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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.128, #3)

Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1 by Devendra Jain; V. Udayasuriyan; P. Indra Arulselvi; Sona S. Dev; P. Sangeetha (pp. 185-194).

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1+orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.

Keywords: Bacillus thuringiensis ; δ-endotoxin; Cry2Ab7; cry2Aa promoter; cloning; Helicoverpa armigera

Screening thermotolerant white-rot fungi for decolorization of wastewaters by P. Chairattanamanokorn; T. Imai; R. Kondo; M. Ukita; P. Prasertsan (pp. 195-204).

To select a thermotolerant fungal strain for decolorization of wastewaters, ligninolytic enzyme production (lignin peroxidase, manganese peroxidase [MnP], and laccase), decolorization, and removal of total phenol and chemical oxygen demand (COD) were detected. Thirty-eight fungal strains were studied for enzyme production at 35 and 43°C on modified Kirk agar medium including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl₂. Thirteen strains grew on manganese-containing agar and provided green color on ABTS-containing agar plates under culture at 43°C. Decolorization of wastewater from alcohol distillery (WAD) by these strains was compared under static culture at 43°C, and Pycnoporus coccineus FPF 97091303 showed the highest potential. Thereafter, immobilized mycelia were compared with free mycelia for WAD decolorization under culture conditions of 43°C and 100 rpm. The immobilized mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as total phenol and color removal. At about the same COD removal, MnP and laccase produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43°C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized by the immobilized mycelia within 1 to 2 d for four cycles.

Keywords: Thermotolerant fungi; Pycnoporus coccineus ; ligninolytic enzyme; decolorization; screening

Ethanol-induced changes in glycolipids of Saccharomyces cerevisiae by Renuka Malhotra; Balwant Singh (pp. 205-213).

Total glycolipid content of Saccharomyces cerevisiae cells increased in ethanol-treated yeast cells. Sialic acid and hexosamine contents of glycolipids from ethanol-treated cells decreased, whereas those of hexoses increased. Increased sialidase activity in the presence of ethanol may be responsible for the decrease in sialic acid content of glycolipids. The saccharide moieties of glycolipids of S. cerevisiae consisted of fucose, mannose, galactose, and glucose. Ethanol treatment of yeast cells caused an increase in glucose and a decrease in galactose content of glycolipids. The changes in glucose content can be related to changes in β-glucosidase activity under alcohol stress. The content of cerebrosides, sulfatides, and monoglucosyldiglycerides was enhanced following ethanol treatment. An increase in cerebroside as well as in sulfatide content during alcohol stress might play an important role in stabilizing the membrane both physically and structurally. Such variations in glycolipid content and composition of S. cerevisiae cells may represent an adaptive response to ethanol stress.

Keywords: Saccharomyces cerevisiae ; glycolipids; sugars; ethanol

Kinetics and thermal stability of two peroxidase isozymes from Eupatorium odoratum by D. Nisha Rani; T. Emilia Abraham (pp. 215-226).

The Eupatorium odoratum leaf peroxidase exists as at least seven distinct isozymes (three cationic, three anionic, and one neutral). These isozymes were identified and separated by preparative iso-electric focusing. Thermal stability, including the activation enthalpy (ΔH ^*), free energy of inactivation (ΔG ^*) and activation entropy (ΔS ^*), and kinetic studies of two isozymes, one having a pI of 5.0 (E5) and another one having a pI of 7.0 (E7) with mol mass of 43 and 50 kD, respectively, were studied in detail. Of the molecular weight of E5 and E7, 25 and 32% correspond to the carbohydrate content of the isozymes. Optimal pH was in the acidic range of 3.6–3.8 for E5 and 3.8 for E7 with the oxidation of ABTS. E7 and E5 showed activation energy for inactivation, 194.8 and 145.4 kJ/mol, respectively. Both the isozymes showed distinct substrate specificity. The catalytic specificity constant for E5 and E7 were 112×10⁵ and 124×10⁵/s·M, respectively, when 2,2′-azino-bis-(3-ethylbenz-thiazoline-6 sulfonic acid) was used as the substrate. Maximum affinity (i.e., lowest K _m value) to H₂O₂ was shown by E5 and E7 along with Pyrogallol and was 0.02 and 0.05/s·M, respectively.

Keywords: Arrhenius equation; Eupatorium odoratum ; kinetics; peroxidase; thermal stability

Production of natural fruity aroma by Geotrichum candidum by Naziha Mdaini; Mohamed Gargouri; Mohamed Hammami; Lotfi Monser; Moktar Hamdi (pp. 227-235).

Based on its aromatic potential, Geotrichum candidum isolated from olive vegetation water was tested for the production of volatile compounds. When G. candidum was cultivated on media with glucose as the carbon source, flavor volatile compounds were produced and accumulated in the broth. Fruity flavoring compounds (pineapple-like) such as esters and alcohols were analyzed by gas chromatography coupled to mass spectrometry, including ethyl esters of acetic acid and butyric acid, methyl-3-butan-1-ol, and methyl-2-propan-1-ol. Their synthesis corresponded to the stationary growth phase of the strain. Production of the volatile compounds reached 9.5 g/L of 2-hexanoic acid ethyl ester and 1.6 g/L of benzaldehyde as the main concentrated molecules. Ethyl alcohol seems to be an intermediate metabolite in this pathway.

Keywords: Benzaldehyde; ethanol; fruity aroma; Geotrichum candidum ; 2-hexanoic acid ethyl ester

Transcription analysis of recombinant Saccharomyces cerevisiae reveals novel responses to xylose by Laura Salusjärvi; Juha-Pekka Pitkänen; Aristos Aristidou; Laura Ruohonen; Merja Penttilä (pp. 237-273).

Lignocellulosic biomass, rich in hexose and pentose sugars, is an attractive resource for commercially viable bioethanol production. Saccharomyces cerevisiae efficiently ferments hexoses but is naturally unable to utilize pentoses. Metabolic engineering of this yeast has resulted in strains capable of xylose utilization. However, even the best recombinant S. cerevisiae strains of today metabolize xylose with a low rate compared to glucose. This study compares the transcript profiles of an S. cerevisiae strain engineered to utilize xylose via the xylose reductase-xylitol dehydrogenase pathway in aerobic chemostat cultures with glucose or xylose as the main carbon source. Compared to the glucose culture, 125 genes were upregulated, whereas 100 genes were downregulated in the xylose culture. A number of genes encoding enzymes capable of nicotinamide adenine dinucleotide phosphate regeneration were upregulated in the xylose culture. Furthermore, xylose provoked increased activities of the pathways of acetyl-CoA synthesis and sterol biosynthesis. Notably, our results suggest that cells metabolizing xylose are not in a completely repressed or in a derepressed state either, indicating that xylose was recognized neither as a fermentable nor as a respirative carbon source. In addition, a considerable number of the changes observed in the gene expression between glucose and xylose samples were closely related to the starvation response.

Keywords: Xylose; transcriptional profiling; ethanol; metabolic engineering; Saccharomyces cerevisiae

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.128, #3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.128, #3)