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

Abstract

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 k_cat/K_m 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.

Keywords: Chit42 3D structure; Chitinase; Chitooligosaccharides; Partially acetylated chitooligosaccharides; Trichoderma harzianum.

Fig. 1

Activity profiles of cultures expressing Chit42. The P. pastoris transformant was grown in flask (a) and in fed-batch fermenter (b) supplemented with methanol. OD₆₀₀ (black circles), pH (empty cycles) and extracellular chitinase activity using colloidal chitin as substrate (blue circles) were measured at the indicated times at 35 °C. Each point of activity represents the average of three independent measurements and standard errors are indicated

Fig. 2

PAGE analyses of Chit42 expressed in P. pastoris. Filtrates (5 μL) from yeasts grown in flask were evaluated after 0, 16, 24, 48, 72, 96 and 120 h of methanol induction (lane 1, 2, 3, 4, 5, 6, 7, respectively) using SDS-PAGE (a). Filtrate (20 μL) was revealed in situ after 96 h of induction (lane 1) (b). Filtrates from yeast grown in fed-batch and induced with methanol during 0, 48 h (0.5 μL; lane 1 and 2), 72 h (0.2 μL; lane 3) and 96 h (0.15 μL; lane 4) were analysed (c). Numbers on the left of panels indicate the positions of molecular mass standards (lane M) in kDa

Fig. 3

Temperature, pH and thermostability dependence profiles. The effect of temperature (a) and pH (b) on the Chit42 chitinase activity was evaluated on colloidal chitin at pH 6 and 35 °C, respectively. c The chitinase was incubated for the indicated temperatures during the referred time periods (in min) prior to the addition of the substrate. Remaining activity was determined at 35 °C as described in the “Methods” section. Results represent the mean of three independent values. Standard errors are indicated

Fig. 4

HPAEC-PAD analyses. Chromatograms of the 24 h reactions catalysed by Chit42 with chitosan QS1 (black) and colloidal chitin (blue) as substrates. Peaks: (1) GlcNAc; (2) (GlcNAc)₂; (3) (GlcNAc)₃; (4) (GlcNAc)₄; (*) Unknown. On the right a schematic representation of polymerization degree and composition of reaction products predicted from mass spectrometry data (Additional file 1: Figure S2 and Tables S1, S2) is presented. Blue circles: GlcN. Green circles: GlcNAc. Peaks correspondence in parenthesis

Fig. 5

Evolution of the COS produced by Chit42 using colloidal chitin as substrate. Only the identified products (fully acetylated COS with DP 1–3) were quantified and their evolution in the reaction mixtures represented. Each point represents the average of two measurements and standard errors are indicated

Fig. 6

The active site of Chit42. Detail of the crystal structure showing the proposed binding of a COS chain. Sugar was modelled into the active site by structural superimposition with the reported complex from S. marcescens ChiA (PDb code 1EIB). Molecular surface of Chit42 showing the sugar in magenta sticks. The catalytic Asp169 and Glu171 are in red (a). Details of atomic interaction between COS and Chit42 relevant residues are represented in sticks. Catalytic residues highlighted and proposed hydrogen links represented as dashed lines (b)