QUITINASE DE Thermothelomyces heterothallicus PA2S4T: PURIFICAÇÃO, CARACTERIZAÇÃO BIOQUÍMICA E APLICAÇÃO BIOTECNOLÓGICA

Abstract

Chitinases are enzymes responsible for degrading chitin, an insoluble polymer present in the exoskeleton of insects, fungi, yeasts, algae, and crustaceans. There are countless organisms that produce these enzymes, and fungi are the main sources of obtaining them. They are used in several areas by the industry such as in the composition of agricultural pesticides, in pharmaceutical products as a preservative and as an adjuvant in therapy with antifungal drugs. In addition, chitooligosaccharides (COS), products generated after the hydrolysis of chitin, have different biological activities, such as antimicrobial and prebiotic. Thus, this study aimed to purify and biochemically characterize the extracellular chitinase of the fungus Thermothelomyces heterothallicus PA2S4T, as well as to evaluate its application in different biotechnological methodologies. The partially purified extracellular chitinase exhibited a molecular mass of 120 kDa and, after analysis of the zymogram and biochemical characteristics, it was found to have a β-N-acetylglucosaminidase (GlcNAcase). GlcNAcase exhibited higher activity at 65 ºC and pH 6.0, remaining stable at temperatures between 40 and 55 ºC and in the range of pH 6.0 to 6.5. Ethyl, isobutyl, isopropyl alcohols, and glycerol increased enzymatic activity, while HgCl2 and FeCl2 at 5 mmol/L completely inhibited it. GlcNAcase showed high catalytic activity with the substrates p-nitrophenyl N-acetyl-β-D-glucosaminide and p-nitrophenyl β-D glucopyranoside and no activity in the presence of colloidal chitin. The values of Vmax, Km, kcat, kcat/Km, and Ea were 971.81 µmol/mL/min, 0.14 mg/mL, 1962.4 s-1 , 4867.06 mM/s, and 43.92 kJ/mol, respectively. The analysis of images obtained by scanning electron microscopy (SEM) indicated that the bioconversion of in natura shrimp shell was effective, and that no pre-treatment is necessary for the degradation to be successful. After analyzing the products obtained, the production of N acetylglucosamine (GlcNAc) and COS with higher degrees of polymerization (DP) was verified, possibly diacetylchitobiose and chitotriose, indicating that the crude extract of T. heterothallicus may contain two types of chitinases: an endochitinase or chitobiase and a GlcNAcase. Furthermore, the COS obtained showed a prebiotic effect on the growth of Lactobacillus paracasei superior to that of glucose. The characteristics presented by the chitinase in this study and by the products of their hydrolysis show the great potential for their use by the biotechnology industry.