The inulin hydrolysis by recombinant exo-inulinases: determination the optimum temperatures and activation energies

The advantages of recombinant enzymes over native include the control in a production environment, product purity and also high yield. The paper presents the determination the optimum temperatures and the activation energies for various origin recombinant exo-inulinases, among others from Aspergillus niger, A. awamori, Kluyveromyces marxianus and K. cicerisporus. The parameters were estimated based on the literature of the activity curves versus temperature for hydrolysis of inulin. It was assumed that both the hydrolysis reaction process and the deactivation process of recombinant exo-inulinase were first-order reactions by the enzyme concentration. A mathematical model describing the effect of temperature on recombinant exo-inulinase activity was used. Based on the comparison analysis, values of the activation energies $${E_{\rm a }}$$ E a were in the range from $${32.01 \pm 7.80}$$ 32.01 ± 7.80 to $${43.83 \pm 4.87}$$ 43.83 ± 4.87 kJ mol$$^{-1}$$ - 1 , the deactivation energies $${E_{\rm d }}$$ E d were in the range from $${83.93 \pm 4.82}$$ 83.93 ± 4.82 to $${352.44 \pm 14.26}$$ 352.44 ± 14.26 kJ mol$$^{-1}$$ - 1 and the optimum temperature $${T_{\rm opt }}$$ T opt were obtained in the range from $${318.91 \pm }$$ 318.91 ± 1.19 to $${328.76 \pm 0.25}$$ 328.76 ± 0.25 K.

Comparative study for obtaining inulinase and invertase by yeasts

The aim of this comparative study was to obtain a model for production of inulinase and invertase by species Saccharomyces, Candida and Hansenula, strains from culture collection of INCDCF-ICCF, using submerged fermentation in a medium containing inulin as source of C. This model explained the data variation and the actual relationships between the parameters and responses. The dry biomass content as well as the production of inulinase and invertase in the bioprocess medium was influenced by inulin concentration and microelement composition. The main parameters for bioprocesses were: inoculum size 2% (v/v), pH 6, temperature 280 C and 220 rpm agitation speed. Following comparative study for production of extracellular inulinase (exo and endo inulinase) and invertase were obtained for Candida arborea the best results, invertase production having significantly higher concentrations than inulinase (35.92 U/mL invertase activity vs. 8.01 U/mL inulinase activity), on M5 medium. These results could be useful for industrial applications such as food industry, pharmaceutical.

The effect of production conditions on gene expression levels of inulinase of Aspergillus wentii NRLL 1778

In this study, the production of inulinase from Aspergillus wentii, the optimum conditions of that production and how those conditions influence gene expression levels of the enzyme were examined. For inulinase of A. wentii, the time of production was determined as 3 days, the temperature of production as 30°C, the starting pH of the production medium as 6.0, and concentration of Jerusalem artichoke added in to production medium as 3%. When the effect of C and N resources added to growth mediums on inulinase activity was investigated, the highest activity was observed in the medium containing 1% maltose. The medium containing 1% (NH4)2HPO4 was determined to be best growth medium. The enzyme was observed to be stable at pH 5.0-6.0 and to maintain its activity at 50°C for 30 minutes. It was found that gene expression was maximum at 2% Jerusalem artichoke concentration, pH 6.0, 35°C on the 1st day of production. The enzyme gene expression levels were higher compared to other studied resources when 1% cellulose was used as the carbon resource and 1% NH4H2PO4 as the nitrogen resource.

Inactivation of Inulinase and Marination of High-Quality Jerusalem Artichoke (Helianthus tuberosus L.) Pickles With Screened Dominant Strains

Freshly harvested Jerusalem artichoke tubers contain inulinase, an enzyme that requires inactivation, because of its ability to hydrolysis inulin into fructose, which can be consumed by microorganism during marination. As the traditional pickling process takes 6 months, and involves the addition of a large amount of salt (18–20%), this production strategy is uneconomical and increases the nitrite intake. Additionally, miscellaneous bacteria produced during pickling affect the product taste. In this study, the enzyme inactivation effects of NaCl, NaHCO3, and ultrasound were evaluated. NaHCO3 treatment results in the highest degree of enzyme inactivation; however, the quality and flavor of the obtained Jerusalem artichoke pickles were not ideal. The Jerusalem artichoke pickles in which the enzymes were inactivated using a combination of NaCl and ultrasound exhibited better flavor than those exposed to NaHCO3; further, this combination reduced the inulinase activity of the Jerusalem artichokes to 2.50 U/mL, and maintained the inulin content at 61.22%. The strains LS3 and YS2, identified as Enterococcus faecalis and the salt-tolerant yeast Meyerozyma guilliermondii, respectively, were the dominant microorganisms isolated from the pickle juice. Jerusalem artichokes with inactivated inulinase were pickled with microbial powder, separated, purified, and dried to remove the natural Jerusalem artichoke sauce. This process shortened the fermentation cycle and improved product quality.

