Iranian Journal of Biotechnology
Year:2009 | Volume:7 | Issue:4|Papers Count:9

The present investigation was aimed at studying the effect of incubation period, media, vitamins and solvents on biotransformation of albendazole by Cunninghamella blakesleeana. The transformation was evaluated and identified by high performance liquid chromatography (HPLC) and the structures of the transformed products were assigned by liquid chromatography- tandem mass spectrometry (LC/MS/MS) analysis. The fungus was found to metabolize albendazole into albendazole sulfoxide (M1), albendazole sulfone (M2) and the N-methyl metabolite of albendazole sulfoxide (M3). Incubation period was found to influence the biotransformation significantly; 4 days was found to be optimum, but the effect was neither linear nor progressive. There was a significant effect of medium on the extent of biotransformation, with the highest substrate depletion produced by the glucose broth. The media also influenced qualitative metabolite formation. Presence of thiamine in the glucose media produced the maximum extent of transformation when compared to other vitamins studied. Dimethylformamide produced a higher extent of biotransformation. The fermentor was found to produce an increased level of biotransformation as compared to that obtained in shake flasks.

In the present study, Streptomyces gulbargensis and its mutant form, S.gulbargensis mu24, immobilized on polyurethane foam were investigated for the production of L-asparaginase using groundnut cake extract as medium. The medium with an initial pH of 8.5 was inoculated with free and immobilized cells separately and then subjected to fermentation by incubation at 40oC and shaking at 200rev/min. In the immobilized cell system, enzyme production was enhanced by approximately 30% compared to the conventional FreeCell fermentation. The immobilized cells were subjected to repeated batch fermentation processes to determine their reusability. These cells retained their ability to produce L-asparaginase over seven cycles and the activities remained between 16.2-41.3 IU/ml and 39-60 IU/ml for S.gulbargensis and S.gulbargensis mu24, respectively. The maximum enzyme titer was obtained during the third batch by both strains. However, the mutant strain was more potent for L-asparaginase production than the prototype. Therefore, the polyurethane foam immobilized cells of S.gulbargensis mu24 can be proposed as an effective biocatalyst, which can be repeatedly used for maximum production of L-asparaginase.

A bacterial strain (designated as Alcaligenes sp. MS-103) isolated from oil sample of the Aghajari oilfield in the south of Iran, was able to produce an effective extracellular lipopolysaccharide biosurfactant (1.2±0.05 g/l) on molasses as a sole carbon source. The highest surface tension reduction to level 20 mN/m was achieved by biosurfactant produced by cells grown on molasses under optimum conditions. The optimum values of carbon to nitrogen ratio (C/N), salinity, pH and temperature for biosurfactant production were determined as 60:1, 7.5%, 7.0 and 50oC, respectively. Biosurfactant flooding experiments were carried out on both fractured and unfractured carbonate cores. The highest recovery of residual oil among different experiments was about 10.7% in the unfractured cores. Oil displacement indicates that recovery of crude oil can be increased by 9.2% from fractured core with a permeability of 12 mD. The results showed that the biosurfactant produced by Alcaligenes sp. MS- 103 has the potential for industrial applications and may be used in microbial enhanced oil recovery (MEOR).

CoQ10 and lycopene are isoprenoid compounds with nutraceutical and pharmaceutical benefits. In this study, the effect of concomitant lycopene biosynthesis on CoQ10 accumulation in transformed Escherichia coli DH5a was studied. A lycopene production pathway including geranylgeranyl diphosphate synthase (crtE), phytoene synthase (crtB), and phytoene desaturase (crtI) from Erwinia herbicola was constructed in two CoQ10-producing E. coli strains. E. coli Ba and E. coli Br containing dds orthologs encoding for decaprenyl diphosphate synthase (Dds), respectively from Agrobacterium tumefaciens and Rhodobacter sphaeroides were transformed by the lycopene pathway resulting in E. coli Ba-lyc and E. coli Br-lyc. The lycopene pathway in E. coli Br-lyc interestingly resulted in a significant increase in CoQ10 production from 564±28 to 989±22mg /g DCW. To confirm that the improvement of CoQ10 production in E. coli Br-lyc was due to lycopene biosynthesis and not just geranylgeranyl diphosphate formation in the lycopene pathway, crtE was only introduced into E. coli Ba and E. coli Br strains. Surprisingly, crtE expression had adverse effects on CoQ10 production in both strains. The results shed light on the Dds-catalyzed reaction as a bottleneck controlled by precursors; and the efficiency of a parallel lycopene pathway to streamline the flow of metabolites.

This study aimed at applying both growth and survival approaches to compare three native strains of lactobacilli, belonging to Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus acidophilus species, with two commercial probiotic strains in their tolerance to acid and bile. The association between the data obtained from the methods was studied. The results of the different methods applied in this study, did not confirm each other for all the examined strains. However, the native strain of L. plantarum and the commercial strain of L. acidophilus repeatedly demonstrated the most and least bile resistances, respectively. The former excelled in all growth approaches but showed moderate acid resistance in the survival studies. Bile stress seemed to have more detrimental effects on all examined strains. The overall results suggest that the growth-rate designed studies and survival studies evaluating transit tolerance, might bring up different results when the examined strains belong to different species of lactobacilli showing different growth and metabolic activities. The strain of L. plantarum examined here could thus be considered as a potential probiotic, regarding its overall resistance to acid and bile.

