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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.82, #3)

Protection of myocardial mitochondria against oxidative damage by selenium-containing abzyme m4G3 by Dan-Hua Qi; Gui-Min Luo; Li Zhou; Tong-Shu Yang (pp. 167-173).

Selenium-containing abzyme (m4G3) was prepared and its protection of myocardial mitochondria against oxidative damage was studied using the swelling of mitochondria, quantity of lipid peroxidation products, and change in cytochrome-c oxidase activity as a measure of mitochondrial damage. The results showed that m4G3 could inhibit mitochondrial damage caused by the hypoxanthine-xanthine oxidase system in vitro. Electronic spin resonance (ESR) studies demonstrated that m4G3 could decrease the amount of free radicals generated in the damage system.

Keywords: Abzyme m4G3; mitochondria; oxidative damage; xanthine oxidase/hypoxanthine system; glutathione peroxidase; enzyme mimics

Xanthan gum production by Xanthomonas campestris w.t. fermentation from chestnut extract by M. Liakopoulou-Kyriakides; S. K. Psomas; D. A. Kyriakidis (pp. 175-183).

Xanthomonas campestris w.t. was used for production of xanthan gum in fermentations with chestnut flour for the first time. Fermentations were carried out with either chestnut flour or its soluble sugars (33.5%) and starch (53.6%), respectively, at 28°C and 200 rpm at initial pH 7.0 in flasks. The effect of agitation rate (at 200, 400, and 600 rpm) on xanthan gum production was also studied in a 2-L batch reactor. It was found that xanthan production reaches a maximum value of 3.3 g/100 mL at 600 rpm and 28°C at 45 h.

Keywords: Xanthomonas campestris ; fermentation; xanthan gum; chestnuts

Lipase-catalyzed production of short-chain acids terpenyl esters of interest to the food industry by Françoise Laboret; Robert Perraud (pp. 185-198).

The production of low molecular weight esters as flavor compounds by biotechnological processes has a potential interest for the food industry. The use of natural available substrates and enzymes is an essential part of the process design, because the products may obtain natural label. In this study, direct esterification of citronellol and geraniol with short-chain fatty acids catalyzed by free lipase from Mucor miehei was performed with high yields in n-hexane. The effects of the acid:alcohol ratio on the bioconversion rate of increasing chain length esters was investigated. To reach the optimum yield, substrates and enzyme concentration were determined. The inhibiting effects of acid are strongly attenuated by reducing the quantity of acid and increasing the amount of enzyme in media following the optimum values. Improvements have been made to increase the ester purity. The consumption of excess substrate by adding calculated amounts of acid gives a 10% yield enhancement, and leads to 100% pure terpenyl esters. The first steps to a scale-up application were attempted using a reactor that allowed us to produce ester quantities up to 100 cm³. Separation and purification of the products were treated with success, underlining the lipase stability and efficiency under the conditions of this study. The ability to recover the enzyme, and reusing it in bioconversions, plays a major role in reducing the cost of the overall process.

Keywords: Lipase; Mucor miehei ; direct esterification; enzymatic synthesis; organic solvents; terpenyl esters

Biosynthesis of poly-β-hydroxybutyrate and exopolysaccharides on azotobacter chroococcum strain 6B utilizing simple and complex carbon sources by Javier C. Quagliano; Silvia S. Miyazaki (pp. 199-208).

Coproduction of poly-β-hydroxybutyrate (PHB) and exopolysaccharides (EPS) was investigated with Azotobacter chroococcum strain 6B isolated from soil samples. The bacterium was cultured using various carbon sources solely or with 0.1 g/L of ammonium sulfate. Ammonium addition resulted in reduced PHB and EPS production with glucose, fructose, and sucrose media, but cellular mass remained constant except for sucrose. Protein was nearly twofold higher in ammonium-grown cultures. Glucose and fructose alone biosynthesized high amounts of EPS (maximum 2.1 and 1.1 g/L, respectively, at 72 h), whereas PHB was accumulated only in glucose-grown cells. Sucrose almost did not produce EPS. Conversely, PHB content was the highest obtained from all experimented conditions (1.1 g/L at 48 h, 40% cell dry wt). When a complex carbon source such as sugar cane molasses was utilized, PHB was accumulated concomitant with EPS production from the initial time to 48 h (0.75 g/L, 37% cell dry wt and 0.6 g/L, respectively), and then PHB decayed at 72 h (0.2 g/L). On the other hand, EPS continued to be biosynthesized (1.1 g/L, 72 h). PHB fractions of total intra- and extracellular biopolymers were calculated. Sucrose-modified Burk’s medium without ammonium addition is suggested as a medium capable of diverting the carbon source for the production of intracellular PHB rather than EPS with A. chroococcum 6B.

