In our Protein Engineering studies we use site-directed and random mutagenesis based on PCR, molecular modeling and computational bioinformatics studies of structure function relationships. A strategy was developed to obtain complete genes of new enzymes which includes screening by PCR mediated by Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) and a improved genome walking method. We are continuing our modeling on the “cryophilicity” of cold active enzymes.
Some of our recent developments in Protein Engineering include the development of rationally optimized protein variants and of an improved xylanase and L-asparginase. The enzyme L-asparaginase hydolyses L-asparagine to aspartic acid and ammonia. The activity of this enzyme can cause cytotoxicity in some cells making it a useful therapeutic agent. We are also improving the thermostabilty of a psycrophilic xylanase, xyl-L, using a combination of random and site directed mutagenesis of domains and loops in order to rigidize its structure. Several improved variants have been obtained.
Our work also includes the use of high-throughput exhaustive prediction tools for studying the effect of mutations and the design and applications of ferritin-based bionanomaterials. We have developed new bioinformatics methodologies to identify amino acid positions in an enzyme that can be modified to alter its thermal stability without significantly affecting its catalytic properties. We demonstrated that non-random partitions of the protein in subsectors allows obtention of better predictors, and also have developed a new method to synthesize silver nanoparticles in ferritin using fast and easy photoreduction.
Other projects include the production of recombinant cellulases and auxiliary enzymes and the characterization of recombinant alginate-lyases for the saccharification of seaweed. The objective of this study is to produce enzymes for wheat straw degradation. Genes of fungal enzymes (endoglucanase TvEg, cellobiohydrolase PcCel7C, xylanase GtXyn10A and polysaccharide monooxigenase GtLPMO) were cloned and the enzymes were characterized, also we have developed the methodology for the expression and selection of the best conditions for production of several endo and exo alginate lyases, expressed in E.coli.
We are also carrying out the scale-up of the production of human adipose-derived mesenchymal stem cells for their differentiation into Insulin and Glucagon expressing cells for the treatment of diabetes.