Physico-chemical properties and substrate specificity of α-(1→3)-d-glucan degrading recombinant mutanase from Trichoderma harzianum expressed in Penicillium verruculosum

Abstract

The gene mutAW encoding Trichoderma harzianum fungus mutanase (MutA, GH71 family, α-1,3-glucanase, EC 3.2.1.59) was cloned and heterologously expressed by the highly productive Penicillium verruculosum fungus. P. verruculosum MutA strain secreted crude enzyme preparations with the recombinant MutA content of 40% of the total secreted protein, and the specific activity increased 150 folds compared to that of enzyme preparation obtained by the host strain. Homogeneous MutA had molecular mass of 70 kDa and displayed maximum of the activity on mutan at pH 5.0 and 50°C, with K_m and k_cat being 1.0 g/L and 30 s^-1, respectively. At 40-50°C, the MutA was stable for at least 3 h. Glucose was the main product of long-term mutan hydrolysis. HPLC analysis of hydrolysis product of oligo-α-(1→3)-D-glucosides bearing UV-detectable N-trans-cinnamoyl residue in the aglycon clearly indicated that MutA has an endo-processive hydrolytic mode of action. It was demonstrated that MutA can destroy the polysaccharide matrix of both gram-positive and gram-negative pathogenic bacteria biofilms.

Importance: The manuscript describes the properties of a novel recombinant GH71 mutanase Mut A from Trichoderma harzianum. Gene mutAW encoding mutanase was heterologously expressed in the host strain Penicillium verruculosum B1-537 (ΔniaD). The recipient strain has a high secretory ability and allowed to obtain preparations containing the target recombinant enzyme up to 80% of the total protein pool. MutA exhibited a high activity against mutan and negligible or zero activity toward other types of glucans including α-(1→4)-, β-(1→3)-, β-(1→4)-, and β-(1→6)-glucans. By using a series of synthetic oligo-α-(1→3)-D-glucosides, we demonstrated that MutA is an endo-processive enzyme, which hydrolyzes the internal glucosidic bonds and releases glucose from the reducing end sliding into the non-reducing end. MutA recognizes tetrasaccharide as a minimal substrate and hydrolyzes it to trisaccharide and glucose. The effectiveness of the use of MutA for the destruction of clinical isolates of gram-positive and gram-negative bacteria is also described.

Keywords: Penicillium verruculosum; bacterial biofilms; mutanase; oligo-α-(1→3)-D-glucoside substrates; α-(1→3)-d-glucan; α-1,3-glucanase.

Fig 1

The structures of synthetic N-trans-cinnamoylated glycoconjugates 1b–7b studied as mutanase substrates and of parent oligosaccharide aminopropyl glycosides 1a–7a. The carbohydrate sequences are represented according to symbol carbohydrate nomenclature (30).

Fig 2

Mutan hydrolysis by the purified MutA. (A) Progress kinetic curves (RS release) for 10 units of MutA per 1 mL of the reaction mixture. (B) HPAEC-PAD evaluation of hydrolysis products after 24 h of reaction (10 U/mL of mutanase activity).

Fig 3

(A) Chromatograms of cinnamoylated substrates 1b–7b and (B) chromatograms of oligosaccharide 6b and the products of its hydrolysis by MutA at a concentration of 0.016 mg/mL (CH₃CN:H₂O 79:21). 1—Cinnamic acid (5.03 OE); 2–1b (5.03 OE); 3–2b (3.52 OE); 4–3b (2.79 OE); 5–4b (4.88 OE); 6–5b (2.38 OE); 7–6b (3.52 OE).

Fig 4

Kinetics of 200 µM 5b (A) and 200 µM 4b (B) hydrolysis by MutA at a concentration of 0.016 mg/mL.

Fig 5

The effect of mutanase enzyme preparation M22 on biofilms of various types of microorganisms (staining with gentian violet in small wells of a 96-well plate): 1—primary biofilm; 2—primary biofilm and distilled water; 3—primary biofilm and inactivated mutanase enzyme preparation M22; 4—primary biofilm and saline solution; 5—primary biofilm and P. verruculosum B1-537 cellulase enzyme preparation produced by recipient strain under inducing conditions (control); 6—primary biofilm and mutanase enzyme preparation M22.

Fig 6

Visualization of the effect of purified MutA on the primary biofilms produced by S. aureus and P. aeruginosa on the surface of glass slides and stained with gentian violet (A) and alcian blue (B) primary biofilms and purified MutA (C).