Extracellular Inulinase Research Articles

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.

Solid-state fermentation enhances inulinase and invertase production by Aspergillus brasiliensis

Extracellular inulinase and invertase production by Aspergillus brasiliensis was evaluated in solid-state fermentation (SSF) and submerged fermentation (SmF) with a high sucrose concentration. The parameters estimated from online CO2 analysis revealed a lower Lag phase in SSF compared to SmF; the specific CO2 production rate (μCO2) decreased in both systems as the sucrose concentration increased. The highest maximum CO2 production rate (10.45 ± 0.72 mg/h mL) and total CO2 production (138.53 ± 6.48 mg/mL) were obtained in SSF with a sucrose concentration of 210 g/L. Inulinase and invertase production (1.13 ± 0.02 and 5.84 ± 0.12 U/mL, respectively) in SmF was independent of the initial sucrose concentration; meanwhile, both activities increased in SSF as the sucrose concentration increased, reaching maximum values of 5.88 ± 0.78 and 41.69 ± 3.60 U/mL, respectively, at the highest sucrose concentration (210 g/L). The zymographic analysis showed one band of inulinase and two bands of invertases involved in sucrose hydrolysis.

Inulinase Activity of Extracellular Protein of <i>Lactobacillus casei </i>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

Enhanced inulinase production by Fusarium solani JALPK from invasive weed using response surface methodology

The present study is the first report of utilizing Tithonia rotundifolia weed as a substrate for inulinase production from Fusarium solani JALPK. It also deals with the statistical optimization of culture conditions to enhance the enzyme yield. Amongst the 11 variables screened by Plackett- Burman design, Inulin in combination with Agave sisalana extract, Tithonia rotundifolia extract and NaNO3 had a significant influence on inulinase production and their concentrations were further optimized employing Box Behnken design. An enhancement of inulinase production from 970 EU/mL to 3261.011 EU/mL was gained after media optimization. Amongst the screened carbon sources Tithonia rotundifolia was found to be very effective in stimulating elevated inulinase synthesis. The Tithonia rotundifolia weed extract was treated with inulinase from Fusarium solani JALPK to form fructose which was estimated spectrophotometrically. This liberated fructose was also confirmed by osazone formation test and FTIR. HPTLC analysis of product revealed the exoinulinase nature of the enzyme produced by Fusarium solani JALPK since fructose was the only end product after hydrolysis of inulin rich weed in fermented broth. Thus the elevated extracellular inulinase yielding novel property of Fusarium solani JALPK (KY914560) contributes in considering it as a potential candidate with food, pharmaceutical and bioremediation applications.

Purification of bacterial inulinase in aqueous two-phase systems.

In this study, extracellular inulinase from Bacillus sp. 11/3 was partially purified and concentrated using aqueous two-phase system (ATPS). Two different phase forming salts and four types of polyethylene glycol (PEG) were used. Binodal curves and tie-length lines (TLLs) for eight ATPS were developed. For inulinase purification, concentrations of PEG and salt according to binodal curves (between 17 and 26%) were chosen. All ATPSs for inulinase purification were characterized. An ATPS consisted of 26% PEG1000 and 26% MgSO4 was found to be the most suitable for inulinase purification. This ATPS has 28.47% TLL, 1.03 of volume ratio, purification factor of 4.65 fold and recovery yield of 66.17%. On the SDS-PAGE electrophoresis two protein bands with molecular weight of around 24 and 56 kDa were observed. The partially purified enzymes had optimal activity at pH 8.0 and 6.5, optimal temperature at 30 and 70°C and kinetic parameters Km = 26.32 mmol and Vmax = 526 mmol/min.

Enhanced production of extracellular inulinase by the yeast Kluyveromyces marxianus in xylose catabolic state

The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome. Inulinase is an enzyme hydrolyzing β-2,1-fructosyl bond in inulin and sucrose and is not required for xylose assimilation. Disruption of INU1 in the strain DMKU 3-1042 lost the production of the extracellular protein and resulted in growth defect in sucrose and inulin media, indicating that the extracellular protein was inulinase (sucrase). In addition, six K.marxianus strains among the 16 strains that were analyzed produced more inulinase in xylose medium than in glucose medium. However, expression analysis indicated that the INU1 promoter activity was lower in the xylose medium than in the glucose medium, suggesting that enhanced production of inulinase is controlled in a post-transcriptional manner. The production of inulinase was also higher in cultures with more agitation, suggesting that oxygen supply affects the production of inulinase. Taken together, these results suggest that both xylose and oxygen supply shift cellular metabolism to enhance the production of extracellular inulinase.

Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals.

