Site-Specific Protein Modifications by an Engineered Asparaginyl Endopeptidase from Viola canadensis

Asparaginyl endopeptidases (AEPs) or legumains are Asn/Asp (Asx)-specific proteases that break peptide bonds, but also function as peptide asparaginyl ligases (PALs) that make peptide bonds. This ligase activity can be used for site-specific protein modifications in biochemical and biotechnological applications. Although AEPs are common, PALs are rare. We previously proposed ligase activity determinants (LADs) of these enzymes that could determine whether they catalyze formation or breakage of peptide bonds. LADs are key residues forming the S2 and S1′ substrate-binding pockets flanking the S1 active site. Here, we build on the LAD hypothesis with the engineering of ligases from proteases by mutating the S2 and S1′ pockets of VcAEP, an AEP from Viola canadensis. Wild type VcAEP yields <5% cyclic product from a linear substrate at pH 6.5, whereas the single mutants VcAEP-V238A (Vc1a) and VcAEP-Y168A (Vc1b) targeting the S2 and S1′ substrate-binding pockets yielded 34 and 61% cyclic products, respectively. The double mutant VcAEP-V238A/Y168A (Vc1c) targeting both the S2 and S1′ substrate-binding pockets yielded >90% cyclic products. Vc1c had cyclization efficiency of 917,759 M−1s−1, which is one of the fastest rates for ligases yet reported. Vc1c is useful for protein engineering applications, including labeling of DARPins and cell surface MCF-7, as well as producing cyclic protein sfGFP. Together, our work validates the importance of LADs for AEP ligase activity and provides valuable tools for site-specific modification of proteins and biologics.

Dealuminated BEA zeolite for selective synthesis of five-membered cyclic acetal from glycerol under ambient conditions

BEA zeolite is modified using phenoldisulfonic acid to change catalyst characteristics, which helps to form a single cyclic product. A new term called volume space acidity (VSA) provides volume space available for dioxane to dioxalane rearrangement.

Cyclodextrin Formation Initiated by Enzyme Debranching Reaction On Amylopectin Branch Chain Of Tapioka

Degradation of starch by the glucosyltransferase enzyme (CGTase) to produce the primary product of chainsplitting undergoes an intramolecular reaction without the participation of water molecule. From this process, α-1,4-Linked cyclic product, known as cyclodextrins, are formed. The aim of the research was to cut amylopectinbranch in order to produce one straight chain, to optimize cyclic reaction formation cyclodextrin by CGTase. Theresearch was devided into 3 stages; (1) debranching enzyme concentration estimation (5,10,15,20, and 25 unit/gram) and the length of otimum reaction to produce straight chain for 5 hours which sample was taken each hour,(2) reaction length time estimation to form cyclodextrin in order to use debranching products (straight chain) assubstrates, the reaction length for 360 minutes and sample taken each for 30 minutes, and (3) the best substrateconcentration for straight chain (20-40% w/v) to produce cyclodextrin. The result showed that enzyme concentrationtreatment and optimal length reaction will produce straight chain with enzyme concentration of 14 units/gram for3 hours and straight chain product of 20 units/gram for 1 hour with straight chain product of 83.5%. The optimumlength of reaction for cyclodextrin formation from amylose produced from the de-branching process was 240minutes. The amount of cyclodextrin produced was 143.45 g/L with conversion value of 47.81% at 30% (w/v)substrate concentration. Highest yield of cyclodextrin (154,28 g/L) and conversion value of 44.08% was obtained at35% (w/v) substrate concentration