RESUMEN
A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background.
Asunto(s)
Bacillus subtilis/enzimología , Erwinia/enzimología , Proteínas Fúngicas/química , Glucosiltransferasas/química , Isomaltosa/análogos & derivados , Esporas Bacterianas/enzimología , Bacillus subtilis/genética , Erwinia/genética , Proteínas Fúngicas/genética , Glucosiltransferasas/genética , Calor , Isomaltosa/síntesis química , Isomaltosa/química , Isomaltosa/genética , Esporas Bacterianas/genética , Sacarosa/químicaRESUMEN
AIMS: Isomaltulose (palatinose) is a slowly digestible sucrose isomer that can reduce both the glycemic and insulinemic response to foods. The aim of this study was to clone and express a sucrose isomerase (SIase) gene and characterize the protein that is responsible for the production of isomaltulose in the micro-organism Enterobacter sp. FMB-1. METHODS AND RESULTS: A cosmid clone containing c. 6 kbp region encoding an SIase gene was identified. The 5969-bp chromosomal DNA fragment covering the SIase (esi) gene in Enterobacter sp. FMB-1 was sequenced. Although this DNA fragment contained several open reading frames other than esi, only the presence of esi was sufficient to produce isomaltulose in recombinant Escherichia coli. The esi gene was expressed in E. coli, leading to the characterization of its SIase activity. CONCLUSIONS: The Enterobacter sp. FMB-1 esi gene was successfully cloned and expressed in E. coli. This gene encoded a functional SIase that produced isomaltulose from sucrose. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first molecular analysis of an SIase gene in an Enterobacter strain. The functional expression of the Enterobacter sp. FMB-1 esi gene in E. coli offers an alternative choice for the industrial production of isomaltulose.
Asunto(s)
Clonación Molecular , Enterobacter/enzimología , Genes Bacterianos , Transferasas Intramoleculares/genética , Isomaltosa/análogos & derivados , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromatografía en Capa Delgada , ADN Bacteriano/genética , Enterobacter/genética , Regulación Bacteriana de la Expresión Génica , Biblioteca de Genes , Transferasas Intramoleculares/metabolismo , Isomaltosa/genética , Isomaltosa/metabolismo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are intestinal microvillus membrane hydrolases that play important roles in carbohydrate digestion. Although the expression of these enzymes during postnatal development has been characterized, the effect of old age on disaccharidase activity is poorly understood. In the present investigation, we examined the effect of aging on lactase and sucrase activities and their mRNA levels in the small intestines of 3-, 12- and 24- mo-old rats by sampling from nine equidistant segments of small intestine. Total intestinal disaccharidase activity or mRNA abundance was determined from areas under the proximal-to-distal curves. Rats 24 mo of age had total intestinal lactase and sucrase activities that were 12 and 38% lower, respectively, than the 3-mo-old animals (P < 0.05). In contrast, total LPH and SI mRNA abundance did not change significantly. Thus, total intestinal lactase and sucrase activities decrease with age in a manner that likely involves a posttranscriptional process. The age-related decline in disaccharidase activity, if extrapolated to humans, may have important implications for the digestion of carbohydrate contained in the diet of the elderly.
