Molecular characteristics of plant UDP-arabinopyranose mutases.

l-arabinofuranose is a ubiquitous component of the cell wall and various natural products in plants, where it is synthesized from cytosolic UDP-arabinopyranose (UDP-Arap). The biosynthetic machinery long remained enigmatic in terms of responsible enzymes and subcellular localization. With the discovery of UDP-Arap mutase in plant cytosol, the demonstration of its role in cell-wall arabinose incorporation and the identification of UDP-arabinofuranose transporters in the Golgi membrane, it is clear that the cytosolic UDP-Arap mutases are the key enzymes converting UDP-Arap to UDP-arabinofuranose for cell wall and natural product biosynthesis. This has recently been confirmed by several genotype/phenotype studies. In contrast to the solid evidence pertaining to UDP-Arap mutase function in vivo, the molecular features, including enzymatic mechanism and oligomeric state, remain unknown. However, these enzymes belong to the small family of proteins originally identified as reversibly glycosylated polypeptides (RGPs), which has been studied for >20 years. Here, we review the UDP-Arap mutase and RGP literature together, to summarize and systemize reported molecular characteristics and relations to other proteins.

Random mutagenesis of super Koji (Aspergillus oryzae): improvement in production and thermal stability of α-amylases for maltose syrup production.

BACKGROUND: Alpha-amylases hydrolyze 1,4 α-glycosidic bonds of starch and produce malto-oligosaccharides. It is an important enzyme generally applied in textile, food and brewing industries. Enhancement in thermal stability and productivity of enzymes are the two most sought after properties for industrial use. The Aspergillus oryzae (Koji) has Generally Recognized as Safe (GRAS) status and safe for use in food industry. Hence, Koji strain's development for the screening of potent mutants, hyper producer of thermostable α-amylases, with desired attributes is the need of the time. RESULTS: A process has been developed to improve super Koji (A. oryzae cmc1) strain through γ-rays treatment. The doses i.e. 0.60, 0.80, 1.00, 1.20 & 1.40 KGy gave more than 3.0 log kill. Initially, 52 Koji mutants resistant to 1% (w/v) Triton X-100 were selected. 2nd screening was based on α-amylases hyper production and 23 mutants were sorted out by measuring clearing zones index (CI). Afterwards nine potent mutants, resistant to 2-deoxy D-glucose, were screened based on CI. These were further analyzed for thermal stability and productivity of α-amylase under submerged conditions. The mutants' M-80(10), M-100(6) & M-120(5) gave about four fold increases in α-amylases productivity. The half life of M-100(6) α-amylase at 55 °C was 52 min and was highest among the mutants. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis confirmed that mutants did not produce aflatoxins. Field Emission Scanning Electron Microscopy (FESEM) of Koji mycelia depicted that exposure to gamma rays increased rigidity of the mycelium. The potent Koji mutant M-100(6) was grown on soluble starch in 10L fermenter and produced 40.0 IU ml-1 of α-amylases with specific activity of 2461 IU mg-1 protein. Growth kinetic parameters were: µ = Specific growth rate= 0.069 h-1, td = Biomass doubling time= 10.0 h, Yp/x = Product yield coefficient with respect to cell mass = 482 U g-1; qp= Specific rate of product formation= 33.29 U g-1 h-1. CONCLUSION: It was concluded that the developed five step screening process has great potential to generate potent mutants for the hyper production of thermostable enzymes through γ-rays mediated physical mutagenesis. The developed thermostable α-amylases of super Koji mutantM-100(6) has immense potential for application in saccharification process for maltose syrup production. Moreover, the developed five step strain's development process may be used for the simultaneous improvement in productivity and thermal stability of other microbial enzymes.

Three dimensional (3D) structure prediction and substrate-protein interaction study of the chitin binding protein CBP24 from B. thuringiensis.

Bacillus thuringiensis is an insecticidal bacterium whose chitinolytic system has been exploited to improve insect resistance in crops. In the present study, we studied the CBP24 from B. thuringiensis using homology modeling and molecular docking. The primary and secondary structure analyses showed CBP24 is a positively charged protein and contains single domain that belongs to family CBM33. The 3D model after refinement was used to explore the chitin binding characteristics of CBP24 using AUTODOCK. The docking analyses have shown that the surface exposed hydrophilic amino acid residues Thr-103, Lys-112 and Ser-162 interact with substrate through H-bonding. While, the amino acids resides Glu-39, Tyr-46, Ser-104 and Asn-109 were shown to have polar interactions with the substrate. The binding energy values evaluation of docking depicts a stable intermolecular conformation of the docked complex. The functional characterization of the CBP24 will elucidate the substrate-interaction pathway of the protein in specific and the carbohydrate binding proteins in general leading towards the exploration and exploitation of the prokaryotic substrate utilization pathways.