Pankaj K. Pawar

Characterization of two coleopteran α-amylases and molecular insights into their differential inhibition by synthetic α-amylase inhibitor, acarbose

Post-harvest insect infestation of stored grains makes them unfit for human consumption and leads to severe economic loss. Here, we report functional and structural characterization of two coleopteran α-amylases viz. Callosobruchus chinensis α-amylase (CcAmy) and Tribolium castaneum α-amylase (TcAmy) along with their interactions with proteinaceous and non-proteinaceous α-amylase inhibitors. Secondary structural alignment of CcAmy and TcAmy with other coleopteran α-amylases revealed conserved motifs, active sites, di-sulfide bonds and two point mutations at spatially conserved substrate or inhibitor-binding sites. Homology modeling and molecular docking showed structural differences between these two enzymes. Both the enzymes had similar optimum pH values but differed in their optimum temperature. Overall, pattern of enzyme stabilities were similar under various temperature and pH conditions. Further, CcAmy and TcAmy differed in their substrate affinity and catalytic efficiency towards starch and amylopectin. HPLC analysis detected common amylolytic products like maltose and malto-triose while glucose and malto-tetrose were unique in CcAmy and TcAmy catalyzed reactions respectively. At very low concentrations, wheat α-amylase inhibitor was found to be superior over the acarbose as far as complete inhibition of amylolytic activities of CcAmy and TcAmy was concerned. Mechanism underlying differential amylolytic reaction inhibition by acarbose was discussed.

A glycoprotein α-amylase inhibitor from Withania somnifera differentially inhibits various α-amylases and affects the growth and development of Tribolium castaneum

BACKGROUND Identification and characterisation of plant defensive molecules enrich our resources to design crop protection strategies. In particular, plant-derived proteinaceous inhibitor(s) of insect digestive enzymes appear to be a safe, sustainable and attractive option. RESULTS A glycoprotein having non-competitive α-amylase inhibitory activity with a molecular weight of 8.3 kDa was isolated and purified from seeds of Withania somnifera α-amylase inhibitor (WSAI). Its mass spectrometry analysis revealed 59% sequence coverage with Wrightide II-type α-amylase inhibitor from Wrightia religiosa. A dose-dependent inhibition of α-amylases from Aspergillus oryzae, Bacillus subtilis, Helicoverpa armigera and Tribolium castaneum was recorded. Interestingly, WSAI did not inhibit human salivary α-amylase significantly. When adults of T. castaneum were fed with WSAI (1.6 mg g-1 ), decrease in consumption, growth and efficiency of conversion of ingested food was evident, along with over fourfold increases in feeding deterrence index. A decline in larval residual α-amylase activity after feeding of WSAI resulted in a reduction in longevity of T. castaneum. CONCLUSION The study reflects the significance of WSAI in affecting the overall growth and development of T. castaneum. Pre- and post-harvest pest resistive capability makes WSAI a potential candidate for insect pest management. Further, the effectiveness of this inhibitor could be explored either in formulations or through a transgenic approach.

Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species

The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3′UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC50. Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.

Herbal augmentation enhances malachite green bio degradation efficacy of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was able to degrade a highly toxic textile dye malachite green (MG) at 100 mg/L concentration. Although 99% decolourization was observed, a tremendous metabolic and oxidative stress was exerted on the cells. Ethanolic extracts of Terminalia chebula, Clitoria ternatea and Boerhaavia diffusa at a concentration of 1 mg/mL were independently supplied to S. cerevisiae cells to counter the stress. T. chebula, C. ternatea and B. diffusa extracts reduced the activities of glutathione peroxidase (67, 8 and 71%), superoxide dismutase (2, 7 and 16%) and catalase (16, 52 and 57%), respectively. Inductions in the activities of laccase (66, 82 and 50%), lignin peroxidase (35, 75 and 10%), NADH-DCIP reductase (43, 52 and 91%) and MG reductase (66, 126 and 117%) were observed respectively. Presence of dye (MG) extended the lag phase of the growth cycle of S. cerevisiae up to 36 h, which was observed to be restored to normal (4 h) after phytoextract supplementation. Scanning electron microscope imaging revealed the restored cell morphology upon exposure to plant extracts. The accumulation of reactive oxygen species (ROS) was observed to be 355% greater in cells exposed to MG, which was significantly reduced after phytoextracts augmentation when compared to control cells. Phytoextracts proved to be beneficial in increasing the viability of S. cerevisiae cells and reduced the intracellular ROS and nuclear damage. Inclusion of plant extracts during decolourization proved to be beneficial and protected the cells so that 20 treatment cycles could be run achieving significant removal of MG.

Chebulinic acid and Boeravinone B act as anti-aging and anti-apoptosis phyto-molecules during oxidative stress

INTRODUCTION Aquatic pollutant Malachite green (MG) induces oxidative stress by producing intracellular H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals in Saccharomyces cerevisiae. These radicals disturb cellular functions leading to early aging. Exogenous supply of natural antioxidants may play a crucial role as anti-aging by ensuring the cellular survival. METHODS Protective effect of Chebulinic acid (CA) and Boeravinone B (BB) was biochemically evaluated by measuring the expression levels of antioxidant enzymes. Intracellular oxidants generation, nuclear damage, necrosis, apoptosis, reduction in caspase 3/7 activity studied microscopically, spectrofluorometrically and biochemically along with growth dynamics and relative quantitation of Yap1, Sir2 and Bir1 expression using RT-PCR. RESULTS Malachite green (MG) showed adverse effect on S. cerevisiae showing 400.83% enhancement in accumulation of intracellular H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals. Independent supplementation of CA (5 μg/ml) and BB (3 μg/ml) significantly reduced the accumulation by 385.78 and 372.68%, respectively. Presence of MG extended the lag phase of growth curve and also reduced colony forming units (CFUs)/ml to 3 × 108 from 15 × 108. Whereas, CA and BB maintained the normal growth curve, CFUs and proved as anti-aging. Elevation in the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) by 241.35, 539.02 and 432.60% was observed after 2 h MG exposure. However, CA and BB significantly reduced the CAT, SOD and GPx activities. Microscopic observation of CA and BB augmented cells revealed protection from H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals accumulation, nuclear disorganization, morphological distortion, apoptosis and necrosis contrary to MG exposed cells. An enhancement of 112.78% in caspase 3/7 activity was noted in MG exposed cells over control. Both CA and BB supplementation reduced the caspase 3/7 activity by 106.06 and 105.82%, respectively which was almost near normal. MG was found to induce the expression of yeast transcription factor Yap1; while presence of CA and BB restored expression of Yap1. Expression of longevity responsible gene Silent Information Regulator (Sir2) was also found to be reduced during MG exposure. However, CA and BB triggered the expression of Sir2. Similarly, MG lowered the expression of Baculoviral IAP repeat (Bir1) which is the inhibitor of apoptosis while CA and BB aided the over expression of Bir1. CONCLUSIONS CA and BB supplementation could significantly decrease oxidative stress, enhance cell viability and ultimately protected S. cerevisiae cells form aging.