RESUMO
Poor solubility is the main drawback of the direct industrial exploitation of chitin, the second most abundant biopolymer after cellulose. Chemical methods are conventional to solubilize chitin from natural sources. Enzymatic hydrolysis of soluble chitinous substrates is a promising approach to obtain value-added by-products, such as N-acetylglucosamine units or low molecular weight chito-oligomers. Protein display on the bacterial membrane remains attractive to produce active enzymes anchored to a biological surface. The Lpp-OmpA system, a gene fusion of the Lpp signal sequence with the OmpA transmembrane region, represents the traditional system for targeting enzymes to the E. coli surface. EhCHT1, the amoebic chitinase, exhibits an efficient endochitinolytic activity and significant biochemical features, such as stability over a wide range of pH values. Using an extended Lpp-OmpA system as a protein carrier, we engineered E. coli to express the catalytic domain of EhCHT1 on the surface and assess the endochitinase activity as a trait. Engineered bacteria showed a consistent hydrolytic rate over a typical substrate, suggesting that the displayed enzyme has operational stability. This study supports the potential of biomembrane-associated biocatalysts as a reliable technology for the hydrolysis of soluble chitinous substrates.
Assuntos
Amoeba/enzimologia , Domínio Catalítico , Quitinases/genética , Quitinases/metabolismo , Escherichia coli/genética , Engenharia Genética , Quitina/metabolismo , Quitinases/química , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , SolubilidadeRESUMO
Stocks of intestinal amoebae isolated from hospital patients in Mexico City and grown in monoxenic culture were compared among themselves and with those already described (SARGEAUNT & WILLIAMS, 1979), using the electrophoretic patterns of four enzymes: glucose phosphate isomerase (GPI), phosphoglucomutase (PGM), L-malate:NADP+ oxido-reductase (oxalacetate-decarboxylating) (ME) and hexokinase (HK). New isoenzyme groups (SARGEAUNT & WILLIAMS, 1979) of all the amoebae, including Entamoeba histolytica have been demonstrated. Amongst these have been found seven more groups of E. histolytica, two new groups of E. hartmanni, one new group of Dientamoeba fragilis and one new group of E. coli. Of the seven new groups of E. histolytica three are known to originate from patients with clinical amoebiasis whilst the remainder are from asymptomatic subjects. Only 11.2% of the 125 isolations were associated with clinical amoebiasis, and these are clearly distinguished from the isolations from asymptomatic patients by their electrophoretic isoenzyme pattern.