Search

Affinity chromatography is a widely used technique for antibody isolation. This article presents the successful synthesis of a novel affinity resin with a mutant form of protein A (BsrtA) immobilized on it as a ligand. The key aspect of the described process is the biocatalytic immobilization of the ligand onto the matrix using the sortase A enzyme. Moreover, we used a matrix with primary amino groups without modification, which greatly simplifies the synthesis process. The resulting resin shows a high dynamic binding capacity (up to 50 mg IgG per 1 mL of sorbent). It also demonstrates high tolerance to 0.1 M NaOH treatment and maintains its effectiveness even after 100 binding, elution, and sanitization cycles. [ABSTRACT FROM AUTHOR]

Results reveal that during the course of yeast pyrophosphatase pathway, enzyme-pyrophosphate intermediate exhibits isomerization. Data indicate that protonation of the isomerized enzyme-pyrophosphate intermediate decreases pyrophosphate hydrolysis but steps-up its release. An ionizable group with a pKa of approximately 9 is needed for structural integrity of the enzyme.

Two enzyme-pyrophosphate intermediates are formed during the reaction of cytoplasmic pyrophosphatase on pyrophosphate. The conversion of the intermediates is the rate-limiting step during synthesis and a partially rate-limiting step during hydrolysis.

The structural and functional consequences of substitutions at the tyrosine 55 - lysine 104 hydrogen bond in Escherichia coli were studied using inorganic pyrophosphatase. Results indicate the highly integrated nature of the active site supporting the hydrolysis of enzyme-bound inorganic pyrophosphatase. This suggests a reflect distortion caused by the substitution that propagates in the integrated active site reaching the trimer-trimer interface.

The Mg(super 2+) - bound form of the family II PPtase from Streptococcus gordonii is reported to be nearly instantly activated by incubation with equimolar Zn2+, but the activity thereafter decays on a time scale of minutes. The inability of Zn2+ to activate apo-PPase suggests that Zn(super 2+) binds more tightly to M2 than to M1, allowing direct binding to M2.