Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues

Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli. Following 6xHis tag purification, characterization revealed the protein to be a covalent flavoprotein of 47 kDa with a remarkably similar reactivity and substrate specificity to that of AldO. A steady-state kinetic analysis with a number of different polyol substrates revealed lower catalytic rates but slightly altered substrate specificity when compared to AldO. Thermostability measurements revealed that the novel AldO is a highly thermostable enzyme with an unfolding temperature of 84 °C and an activity half-life at 75 °C of 112 min, prompting the name HotAldO. Inspired by earlier studies, we attempted a straightforward, exploratory approach to improve the thermostability of AldO by replacing residues with high B-factors with corresponding residues from HotAldO. None of these mutations resulted in a more thermostable oxidase; a fact that was corroborated by in silico analysis

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering

Engineering a hyperstable enzyme by manipulation of early steps in the unfolding process

Protein engineering experiments have recently yielded hyperstable variants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). These variants contain mutations suggested by comparison of TLP-ste with its more thermostable counterpart thermolysin, as well as rationally designed mutations. The key to the successful stabilization strategy was the identification of a "weak" region that is involved in early unfolding events ("unfolding region"). Mutations in this region had large effects on stability, whereas mutations in other parts of the protein generally had minor effects. The mutational strategies that were used as well as characteristics of the engineered hyperstable biocatalysts are reviewed below