In silico recombinant plasmid design of pHA171 with phdABCD insertion for ethidium bromide degradation

Background: Ethidium bromide is a common reagent that is used in nucleic acid staining. However, ethidium bromide has toxic and carcinogenic properties that are harmful to the environment. Phenanthrene dioxygenase (encoded by phdA, phdB, phdC, and phdD genes) in Nocardioides sp. KP7 can oxidize the phenanthridine structure aim to eliminate carcinogenic properties. Objective: This study aims to visualize and predict the structure, active site, and characteristics of the phenanthrene dioxygenase using bioinformatics tools. Methods: Plasmid design were prepared by inserting genes of interest phdA, phdB, phdC, and phdD from the NCBI database. Furthermore, several protein analysis tools were used for structure visualization, active site enzyme improvement, and protein characteristic of phenanthrene dioxygenase. Results: The prediction results found that phenanthrene dioxygenase reacts with the ethidium bromide substrate through the interaction of Fe3+ ions with water. The solubility level of phenanthrene dioxygenase protein is 0.404, suggesting that the protein has low solubility. The protein isoelectric point (pI) is between 5.17 to 5.36, and the protein molecular weight is 121.143 kDa. Conclusion: In silico analysis has supported that recombinant plasmid met characteristics for the construct which consists of gene interest and protein library.

Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review

Structural and functional catalytic characteristics of cross-linked enzyme aggregates (CLEA) are reviewed. Firstly, advantages of enzyme immobilization and existing types of immobilization are described. Then, a wide description of the factors that modify CLEA activity, selectivity and stability is presented. Nowadays CLEA offers an economic, simple and easy tool to reuse biocatalysts, improving their catalytic properties and stability. This immobilization methodology has been widely and satisfactorily tested with a great variety of enzymes and has demonstrated its potential as a future tool to optimize biocatalytic processes.

From Structure to Catalysis: Recent Developments in the Biotechnological Applications of Lipases

Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to “order” a “customized” enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts.