RESUMO
Enhancing functional gene expression is key to high-level production of active chitinases. For this purpose, the effects of culture cell density, inducer concentration, post-induction time and induction temperatures on the functional expression of two different chitinases (HsChiA1p, a family 18 archaeal chitinase and PtChi19p, a family 19 bacterial chitinase) were comparatively investigated. Results showed that the effect of each parameter on the activity of both chitinases was specific to each enzyme. In addition, different Escherichia coli host strains compatible with the expression in pET systems were assayed for active protein overexpression. When using BL21 Star (DE3), a significant increase of 60% in expression was observed for the active archaeal chitinase HsChiA1p as compared to that found when using BL21 (DE3), indicating that the rne131 gene mutation efficiently stabilizes the mRNA for HsChiA1p. Using the Codon Adaptation Index value, rare codon analysis of the archaeal HschiA1 and bacterial Ptchi19 genes revealed that both DNA sequences were not optimal for maximal expression in E. coli. Different E. coli host strains possess extra copies of some of the tRNA genes for rare codons. For the Rosetta 2 (DE3) and the BL21 RP (DE3) strains, a significant increase of 40% was reached for the activity of HsChiA1p and PtChi19p. Finally, as part of the protein still remained insoluble, the best conditions for recovering biologically active protein from inclusion bodies were established for each enzyme.
Assuntos
Quitina/metabolismo , Quitinases/metabolismo , Escherichia coli/enzimologia , Halobacterium salinarum/enzimologia , Pseudoalteromonas/enzimologia , Eletroforese em Gel de Poliacrilamida , Proteínas Recombinantes/metabolismoRESUMO
The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6-8.5) and temperature (25-45 °C) ranges. Protein activity was stimulated by the metal ions Mg(+2), K(+), and Ca(+2) and strongly inhibited by Mn(+2). HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20% of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc)3, p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.
Assuntos
Organismos Aquáticos/enzimologia , Quitinases/metabolismo , Halobacterium salinarum/enzimologia , Organismos Aquáticos/genética , Quitinases/química , Quitinases/genética , Quitinases/isolamento & purificação , Clonagem Molecular , Ativadores de Enzimas/metabolismo , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Halobacterium salinarum/genética , Concentração de Íons de Hidrogênio , Íons/metabolismo , Metais/metabolismo , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The biodiversity of lactic acid bacteria in musts and wines of Albariño variety has been studied. The identification of species was addressed through a combination of biochemical and genetic methods (API® 50 CHL test, 16S rDNA and recA gene sequences, Amplified Ribosomal DNA Restriction Analysis -ARDRA- and 16S-26S intergenic region analysis). The results grouped the isolates into six species predominating those of the genus Lactobacillus and showing a typical biogeographical distribution. Among sixteen strains evaluated, eight of them showed malolactic activity. The study of the presence of genes hdc, odc, and tdc, along with the LC/MS-MS analysis of biogenic amines in wine, showed five strains lacking aminogenic ability. The absence of the pad gene in the above-mentioned strains discards its ability to produce volatile phenols that may adversely affect the aroma. Finally, all malolactic strains showed ß-glucosidase activity so that they could contribute to enhance and differentiate the aromatic profile of Albariño wines.
RESUMO
Many relevant applications have been demonstrated for chitinolytic enzymes. However, their successful exploitation depends upon the availability of strains and expression conditions that allow the production of active forms and large quantities of these enzymes. Escherichia coli has been commonly used to express and overproduce different proteins, among them chitinases. Improving the functional gene expression of chitinases is key to exploiting their potential. In a recent study, we described the effect of various parameters on the functional expression of 2 chitinases from different families, demonstrating that the effect of each of these parameters on the activity of both chitinases was specific to each enzyme. In this study, the expression of a Lactococcus lactis chitinase encoded by a new allele, ChiA1-2, was optimized. The results showed that not only the expression parameters seemed to influence protein production, solubility and activity but also the plasmid used for the expression. Herein, we describe the effect of 2 different promoters, tac and T7, on the expression of the active form of the chitinolytic enzyme.