Application of Thermomyces lanuginosus polygalacturonase produced in Komagataella phaffii in biomass hydrolysis and textile bioscouring.

In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 µmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.

Purification and characterization of a beta-Glucanase produced by Trichoderma harzianum showing biocontrol potential

A beta-1,3-glucanase was produced by Trichoderma harzianum in cultures containing chitin as the sole substrate. Two proteins showing beta-1,3-glucanase activity were purified to apparent homogeneity by hydrophobic chromatography. The molecular masses of these proteins were 29 and 36 kDa. The 36 kDa protein was further characterized. It was active on a broad pH range, and maximal activity was detected at pH 5.0. The optimum temperature of the 36 kDa beta-1,3-glucanase was 50°C, but the purified enzyme was very sensitive to temperature. It lost about 60 percent or more of the activity after incubation for 30 min at 45, 50 and 60°C. The apparent K M and Vmax for hydrolysis of laminarin at pH 5.0 and 37°C, were 0.099 mg of reducing sugar/mL and 0.3 mg of reducing sugar/min.mL, respectively. The enzyme was insensitive to organic compound and metal ions, except for the ferric ion which inhibited about 100 percent of the original activity at the concentration of 1 mM. In contrast to other hydrolytic enzymes (a chitinase and a protease) produced by the same T. harzianum isolate (1051), the beta-1,3-glucanase showed no effect on the cell wall of the phytopathogenic fungus Crinipellis perniciosa.

Uma beta-1,3-glucanase foi produzida por Trichoderma harzianum em cultura contendo quitina como fonte de carbono. Duas proteínas com atividade de beta-1,3-glucanase foram purificadas através de cromatografia de interação hidrofóbica. As massas moleculares destas proteínas foram de 29 kDa e 36 kDa. A proteína de 36 kDa foi caracterizada quanto à influência das condições de pH e temperatura. A atividade máxima foi encontrada em pH 5,0 e temperatura de 50°C. A proteína purificada mostrou-se muito sensível à temperatura. Aproximadamente 60 por cento da atividade original foi perdida por incubação da proteína a 45°C, 50°C e 60°C, por 30 min. O K M aparente e a Vmax para hidrólise de laminarina em pH 5,0 à 37°C, foram de 0,099 mg de açúcar redutor/mL e 0,3 mg de açúcar redutor/min.mL, respectivamente. Esta enzima mostrou-se insensível a compostos orgânicos e íons metálicos, exceto íon férrico o qual em uma concentração de 1 mM, inibiu em aproximadamente 100 por cento a atividade da enzima. Ao contrário de outras enzimas hidrolíticas (quitinase e protease) produzidas pelo mesmo isolado 1051 de T. harzianum, a beta-1,3-glucanase descrita aqui não afetou a integridade da parede celular do fitopatógeno Crinipellis perniciosa.