Cyclodextrin glucanotransferase: fundamentals and biotechnological implications.

Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme of the GH13 α-amylase family that catalyzes a unique intramolecular reaction known as cyclization to transform α-1, 4-glucans and similar starches into cyclodextrins. They also catalyze intermolecular transglycosylation reactions namely coupling, disproportionation, and some hydrolyzing effects on starch. The monomeric structures of the CGTase exhibit five domains (A, B, C, D, and E domains) with different molecular weights and amino acid sequences depending on the source. Among bacteria, Bacillus genus covers approximately 90% of the CGTase producers, while other genera like Klebsiella, Paenibacillus, and Thermoanaerobacter also shown decent contributions in recent studies. CGTase production is highly supported by alkaliphilic bacteria under submerged fermentation rather than solid-state fermentation. The bacterial sources, biochemical properties, production conditions, and structure of CGTases are compiled in this review. Cyclodextrins have the unique property of making inclusion complexes with various compounds, hence widely used in the food, pharmaceutical, cosmetics, laundry, and chemical sectors. This review presents a comprehensive view of CGTase produced by Bacillus spp., and other bacterial genera like Klebsiella, Paenibacillus, and Microbacterium. It also gives insight of the properties and recent biotechnological applications of cyclodextrins. KEY POINTS: • Transglycosylation reactions catalyzed by CGTase and their structural properties. • Comparative data of CGTase production by various genera and Bacillus spp. • Structures, properties, and applications of different cyclodextrins.

Microbial surfactants: A journey from fundamentals to recent advances.

Microbial surfactants are amphiphilic surface-active substances aid to reduce surface and interfacial tensions by accumulating between two fluid phases. They can be generically classified as low or high molecular weight biosurfactants based on their molecular weight, whilst overall chemical makeup determines whether they are neutral or anionic molecules. They demonstrate a variety of fundamental characteristics, including the lowering of surface tension, emulsification, adsorption, micelle formation, etc. Microbial genera like Bacillus spp., Pseudomonas spp., Candida spp., and Pseudozyma spp. are studied extensively for their production. The type of biosurfactant produced is reliant on the substrate utilized and the pathway pursued by the generating microorganisms. Some advantages of biosurfactants over synthetic surfactants comprise biodegradability, low toxicity, bioavailability, specificity of action, structural diversity, and effectiveness in harsh environments. Biosurfactants are physiologically crucial molecules for producing microorganisms which help the cells to grasp substrates in adverse conditions and also have antimicrobial, anti-adhesive, and antioxidant properties. Biosurfactants are in high demand as a potential product in industries like petroleum, cosmetics, detergents, agriculture, medicine, and food due to their beneficial properties. Biosurfactants are the significant natural biodegradable substances employed to replace the chemical surfactants on a global scale in order to make a cleaner and more sustainable environment.

Degrons of yeast and mammalian ornithine decarboxylase enzymes make potent combination for regulated targeted protein degradation.

Elevation of polyamine levels in eukaryotes leads to rapid degradation of ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis pathway. ODC in yeast (yODC) has two domains, the Nα/ß domain consisting of α/ß barrel domain (α/ß) preceded by an overhang of 50 residues at its N-terminus (N50) and ß sheet domain at its C-terminus. Two degradation determinant signals or degrons in yODC sequence, namely the N50 and the antizyme-binding element (AzBE) housed in the α/ß domain, are responsible for its degradation by proteasomes. Antizyme (Az) induced under polyamine excess binds to AzBE and delivers ODC to proteasome, while the N50 threads the protein into proteasome. It was previously reported by us that the peptide Nα/ß of yODC acts as an independent transplantable degron, whose action can be modulated with the help of antizyme by varying polyamine levels. Mammalian ODC (mODC), in spite of its 40% sequence homology with yODC, is devoid of N50 of yODC and instead sports a C-terminal tail of 37 residues (CmODC). CmODC acts as an independent transplantable degron with no equivalent in yODC. The present study investigates the merits of employing the two degrons Nα/ß and CmODC together for targeted protein degradation by expressing them in a chimeric fusion with green fluorescent protein (GFP). Our results establish that under the regulation of antizyme, the signals Nα/ß and CmODC acting together enhance degradation better than either degron in isolation. The combination of Nα/ß and CmODC can be employed to study the function of novel proteins through their rapid removal.

Engineering degrons of yeast ornithine decarboxylase as vehicles for efficient targeted protein degradation.

BACKGROUND: Ornithine decarboxylase (ODC), which catalyzes the first step of polyamine biosynthesis, undergoes rapid targeted degradation (TPD) with the help of its two degron sequences, namely the N-terminal 50 residues (N50) and α/ß domain (α/ß) housing antizyme binding element (AzBE), in response to increased polyamine levels. Antizyme binds to AzBE of ODC and delivers it to proteasome for degradation. Entire ODC was used as a tag to demonstrate TPD of chimeric proteins. METHODS: Here we fashioned three peptide sequences from yeast ODC to test their capability to act as degrons, namely N50, α/ß and Nα/ß (a combination of N50 and α/ß), and monitored their degradation potentials in chimeric proteins. We have examined the correlation between degradation potentials and structural integrity of the peptides, to find mechanistic explanations. RESULTS: Nα/ß with two signals in tandem is a better degron, under the regulation of antizyme. N50 like N44 reported earlier could drive chimeric proteins to degradation, while α/ß could not act as an independent degron. Strong correlation was observed between functional efficacy of the peptides and their structural integrity. N50, which was believed to be unstructured, displayed propensity for helical conformation. Nα/ß exhibited optimal structure, while α/ß failed to adopt native like conformation. CONCLUSIONS AND GENERAL SIGNIFICANCE: Functional efficacy of the degron Nα/ß is a consequence of its structural integrity. Nα/ß and N50 could target chimeric proteins to degradation. However, α/ß failed in the quest. Nα/ß, regulated by antizyme, is better suited than N50 for TPD to understand the function of novel proteins.

Synthesis, spectroscopic characterization and DNA nuclease activity of Cu(II) complexes derived from pyrazolone based NSO-donor Schiff base ligands.

Two neutral mononuclear Cu(II) complexes have been prepared in EtOH using Schiff bases derived from 4-toluoyl pyrazolone and thiosemicarbazide. Both the ligands have been characterized on the basis of elemental analysis, IR, (1)H NMR, (13)C NMR and mass spectral data. The molecular geometry of one of these ligands has been determined by single crystal X-ray study. It reveals that these ligands exist in amine-one tautomeric form in the solid state. Microanalytical data, Cu-estimation, molar conductivity, magnetic measurements, IR, UV-Visible, FAB-Mass, TG-DTA data and ESR spectral studies were used to confirm the structures of the complexes. Electronic absorption and IR spectra of the complexes suggest a square-planar geometry around the central metal ion. The interaction of complexes with pET30a plasmid DNA was investigated by spectroscopic measurements. Results suggest that the copper complexes bind to DNA via an intercalative mode and can quench the fluorescence intensity of EB bound to DNA. The interaction between the complexes and DNA has also been investigated by agarose gel electrophoresis, interestingly, we found that the copper(II) complexes can cleave circular plasmid DNA to nicked and linear forms.