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Physicochemical, enzymatic and molecular characterisation of the storage protein of aerial tuber, Dioscorea bulbifera Linn.

ABSTRACT: BACKGROUND:The storage protein of the aerial tuber of Dioscorea bulbifera was purified and its physicochemical, enzymatic and molecular properties determined with a view to comparing its functionality and genetic relatedness with other storage proteins. RESULTS:The purified protein had molecular weight of 21?kDa. The protein showed carbonic anhydrase, trypsin inhibitory, dehydroascorbate reductase and monodehydroascorbate reductase activities. Amplifications with polymerase chain reactions resulted in the detection of two genes encoding the storage protein. The deduced amino acid sequence of the shorter and larger genes had homologies with the storage proteins of members of the Dioscorea family. CONCLUSION:The study concluded that the storage protein of the aerial tuber of D. bulbifera had similar properties with those of other Dioscorea species and may be suitable for development as functional food.

SUBMITTER: Osukoya OA

PROVIDER: S-EPMC7358276 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Physicochemical, enzymatic and molecular characterisation of the storage protein of aerial tuber, Dioscorea bulbifera Linn.

Osukoya Olukemi Adetutu OA Kuku Adenike A

Journal, genetic engineering & biotechnology 20200713 1

<h4>Background</h4>The storage protein of the aerial tuber of Dioscorea bulbifera was purified and its physicochemical, enzymatic and molecular properties determined with a view to comparing its functionality and genetic relatedness with other storage proteins.<h4>Results</h4>The purified protein had molecular weight of 21 kDa. The protein showed carbonic anhydrase, trypsin inhibitory, dehydroascorbate reductase and monodehydroascorbate reductase activities. Amplifications with polymerase chain ...[more]

PMID: 32661737

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Project description:BackgroundDioscorea bulbifera is an African medicinal plant used to treat microbial infections. In the present study, the methanol extract, fractions (DBB1 and DBB2) and six compounds isolated from the bulbils of D. bulbifera, namely bafoudiosbulbins A (1), B (2), C (3), F (4), G (5) and 2,7-dihydroxy-4-methoxyphenanthrene (6), were tested for their antimicrobial activities against Mycobacteria and Gram-negative bacteria involving multidrug resistant (MDR) phenotypes expressing active efflux pumps.MethodsThe microplate alamar blue assay (MABA) and the broth microdilution methods were used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the above samples.ResultsThe results of the MIC determinations indicated that when tested alone, the crude extract, fractions DBB1 and DBB2 as well as compounds 2 to 5 were able to prevent the growth of all the fifteen studied microorganisms, within the concentration range of 8 to 256 ?g/mL. The lowest MIC value for the methanol extract and fractions (16 ?g/mL) was obtained with DBB1 and DBB2 on E, coli AG100A and DBB2 on Mycobacterium tuberculosis MTCS2. The lowest value for individual compounds (8 ?g/mL) was recorded with compound 3 on M. smegmatis and M. tuberculosis ATCC and MTCS2 strains respectively. The activity of the samples on many MDR bacteria such as Enterobacter aerogenes EA289, CM64, Klebsiella pneumoniae KP63 and Pseudomonas aeruginosa PA124 was better than that of chloramphenicol. When tested in the presence of the efflux pump inhibitor against MDR Gram-negative bacteria, the activity of most of the samples increased. MBC values not greater than 512 ?g/mL were recorded on all studied microorganisms with fraction DBB2 and compounds 2 to 5.ConclusionsThe overall results of the present investigation provided evidence that the crude extract D. bulbifera as well as some of the compounds and mostly compounds 3 could be considered as potential antimicrobial drugs to fight against MDR bacteria.

