Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Administration, Oral
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Anabaena variabilis / enzymology
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Bacterial Proteins / administration & dosage
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Bacterial Proteins / metabolism
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Bacterial Proteins / therapeutic use*
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Binding Sites / genetics
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Chymotrypsin / metabolism
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Enzyme Replacement Therapy / methods*
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Enzyme Stability
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Hot Temperature
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Humans
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Hydrogen-Ion Concentration
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Injections
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Models, Molecular
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Mutagenesis, Site-Directed
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Phenylalanine Ammonia-Lyase / genetics
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Phenylalanine Ammonia-Lyase / metabolism
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Phenylalanine Ammonia-Lyase / therapeutic use*
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Phenylketonurias / drug therapy*
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Polyethylene Glycols / chemistry
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Protein Engineering / methods
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Protein Multimerization
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Protein Structure, Quaternary
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Silicon Dioxide / chemistry
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Structure-Activity Relationship
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Technology, Pharmaceutical / methods
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Trypsin / metabolism
Substances
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Bacterial Proteins
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Polyethylene Glycols
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Silicon Dioxide
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Chymotrypsin
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Trypsin
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Phenylalanine Ammonia-Lyase