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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.125, #2)

Industrial scale-up of pH-controlled liquid hot water pretreatment of corn fiber for fuel ethanol production by Nathan S. Mosier; Richard Hendrickson; Mark Brewer; Nancy Ho; Miroslav Sedlak; Richard Dreshel; Gary Welch; Bruce S. Dien; Andy Aden; Michael R. Ladisch (pp. 77-97).

The pretreatment of cellulose in corn fiber by liquid hot water at 160°C and a pH above 4.0 dissolved 50% of the fiber in 20 min. The pretreatment also enabled the subsequent complete enzymatic hydrolysis of the remaining polysaccharides to monosaccharides. The carbohydrates dissolved by the pretreatment were 80% soluble oligosaccharides and 20% monosaccharides with º1% of the carbohydrates lost to degradation products. Only a minimal amount of protein was dissolved, thus enriching the protein content of the un dissolved material. Replication of laboratory results in an industrial trial at 43 gallons per minute (163 L/min) of fiber slurry with a residence time of 20 min illustrates the utility and practicality of this approach for pretreating corn fiber. The added costs owing to pretreatment, fiber, and hydrolysis are equivalent to less than $0.84/gal of ethanol produced from the fiber. Minimizing monosaccharide formation during pretreatment minimized the formation of degradation products; hence, the resulting sugars were readily fermentable to ethanol by the recombinant hexose and by pentose-fermenting Saccharomyces cerevisiae 424A (LNH-ST) and ethanologenic Escherichia coli at yields >90% of theoretical based on the starting fiber. this cooperative effort and first successful trial opens the door for examining the robustness of the pretreatment system under extended run conditions as well as pretreatment of other cellulose-containing materials using water at controlled pH.

Keywords: Corn fiber; Cellulose; ethanol; stillage; degradation products; pretreatment

Purification and characterization of two low molecular weight endoglucanases produced by Penicillium occitanis mutant pol 6 by Semia Ellouz Chaabouni; Tahar Mechichi; Ferid Limam; Nejib Marzouki (pp. 99-112).

Two endoglucanases (EGs), EG A and EG B, were purified to homogeneity from Penicillium occitanis mutant Pol6 culture medium. The molecular weights of EGA and EGB were 31,000 and 28,000 kDa, respectively. The pI was about 3 for EG A and 7.5 for EG B. Optimal activity was obtained at pH 3.5 for both endoglucanases. Optimal temperature for enzyme activity was 60°C for EG A and 50°C for EG B. EG A was thermostable at 60°C and remained active after 1 h at 70°C. EGs hydrolyzed carboxymethylcellulose, phosphoric acid swollen cellulose, and ß-glucan efficiently, whereas microcrystalline cellulose (Avicel) and laminarin were poorly hydrolyzed. Only EG B showed xylanase activity. Furthermore, these EGs were insensitive to the action of glucose and cellobiose but were inhibited by the divalent cations Hg²⁺, Co²⁺, and Mn²⁺.

Keywords: Penicillium occitanis ; mutant Pol 6; cellulase; endoglucanases; purification

Stimulation of Erwinia sp. Fumarase and aspartase synthesis by changing medium components by Z. N. Bagdasaryan; G. A. Aleksanyan; A. M. Mirzoyan; J. C. Roseiro; S. N. Bagdasaryan (pp. 113-126).

The optimal concentrations of nutrient medium components, aeration conditions, and pH providing for maximum biomass yields, as well as fumarase and l-aspartase activities, during submerged cultivation of Erwinia sp. were determined. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum fumarase and l-aspartase activities. Calculations performed by application of the additive lattice model suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases as compared to the experimental means in Erwinia sp. biomass, and l-aspartase and fumarase activities, respectively. The conditions of the fumaric acid biotransformations into l-malic and l-aspartic acids were optimized on the basis of intact Erwinia sp. cells, a fumarase and l-aspartase producer. In the cases of fumarate transformation into l-malic acid and of fumarate transformation into l-aspartic acids, fumarase and l-aspartase activities increased 1.5- and 1.7-fold, respectively. The experimental data were consistent with these estimates to 80% accuracy. In comparison with the additive lattice model, the application of polynomial nonlinear model allowed the between-factor relations to be considered and analyzed, which resulted in 1.1-, 1.27-, and 1.1-fold increases in Erwinia sp. biomass and fumarase and l-aspartase activities for the case of cultivation. In the case of fumarate transformation into l-malic acid, this model demonstrated a 1.7-fold increase in fumarase activity, whereas during fumarate transformation into l-aspartic acid no significant change in aspartase activity was observed.

Keywords: Erwinia ; fumarase; aspartase; additive lattice model; polynomial nonlinear model

Purification of glutathione reductase from chicken liver and investigation of kinetic properties by Mustafa Erat; Hülya Demir; Halis Šakiroglu (pp. 127-138).

Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K _M and V _max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.

Keywords: Glutathione reductase; purification; chicken liver; enzyme; gel filtration chromatography; affinity chromatography

Purification and partial characterization of a lipase from Bacillus coagulans ZJU318 by Tang Lianghua; Xia Liming (pp. 139-146).

An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with 18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme activity was strongly inhibited by Ag⁺ and Cu²⁺, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly inhibit lipase activity.

Keywords: Lipase; Bacillus coagulans ; purification; characterization; stability

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.125, #2)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.125, #2)