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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #3)

Assessment of Physical Process Conditions for Enhanced Production of Novel Glutaminase-Free L-Asparaginase from Pectobacterium carotovorum MTCC 1428 by Sanjay Kumar; Venkata Dasu Veeranki; Kannan Pakshirajan (pp. 327-337).

Statistically based experimental design was applied to maximize the production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. The effect of physical process parameters (initial pH of the medium, temperature, rpm of the shaking incubator, and inoculum size) on the production of L-asparaginase from P. carotovorum MTCC 1428 was studied using central composite design technique. The individual optimum levels of initial pH of the medium, temperature, rpm of shaking incubator, and inoculum size were found to be 6.90, 29.8 °C, 157 rpm, and 2.61% (v/v), respectively, for the production of L-asparaginase. After physical process parameters optimization, the production and productivity of L-asparaginase was enhanced by 26.39% (specific activity) and 10.19%, respectively. Maximization of L-asparaginase production was achieved at 12 h under optimal levels of physical process parameters in shake flask level.

Keywords: Pectobacterium carotovorum ; L-asparaginase; Physical process parameters; Central composite design; Response surface methodology

Development of DNA-Designed Avian IgY Antibodies for Quantitative Determination of Bovine Interferon-Gamma by Gholamreza Nikbakht Brujeni; Sayed Amir Hossein Jalali; Mohammad Kazem Koohi (pp. 338-345).

Interferon-gamma (IFN-γ), a cytokine produced by sensitized T lymphocytes, is one of the key elements in defining T helper 1 lymphocyte immune responses. Quantitative evaluation of IFN-γ expression could provide an important analytical tool for measurement of cell-mediated immunity and investigating immune responses to infectious diseases. Method of DNA-designed avian IgY antibodies was used for production of monospecific polyclonal antibodies that allows quantification of the recombinant bovine IFN-γ protein. IFN-γ cDNA was subcloned and expressed in mammalian expression plasmid (pcDNA3.1(+)) under the control of the human cytomegalovirus promoter. Chickens were immunized by plasmid DNA, and eggyolk antibodies extracted from eggs were collected after immunization. IgY-specific antibodies were evaluated by an antigen capture enzyme-linked immunosorbent assay (ELISA) using recombinant IFN-γ. Based on the results, developed bovine IFN-γ capture ELISA could detect up to 1 ng/ml of IFN-γ by 64-fold diluted IgY. Monospecific anti-bovine IFN-γ antibodies generated in chickens are useful for quantifying different concentrations of recombinant bovine IFN-γ, which is expressed in cell culture.

Keywords: Bovine interferon gamma; Capture enzyme-linked immunosorbent assay (ELISA); DNA-designed IgY

Decolorization Potential of Some Reactive Dyes with Crude Laccase and Laccase-Mediated System by Samet Şaşmaz; Serap Gedikli; Pınar Aytar; Gökhan Güngörmedi; Ahmet Çabuk; Evrim Hür; Arzu Ünal; Nazif Kolankaya (pp. 346-361).

In this study, decolorization of dyestuffs, such as Reactive Red 198, Rem Blue RR, Dylon Navy 17, Rem Red RR, and Rem Yellow RR was studied using laccase and laccase-mediated system. The laccases are known to have an important potential for remediation of pollutants. Among these dyestuffs, decolorization of Rem Blue RR and Dylon Navy 17 was performed with crude laccase under optimized conditions. Vanillin was selected as laccase mediator after screening six different compounds with Rem Yellow RR, Reactive Red 198, and Rem Red RR as substrates. However, Rem Yellow RR was not decolorized by either laccase or laccase-mediated system. It is observed that the culture supernatant contained high laccase activity after treatment with catalase that was responsible for the decolorization. Besides, culture supernatant with high laccase activity as enzyme source was treated with catalase; in this way, the hypothesis that laccase was the enzyme responsible for decolorization was supported. The Rem Blue RR was decolorized with 64.84% under the optimum conditions and Dylon Navy 17 with 75.43% with crude laccase. However, using the laccase and vanillin, the decolorization of Reactive Red 198 and Rem Red RR was found to be 62% and 68%, respectively. Our study demonstrated that the decolorization abilities of laccase and/or laccase mediator systems were based on the types of mediator, the dye structure, and the standard experimental conditions. Also, the electrochemical behaviors of some samples were studied. The redox potentials of these samples were determined using cyclic voltammetry on glassy carbon electrode in phosphate buffer (pH 6) solution.

