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The hydrolysis of milk proteins by the recombinant AprX-SK37 protease and the changes in the rheological properties of the milk gel generated with AprX-SK37 and glucono-δ-lactone (GDL) were investigated. The AprX-SK37 and rennet selectively hydrolysed κ-casein to yield a 16-kDa band, while subtilisin hydrolysed all of the casein components. Milk treated only with AprX-SK37 formed softer gel. Storage modulus (G′) values of the combined gels increased with GDL concentrations up to 7 g/L. High tan δ was observed in the combined gel at 8.75 g/L GDL alongside syneresis. AprX-SK37 is a promising milk-clotting enzyme when combined with an optimal GDL concentration.

In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 172], the variant oxidizes both sugars equally well [(k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K(M) values and approximately ten-fold higher k(cat) values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60 degrees C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D-glucose/D-galactose mixtures at both 30 and 50 degrees C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 A resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.

Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito-oligosaccharide-based value-added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error-prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p-nitrophenyl-beta-1,4-N, N'-diacetyl-chitobiose at various pH in microtiter plates. From a medium-throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p-nitrophenyl-beta-1,4-N, N'-diacetyl-chitobiose as substrate, the overall catalytic efficiency, k(cat)/K(m) of the improved chitinase was 2.7- and 2.3-fold higher than the average k(cat)/K(m) of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications.

We have identified a approximately 140 amino acid domain that is shared by a variety of proteins in budding and fission yeast, nematode, rat, mouse, frog, oat, and man. Typically, this domain is located within 20 residues of the N-terminus of the various proteins. The percent identity among the domains in the 12 proteins ranges from 42 to 93%, with 16 absolutely conserved residues: N-x(11-13)-V-x2-A-T-x(34-36)-R-x(7-8)-W-R-x3-K-x12-G-x-E-x15 -L-x11-12-D-x-G-R-x11-D-x7-R. Even though these proteins share little beyond their segment of homology, data are emerging that several of the proteins are involved in endocytosis and or regulation of cytoskeletal organization. We have named this protein segment the ENTH domain, for Epsin N-terminal Homology domain, and hypothesize that it is a candidate for binding specific ligands and/or enzymatic activity in the cell.