Inulins are polysaccharides that belong to an important class of carbohydrates known as fructans and are used by many plants as a means of storing energy. Inulins contain 20 to several thousand fructose units joined by β-2,1 glycosidic bonds, typically with a terminal glucose unit. Plants with high concentrations of inulin include: agave, asparagus, coffee, chicory, dahlia, dandelion, garlic, globe artichoke, Jerusalem artichoke, jicama, onion, wild yam, and yacón. To utilize inulin as its carbon and energy source directly, a microorganism requires an extracellular inulinase to hydrolyze the glycosidic bonds to release fermentable monosaccharides. Inulinase is produced by many microorganisms, including species of Aspergillus, Kluyveromyces, Penicillium, and Pseudomonas. We review various inulinase-producing microorganisms and inulin feedstocks with potential for industrial application as well as biotechnological efforts underway to develop sustainable practices for the disposal of residues from processing inulin-containing crops. A multi-stage biorefinery concept is proposed to convert cellulosic and inulin-containing waste produced at crop processing operations to valuable biofuels and bioproducts using Kluyveromyces marxianus, Yarrowia lipolytica, Rhodotorula glutinis, and Saccharomyces cerevisiae as well as thermochemical treatments.

Immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system

An extracellular inulinase was partially purified by ethanol precipitation and gel exclusion chromatography from a cell free extract of Kluyveromyces marxianus. Partially purified inulinase exhibited 420 IU/mg specific activity and it was immobilized on chitosan beads. Activity yield of immobilized inulinase was optimized with glutaraldehyde concentration (1–5%), glutaraldehyde treatment time (30–240min), enzyme coupling-time (2–16h) and enzyme loading (5–30 IU) as functions. Under the optimized conditions maximum yield 65.5% of immobilized inulinase was obtained. Maximum hydrolysis of inulin 84.5% and 78.2% was observed at 125rpm after 4h by immobilized and free enzyme, respectively. A retention-time of 4h and 5h was found optimal for the hydrolysis of inulin under agitation (125rpm) by free and immobilized enzyme, respectively. The recycling of the developed immobilized biocatalyst was carried out after 5h of inulin hydrolysis in a batch system. The developed immobilized biocatalyst was successfully used for the hydrolysis of inulin for 14 batches. This is the first report on the immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system.

Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.Electronic supplementary materialThe online version of this article (doi:10.1007/s10295-016-1771-5) contains supplementary material, which is available to authorized users.

Engineering a natural Saccharomyces cerevisiae strain for ethanol production from inulin by consolidated bioprocessing.

BackgroundThe yeast Saccharomyces cerevisiae is an important eukaryotic workhorse in traditional and modern biotechnology. At present, only a few S. cerevisiae strains have been extensively used as engineering hosts. Recently, an astonishing genotypic and phenotypic diversity of S. cerevisiae was disclosed in natural populations. We suppose that some natural strains can be recruited as superior host candidates in bioengineering. This study engineered a natural S. cerevisiae strain with advantages in inulin utilization to produce ethanol from inulin resources by consolidated bioprocess. Rational engineering strategies were employed, including secretive co-expression of heterologous exo- and endo-inulinases, repression of a protease, and switch between haploid and diploid strains.ResultsResults from co-expressing endo- and exo-inulinase genes showed that the extracellular inulinase activity increased 20 to 30-fold in engineered S. cerevisiae strains. Repression of the protease PEP4 influenced cell physiology in late stationary phase. Comparison between haploid and diploid engineered strains indicated that diploid strains were superior to haploid strains in ethanol production albeit not in production and secretion of inulinases. Ethanol fermentation from both inulin and Jerusalem artichoke tuber powder was dramatically improved in most engineered strains. Ethanol yield achieved in the ultimate diploid strain JZD-InuMKCP was close to the theoretical maximum. Productivity achieved in the strain JZD-InuMKCP reached to 2.44 and 3.13 g/L/h in fermentation from 200 g/L inulin and 250 g/L raw Jerusalem artichoke tuber powder, respectively. To our knowledge, these are the highest productivities reported up to now in ethanol fermentation from inulin resources.ConclusionsAlthough model S. cerevisiae strains are preferentially used as hosts in bioengineering, some natural strains do have specific excellent properties. This study successfully engineered a natural S. cerevisiae strain for efficient ethanol production from inulin resources by consolidated bioprocess, which indicated the feasibility of natural strains used as bioengineering hosts. This study also presented different properties in enzyme secretion and ethanol fermentation between haploid and diploid engineering strains. These findings provided guidelines for host selection in bioengineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0511-4) contains supplementary material, which is available to authorized users.

ENHANCEMENT OF INULINASE PRODUCTION FROM AN Acetobacter sp. IN SUBMERGED CULTURES

Recently, inulinases one of the most important enzymes which have gained much attention in industrial purposes. They can be applied for production of high fructose syrup from inulin and could reduce the process steps, resulting in cost reduction and increasing the amount of products. Acetobacter sp. produced extracellular inulinase in vitro. Maximum inulinase production was obtained at pH 7.0, 300C temperature, ammonium sulphate as nitrogen source, inoculum volume 5% (v/v), 1% sucrose as additional carbon source and 48 hr after inoculation.The enzyme showed maximum activity at pH 7 and 50 0C. The enzyme was stable more than 80% of its maximal activity at 30-50 0C after 30 min heat treatment.