Keywords: Laccase; Dyestuffs; Mediator; Decolorization; Trametes versicolor

The Stability of Accumulating Nitrite from Swine Wastewater in a Sequencing Batch Reactor by Liang Wang; Jun Zhu; Curtis Miller (pp. 362-372).

Shortcut nitrification is the first step of shortcut nitrogen removal from swine wastewater. Stably obtaining an effluent with a significant amount of nitrite is the premise for the subsequent shortcut denitrification. In this paper, the stability of nitrite accumulation was investigated using a 1.5-day hydraulic retention time in a 10-L (working volume) activated sludge sequencing batch reactor (SBR) with an 8-h cycle consisted of 4 h 38 min aerobic feeding, 1 h 22 min aerobic reaction, 30 min settling, 24 min withdrawal, and 1 h 6 min idle. The nitrite production stability was tested using four different ammonium loading rates, 0.075, 0.062, 0.053, and 0.039 g NH₄-N/g (mixed liquid suspended solid, MLSS) day in a 2-month running period. The total inorganic nitrogen composition in the effluent was not affected when the ammonium load was between 0.053 and 0.075 g NH₄-N/g MLSS · day (64% NO₂-N, 16% NO₃-N, and 20% NH₄-N). Under 0.039 g NH₄-N/g MLSS · day, more NO₂-N was transformed to NO₃-N with an effluent of 60% NO₂-N, 20% NO₃-N, and 20% NH₄-N. The reducing load test was able to show the relationship between a declining free nitrous acid (FNA) concentration and the decreasing nitrite production, indicating that the inhibition of FNA on nitrite oxidizing bacteria depends on its levels and an ammonium loading rate around 0.035 g NH₄-N/g MLSS · day is the lower threshold for producing a nitrite dominance effluent in the activated sludge SBR under the current settings.

Keywords: Swine manure; Nitrite accumulation; SBR; Nitrogen removal; Shortcut nitrification and denitrification

Biosynthesis of Ribostamycin Derivatives by Reconstitution and Heterologous Expression of Required Gene Sets by Nagendra Prasad Kurumbang; Kwangkyoung Liou; Jae Kyung Sohng (pp. 373-382).

Ribostamycin is a 4,5-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics and naturally produced by Streptomyces ribosidificus ATCC 21294. It is also an intermediate in the biosynthesis of butirosin and neomycin. In the biosynthesis of ribostamycin, DOS is glycosylated to generate paromamine which is converted to neamine by successive dehydrogenation followed by amination, and finally ribosylation of neamine gives ribostamycin. Here, we report the biosynthesis of 6′-deamino-6′-hydroxyribostamycin (a ribostamycin derivative or pseudoribostamycin) in Streptomyces venezuelae YJ003 by reconstructing gene cassettes for direct ribosylation of paromamine. A trace amount of pseudoribostamycin was detected with ribostamycin in the isolates of ribostamycin cosmid heterologously expressed in Streptomyces lividans TK24. It has also indicated that the ribosyltransferase can accept both neamine and paromamine. Thus, the present in vivo modification of ribostamycin could be useful for the production of hybrid compounds to defend against bacterial resistance to aminoglycosides.

Keywords: Aminoglycoside; Ribostamycin derivatives; Biosynthesis; Ribosyltransferase; Heterologous expression; Streptomyces

Screening of Pectinase-Producing Microorganisms with Polygalacturonase Activity by Jamile Zeni; Karine Cence; Camila Elis Grando; Lídia Tiggermann; Rosicler Colet; Lindomar A. Lerin; Rogério L. Cansian; Geciane Toniazzo; Débora de Oliveira; Eunice Valduga (pp. 383-392).

The aim of this work was to perform the screening of microorganisms, previously isolated from samples of agro-industrial waste and belonging to the culture collection of our laboratory, able to produce polygalacturonases (PG). A total of 107 microorganisms, 92 newly isolated and 15 pre-identified, were selected as potential producers of enzymes with PG activity. From these microorganisms, 20 strains were able to synthesize PG with activities above 3 U mL⁻¹. After the kinetic study, the enzyme activity was increased up to 13 times and the microorganism identified as Aspergillus niger ATCC 9642 and the newly isolated W23, W43, and D2 (Penicillium sp.) after 24 h of fermentation led to PG activities of 30, 41, 43, and 45 U mL⁻¹, respectively. The RAPD analysis demonstrated that the selected strains differs genetically, indicating that no duplication of strains among them in the experiments for polygalacturonases production was verified.

