 Hello, and welcome to our biotechnology and bi-engineering video highlight. We would like to introduce our recent article entitled The Role of Substrate-Binding Pocket Residues, phenylalanine 176, and phenylalanine 196 on pseudomonas sp0x1 toluene-ortozylene monooxygenase activity and reduced specificity. Now in this paper, we work with the remarkable biocatalis toluene-ortozylene monooxygenase, or TOMO. The enzyme TOMO belongs to the family of bacterial multi-component monooxygenases, and it has been shown to have great potential for biotechnological and environmental applications. Now, TOMO residues, phenylalanine 176 here shown in red, and phenylalanine 196 shown in blue, locate in the substrate binding pocket, and both are conserved in all other related toluene monooxygenase. Now in this work, we subjected these two positions, so saturation mutagenesis, to investigate their role in catalysis and generate further improvements in TOMO activity and reduced specificity. Now we perform saturation mutagenesis because we wanted to explore all the substitutions for these codowns, and this approach enabled us to isolate beneficial substitutions which were not predicted or isolated before. So, we isolated 11 substitutions, among which 9 were novels in TOMO, and 8 among the related family enzymes. Specifically, we identified variant histidine 176 that had 4.7 faster hydroxylation activity towards phenol, compared to the native enzyme. This variant also produced 61% of the novel product hydroquinone from phenol. It also made two-fold more two-nastol from nastolin, and its reduced specificity of toluene changed from 51% to 73% paracryso. Overall, we show in the study that alpha subunit residues phenylalanine 176 and 196 affect catalytic activity and or reduced specificity towards phenol, toluene and nastolin. To our knowledge, both of these positions have never been studied through the saturation mutagenesis approach before. Our results in this study also show that combination of mutations from these positions affects the reduced specificity as well as activity too. So, in the future, it would be interesting to examine the effect of combining different beneficial mutations and extend the work to other substrates. Finally, we would like to thank Mary Curie reintegration program for funding. Please contact us if you would like more information. Thank you for watching and happy reading.