Karsten Matthias Kragh

Biomimetic silica encapsulation of enzymes for replacement of biocides in antifouling coatings.

Current antifouling technologies for ship hulls are based on metals such as cuprous oxide and co-biocides like zinc pyrithione. Due to the persistent adverse environmental effects of these biocides, enzyme-based antifouling paints are proposed as a bio-based, non-accumulating alternative. Here, a hydrogen peroxide-producing system composed of hexose oxidase (HOX, EC 1.1.3.5), glucoamylase (GA, EC 3.2.1.3) and starch is tested for the chemical and physical functionalities necessary for successful incorporation into a marine coating. The activity and stability of the enzymes in seawater was evaluated at different temperatures, and paint compatibility was assessed by measuring the distribution and activity of the enzymes incorporated into prototype coating formulations. We used a biomimetic encapsulation procedure for HOX through polyethylenimine-templated silica co-precipitation. The co-precipitation and formulation of a powder for mixing into a marine paint was performed in a one-step economical and gentle formulation process, in which silica co-precipitated HOX was combined with GA and starch to form the antifouling system. Silica co-precipitation significantly improved the stability and performance of the antifouling system in marine-like conditions. For example, encapsulation of HOX resulted in 46% higher activity at pH 8, and its stability in artificial seawater increased from retaining only 3.5% activity after 2 weeks to retaining 55% activity after 12 weeks. A coating comprising the full enzyme system released hydrogen peroxide at rates exceeding a target of 36 nmol cm−2 d−1 for 3 months in a laboratory assay, and had potential for prolonged action through incorporation in a self-polishing coating.

Efficient purification, characterization and partial amino acid sequencing of two α-1,4-glucan lyases from fungi

alpha-1,4-Glucan lyases from the fungi Morchella costata and M. vulgaris were purified by affinity chromatography on beta-cyclodextrin-sepharose, followed by ion exchange and gel filtration. The purified enzymes produced 1,5-anhydro-D-fructose from glucose oligomers and polymers with alpha-1,4-glucosidic linkages, such as maltose, maltosaccharides, amylopectin, and glycogen. The lyases were basically inactive towards glucans linked through alpha-1,1, alpha-1,3 or alpha-1,6 linkages. For both enzymes the molecular mass was around 121,000 Da as determined by matrix-assisted laser desorption mass spectrometry. The pI for the lyases from M. costata and M. vulgaris was 4.5 and 4.4, respectively. The lyases exhibited an optimal pH range of pH 5.5 to pH 7.5 with maximal activity at pH 6.5. Optimal temperature was between 37 degrees C and 48 degrees C for the two lyases, depending on the substrates. The lyases were examined with 12 inhibitors to starch hydrolases and it was found that they were inhibited by the -SH group blocking agent PCMB and the following sugars and their analogues: glucose, maltitol, maltose, 1-deoxynojirimycin and acarbose. Partial amino acid sequences accounting for about 35% of the lyase polypeptides were determined. In the overlapping region of the sequences, the two lyases showed 91% identity. The two lyases also cross-reacted immunologically.

Purification and characterisation of a malto-oligosaccharide-forming amylase active at high pH from Bacillus clausii BT-21.

Bacillus clausii BT-21 produced an extracellular malto-oligosaccharide-forming amylase active at high pH when grown on starch substrates. The enzyme was purified to homogeneity by affinity and anion-exchange chromatography. The molecular weight of the enzyme estimated by sodium dodecyl sulfate polyacrylamide electrophoresis was 101 kDa. The enzyme showed an optimum of activity at pH 9.5 and 55 degrees C. Maltohexaose was detected as the main initially formed starch hydrolysis product. Maltotetraose and maltose were the main products obtained after hydrolysis of starch by the enzyme for an extended period of time and were not further degraded. The enzyme readily hydrolysed soluble starch, amylopectin and amylose, while cyclodextrins, pullulan or dextran were not degraded. The mode of action during hydrolysis of starch indicated an exo-acting type of amylolytic enzyme mainly producing maltohexaose and maltotetraose. Amino acid sequencing of the enzyme revealed high homology with the maltohexaose-forming amylase from Bacillus sp. H-167.

A group of α-1,4-glucan lyases and their genes from the red alga Gracilariopsis lemaneiformis: purification, cloning, and heterologous expression

We present here the first report of a group of alpha-1,4-glucan lyases (EC 4.2.2.13) and their genes. The lyases produce 1, 5-anhydro-D-fructose from starch and related oligomers and polymers. The enzymes were isolated from the red alga Gracilariopsis lemaneiformis from the Pacific coasts of China and USA, and the Atlantic Coast of Venezuela. Three lyase isozymes (GLq1, GLq2 and GLq3) from the Chinese subspecies, two lyase isozymes (GLs1 and GLs2) from the USA subspecies and one lyase (GLa1) from the Venezuelan subspecies were identified and investigated. GLq1, GLq3, GLs1 and GLa1 were purified and partially sequenced. Based on the amino acid sequences obtained, three lyase genes or their cDNAs (GLq1, GLq2 and GLs1) were cloned and completely sequenced and two other genes (GLq3 and GLs2) were partially sequenced. The coding sequences of the lyase genes GLq1, GLq2 and GLs1 are 3267, 3276 and 3279 bp, encoding lyases of 1088, 1091 and 1092 amino acids, respectively. The deduced molecular masses of the mature lyases from the coding sequences are 117030, 117667 and 117790 Da, respectively, close to those determined by mass spectrometry using purified lyases. The amino acid sequence identity is more than 70% among the six algal lyase isozymes. The algal GLq1 gene was expressed in Pichia pastoris and Aspergillus niger, and the expression product was identical to the wild-type enzyme.

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.

Implementation of cross-linked enzyme aggregates of proteases for marine paint applications

Cross-linked enzyme aggregates (CLEAs) of proteases were tested in artificial seawater (ASW) both as it is and as a component of the paint. It is found that all CLEAs have tolerance to xylene and have great stability in dried paint. Moreover, CLEA Bacillus licheniformis shows 900% activation during the storage in ASW in dried paint.