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

Abstract

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.

Keywords: Bioactive chito-oligomers; COS, chitooligosaccharides; Chitin; Chitosan; DD, degree of deacetylation; DP, degree of polymerization; FRAP, Ferric Reducing Antioxidant Power; GH, Glycoside Hydrolase; GlcN, D-glucosamine; GlcNAc, N-Acetyl-d-glucosamine; Heterologous expression; Pichia pastoris; RSA, Radical Scavenging Activity; paCOS, partially acetylated COS.

Graphical abstract

Fig. 1

Activity profile and PAGE analyses of P. pastoris culture expressing chitinase Chit33. (A) The yeast transformant including plasmid CHIT33-pIB4 was cultivated in BMM using 1 L flask. The extracellular chitinase activity (red line; big circles), cell growth (OD₆₀₀; blue line; squares) and pH (black line; small circles) were measured at the indicated times. Each point of activity represents the average of three independent measurements and standard errors are indicated. (B) Culture filtrates (11 μL) were evaluated after 0, 24, 48, 72, 96, 120 h and 144 h of methanol induction (lane 1, 2, 3, 4, 5, 6, 7, respectively) using SDS-PAGE. (C) Culture filtrate-144 h induction (500 μL) was 25-times concentrated and revealed in situ (lane 1). (D) Culture filtrate (1 μL) from yeast grown in fed-batch and induced with methanol during 4 days. Numbers on the left of panels (B, C, D) indicate positions of molecular mass standards (lane M) in kDa.

Fig. 2

Temperature, pH and thermostability dependence profiles of the heterologous Chit33 activity. (A) The effect of temperature (black line) and pH (light blue line) on the heterologous Chit33 activity was evaluated using colloidal chitin as substrate at pH 6.0 and 45 °C, respectively. (B) Chitinase was incubated for the indicated temperatures and times prior to the addition of substrate. Remaining activity was determined at 45 °C. Lines from top to botton corresponding progressively to the intervals ranging from 10 to 90 min.

Fig. 3

Analyses of reaction based on Chit33 and colloidal chitin. (A) HPAEC-PAD-chromatogram of the 24 h reaction. Peaks: (1) GlcNAc; (2) (GlcNAc)₂; (3) (GlcNAc)₃; (4) (GlcNAc)₄; (5) Possible GlcN-(GlcNAc)₂; (*) Unknown due to lack of the corresponding commercial standard. A schematic representation of DP and composition of reaction products predicted from mass spectrometry data is presented. Blue hexagons; dark symbols): GlcN. Green hexagons (clear symbols): GlcNAc. Peaks correspondence in brackets. (B) Evolution of the referred COS in the reaction mixture. Only the identified products (fully acetylated COS with DP 1-4) were quantified and their evolution represented. Each point represents the average of two measurements and standard errors are indicated. Lines from top to bottom corresponding to (GlcNAc)₄, (GlcNAc)₂, (GlcNAc)₃, GlcNAc.

Fig. 4

Analyses of reactions based on Chit33 and different chitosan types. (A) HPAEC-PAD-chromatograms of 24 h reactions including (Lines from bottom to top) chitosan CHIT600 (black), QS2 (red) and CHIT100 (blue), all 0.8 % (w/v) final concentration. Peaks: (1) GlcNAc; (2) (GlcNAc)₂; (3) (GlcNAc)₃; (4) (GlcNAc)₄; (5) Possible GlcN-(GlcNAc)₂; (*) Unknown. A schematic representation of DP and composition of reaction products predicted from mass spectrometry data is presented. Symbols and colours as in Fig. 3. (B) Evolution of the referred COS produced from chitosan QS2. Values are means of two measurements and standard errors are indicated. Lines from top to bottom: GlcN-(GlcNAc)₂ and (GlcNAc) in clear and dark lines, respectively; (GlcNAc)₃; (GlcNAc)₂.

Fig. 5

Free Radical Scavenging Activity of the referred chitinolytic materials. Ascorbic acid was used as positive control. (A) Percentage of radical scavenging activity (RSA) using ABTS, relative to ascorbic acid 8 μg/mL (100 % RSA) used as control is showed. RSA of ascorbic acid at 4.2 μg/mL (50 % RSA) is indicated. (B) Percentage of RSA %) using DPPH. All data are means of three independent assays and standard errors are indicated. For each substrate 3 bars representing values obtaines with 0.5–2 kDa, 2–10 kDa and polymeric samples, from left to right, respectively.