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The present study was conducted to evaluate the effect of three different temperatures on the antibacterial potential of fermented goat milk.Goat milk is one of the naturalfoods to complete perfection.Bioactive peptides, the specific protein fragments that have a positive impact on body functions, can be produced during milk fermentation. Thanks to the starter cultures’ proteolytic activity.Fresh pooled goat milk sample collected was divided into four lots. One set was used for raw milk analysis and the remaining three were subjected to heat treatment at three different timetemperature combinations namely;72°C for 15 sec, 85°C for 10 min, and 121° C for 15 min. These samples were cooled immediately to 42ºC, inoculated with 0.04 % DVS yogurt culture,incubated at 42º C for 4 hrs, and then stored at 5±2°C for 21 days for storage studies. The analysis was carried out at 7-day intervals. An agar well diffusion assay was performed to assess the inhibitory activity ofWater-soluble peptide extracts (WSPE) of these samples against Bacillus cereus ATCC 10876, Escherichia coli NCIM 2685, Salmonella enterica ATCC 6017, and Staphylococcus aureus ATCC 25923. The milk that was heated to 85°C for 10 min before fermentation showed the highest antibacterial activity against E.coli during the 7th and 14th day of storage, withthe zone of inhibition measuredat 17±0.433mm and 21±0.55mm respectively.Thus, it can be concluded that milk heated at 85°C for 10 min had better antibacterial activity than the other two treatments.

Biogenic synthesis of papain-conjugated copper metallic Nanoparticles and their antibacterial and antifungal activities Papain metallic conjugated nanoparticles (Papain-CuNPs) were synthesised using Papain and CuSO4.5H2O. Papain-CuNPs were characterized using UV-visible spectroscopy, FT-IR (Fourier-Transform Infrared), HR-TEM (High-Resolution Transmission Electron Microscopy), XRD (X-ray diffraction), FE-SEM (Field-emission scanning electron microscopy), zeta potential, and a zeta sizer. The antibacterial activity of papain-CuNPs against human infectious microorganisms (Citrobacter spp, Pseudomonas aeruginosa and Candida albicans) was investigated. The mechanism of action of papain-CuNPs was evaluated using FE-SEM and HRTM. UV spectroscopy confirmed the plasma resonance (SPR) at 679 nm, which indicated the formation of papain-CuNPs. The FT-IR spectrum absorbance peaks at 3927, 3865, 3842, 3363, 2978, and 2900 cm-1 indicate the presence of O-H and N-H of the secondary amine, and peaks at 1643 and 1572 cm-1 represent C=O functional groups in Papain-CuNPs. EDAX analysis confirmed the presence of copper in the papain-CuNPs. The zeta potential (-42.6 mV) and zeta size (99.66 d. nm) confirmed the stability and size of the nanoparticles. XRD confirmed the crystalline nature of the papain-CuNPs. FE-SEM and HRTM showed an oval structure and the nano particles' 16.71244-34.84793 nm. The synthesized papain-NPs showed significant antibacterial activity against clinical P. aeruginosa (15 mm). MIC 125 µg/ml) showed bactericidal activity against P. aeruginosa and the mechanism of action of Papain-NPs was confirmed using an electron microscope by observing cell damage and cell shrinking. Papain-CuNPs have significant antibacterial activity and are thus used in the treatment of P. aeruginosa infections. [ABSTRACT FROM AUTHOR]

Quinolone synthase from Aegle marmelos (AmQNS) is a Rutacean-specific plant type III polyketide synthase that synthesizes quinolone, acridone, and benzalacetone with therapeutic potential. Simple architecture and broad substrate affinity of AmQNS make it as one of the target enzymes to produce novel structural scaffolds. Another unique feature of AmQNS despite its high similarity to acridone forming type III polyketide synthase from Citrus microcarpa is the variation in the product formation. Hence, to explore the characteristic features of AmQNS, an in-depth sequence and structure-based bioinformatics analyses were performed. Our studies indicated that AmQNS and its nearest homologs have evolved by a series of gene duplication events and strong purifying selection pressure constrains them in the evolutionary process. Additionally, some amino acid alterations were identified in the functionally important region(s), which can contribute to the functional divergence of the enzyme. Prediction of favorable amino acid substitutions will be advantageous in the metabolic engineering of AmQNS for the production of novel compounds. Furthermore, comparative modeling and docking studies were utilized to investigate the structural behavior and small molecule interaction pattern of AmQNS. The observations and results reported here are crucial for advancing our understanding of AmQNS's phylogenetic position, selection pressure, evolvability, interaction pattern and thus providing the foundation for further studies on the structural and reaction mechanism.

Key Message: Functional characterization and ectopic expression studies of chalcone synthase mutants implicate the role of phenylalanine in tailoring the substrate specificity of type III polyketide synthase. Chalcone synthase (CHS) is a plant-specific type III polyketide synthase that catalyzes the synthesis of flavonoids. Native CHS enzyme does not possess any functional activity on N-methylanthraniloyl-CoA, which is the substrate for acridione/quinolone alkaloid biosynthesis. Here, we report the functional transformation of chalcone synthase protein from Emblica officinalis (EoCHS) to quinolone and acridone synthase (ACS) with single amino acid substitutions. A cDNA of 1173 bp encoding chalcone synthase was isolated from E. officinalis and mutants (F215S and F265V) were generated by site-directed mutagenesis. Molecular modeling studies of EoCHS did not show any active binding with N-methylanthraniloyl-CoA, but the mutants of EoCHS showed strong affinity to the same. As revealed by the modeling studies, functional analysis of CHS mutants showed that they could utilize p-coumaroyl-CoA as well as N-methylanthraniloyl-CoA as substrates and yield active products such as naringenin, 4-hydroxy 1-methyl 2(H) quinolone and 1,3-dihydroxy-n-methyl acridone. Exchange of a single amino acid in EoCHS (F215S and F265V) resulted in functionally active mutants that preferred N-methylanthraniloyl-CoA over p-coumaroyl-CoA. This can be attributed to the increase in the relative volume of active sites in mutants by mutation. Moreover, metabolomic and MS analyses of tobacco leaves transiently expressing mutant genes showed high levels of naringenin, acridones and quinolone derivatives compared to wild-type CHS. This is the first report demonstrating the functional activity of EoCHS mutants with N-methylanthraniloyl-CoA and these results indicate the role of phenylalanine in altering the substrate specificity and in the evolution of type III PKSs.

Zingiberaceae or 'ginger family' is the largest family in the order 'Zingiberales' with more than 1300 species in 52 genera, which are mostly distributed throughout Asia, tropical Africa and the native regions of America with their maximum diversity in Southeast Asia. Many of the members are important spice, medicinal or ornamental plants including ginger, turmeric, cardamom and kaempferia. These plants are distinguished for the highly valuable metabolic products, which are synthesised through phenylpropanoid pathway, where type III polyketide synthase is the key enzyme. In our present study, we used sequence, structural and evolutionary approaches to scrutinise the type III polyketide synthase (PKS) repertoire encoded in the Zingiberaceae family. Highly conserved amino acid residues in the sequence alignment and phylogram suggested strong relationships between the type III PKS members of Zingiberaceae. Sequence and structural level investigation of type III PKSs showed a small number of variations in the substrate binding pocket, leading to functional divergence among these PKS members. Molecular evolutionary studies indicate that type III PKSs within Zingiberaceae evolved under strong purifying selection pressure, and positive selections were rarely detected in the family. Structural modelling and protein-small molecule interaction studies on Zingiber officinale PKS 'a representative from Zingiberaceae' suggested that the protein is comparatively stable without much disorder and exhibited wide substrate acceptance.