In silico study of Citrobacter amalonaticus phytase enzyme to increase thermal stability

BACKGROUND AND OBJECTIVESEnzymes are natural nanoscale biocatalysts that catalyze chemical reactions in living cells. Phytase is the most widely used food enzyme in the world. Phytases are a subgroup of phosphatases that are capable of phosphorylating phytate, the most abundant inositol phosphate in nature.In order to be used in industrial processes, phytases must have high specific activity and the ability to function in the conditions of the digestive system of animals and to withstand short-term exposure to high temperatures during feed granulation (۶۰-۸۰ degrees Celsius). The aim of this study is to increase the thermal stability of phytase enzyme by applying stabilizing mutations.MATERIALS AND METHODSThe phytase gene of Citrobacter amalonaticus with a sequence of ۱۳۱۱ base pairs, which encodes an enzyme with ۴۳۶ amino acids, was selected in order to improve stability. This phytase belongs to the family of histidine acid phosphatases. In the first step, the main protein was cloned and its biochemical indicators were investigated. Then, using MUPRO software (https://mupro.proteomics.ics.uci.edu), the desired mutations were measured to increase the thermal stability of the enzyme, and ۶۴ mutations were selected among the ۹۹۰ mutations examined. The selection of amino acids for the mutation was based on the hydropathy index and the amount of presence in the protein. Amino acids glycine, alanine, valine, isoleucine, leucine and serine are suitable candidates for mutation.RESULTS AND DISCUSSIONFinally, based on the three-dimensional structure of the protein, after applying each of these ۶۴ mutations using the I-TASSER server, ۳ mutations were selected as the final candidates, and the simulation of each of these mutations was done separately, and in terms of biochemical indicators with enzymes The original was compared.CONCLUSIONAfter checking the secondary simulation results, Q۱۹۰L and T۳۰۵L mutations were applied. Of course, our work is at the level of prediction and it should be checked experimentally to prove its correctness.

کلیدواژه ها:

Phytase ، Citrobacter Amalonaticus ، Increased Thermal Stability ، Mutation