A review on recent upgradation and strategies to enhance cyclodextrin glucanotransferase properties for its applications.

Cyclodextrin glycosyltransferase (CGTase) is a significant extracellular enzyme with diverse functions. CGTase is widely used in production of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch via transglycosylation reaction. Recent discoveries of novel CGTases from different microorganisms have expanded its applications but natural CGTase have lower yield, leading to heterologous expression for increased production to meet various needs. Moreover, significant advancements in directed evolution approach have been explored to alter the molecular structure of CGTase to enhance its performance. This review comprehensively summarizes the strategies employed in heterologous expression to boost CGTase production and secretion in various host. It also outlines molecular engineering approaches aimed to improving CGTase properties, including product and substrate specificity, catalytic efficiency, and thermal stability. Additionally, a considerable stability against changes in temperature and organic solvents can be obtained by immobilization.

Uncovering the Interaction Interface Between Harpin (Hpa1) and Rice Aquaporin (OsPIP1;3) Through Protein-Protein Docking: An In Silico Approach.

Hpa1 (a type of harpin) is involved in T3SS (Type III Secretion System) assembly in the infection mechanism by Xanthomonas Oryzae pv. oryzae (Xoo). Hpa1 interacts with the plasma membrane components of plants thereby assisting effector proteins toward the cytoplasm, wherein effectors execute their pathological functions. Independently, harpins also induce hypersensitive response and systemic acquired resistance in plants. However, lack of knowledge regarding the plant-harpin interaction mechanism constrains the pathway of its agricultural application. Although an in vitro study proved that Hpa1 protein can interact with OsPIP1;3, a rice aquaporin, the structural basis of the interaction is yet to be discovered. The presented work is the first of its kind where an in silico approach is used for the PPI (protein-protein interaction) of harpin protein. The study discovered participation of Hpa1 N-terminal amino acids at the interface. Besides, MD simulation studies were performed to assess the stability. RMSD values were 0.35 ± 0.049, 0.73 ± 0.11, and 0.50 ± 0.065 nm for OsPIP1;3, Hpa1, and Hpa1-OsPIP1;3 complex, respectively. Additionally, Residue-wise fluctuations have also been studied post-MDS. Taken together, these findings not only give a solid foundation for a deeper knowledge of various interacting target molecules with Harpin protein orthologs but also bring a new avenue for the structural-functional relationship study of harpin proteins.

An in-silico approach to unravel the structure of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS): a critical enzyme for sennoside biosynthesis in Cassia angustifolia Vahl.

The laxative properties of senna are attributed to the presence of sennosides produced in the plant. The low production level of sennosides in the plant is an important impediment to their growing demand and utilization. Understanding biosynthetic pathways helps to engineer them in terms of enhanced production. The biosynthetic pathways of sennoside production in plants are not completely known yet. However, attempts to get information on genes and proteins engaged in it have been made which decode involvement of various pathways including shikimate pathway. 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) is a key enzyme involved in sennosides production through the shikimate pathway. Unfortunately, there is no information available on proteomic characterization of DAHPS enzyme of senna (caDAHPS) resulting in lack of knowledge about its role. We for the first time characterized DAHPS enzyme of senna using in-silico analysis. To the best of our knowledge this is the first attempt to identify the coding sequence of caDAHPS by cloning and sequencing. We found Gln179, Arg175, Glu462, Glu302, Lys357 and His420 amino acids in the active site of caDAHPS through molecular docking. followed by molecular dynamic simulation. The amino acid residues, Lys182, Cys136, His460, Leu304, Gly333, Glu334, Pro183, Asp492 and Arg433 at the surface interact with PEP by van der Waals bonds imparting stability to the enzyme-substrate complex. Docking results were further validated by molecular dynamics. The presented in-silico analysis of caDAHPS will generate opportunities to engineer the sennoside biosynthesis in plants.Communicated by Ramaswamy H. Sarma.