Leptin (LEP), the expression product of the obese gene produced primarily in the adipose tissue, is related to feed intake, growth and lipid metabolism. The aim of this study was to study the possible association between polymorphism of the LEP gene with growth and carcass traits among three Iranian sheep breeds of the Shal, Zandi and Zel varieties. A total of 180 purebred animals of the Shal, Zandi and Zel, breeds were chosen for this study. Three flocks, each comprising of 60 ewes of three breeds, were derived from Aboureyhan sheep populations. The Shal (n=18), Zandi (n=24) and Zel (n=17) lambs were screened for polymorphism of the LEP gene. Following genomic DNA extraction from whole blood samples, polymerase chain reaction (PCR) was carried out in order to amplify a 260 bp fragment of the target gene. Polymorphisms were detected using the single strand conformational polymorphism (SSCP) technique. Two genotypes of AA and AG with frequencies of 0.53 and 0.47 in the Shal 0.70 and 0.30 in the Zandi and 0.65, 0.35 in the Zel breed were observed, respectively. The frequencies of alleles A and G were 0.74, 0.26 in the Shal breed, 0.85, 0.15 in the Zandi breed and 0.82, 0.18 in the Zel breed, respectively. Chi-Square test (x2) confirmed the Hardy-Weinberg equilibrium for the LEP loci. Average heterozygosity (30%) of the LEP locus for the three breeds was slightly low. Comparison of the sequence of the target gene available in the GeneBank with the results of the present study showed two single nucleotide polymorphisms (SNP) A®G and T®C transitions at 113 and 165 bp positions, respectively. In the Shal breed, the A113G SNP associated with an increase in cold carcass weight (p<0.01), fat-tail percent (p<0.05) and total body fat weight (p<0.05). In the Zel breed, the A113G SNP was associated with an increase in fat-tail percent (p<0.05) and reduction in slaughter weight (p<0.05), cold carcass weight (p<0.01) and lean meat weight (p<0.01). Therefore, a significant association between SNP within the LEP gene and certain carcass traits in the Shal and Zel breeds is proposed. In the Zandi breed the A113G SNP was not associated with carcass traits but showed a reduction in weaning weight (P<0.05).

Presence of antibiotic resistance markers has always been considered as one of the main safety concerns in transgenic plants and their derived products. Elimination of antibiotic selectable markers from transgenics is a major hurdle for finding efficient and safe candidates. Herbicide tolerance genes might be attractive alternatives. In this study, a variant form of the 5- enoylpyruvyl shikimate-3-phosphate synthase (EPSPS) gene that harbors glycine at position 96 to alanine and alanine 183 to threonine substitutions and confers higher resistance to the broad-spectrum herbicide, glyphosate, was substituted against the spectinomycin resistant gene as a sole selectable marker for plastid transformation of Nicotiana tabacum. Plastid transformation was carried out using the biolistic delivery procedure while delivery parameters such as rupture disk pressure, bombardment distance, etc had been optimized first. A previous study showed that the glyphosate herbicide imposes lethal effects on the structure and integrity of the plastid membrane, even at low concentrations. In order to overcome this problem, a modified procedure for selection of transplastomic cells was used. A long preculture incubation period followed by a gradual increased in glyphosate concentration led to sufficient expression of the transgene. Tolerant calli were thus regenerated through direct selection of transformed plastids in the presence of the glyphosate.

Polymorphism of the b-lactoglobulin (b-LG) gene in 101 cows belonging to the Holstein herd and a superior cow, producing more than 150 Kg milk/day, together with four offsprings was investigated by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. In the Holstein herd, the alleles A and B of the b-LG gene had frequencies of 0.53 and 0.47, respectively. The genotypes AA, AB and BB of the b-LG gene were estimated to have frequencies of 0.257, 0.544 and 0.198, respectively. Genotypes were distributed according to the Hardy-Weinberg equilibrium. Results indicated that the b-LG genotypes significantly affected (P<0.01) milk yield (genotype AA being more effective than genotype BB). The superior cow and her progenies were all heterozygotes (AB).

A pair of degenerate primers, GMPF1 and GMPR1, was designed on the basis of alignment of previously reported Grapevine fanleaf virus (GFLV) movement protein (MP) nucleotide sequences from Iran and other parts of the world. cDNA was synthesized by the use of Oligo d(T)18 from total RNA extraction from each diseased grapevine leaf sample and subjected to polymerase chain reaction (PCR) with the degenerate primers under a range of annealing temperatures from 48 to 62oC. It was revealed that 55oC gave the best result in terms of producing exactly the expected fragment (1044 bp) from as many samples as possible although accompanied by few fade non specific fragments. However, by application of "hot-start" PCR and annealing at 60oC the specific fragment was amplified from 41 out of 86 samples. This was the first amplification of the precise MP cDNA from GFLVs in Iran which is very important as to preparation of recombinant anti- GFLV MP antibody to use in studying the GFLVgrapevine interaction, and also for generating pathogen-derived resistant vines.