Reduction of L-phenylalanine in protein hydrolysates using L-phenylalanine ammonia-lyase from Rhodosporidium toruloides.

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.25) from Rhodosporidium toruloides was utilized to remove L-phenylalanine (L-Phe) from different commercial protein hydrolysates. A casein acid hydrolysate (CAH, L-Phe ~2.28 %) was employed as a model substrate. t-Cinnamic acid resulting from deamination of L-Phe was extracted, analyzed at λ = 290 nm, and used for PAL activity determination. Optimum reaction conditions, optimized using successive Doehlert design, were 35 mg mL(-1) of CAH and 800 mU mL(-1) of PAL, while temperature and pH were 42 °C and 8.7, respectively. Reaction kinetics of PAL with CAH was determined under optimized conditions. Then, removal of L-Phe from CAH was tested. Results showed that more than 92 % of initial L-Phe was eliminated. Similar results were obtained with other protein hydrolysates. These findings demonstrate that PAL is a useful biocatalyst for L-Phe removal from protein hydrolysates, which can be evaluated as potential ingredients in foodstuffs for PKU patients.

Kinetic mechanism determination and analysis of metal requirement of dehydroquinate synthase from Mycobacterium tuberculosis H37Rv: an essential step in the function-based rational design of anti-TB drugs.

The number of new cases of tuberculosis (TB) arising each year is increasing globally. Migration, socio-economic deprivation, HIV co-infection and the emergence of drug-resistant strains of Mycobacterium tuberculosis, the main causative agent of TB in humans, have all contributed to the increasing number of TB cases worldwide. Proteins that are essential to the pathogen survival and absent in the host, such as enzymes of the shikimate pathway, are attractive targets to the development of new anti-TB drugs. Here we describe the metal requirement and kinetic mechanism determination of M. tuberculosis dehydroquinate synthase (MtDHQS). True steady-state kinetic parameters determination and ligand binding data suggested that the MtDHQS-catalyzed chemical reaction follows a rapid-equilibrium random mechanism. Treatment with EDTA abolished completely the activity of MtDHQS, and addition of Co(2+) and Zn(2+) led to, respectively, full and partial recovery of the enzyme activity. Excess Zn(2+) inhibited the MtDHQS activity, and isotitration microcalorimetry data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. We also report measurements of metal concentrations by inductively coupled plasma atomic emission spectrometry. The constants of the cyclic reduction and oxidation of NAD(+) and NADH, respectively, during the reaction of MtDHQS was monitored by a stopped-flow instrument, under single-turnover experimental conditions. These results provide a better understanding of the mode of action of MtDHQS that should be useful to guide the rational (function-based) design of inhibitors of this enzyme that can be further evaluated as anti-TB drugs.