Chemically stabilized subtilisins in peptide synthesis

We have stabilized alcalaseTM and subtilisin Carlsberg (SC) against heat by chemical modification with ethylene glycol bis-succinimidyl succinate (EGNHS), a procedure not previously reported for subtilisins. The increases in thermal stability at 65oC were 1.8-fold and 4.7-fold respectively. Caseinolytic activity of alcalase in aqueous buffer was unchanged following modification but apparent Km of SC decreased 2.5-fold. Native and modified forms of both enzymes synthesized the tripeptide Z-Tyr-Gly-Gly-NH2 under kinetic control in mixtures based on 0.2M barbitone buffer, pH 9.0 and 50% v/v dimethyl formamide/ barbitone buffer. Native enzymes gave faster rates of product formation than their modified counterparts in buffer but differences were much less pronounced in the mixed solvent. We also compared native alcalase and SC in terms of thermal stability, tolerance of organic solvents and autolysis. Alcalase was approx. 4.6-fold more stable than SC at 65oC and was more tolerant of acetone, acetonitrile and 1,4-dioxane. Alcalase underwent autolysis at approx. half the rate of SC. Against succinyl- Ala-Ala-Pro-Phe substrates, alcalase showed a much higher esterase/ amidase ratio (567) than SC (29) in aqueous buffer but this was reversed in 50% v/v dimethylformamide, where the esterase/ amidase ratios were 43 and 113 respectively

Gel filtration chromatography

Gel-filtration chromatography is a popular and versatile technique that permits the effective separation of proteins and other biological molecules in high yield. Here, the basis of the method is described and typical matrix types are contrasted. The selection of suitable operating conditions and applications of the method are also discussed

Stability and kinetic studies on recombinant and variant forms of human pyroglutamyl peptidase I and a single-site variant.

Human brain pyroglutamyl peptidase (PAPI; EC 3.4.19.3) is an omega exopeptidase which cleaves pyroglutamic acid from the N-terminus of bioactive peptides and proteins. It plays an important role in the processing and degradation of regulatory peptides such as thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH). To gain further insights into its performance in vivo and suggest possible applications, such as peptide processing or sequencing, this study focuses on the in vitro stability properties and Michaelis- Menten kinetics of the recombinant wild type enzyme and a single-site mutant, Tyr147→Phe (Y147F)

Stability properties of an ancient plant peroxidase

Plant (Class III) peroxidases have numerous applications throughout biotechnology but their thermal and oxidative stabilities may limit their usefulness. Horseradish peroxidase isoenzyme C (HRPC) has good catalytic turnover and is moderately resistant to heat and to excess (oxidizing) concentrations of hydrogen peroxide. In contrast, HRP isoenzyme A2 (HRPA2) has better oxidative but poorer thermal stability, while soybean peroxidase (SBP) displays enhanced thermal stability. Intrigued by these variations amongst closely related enzymes, we previously used maximum likelihood methods (with application of Bayesian statistics) to infer an amino acid sequence consistent with their most recent common ancestor, the ‘Grandparent’ (GP). Here, we report the cloning and expression of active recombinant GP protein in Escherichia coli. GP’s half-inactivation temperature was 45 oC, notably less than HRPC’s, but its resistance to excess H2O2 was 2-fold greater. This resurrected GP protein enables a greater understanding of plant peroxidase evolution and serves as a test-bed to explore their ancestral properties