C. Schiraldi

Effective production of a thermostable α-glucosidase from Sulfolobus solfataricus in Escherichia coli exploiting a microfiltration bioreactor

A microfiltration (MF) membrane bioreactor was developed for an efficient production of a recombinant thermostable alpha-glucosidase (rSsGA) from Sulfolobus solfataricus MT-4. The aim of the membrane bioreactor was to improve the control of the concentration of key components in the growth of genetic engineered microorganisms, such as Escherichia coli. The influence of medium composition was studied in relation to cell growth and alpha-glucosidase production. The addition of components such as yeast extract and tryptone resulted in a higher enzyme production. High cell density cultivation of E. coli BL21(DE3) on semidefined medium, exploiting a microfiltration bioreactor, was studied in order to optimize rSsGA production. In addition to medium composition, the inducer employed (either isopropyl beta-D-thiogalactopyranoside or lactose), the induction duration, and the cultivation mode influenced both the final biomass and the enzyme yield. The MF bioreactor allowed a cell concentration of 50 g/L dry weight and a corresponding alpha-glucosidase production of 11,500 U/L. The improvement obtained in the enzyme production combining genetic engineering and the microfiltration strategy was estimated to be 2,000-fold the wild-type strain.

Improvement of the flavour of Falanghina white wine using a purified glycosidase preparation from Aspergillus niger

Abstract Glycosidases are enzymes of particular interest for the wine-making and fruit-juice processing industry as the hydrolysis of glycoside-terpenols generates the free terpenols which contribute to the bouquet. However, the crude industrial preparations available on the market contain different types of glycosidases and contaminating enzymes whose contemporary action can produce undesired effects. This paper describes an effective and cheap procedure for purifying and separating different glycosidases present in an industrial crude extract and their use in comparative tests for flavour improvement of Falanghina wines. The methodology, which can be scaled up to an industrial level, allows the separation of α- l -rhamnopyranosidase, β- d -glucopyranosidase and α- l -arabinofuranosidase on partial cross-linked chitosan, with a recovery of 90, 74 and 70%, respectively. These activities were characterized for their optimal pH and temperature and stability in wine. The crude enzymatic preparation and the purified glycosidases were used and compared for the flavour improving of Falanghina white wine.

An enzymatic process for the production of the pharmacologically active glycoside desglucodesrhamnoruscin from Ruscus aculeatus L.

The pharmacological properties of the extract of Ruscus aculeatus L. have been well established for many years now. The compounds which possess these properties are the steroid glycosides ruscin and ruscoside and their hydrolysis products desglucoruscin, desglucodesrhamnoruscin and desglucoruscoside. As the pharmacological action increases with the decrease of the amount of the sugar molecules, the plant extracts must be submitted to chemical or enzyme hydrolysis in order to obtain the most active compounds. In our laboratory, a bioconversion process to produce the monoglycoside desglucodesrhamnoruscin from dry extracts of the rhizome of R. aculeatus has been developed using enzyme preparations containing a β-glucopyranosidase and an α-rhamnopyranosidase. Identifying the concentrations of substrate, enzyme and ethanol that are most advantageous for the bioconversion has optimized the process. The developed process gave a final product containing 19% of desglucodesrhamnoruscin.

Discovery of novel multi-target indole-based derivatives as potent and selective inhibitors of chikungunya virus replication

We recently identified indole derivatives (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities at lower micro molar concentrations and a selective index of inhibition higher than the lead compound Arbidol. Here we highlight new structural information for the optimization of the previously identified lead compounds that contain the indole chemical core. Based on the structural data, a series of indole derivatives was synthesized and tested for their antiviral activity against chikungunya virus in Vero cell culture by a CPE reduction assay. Systematic optimization of the lead compounds resulted in tert-butyl-5-hydroxy-1-methyl-2-(2-trifluoromethysulfynyl)methyl)-indole-3-carboxylate derivative IIc with a 10-fold improved anti-CHIKV inhibitory activity (EC50=6.5±1μM) as compared to Arbidol demonstrating a potent, selective and specific inhibition of CHIKV replication with only a moderate cell protective effect against other related alphaviruses. The reported computational insights, together with the accessible synthetic procedure, pave the road towards the design of novel synthetic derivatives with enhanced anti-viral activities.