alpha-chymotrypsin and Phenylketonurias

Phenylalanine ammonia lyase (PAL) has long been recognized as a potential enzyme replacement therapeutic for treatment of phenylketonuria. However, various strategies for the oral delivery of PAL have been complicated by the low intestinal pH, aggressive proteolytic digestion and circulation time in the GI tract. In this work, we report 3 strategies to address these challenges. First, we used site-directed mutagenesis of a chymotrypsin cleavage site to modestly improve protease resistance; second, we used silica sol-gel material as a matrix to demonstrate that a silica matrix can provide protection to entrapped PAL proteins against intestinal proteases, as well as a low pH of 3.5; finally, we demonstrated that PEGylation of AvPAL surface lysines can reduce the inactivation of the enzyme by trypsin.

Topics: Administration, Oral; Anabaena variabilis; Bacterial Proteins; Binding Sites; Chymotrypsin; Enzyme Replacement Therapy; Enzyme Stability; Hot Temperature; Humans; Hydrogen-Ion Concentration; Injections; Models, Molecular; Mutagenesis, Site-Directed; Phenylalanine Ammonia-Lyase; Phenylketonurias; Polyethylene Glycols; Protein Engineering; Protein Multimerization; Protein Structure, Quaternary; Silicon Dioxide; Structure-Activity Relationship; Technology, Pharmaceutical; Trypsin

Phenylalanine ammonia-lyase was entrapped in silk fibroin. The entrapped enzyme showed a similar Km for Phe and pH optimum to the free enzyme. It was resistant against chymotrypsin and trypsin in vitro. To assess the activity in vivo, the free or entrapped enzymes and then Phe were injected into rat duodenum, and cinnamate, a product, in plasma was determined as the most direct evidence of the enzyme activity. The entrapped enzyme but not the free form caused a marked raise of plasma cinnamate. It declined with a half life of about 45 min, which was significantly longer than that (10-15 min) observed upon i.v. administration of cinnamate. These results indicated that the entrapped enzyme was actively degrading Phe in the intestinal tract. Entrapment of phenylalanine ammonia-lyase in fibroin thus provides a new prospect for oral enzyme therapy of phenylketonuria.

Topics: Ammonia-Lyases; Animals; Chymotrypsin; Cinnamates; Fibroins; Phenylalanine; Phenylalanine Ammonia-Lyase; Phenylketonurias; Rats; Trypsin

Low-phenylalanine-peptides for dietotherapy of phenylketonuria (PKU) were prepared from soybean protein isolate. Soluble fraction of soybean protein isolate was hydrolysed by alpha-chymotrypsin then followed by carboxypeptidase-A. Molecular weight distribution and amino acid analysis were made on the resultant polypeptides. The chymotrypsin hydrolysate was divided into two fractions, Fraction I (molecular weight greater than 2500) and Fraction II (molecular weight between 1000 and 2500). The phenylalanine content of Fraction I (3.1%) was lower than that of Fraction II (5%), indicating the nonuniform distribution of phenylalanine in soy bean protein. Carboxypeptidase hydrolysis of Fraction I further reduced the phenylalanine concentration to 2.3%, approximately half of the original concentration in soybean protein isolate.

Topics: Amino Acids; Carboxypeptidases; Carboxypeptidases A; Child; Chymotrypsin; Dietary Proteins; Glycine max; Humans; Peptides; Phenylalanine; Phenylketonurias; Plant Proteins