Inulinase Activity of Extracellular Protein of Lactobacillus casei AP in Different Growth Conditions

Inulinase is an enzyme that catalyzes the reaction involving the hydrolysis of inulin into fructose and/or small fructooligosaccharides by cutting the β-2,1 terminal. The present study aimed to determine the inulolytic activity of extracellular inulinase, extracted from L. casei AP, when grown under different conditions. Extracellular inulinase was extracted from the growth supernatant of L. casei AP, following which the extract was subjected to the qualitative inulinase test; inulinase activity was determined under growth conditions involving different combinations of pH and temperature. The results showed that L. casei AP had inulinolytic ability; therefore, it degraded inulin both in inulin media and inulin extract. The optimum activity of inulinase occurred at the combination of a temperature of 41 °C and pH of 5, with the activity of 20.53 in inulin media and 17.73 in inulin extract.

PARAMETERS OPTIMIZATION FOR INCREASED INTRACELLULAR INULINASE ACTIVITY OF A YEAST STRAIN

This study reveals the selection of a yeast strain, possessing inulinase activity and finding the optimal conditions of cultivation. Intra- and extracellular activity assay was performed after cultivation on media, containing inulin as a sole source of carbon. Optimization of the cultivation conditions was carried out for establishing the favorable conditions for biosynthesis of inulinase. Modifying the physicochemical and nutritional parameters of a cultivation process lead to major improvement of the enzyme activity. Highest intra- and extracellular inulinase activity was registered when 1.5% inulin was used, 5 % inoculum, temperature 28°C, pH=6.5 and agitation of 200rpm. The selected strain Kluyveromyces sp. C showed higher values for the intracellular inulinase activity, making it suitable for immobilization and further use. Key words: Kluyveromyces sp., inulinase activity, parameters optimization

PARAMETERS OPTIMIZATION FOR INCREASED INTRACELLULAR INULINASE ACTIVITY OF A YEAST STRAIN

This study reveals the selection of a yeast strain, possessing inulinase activity and finding the optimal conditions of cultivation. Intra- and extracellular activity assay was performed after cultivation on media, containing inulin as a sole source of carbon. Optimization of the cultivation conditions was carried out for establishing the favorable conditions for biosynthesis of inulinase. Modifying the physicochemical and nutritional parameters of a cultivation process lead to major improvement of the enzyme activity. Highest intra- and extracellular inulinase activity was registered when 1.5% inulin was used, 5 % inoculum, temperature 28°C, pH=6.5 and agitation of 200rpm. The selected strain Kluyveromyces sp. C showed higher values for the intracellular inulinase activity, making it suitable for immobilization and further use. Key words: Kluyveromyces sp., inulinase activity, parameters optimization

Overexpression of an Inulinase Gene in an Oleaginous Yeast, Aureobasidium melanogenum P10, for Efficient Lipid Production from Inulin

In this study, in order to directly and efficiently convert inulin into a single-cell oil (SCO), an <i>INU1</i> gene encoding inulinase from<b><i></i></b> <i>Kluyveromyces marxianus</i> was integrated into the genomic DNA and actively expressed in an SCO producer <i>Aureobasidium</i> <i>melanogenum</i> P10. The transformant API41 obtained produced 28.5 U/mL of inulinase and its wild-type strain P10 yielded only 8.62 U/mL. Most (97.5%) of the inulinase produced by the transformant API41 was secreted into the culture. During a 10-L fermentation, 66.2% (w/w) lipid in the yeast cells of the transformant API41 and 14.38 g/L of cell dry weight were attained from inulin of 80.0 g/L within 120 h, high inulinase activity (23.7 U/mL) was also produced within 72 h, and the added inulin was actively hydrolyzed. This confirmed that the genetically engineered yeast of <i>A. melanogenum</i> P10 is suitable for direct production of lipids from inulin. The lipids produced could be used as feedstocks for biodiesel production.