Project description:BackgroundAt present, the emergence and spread of antimalarial drug resistance has become a significant problem worldwide. There has been a challenge in searching for natural products for the development of novel antimalarial drugs. Therefore, this study aims to evaluate compounds from Dioscorea bulbifera responsible for antimalarial properties and investigate potential interactions of the compounds with Plasmodium falciparum lactate dehydrogenase (PfLDH), an essential glycolytic enzyme in the parasite's life cycle.MethodsAn in vitro study of antimalarial activity against chloroquine (CQ)-resistant Plasmodium falciparum (K1 strain) and CQ-sensitive P. falciparum (3D7 strain) was performed using the 3H-hypoxanthine uptake inhibition method. The cytotoxic effects of the pure compounds were tested against Vero cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The interactions of the compounds with the PfLDH active site were additionally investigated using a molecular docking method.ResultsQuercetin (6) exhibited the highest antimalarial activity against the P. falciparum K1 and 3D7 strains, with IC50 values of 28.47 and 50.99 μM, respectively. 2,4,3',5'-Tetrahydroxybibenzyl (9), 3,5-dimethoxyquercetin (4) and quercetin-3-O-β-D-galactopyranoside (14) also possessed antimalarial effects against these two strains of P. falciparum. Most pure compounds were nontoxic against Vero cells at a concentration of 80 μg/ml, except for compound 9, which had a cytotoxic effect with a CC50 value of 16.71 μM. The molecular docking results indicated that 9 exhibited the best binding affinity to the PfLDH enzyme in terms of low binding energy (- 8.91 kcal/mol) and formed strong hydrogen bond interactions with GLY29, GLY32, THR97, GLY99, PHE100, THR101 and ASN140, amino acids as active sites. In addition, 6 also possessed remarkable binding affinity (- 8.53 kcal/mol) to PfLDH by interacting with GLY29, ILE31, ASP53, ILE54, THR97 and THR101.ConclusionQuercetin is a major active compound responsible for the antimalarial activity of D. bulbifera and is an inhibitor of PfLDH. These findings provide more evidence to support the traditional use of D. bulbifera for malaria treatment. Structural models of its interactions at the PfLDH active site are plausibly useful for the future design of antimalarial agents.

Project description:BACKGROUND: Leucine aminopeptidase (LAP) has been known to be a housekeeping protease, DNA-binding protein and repressor or activator in the operon regulation of virulence-associated genes in several bacterial species. LAP activity was consistently detected in overnight cultures of Burkholderia pseudomallei, the causative agent of melioidosis and this enzyme was partially purified and characterised in this study. The intra- and inter-species nucleotide and deduced amino acid sequence variation of LAP encoding gene (pepA) was determined. A pepA/PCR-RFLP assay was designed to facilitate the identification of major LAP sequence types amongst clinical and environmental isolates of B. pseudomallei. RESULTS: LAP activity was detected in B. pseudomallei culture supernantants by zymographic analysis. Optimum activity was at pH 9 and stable at 50[degree sign]C. Enhanced enzymatic activity was observed in the presence of metallic ions Mg2+, Ca2+, Na+ and K+. LAP activity was inhibited by EDTA, 1,10-phenanthroline, amastatin, Mn2+ and Zn2+. Sequence analysis of the complete nucleotide and deduced amino acid sequences of LAP-encoding (pepA) gene showed close genetic relatedness to B. mallei (similarity 99.7%/99.6%), but not with B. thailandensis (96.4%/96.4%). Eight pepA sequence types were identified by comparison with a 596 bp DNA fragment encompassing central regions of the pepA gene. A pepA/PCR-RFLP was designed to differentiate pepA sequence types. Based on restriction analysis with StuI and HincII enzymes of the amplified pepA gene, clinical and environmental isolates showed different predominant RFLP types. Type I was the most predominant type amongst 71.4% (65/91) of the clinical isolates, while Type II was predominant in 55.6% (5/9) of the environmental isolates. CONCLUSIONS: This study showed that LAP is a secretory product of B. pseudomallei with features similar to LAP of other organisms. Identification of major LAP sequence types of B. pseudomallei was made possible based on RFLP analysis of the pepA gene. The high LAP activity detected in both B. pseudomallei and B. thailandensis, suggests that LAP is probably a housekeeping enzyme rather than a virulence determinant.

Dataset Information

Physicochemical, enzymatic and molecular characterisation of the storage protein of aerial tuber, Dioscorea bulbifera Linn.

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Physicochemical, enzymatic and molecular characterisation of the storage protein of aerial tuber, Dioscorea bulbifera Linn.

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