Keywords: Poly-β-hydroxybutyrate; exopolysaccharides; coproduction; Azotobacter chroococcum ; complex carbon

Molecular analysis and heterologous expression of the gene encoding methylmalonyl—coenzyme a mutase from rifamycin SV-producing strain Amycolatopsis mediterranei U32 by Weiwen Zhang; Ling Yang; Weihong Jiang; Guoping Zhao; Yinliu Yang; Juishen Chiao (pp. 209-225).

The conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM), represents an important source of building blocks for rifamycin SV biosynthesis. The structural gene for MCM from rifamycin SV—producing strain Amycolatopsis mediterranei U32 was isolated by using a heterologous gene probe encoding the MCM of Streptomyces cinnamonensis. A 7.8-kbp fragment was sequenced and four complete open reading frames (ORFs) and two incomplete ORFs were found. Two central ORFs, ORF3 and ORF4, overlap by four nucleotides and were found to encode MCM small (602 residues) and large (721 residues) subunits, respectively. Comparison showed that the MCM gene of A. mediterranei U32 was quite similar to those from other sources. The functionally unknown ORF5, immediately downstream of the mut AB gene, was quite similar to the ORFs downstream of mut AB from S. cinnamonensis and Mycobacterium tuberculosis. Such a striking cross-species conservation of gene order suggested that ORF5 could also be involved in the metabolism of methylmalonyl-CoA. MCM gene was overexpressed in Escherichia coli under T7 promoter, and MCM activity could be detected in the recombinant E. coli clone harboring MCM gene after the addition of coenzyme B₁₂. A purification procedure based on the B₁₂ affinity column was established to purify the MCM from E. coli. The molecular weight of purified MCM from E. coli was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to that calculated from the MCM protein sequence and is also the same size as that of the enzyme purified directly from A. mediterranei U32. MCM gene was overexpressed in polyketide monensin producing S. cinnamonensis, and the total monensin production was increased by 32%.

Keywords: Methylmalonyl—Coenzyme A mutase; cloning; expression; rifamycin SV; monensin biosynthesis

Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time by Isabella Savini; Roberto Santucci; Almerinda Di Venere; Nicola Rosato; Giorgio Strukul; Francesco Pinna; Luciana Avigliano (pp. 227-241).

In this study, we investigated the optical features of the redox metal-dependent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate oxidase embedded in a sol-gel-processed silica matrix as a function of gelation time. Circular dichroism, absorbance, and fluorescence spectroscopies revealed that the sol-gel process affects the complex structure of the dimeric ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-c and HRP. Any modifications in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for months. Unfolding-refolding experiments on cytochrome-c revealed severely restricted motility in the protein moiety in the xerogel, the concentrated matrix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.

Keywords: Sol-gel; tetramethoxysilane; cytochrome-c ; ascorbate oxidase; horseradish peroxidase

Hydrolysis of steam-pretreated lignocellulose by Johan Karlsson; József Medve; Folke Tjerneld (pp. 243-258).

The mechanism of hydrolysis of cellulose is important for improving the enzymatic conversion in bioprocesses based on lignocellulose. Adsorption and hydrolysis experiments were performed with cellobiohydrolase I (CBH I) and endoglucanase II (EG II) from Trichoderma reesei on a realistic lignocellulose substrates: steam-pretreated willow. The enzymes were studied both alone and in equimolar mixtures. Adsorption isotherms were determined at 4 and 40°C during 90-min reaction times. Both CBH I and EG II adsorbed stronger at 40 than at 4°C. The time course of adsorption and hydrolysis, 3 min to 48 h, was studied at 40°C. About 90% of the cellulases were adsorbed within 2 h. The hydrolysis rate was high in the beginning but decreased during the time course. Based on adsorption data, the hydrolysis and synergism were analyzed as function of adsorbed enzyme. CBH I showed a linear correlation between hydrolysis and adsorbed enzyme, whereas for EG II the corresponding curve leveled off at both 4 and 40°C. At low conversion, below 1%, EG II produced as much soluble sugars as CBH I. At higher conversion, CBH I was more efficient than EG II. The synergism as function of adsorbed enzyme increased with bound enzyme before reaching a stable value of about 2. The effect of varying the ratio of CBH I:EG II was studied at fixed total enzyme loading and by changing the ratio between the enzymes. Only a small addition (5%) of EG II to a CBH I solution was shown to be sufficient for nearly maximal synergism. The ratio between EG II and CBH I was not critical. The ratio 40% EG II:60% CBH I showed similar conversion to 5% EG II:95% CBH I. Modifications of the conventional endo-exo synergism model are proposed.

Keywords: Trichoderma reesei ; cellulase; cellulose hydrolysis; lignocellulose; cellobiohydrolase; endoglucanase; synergism; adsorption

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.82, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.82, #3)