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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.117, #1)
Evaluation of wet oxidation pretreatment for enzymatic hydrolysis of softwood by Hetti Palonen; Anne Belinda Thomsen; Maija Tenkanen; Anette S. Schmidt; Liisa Viikari (pp. 1-17).
The wet oxidation pretreatment (water, oxygen, elevated temperature, and pressure) of softwood (Picea abies) was investigated for enhancing enzymatic hydrolysis. The pretreatment was preliminarily optimized. Six different combinations of reaction time, temperature, and pH were applied, and the compositions of solid and liquid fractions were analyzed. The solid fraction after wet oxidation contained 58–64% cellulose, 2–16% hemicellulose, and 24–30% lignin. The pretreatment series gave information about the roles of lignin and hemicellulose in the enzymatic hydrolysis. The temperature of the pretreatment, the residual hemicellulose content of the substrate, and the type of the commercial cellulase preparation used were the most important factors affecting the enzymatic hydrolysis. The highest sugar yield in a 72-h hydrolysis, 79% of theoretical, was obtained using a pretreatment of 200°C for 10 min at neutral pH.
Keywords: Wet oxidation; enzymatic hydrolysis; lignocellulose; cellulases
Purification and characterization of an exoinulinase from Aspergillus fumigatus by Prabhjot Kaur Gill; Rajesh Kumari Manhas; Jatinder Singh; Prabhjeet Singh (pp. 19-32).
An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K m (0.25 mM) and higher V max (333.3 IU/mg) values for inulin.
Keywords: Inulin; exoinulinase; fructose; Aspergillus fumigatus ; concanavalin A
Characterization and comparison of metal accumulation in two Escherichia coli strains expressing either CopA or MntA, heavy metal-transporting bacterial P-type adenosine triphosphatases by Nick Zagorski; David B. Wilson (pp. 33-48).
MntA from Lactobacillus plantarum and copA from Enterococcus hirae both encode membrane proteins that are members of the P-type family of adenosine triphosphatases (ATPases). Both transporters act as metal importers to take up nutritionally required substrates; MntA translocates Mn(II) and CopA translocates Cu(I). Both ATPases can also translocate secondary substrates, Cd(II) and Ag(I), respectively. Although functionally and sequentially similar, these ATPases differ in several key residues and in their membrane topologies. The bioaccumulation properties of these two proteins were examined by coexpressing the transporters with overexpressed metallothionein in Escherichia coli cells, a system that has previously shown high levels of substrate-specific uptake. Both strains exhibited rapid metal accumulation, both saturated at around 50 µM metal, and both displayed temperature-sensitive uptake. However, the transporters responded differently when external conditions were varied; MntA displayed increased sensitivity to ionic strength, while CopA was more pH sensitive and more inhibited by chelating agents. The differences in accumulation are likely owing to structural differences in the transmembrane region of these two ATPases.
Keywords: Ion transport; P-type adenosine triphosphatase; copper; cadmium; genetic engineering; metallothionein; lactobacillus ; enterococcus ; Escherichia coli
Polygalacturonase and ethanol production in Kluyveromyces marxianus by Manuel Serrat; Rosa C. Bermúdez; Tomás G. Villa (pp. 49-64).
The coproduction of ethanol and polygalacturonase (PG) in a pilot-scale batch fermentor using yeast extract—glucose (YD)—and sugar beet molasses (SBM)-based media was implemented utilizing a new high-PG-producing strain of Kluveromyces marxianus. A certain growth inhibition was observed in SBM medium, causing ethanol and PG production to be lower. Ethanol productivity and accumulation values of 1.94 g/(L·h) and 40 g/L, respectively, were attained in YD, whereas the best fermentation efficiency (95.1%) was achieved with SBM medium. Maximal PG synthesis occurred at the end of cell growth, with values of 1.08 and 0.46 U/(mg·h) for the YD and SBM media, respectively. When the cultures reached stationary phase, PG production stopped. The highest accumulation level (17 U/mL) occurred in YD medium, in agreement with previous laboratory-scale studies carried out for this strain. The potential applications of the crude enzyme preparations were evaluated with different fruit juices and vegetable slices. The enzyme was able to increase the filtration rate of orange, pear, and apple juices by twofold. Additionally, complete clarification of apple juice was readily accomplished, whereas cucumber, carrot, and banana tissues were macerated to a lesser extent.
Keywords: Yeast; Kluyveromyces marxianus ; Polygalacturonases; food processing; ethanol; beet molasses