Keywords: Screening; Microorganisms; Polygalacturonase; Kinetic study

The Relationships Between Sulphate Reduction and COD/VFA Utilisation Using Grass Cellulose as Carbon and Energy Sources by Jean Mulopo; Harma Greben; Julia Sigama; Vivian Radebe; Mlawule Mashego; Lisa Burke (pp. 393-403).

The release of mine effluents can have a damaging impact on receiving water bodies. Therefore, treatment of mine waters before discharge is imperative. A novel biological $$ {hbox{SO}}_4^{2 - } $$ removal technology has been developed whereby the degradation/fermentation products of grass cellulose, volatile fatty acids (VFA), function as the electron donors and $$ {hbox{SO}}_4^{2 - } $$ as the electron acceptor. The aim of the study presented here was to elucidate the interactions between the cellulose degradation rate, the chemical oxygen demand (COD), VFA production and its/utilisation rate as well as the sulphate reduction rate. To this end, two stirred batch reactors were operated: a test and a control reactor. The results showed that high COD and VFA concentrations were achieved after cellulose degradation, which resulted in a rapid decrease in the $$ {hbox{SO}}_4^{2 - } $$ concentration in the test reactor. The VFA results indicated that propionic and butyric acids were preferentially utilised, producing acetate. In the control reactor, the VFA and the COD production increased initially at the same rate, followed later by a decrease at a similar rate. These results suggest that the degradation products formed were utilised by the methanogenic bacteria to produce methane rather than by the sulphate-reducing bacteria, since the control reactor contained no sulphate (Visser 1995). Furthermore, these results showed a clear relationship between the COD/VFA production and the $$ {hbox{SO}}_4^{2 - } $$ reduction in the test reactor and between the COD and VFA pattern in the control reactor.

Keywords: Biological sulphate removal; Batch reactors; COD/VFA production and utilisation; Relationship; Rumen microorganisms; Sulphate-reducing bacteria; Syntrophy

Probing Reactivity of PQQ-Dependent Carbohydrate Dehydrogenases Using Artificial Electron Acceptor by Lidija Tetianec; Irina Bratkovskaja; Juozas Kulys; Vida Casaite; Rolandas Meskys (pp. 404-414).

The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were determined using various electron acceptors. The radical cations of organic compounds and 2,6-dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose. The reactivity of dioxygen with ASDH is low; the bimolecular constant k _ox = 660 M⁻¹ s⁻¹, while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of dehydrogenases carbohydrates specificity. Mono- and disaccharide reactivity with GDH is higher than the reactivity of oligosaccharides. For ASDH, the reactivity increased with the carbohydrate monomer number increase. The specificity of quinoproteins was compared with specificity of flavoprotein Microdochium nivale carbohydrate oxidase due to potential enzymes application for lactose oxidation.

Keywords: Carbohydrate; Lactose; PQQ; Glucose dehydrogenase; Electron acceptor

Enhanced Laccase Production in White-Rot Fungus Rigidoporus lignosus by the Addition of Selected Phenolic and Aromatic Compounds by Maria Teresa Cambria; Santa Ragusa; Vittorio Calabrese; Antonio Cambria (pp. 415-422).

The white rot fungus Rigidoporus lignosus produces substantial amounts of extracellular laccase, a multicopper blue oxidase which is capable of oxidizing a wide range of organic substrates. Laccase production can be greatly enhanced in liquid cultures supplemented with various aromatic and phenolic compounds. The maximum enzyme activity was reached at the 21st or 24th day of fungal cultivation after the addition of inducers. The zymograms of extracellular fluid of culture preparation in the presence of inducers, at maximum activity day, revealed two bands with enzymatic activity, called Lac1 and Lac2, having different intensities. Lac2 band shows the higher intensity which changed with the different inducers. Laccase induction can be also obtained by adding to the culture medium olive mill wastewaters, which shows a high content of phenolic compounds

Keywords: Rigidoporus lignosus ; Laccase; Induction; Aromatic compounds; Wastewater

Induction and Purification by Three-Phase Partitioning of Aryl Alcohol Oxidase (AAO) from Pleurotus ostreatus by Vaidyanathan Vinoth Kumar; Vinohar Stephen Rapheal (pp. 423-432).

