El-Badry; Afaf S. Fahmy (2054-2065).
α-Amylase activity was screened in the peel, as waste fruit, of 13 species and cultivars of Egyptian citrus. The species Citrus sinensis cv. Abosora had the highest activity. α-Amylase AI from Abosora peel was purified to homogeneity using anion and cation-exchange, and gel filtration chromatographies. Molecular weight of α-amylase AI was found to be 42 kDa. The hydrolysis properties of α-amylase AI toward different substrates indicated that corn starch is the best substrate. The α-amylase had the highest activity toward glycogen compared with amylopectin and dextrin. Potato starch had low affinity toward α-amylase AI but it did not hydrolyze β-cyclodextrin and dextran. Apparent Km for α-amylase AI was 5 mg (0.5%) starch/ml. α-Amylase AI showed optimum activity at pH 5.6 and 40 °C. The enzyme was thermally stable up to 40 °C and inactivated at 70 °C. The effect of mono and divalent metal ions were tested for the α-amylase AI. Ba2+ was found to have activating effect, where as Li+ had negligible effect on activity. The other metals caused inhibition effect. Activity of the α-amylase AI was increased one and half in the presence of 4 mM Ca2+ and was found to be partially inactivated at 10 mM Ca2+. The reduction of starch viscosity indicated that the enzyme is endoamylase. The results suggested that, in addition to citrus peel is a rich source of pectins and flavanoids, α-amylase AI from orange peel could be involved in the development and ripening of citrus fruit and may be used for juice processing.
Keywords: α-Amylase; Citrus sinensis cv. Abosora; Peel; Purification; Properties

Structure and Applications of a Rhamnolipid Surfactant Produced in Soybean Oil Waste by Marcia Nitschke; Siddhartha G. V. A. O. Costa; Jonas Contiero (2066-2074).
Soybean oil soapstock was utilized as an alternative carbon source for the production of rhamnolipids by Pseudomonas aeruginosa LBI strain. The chemical composition and properties of the rhamnolipid mixture obtained were determined to define its potential applications. The chemical characterization of the rhamnolipid has revealed the presence of ten different homologues. The monorhamnolipid RhaC10C10 and the dirhamnolipid Rha2C10C10 were the main components of the mixture that showed predominance of 44% and 29%, respectively, after 144-h of cultivation. The biosurfactant was able to form stable emulsions with several hydrocarbons and showed excellent emulsification for soybean oil and chicken fat (100%). The rhamnolipid removed 67% of crude oil present in sand samples and presented antimicrobial activity against Bacillus cereus and Mucor miehei at 64 μg/mL and inhibition of Neurospora crassa, Staphylococcus aureus, and Micrococcus luteus at 256 μg/mL. The results demonstrated that the rhamnolipid produced in soybean oil soapstock can be useful in environmental and food industry applications.
Keywords: Biosurfactants; Rhamnolipids; Soybean oil waste; Soapstock; Pseudomonas aeruginosa

Biotransformation of Celecoxib Using Microbial Cultures by Keshetty Srisailam; Ciddi Veeresham (2075-2089).
Microbial transformation studies can be used as models to simulate mammalian drug metabolism. In the present investigation, biotransformation of celecoxib was studied in microbial cultures. Bacterial, fungal, and yeast cultures were employed in the present study to elucidate the metabolism of celecoxib. The results indicate that a number of microorganisms metabolized celecoxib to various levels to yield eight metabolites, which were identified by high-performance liquid chromatography diode array detection and liquid chromatography tandem mass spectrometry analyses. HPLC analysis of biotransformed products indicated that majority of the metabolites are more polar than the substrate celecoxib. The major metabolite was found to be hydroxymethyl metabolite of celecoxib, while the remaining metabolites were produced by carboxylation, methylation, acetylation, or combination of these reactions. The methyl hydroxylation and further conversion to carboxylic acid was known to occur in metabolism by mammals. The results further support the use of microorganisms for simulating mammalian metabolism of drugs.
Keywords: Celecoxib; Biotransformation; HPLC; LC–MS/MS; Metabolite; Microorganisms

In this study, effect of various parameters on sophorolipid (SL) production by the yeast Candida bombicola was investigated for the enhancing of its production by employing L18 orthogonal array design of experiments. At optimum conditions of sugarcane molasses 50 g l−1, soybean oil 50 g l−1, inoculum size 5% (v/v), temperature 30 °C, inoculum age 2 days, and agitation 200 rpm, the yeast produced almost equal amounts of the product in batch shake flasks and in a 3-l fermentor without any pH control (45 and 47 g l−1, respectively). However, the yield increased to 60 g l−1 in the fermentor under controlled pH environment. Time course of SL production, yeast biomass growth, and utilization of sugarcane molasses and soybean oil at these optimized conditions were fitted to existing kinetic models reported in the literature. Estimated kinetic parameters from these models suggested that conventional medium containing glucose can very well be replaced with the present low-cost fermentative medium.
Keywords: Sophorolipids; Candida bombicola ; Design of experiments; Sugarcane molasses; Kinetics

