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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.95, #3)
Purification and characterization of an intracellular β-glucosidase from a Candida sake strain isolated from fruit juices by Yannick Gueguen; Patrick Chemardin; Alain Arnaud (pp. 151-162).
A yeast strain isolated in the laboratory from fruit juices was studied and classified as Candida sake. The strain produces an intracellular β-glucosidase when grown with cellobiose as the carbon source. The enzyme was purified by ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, estimated by gel filtration, was 240 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with sodium dodecyl sulfate. The enzyme exhibited optimum activity at 52°C and pH 4.25 with citrate-phosphate buffer. The enzyme was active against soluble glycosides with the (1→4)-β configuration, and from Lineweaver Burk plots, a K m value of 6.9 mmol/L was found for p-nitrophenyl-β-d-glucopyranoside. The β-glucosidase was found to be tolerant to glucose inhibition with a K i value of 0.2 mol/L.
Keywords: β-Glucosidase; purification; Candida sake
Fluorescence investigation of affinity interaction between bovine serum albumin and triazine dye in reversed micelles by Tian-Xi Zhang; Hui-Zhou Liu; Jia-Yong Chen (pp. 163-174).
The effect of Cibacron Blue 3GA (CB) on the fluorescence emission spectra of bovine serum albumin (BSA) was investigated in cationic reversed micelles formed with cetyltrimethylammonium bromide (CTAB) compared with that in aqueous phase. The anionic CB had electrostatic interactions with cationic CTAB and affinity interactions with BSA in the reversed micelles. The addition of CB in the reversed micellar phase led to a great decrease in the fluorescence intensity of BSA and a remarkable red shift of the wavelength of emission maximum (λmax). The fluorescence intensity of BSA decreased and the λmax decreased 5 nm (blue shift) without the addition of CB in the reversed micellar phase. The fluorescence intensity of BSA with the addition of CB had the strongest value in the aqueous phase with the presence of CTAB, a less strong value in the reversed micellar phase, and a weak value in the aqueous phase without the presence of CTAB. The increase in λmax of BSA with the addition of CB in the reversed micellar phase might indicate the decrease in the hydrophobic microenvironment of the Trp residue of BSA, contrary to those microenvironments in the absence of CB.
Keywords: Affinity interaction; reversed micelles; bovine serum albumin; Cibacron Blue 3GA; fluorescence emission spectra
Isolation of a bacterium from mangrove soil for degradation of sea sludge by Yoshihiro Ando; Naoya Mitsugi; Kazuyoshi Yano; Isao Karube (pp. 175-182).
Sea sludge, which is sediment of fish excrement and sewage on the sea bottom, continues to be a serious environmental problem. It has the potential to cause eutrophication and red tide, resulting in the death of shellfish and leading to an offensive odor. Soil taken from a mangrove swamp was added to sea sludge, which promoted an initial fermentation of the sludge components. This article reports on the isolation of a bacterium from mangrove soil that is involved in that fermentation. Three bacteria were isolated on a marine agar plate after incubating for 12 h at 60°C. One of these bacteria fermented sea sludge. 16S rDNA of this bacterium was sequenced, and it had a high homology with that of Bacillus fumarioli LMG17489T (AJ250056).
Keywords: Sea sludge; mangrove; fermentation; isolation; bacterium
Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis by Dae Haeng Cho; Hee Jeong Chae; Eui Yong Kim (pp. 183-193).
The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×105 Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.
Keywords: Rhodotorula glutinis ; exopolysaccharide; extracellular polysaccharide; characterize; uronic acid; molecular weight; culture conditions
Manufacture of xylose-based fermentation media from corncobs by posthydrolysis of autohydrolysis liquors by Gil Garrote; Herminia Domínguez; Juan C. Parajó (pp. 195-207).
Milled corncob samples were mixed with water and heated to obtain a liquid phase containing oligosaccharides, sugars, and acetic acid as main reaction products (autohydrolysis reaction). To hydrolyze the sugar oligomers to the correspondent monomers, sulfuric acid was added to the autohydrolysis liquors to reach 0.5–2 wt% of solution, and the reaction media were heated at 101.5–135°C. With this operational procedure, sugar solutions suitable as fermentation media (containing xylose as the major component) were obtained. The kinetics of the posthydrolysis step was characterized on the basis of experimental data concerning the time courses of the concentrations of xylooligosaccharides, xylose, furfural, and acetic acid. The concentrations of other reaction byproducts (glucose or arabinose) were also measured.
Keywords: Autohydrolysis; corncob; fermentation media; furfural; oligosaccharides; posthydrolysis; xylose
Characterization of yeast strains for wine production by R. N. Ndip; J. -F. K. T. Akoachere; L. L. Dopgima; L. M. Ndip (pp. 209-220).
Sixteen yeast strains isolated from grapefruit (Citrus paradis), orange (Citrus sinensis) and pineapple (Ananas comosus) were characterized using standard microbiological procedures. The species were identified as Saccharomyces uvarum, S. cerevisiae, S. carlbergensis, and S. ellipsoideus. Their abilities for wine production were tested by using sugar and ethanol tolerance tests. The best biochemically active strain, S. ellipsoideus, was used along with commercially available baker’s yeast (S. cerevisiae) to produce wine from grapefruit, orange, and pineapple juices. After fermentation for 14 d with S. cerevisiae and 21 d with S. ellipsoideus, wines produced were compared with Baron de Valls (standard). The highest (10.47% [v/v]) and lowest (7.68% [v/v]) alcohol concentrations with corresponding residual sugar concentrations of 1.88% (w/v) and 7.7% (w/v) were produced from orange after fermentation with S. cerevisiae and S. ellipsoideus, respectively. S. ellipsoideus was found to be the best yeast strain producing wine with the highest acceptable score of 7.41 from orange. The study revealed the possibility of producing wine from our locally available fruits using simple, cheap, and adaptable technology with biochemically characterized yeast strains.
Keywords: Yeast strains; fermentation; wine production
Production of native and recombinant lipases by Candida rugosa by Pau Ferrer; José Luis Montesinos; Francisco Valero; Carles Solà (pp. 221-255).
The yeast Candida rugosa produces multiple lipase isoenzymes sharing high sequence homology but with some differences in their catalytic properties. The regulation of C. rugosa lipase (CRL) synthesis and secretion in C. rugosa obeys a complex pattern. Fermentation processes for both wildtype and mutant C. rugosa strains are available for lipase production. Native CRL preparations have been extensively used for biotransformations. However, their inherent mixture of isoforms with variable profiles complicates interpretation and brings into question the reproducibility achieved between preparations. Although heterologous CRLs gene expression had been hampered owing to a nonuniversal codon usage, recent advances have made heterologous CRLs available. This will expand and improve the industrial utility of CRLs even further. The purpose of this review is to provide a summary of the recent advances on the production of native and recombinant lipases by C. rugosa.
Keywords: Candida rugosa ; lipase; isoenzymes; fermentation; heterologous expression