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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.111, #1)
Effect of some process parameters in enzymatic dyeing of wool by Tzanko Tzanov; Carla Joana Silva; Andrea Zille; Jovita Oliveira; Artur Cavaco-Paulo (pp. 1-13).
This article reports on the dyeing of wool using an enzymatic system comprising laccase; dye precursor, 2,5-diaminobenzenesulfonic acid; and dye modifiers, catechol and resorcinol. Enzymatic dyeing was performed as a batchwise process at the temperature and pH of maximum enzyme activity. The effects of the process variables reaction time, enzyme, and modifier concentration on fabric color were studied, according to an appropriate experimental design. Different hues and depths of shades could be achieved by varying the concentration of the modifiers and the time of laccase treatment. The duration of the enzymatic reaction appeared to be the most important factor in the dyeing process. Thus, the dyeing process, performed at low temperature and mild pH, was advantageous in terms of reduced enzyme and chemical dosage.
Keywords: Enzymatic dyeing; laccase; dye precursors; modifier; full factorial design
Growth and pectinase production by Aspergillus mexican strain protoplast regenerated under acidic stress by Leonardo Peraza; Marco Antonio Ortiz; John F. Peberdy; Guillermo Aguilar (pp. 15-27).
Protoplasts from Aspergillus sp. FP-180 and Aspergillus awamori NRRL-3112 were released and regenerated at extreme acidic conditions. The best conditions for protoplast release were 0.8 M KCI, pH 5.8, and 3h of digestion using mycelia from 12- to 16-h cultures from either Aspergillus sp. FP-180 or A. awamori NRRL-3112. The addition of fresh mycelia to an ongoing digestion after 1 h increased protoplast 4.5–5 times. A regeneration efficiency of 90% was attained at pH 6.0, and it was possible to regenerate protoplasts at pH 1.7 with a regeneration efficiency of 0.5% for Aspergillus sp. FP-180. The LpH-10 strain, derived from protoplast from Aspergillus sp FP-180, was able to regenerate at pH 1.7 and grow at pH values as low as 1.5, values at which the original strain is unable to grow. Regeneratio at extreme pH improved the performance of LpH-10 strain. It showed atwofold increase in cell growth at pH 2.0 in liquid culture and a higher pectinolytic activity in relation to that produced by the original strian.
Keywords: Aspergillus ; protoplast formation; regeneration under acid stress; protoplast storage; pectinases
Production, purification, and biochemical characterization of two β-glucosidases from Sclerotinia sclerotiorum by Smaali Mohamed Issam; Gargouri Mohamed; Limam Farid; Fattouch Sami; Maugard Thierry; Legoy Marie Dominique; Marzouki Nejib (pp. 29-39).
The filamentous fungus Sclerotinia sclerotiorum prudces ß-glucosidases in liquid culture with a variety of carbon sources, including cellulose (filter paper), xylan, barley straw, oat meal, and xylose. Analysis by native polyacrylamide gel electrophoresis (PAGE) followed by an activity staining with the specific chromogenic substrate, 5-bromo 4-chloro 3-indolyl ß-1,4 glucoside (X-glu) showed that two extracellular β-glucosidases, designated as ß-glul1 and -glu2, were in the filter paper culture filtrate. Only one enzyme designated as ß-glus was revealed by the same method in the xylose culture filtrate. ß-glu1 and ß-glu2 were purified to homogeneity. The purification procedure consist of a common step of anion-exchange chromatography on DEAE-Sepharose CL6B, both high-performance liquid chromatography (HPLC) anion-exchange and gel filtration columns for ß-glu1 and only HPLC gel filtration for ß-glu2. ß-glu1 has a molecular mass of 196 kDa and 96.5 kDa, as estimated by gel filtration and sodium dodecyl sulfate (SDS)-PAGE, respectively, suggesting that the native enzyme may consist of two identical subunits. The same analysis showed that ß-glu2 is a monomeric protein with an apparent molecular mass of about 76.5 kDa. ß-glu1 and ß-glu2 hydrolyses PNPG1c and cellobiose, with apparent K m values respectively for PNPGlc and cellobiose of 0.1 and 1.9 mM for ß-glu1 and 2.8 and 8 mM for ß-glu2. Both enzymes exhibit the same temperature and pH optima for PNPG1c hydrolysis (60°C and pH 5.0). ß-glu1 was stable over a pH range of 3–8 and kept 50% of its activity after 30 min of heating at 60°C without substrate. It was further characterized by studying the effect of some cations and various reagents on its activity.
Keywords: ß-Glucosidase; induction; purification; Sclerotinia sclerotiorum
Bioelectrocatalysts by Doo Hyun Park; C. Vieille; J. G. Zeikus (pp. 41-53).
Fumarate reductase was used as a model oxidoreductase to demonstrate continuous electrical cofactor reduction-oxidation during the bioelectrochemical synthesis and detection of chemicals. The enzyme preparation was immobilized onto a graphite felt electrode that was modified with carboxymethylcellulose (CMC). Nicotinamide adenine dinucleotide (NAD), neutral red, and fumarate reductase (which contained menaquinone) were covalently linked by peptide bonds to the CMC. The electron mediator neutral red allowed NAD and menaquinone to be recycled electrically during enzymatic chemical synthesis. Succinate detection by the bioelectrocatalyst was linear from 5 μM to 10 mM succinate. Fumarate synthesis using this bioelectrode was dependent on succinate utilization and resulted in proportional production of electricity and fumarate. Succinate synthesis using this bioelectrocatalyst was dependent on current and fumarate concentration. This bioelectrocatalyst system may enhance the utility of menaquinone- and/or pyridine nucleotide-linked oxidoreductases in diverse enzymatic fuel cells and sensors. It may also enhance the utility of oxidoreductase-based chemical synthesis systems because it eliminates the problem of cofactor recycling.
Keywords: Bioelectrochemistry; fumarate reductase; biosensor; cofactor recycling
An integrated process for purification of lysozyme, ovalbumin, and ovomucoid from hen egg white by Ipsita Roy; M. V. S. Rao; Munishwar N. Gupta (pp. 55-63).
This article describes an integrated process for simultaneous purification of lysozyme, ovalbumin, and ovomucoid from hen egg white. The crude egg white extract was passed through a cation exchanger Streamline™ SP and the bound lysozyme was eluted with 5% ammonium carbonate, pH 9.0, containing 1 M NaCl after elution of avidin. This partially purified lysozyme was further purified 639-fold on dye-linked cellulose beads. Ovalbumin and ovomucoid did not bind to Streamline SP. Ovalbumin could be precipitated from this unbound fraction by 5% trichloroacetic acid, and ovomucoid was removed from the supernatant by precipitation with ethanol. The yields of lysozyme, ovomucoid, and ovalbumin were 77, 94, and 98%, respectively. All the purified proteins showed single bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. All the steps are easily scalable, and the process described here can be used for large-scale simultaneous purification of these proteins in the pure form.
Keywords: Egg white; lysozyme; ovalbumin; ovomucoid; precipitation; dye-linked chromatography