Endo-chitinase Chit33 specificity on different chitinolytic materials allows the production of unexplored chitooligosaccharides with antioxidant activity.

The biological activity of chitooligosaccharides (COS) has made them targets for industrial and medical sectors. In this work, endo-chitinase Chit33 from Trichoderma harzianum CECT 2413 was expressed in Pichia pastoris GS115 to levels never achieved before (630 mg/L; 3.3 U/mL), without its biochemical characteristics being substantially affected. Chit33 produced a mixture of fully and partially acetylated COS from different chitin derivatives. HPAEC-PAD Chromatography and mass spectrometry analyses showed that (GlcNAc)4 and GlcN-(GlcNAc)2 were mainly produced from colloidal chitin and chitosan, respectively. COS in reaction mixtures were fragmented according to their size and their antioxidant activity analyzed by reducing power and free radical scavenging activity essays. The highest antioxidant activity was achieved with COS in the range of 0.5-2 and 2-10 kDa produced from colloidal chitin and chitosan, respectively, which gives biotechnological potential to both the chitin derivatives of 0.5-10 kDa and the biocatalyst producing them.

Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity.

BACKGROUND: Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18. RESULTS: Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5-6.5 and 30-40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the kcat/Km ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (D-glucosamine)1-8-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates. CONCLUSIONS: Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities.