Claus Crone Fuglsang

Antimicrobial enzymes: Applications and future potential in the food industry

Antimicrobial enzymes are ubiquitous in nature, playing a significant role in the defense mechanisms of living organisms against infection by bacteria and fungi. Hydrolytic antimicrobial enzymes function by degrading key structural components of the cell walls of bacteria and/or fungi, whereas antimicrobial oxidoreductases exert their effects by the generation in situ of reactive molecules. The potential of these enzymes in food preservation is still far from realized at present.

Biochemical Analysis of Recombinant Fungal Mutanases A NEW FAMILY OF α1,3-GLUCANASES WITH NOVEL CARBOHYDRATE-BINDING DOMAINS

Nucleotide sequence analysis shows thatTrichoderma harzianum and Penicillium purpurogenum α1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH2-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong α-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50–55 °C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K D around 0.11 and 0.13 μm at pH 7 for the P. purpurogenum andT. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.

Expression and characterization of a recombinant Fusarium spp. galactose oxidase.

The Fusarium spp. (Dactylium dendroides) galactose oxidase was expressed in Aspergillus oryzae and Fusarium venenatum hosts. Under the control of an A. niger α-amylase or a Fusarium trypsin promoter, high level galactose oxidase expression was achieved. The recombinant oxidase expressed in the A. oryzae host was purified and characterized. The purified enzyme had a molecular weight of 66 k Da on sodium dodecyl sulfate-polymerase gel electrophoresis (SDS-PAGE) and 0.4 mol copper atom per mole protein. The stoichiometry increased to 1.2 after a Cu saturation. Based on a peroxidase-coupled assay, the enzyme preparation showed an activity of 440 turnover per second toward d-galactose (0.1 M) at pH7 and 20°C. The enzyme had an optimal temperature of 60°C at pH 6.0 and an activation free Gibbs energy of 33 kJ/mol. A series of d-galactose derivatives was tested as the reducing substrate for the oxidase. The difference in activity was interpreted by the stereospecificity of the oxidase toward the substituents in the pyranose substrate, particularly on the C5 and the cyclic hemiacetal O sites. The recombinan toxidase could act on some galactose-containing polysaccharides, such as guar gum, but was not able to oxidize several common redox compounds that lacked a primary alcohol functional group.

Chemoenzymatic synthesis and characterization of a new type of carbohydrate-based cationic antimicrobial agents

Abstract A simple chemoenzymatic synthesis providing 6- O -monoesters of 3-(trimethylammonio)propyl d -glucopyranoside in high yields is described. These new compounds are very effective cationic surfactants exhibiting both antibacterial and antifungal activity and have a good compatibility with anionic surfactants.