Partial purification of extracellular exo-inulinase from Ulocladium atrum

Abstract Eight fungal species were cultivated on the Czapek liquid medium and a good starting extracellular and intracellular exo-inulinase were selected. Extracellular inulinase from Ulocladium atrum was prepared in the presence of 1% inulin source and 0.2% sodium nitrate as the best carbon and nitrogen sources. Incubation for the U. atrum was increased till it reached its maximum (36 U/ml) at the sixth day of incubation at 30 °C which was the best temperature for the production of exo-inulinase . Effect of all metal ions inhibited inulase production by U. atrum. Exo-inulinase was purified by using ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose. Three active inulinase forms INI, INII and INIII were resolved, each for DEAE cellulose. The specific activity of INI was 1915 U/mg protein which represented 2.65-fold purification over the crude extract with 42.8% recovery pooling of INI placed on CM cellulose chromatography and INI was resolved into INIa, INIb and INIc. The specific activity of INIa was 2479.2 U/mg protein which represented 3.43-fold purification over the crude extract with 24.2% recovery.

Production of novel alkalitolerant and thermostable inulinase from marine actinomycete Nocardiopsis sp. DN-K15 and inulin hydrolysis by the enzyme

An actinomycete strain Nocardiopsis sp. DN-K15 showing high inulinolytic activity was isolated from marine sediment of Jiaozhou Bay in China. Under optimal conditions, Nocardiopsis sp. DN-K15 produced 25.1 U/ml of inulinase within 60 h of fermentation at shake flask level, which was 2.7-fold higher than the level in the basal medium. The optimal pH and temperature of the inulinase from strain DN-K15 were determined to be 60 °C and pH 8.0, respectively. The inulinase was highly active over a wide pH range (5.0–11.0) and retained more than 81 % of residual activity after incubation at 60 °C for 1 h, indicating its alkali-tolerant and thermostable nature. Thin layer chromatography analysis revealed that fructose was the main product of inulin hydrolysis, indicating its exoinulinase activity. The high yield of extracellular inulinase combined with its novel enzymatic property made Nocardiopsis sp. DN-K15 a potential candidate in biotechnological and industrial applications.

Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase fromAspergillus nigerATCC 20611

The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO3, Zn+2, and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R 2) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO3, 1.5 mM (v/v) Zn+2, and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry.

Bioconversion ofAgave tequilanafructans by exo-inulinases from indigenousAspergillus nigerCH-A-2010 enhances ethanol production from rawAgave tequilanajuice

Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme(®) (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.

Increase in extracellular inulinase production for a new Rhizoctonia ssp. strain by using buckwheat (Fagopyrum esculentum) flour as a single carbon source

A newly isolated strain of Rhizoctonia ssp. was used for the production of extracellular inulinase. Previously, the qualitative effects of some carbon and nitrogen sources from fermentative media and the physicochemical parameters for growth were established by Plackett-Burman analysis, and the main parameters that affect extracellular inulinase yield were identified. In this study, the quantitative effect of the carbon to nitrogen ratio in the fermentative medium and the growth temperature were studied and optimized using central composite design and response surface methodology. On the basis of optimization, the maximum extracellular inulinase activity was achieved when 2·5-6·5% buckwheat flour was used as a single carbon source and 4·6-5·0% yeast extract was used as nitrogen source, by submerged cultivation, after 48 h at an incubation temperature between 15 and 27·5°C. Under the fermentative conditions established in this study, a maximum extracellular inulinase yield of 1·8 UI ml⁻¹ was achieved. Rhizoctonia ssp. strain can be used for extracellular inulinase production. Also, buckwheat flour proved to be an inexpensive and abundant substrate suitable for obtaining inulinase. Inulinases are versatile tools for biotechnology as they can be used for a wide range of applications, including production of bioethanol, fructose syrup and inulo-oligosaccharides, lactic acid, citric acid and butanediol.

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40°C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200g L(-1)) at 40°C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2g L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7%, respectively. In the range of 30 to 40°C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.

Effect of extrinsic and intrinsic parameters on inulinase production by Aspergillus niger ATCC 20611

Background: Inulinase is a versatile enzyme from glycoside hydrolase family which targets the β-2, 1 linkage of fructopolymers. In the present study, the effect of medium composition and culture conditions on inulinase production by Aspergillus niger ATCC 20611 was investigated in shake-flasks. Results: The highest extracellular inulinase (3199 U/ ml) was obtained in the presence of 25% (w/v) sucrose, 0.5% (w/v) meat extract, 1.5% (w/v) NaNO 3 and 2.5 mM (v/v) Zn 2+ , at initial pH of 6.5, temperature 35ºC and 6% (v/v) of spores suspension in the agitation speed of 100 rpm. Surfactants showed an inhibitory effect on enzyme production. The optimum temperature for inulinase activity was found to be 50ºC. TLC analysis showed the presence of both exo- and endo-inulinase. Conclusion: Sucrose, Zn 2+ , and aeration were found to be the most effective elements in inulinase production by A. niger ATCC 20611. TLC analysis also showed that the crude enzyme contained both endo and exo-inulinases. The strain is suggested as a potential candidate for industrial enzymatic production of fructose from inulin.