A mesophilic bacterial culture producing a novel thermostable alkaline lipase was isolated from oil rich soil sample and identified as Bacillus subtilis EH 37. The lipase was partially purified by ammonium sulfate precipitation and hydrophobic interaction chromatography with 17.8-fold purification and 41.9 U/ml specific activity. The partially purified enzyme exhibited maximum activity at pH 8.0 and at 60 °C. It retained 100% of activity at 50 °C and 60 °C for 60 min. The presence of Ca+2, Mg+2, and Zn2+ exhibited stimulatory effect on lipase activity, whereas Fe+3 and Co+2 reduced its activity. The enzyme retained more than 80% of its initial activity upon exposure to organic solvents, exhibited 107% and 115% activity in the presence of 15% isopropyl alcohol and 30% n-hexane, respectively. The EH 37 lipase also proved to be an efficient catalyst in synthesis of ethyl caprylate in organic solvent, thus providing a concept of application of B. subtilis lipase in non-aqueous catalysis.
Keywords: Lipase; Esterification; Thermostable

Influences of Different Enzymatic Treatment on Denim Garment by Majid Montazer; Ali Sadeghian Maryan (2114-2128).
In current study, the effects of different enzymatic treatment including acid cellulases, neutral cellulases, and combination of laccases with cellulases on denim garment were investigated. The color changes of different samples were compared by colorimetric indices for the garment surface and back and also white pocket. Abrasion resistance, tensile strength, and crease recovery angle of the samples were measured within the standard methods. Also, surfaces of the treated samples were observed by SEM. The results showed that the combination of laccases with cellulases help to improve the lightness and decrease staining on both back of garment and on white pocket.
Keywords: Cellulases; Laccases; Lightness; Washing; Denim

Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique by Mário A. P. Nunes; Hélder Vila-Real; Pedro C. B. Fernandes; Maria H. L. Ribeiro (2129-2147).
A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA–alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 °C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)–alginate beads with three different sizes (1–3 mm), at three different alginate concentrations (0.2–1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 °C for the PVA–alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)–alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 °C. The Michaelis constant (K Mapp) and the maximum reaction velocity (V maxapp) were evaluated for both free (K Mapp = 0.233 mM; V maxapp = 0.13 mM min−1) and immobilized naringinase (K Mapp = 0.349 mM; V maxapp = 0.08 mM min−1). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 °C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.
Keywords: Naringin; Naringinase entrapment; Poly(vinyl alcohol); PVA; DNS micromethod; Microassay; Hydrolysis; Boric acid; Alginate

Evaluation of Immobilized Metal-Ion Affinity Chromatography (IMAC) as a Technique for IgG1 Monoclonal Antibodies Purification: The Effect of Chelating Ligand and Support by I. T. L. Bresolin; M. Borsoi-Ribeiro; W. M. S. C. Tamashiro; E. F. P. Augusto; M. A. Vijayalakshmi; S. M. A. Bueno (2148-2165).
Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG1 mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand—CM-Asp—was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG1 using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.
Keywords: Monoclonal antibodies; Purification; Adsorption; IMAC; Affinity membrane; Downstream processing

Inactivation Kinetics of Polyphenol Oxidase from Pupae of Blowfly (Sarcophaga bullata) in the Dimethyl Sulfoxide Solution by Chao-Qi Chen; Zhi-Cong Li; Zhi-Zhen Pan; Yu-Jing Zhu; Ruo-Rong Yan; Qin Wang; Jiang-Hua Yan; Qing-Xi Chen (2166-2174).
The effects of dimethyl sulfoxide (DMSO) on the activity of polyphenol oxidase (PPO, EC from blowfly pupae for the oxidation of l-3,4-dihydroxyphenylalanine were studied. The results showed that low concentrations of DMSO could lead to reversible inactivation to the enzyme. The IC50 value, the inactivator concentration leading to 50% activity lost, was estimated to be 2.35 M. Inactivation of the enzyme by DMSO was classified as mixed type. The kinetics of inactivation of PPO from blowfly pupae in the low concentrations of DMSO solution was studied using the kinetic method of the substrate reaction. The rate constants of inactivation were determined. The results show that k +0 was much larger than $$ k_{ + 0}^prime $$ , indicating that the free enzyme molecule was more fragile than the enzyme–substrate complex in the DMSO solution. It was suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.
Keywords: Polyphenol oxidase; Blowfly pupae; Inactivation; Kinetics; Dimethyl sulfoxide

The N-Terminal End Truncated Mu-Opioid Receptor: from Expression to Circular Dichroism Analysis by Isabelle Muller; Valérie Sarramégna; Alain Milon; Franck Jean Talmont (2175-2186).
In order to evaluate the biochemical, biophysical, and pharmacological implication of the N-terminal domain of the human mu-opioid receptor (HuMOR), deletion mutants lacking 64 amino acids from the amino terminus of HuMOR were constructed and expressed in the yeast Pichia pastoris. The recombinant proteins differed with respect to the presence of the Saccharomyces cerevisiae α-factor prepropeptide and the enhanced green fluorescent protein fused to the N terminus of the receptor. Pharmacological studies indicated that deletion of the N-terminal domain produced little effect on ligand affinities. The N-terminal end truncated and c-myc/6his-tagged receptor was subsequently purified to homogeneity and a yield of 5 mg/l was obtained after purification. The N-terminal end truncated receptor was further characterized by circular dichroism in trifluoroethanol and showed a characteristic pattern of α-helical structure. A pH effect on the structure of the receptor was observed when it was solubilized in sodium dodecyl sulfate micelles, with an increase of helicity at low pH.
Keywords: G-protein coupled receptor; Solubilization; Purification; Pichia pastoris ; Mu-opioid receptor