ABSTRACT
In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.
Subject(s)
Bacteria/classification , Bacteria/isolation & purification , Plant Roots/microbiology , Soil Microbiology , Bacterial Typing Techniques , Bacteria/genetics , Cluster Analysis , Cytosol/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Genes , Glucose 1-Dehydrogenase/genetics , Molecular Sequence Data , Pakistan , Phylogeny , Plants , Quinones/analysis , Rhizosphere , /genetics , Sequence Analysis, DNAABSTRACT
Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p < 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.
Subject(s)
Enterobacteriaceae/enzymology , Enterobacteriaceae/physiology , Glucose 1-Dehydrogenase/genetics , Nerve Growth Factors , Phaseolus/growth & development , Phaseolus/microbiology , Cluster Analysis , Computational Biology , Cytosol/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Glucose 1-Dehydrogenase/chemistry , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Conformation , Protein Structure, Tertiary , Phosphorus/metabolism , Plant Roots/growth & development , Plant Shoots/growth & development , Quinones/analysis , Sequence Analysis, DNA , Sequence HomologyABSTRACT
In order to successfully express the carbonyl reductase gene mldh in Bacillus subtilis and complete coenzyme regeneration by B. subtilis glucose dehydrogenase, the promoter PrpsD and the terminator TrpsD from B. subtilis rpsD gene were used as the expression cassette to be a recombinant plasmid pHY300plk-PrpsD-TrpsD. After that, the carbonyl reductase gene mldh was inserted into the previous plasmid and a plasmid pHY300plk-PrpsD-mldh-TrpsD was achieved, followed by transformed into B. subtilis Wb600 to obtain a recombinant B. subtilis Wb600 (pHY300plk-PrpsD-mldh-TrpsD). Subsequently, the results for whole-cell biotransformation from recombinant B. subtilis showed that it could be used to catalyze MAK (1-phenyl- 1-keto-2-methylaminopropane) to d-pseudoephedrine in the presence of glucose. The yield of d-pseudoephedrine could be up to 97.5 mg/L and the conversion rate of MAK was 24.1%. This study indicates the possibility of biotransformation production of d-pseudoephedrine from recombinant B. subtilis.
Subject(s)
Alcohol Oxidoreductases , Genetics , Bacillus subtilis , Genetics , Metabolism , Glucose 1-Dehydrogenase , Chemistry , Metabolism , Mutagenesis, Insertional , Pseudoephedrine , Metabolism , Recombinant Proteins , Genetics , Recombination, GeneticABSTRACT
BACKGROUND/AIMS: Icodextrin (glucose polymer) is metabolized by a-amylase to oligosaccharides such as maltose and maltotriose. The presence of these metabolites could have an effect on the enzymatic glucose measurement especially the glucose dehydrogenase pyrroloquinolinequinone (GDH-PQQ) based method. Patients treated with icodextrin are at risk for inaccurate blood glucose measurements. In this study we measured the blood glucose with different methods and analyzed the results to determine the test accuracy. METHODS: The blood glucose was measured, in seven outpatients and in seven inpatients using icodextrin, by the glucose hexokinase laboratory technique method as well as the GDH-PQQ method (Accu Chek Active)at the same time. To estimate an icodextrin residual effect, after discontinuing icodextin, the blood glucose was measured by the two methods after 48 hours in 4 inpatients. RESULTS: In seven outpatients the blood glucose was overestimated by the Accu Chek Active method (mean difference 68 mg/dL, p value 0.012). In seven inpatients the mean difference in the glucose was 56 mg/dL at 6am, 52 mg/dL at 11am, 52 mg/dL at 4pm, and 50 mg/dL at 9pm by the two different methods. In the four inpatients after changing their dialysate, the mean difference in the glucose was 58 mg/dL after 10 hours, 45 mg/dL after 24 hours, 24 mg/dL after 34 hours, and 26 mg/dL after 48 hours. CONCLUSION: Blood glucose was overestimated by the GDH-PQQ method and the inaccuracies were observed for more than 48 hours.
Subject(s)
Humans , Blood Glucose , Glucans , Glucose , Glucose 1-Dehydrogenase , Hexokinase , Hypoglycemia , Inpatients , Maltose , Oligosaccharides , Outpatients , Peritoneal Dialysis, Continuous Ambulatory , TrisaccharidesABSTRACT
The kinetic mechanism of glucose dehydrogenase (EC 1.1.1.47) from Halobacterium salinarum was studied by initial velocity and product inhibition methods. The results suggest that both, in the forward and reverse direction, the reaction mechanism is of Bi Bi sequential ordered type involving formation of ternary complexes. NADP+ adds first and NADPH formed dissociates from the enzyme last. For the reverse direction, NADPH adds first and NADP+ leaves last. Product inhibition experiments indicate that (a), the coenzymes compete for the same site and form of the enzyme and (b), ternary abortive complexes of enzyme-NADP(+)-glucono-delta-lactone and enzyme-NADPH-glucose are formed. All the other inhibitions are noncompetitive.