Aryl alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin degradation by white rot fungi. Screening of lignolytic and AAO activity from twenty different fungal species were carried out. Among them, seven species showed lignolytic activity and three of them (Pleurotus ostreatus, Pleurotus eous, and Pleurotus platypus) were found to be AAO positive. Maximal AAO activity was observed in batch cultures of P. ostreatus and was found to be induced by aromatic amino acids and aryl alcohols up to a level of 289 U/l. Purification of AAO was carried out by three-phase partitioning (TPP). The 67 kDa enzyme was purified up to 10.19-fold by TPP with an overall recovery of 10.95%. Optimum pH and temperature for P. ostreatus AAO activity was found to be around 6 and 40 °C, respectively. From the LB plot, K _m value of AAO for oxidizing veratryl alcohol was determined to be 0.6 mM. Results of the study indicate that P. ostreatus is the best producers of AAO, and they could be employed as promising fungal species for biotechnological applications.

Keywords: Aryl alcohol oxidase; Inducer; Pleurotus ostreatus ; Three-phase partitioning

Decolorization of Naphthol Blue Black using the Horseradish Peroxidase by Selva Onder; Mithat Celebi; Melda Altikatoglu; Arzu Hatipoglu; Huriye Kuzu (pp. 433-443).

This study evaluates the potential of the enzyme horseradish peroxidase in the decolorization of one common industrial azo dye, naphthol blue black. Studies are carried out to understand the process parameters such as pH, temperature and reaction time. The enzymatic decolorization of the dye was examined by UV-Vis spectrophotometer and LC-MS measurements. Temperature and pH conditions were optimized for obtaining high azo-dye decolorization. Azo-dye removal at a pH range 4-6 was found to be the highest for all temperatures. After 5 minutes of treatment, the color removal of dye was ca. 80-90%. The LC-MS and spectrophotometric analyses indicated that the decolorization of the azo dye with enzyme was due to the reduction of the azo bonds. This study verifies the viability of the use of the horseradish peroxidase in the decolorization of naphthol blue black.

Keywords: Dye decolorization; Horseradish peroxidase; Naphthol blue black; Spectrophotometer; LS-MS

Novel Activity of UDP-Galactose-4-Epimerase for Free Monosaccharide and Activity Improvement by Active Site-Saturation Mutagenesis by Hye-Jung Kim; Sueng Yeun Kang; Jong Jin Park; Pil Kim (pp. 444-451).

Uridine diphosphogalactose-4-epimerase (UDP-galactose-4-epimerase, GalE, EC 5.1.3.2) mediates the 4-epimerization of nucleic acid-activated galactose into UDP-glucose. To date, no enzyme is known to mediate 4-epimerization of free monosaccharide substrates. To determine the potential activity of GalE for free monosaccharide, Escherichia coli GalE was expressed and purified using Ni-affinity chromatography, and its ability to mediate 4-epimerization of a variety of free keto- and aldohexoses was assessed. Purified GalE was found to possess 4-epimerization activity for free galactose, glucose, fructose, tagatose, psicose, and sorbose at 0.47, 0.31, 2.82, 9.67, 15.44, and 2.08 nmol/mg protein per minute, respectively. No 4-epimerization activity was found for allose, gulose, altrose, idose, mannose, and talose. The kinetic parameters of 4-epimerization reactions were K _m = 26.4 mM and k _cat = 0.0155 min⁻¹ for d-galactose and K _m = 237 mM and k _cat = 0.327 min⁻¹ for d-tagatose. The 4-epimerization of tagatose, a reaction of commercial interest, was enhanced twofold (19.79 nmol/mg protein per minute) when asparagine was exchanged with serine at position 179. The novel activity of GalE for free monosaccharide may be beneficial for the production of rare sugars using cheap natural resources. Potential strategies for developing enhanced GalE with increased 4-epimerization activity are discussed in the context of the above findings and an analysis of a 3D structural model.

Keywords: UDP-galactose-4-epimerase (GalE); Free monosaccharide; Substrate specificity

Erratum: Properties of a Cationic Peroxidase from Citrus jambhiri cv. Adalia by Saleh A. Mohamed; Mohamed O. El-Badry; Ehab A. Drees; Afaf S. Fahmy (pp. 452-452).

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #3)