Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 2. Application for high fructose syrup production

Batch and continuous production of high fructose syrup from Jerusalem artichoke tubers has been studied using yeast cells immobilized in open pore gelatin matrix. In a batch reactor, the hydrolysis was 93% ($\text{d}\text{-fructose/}\text{d}\text{-glucose}\text{= 90/10}$) and 42 mg d-fructose per ml was produced from the artichoke tuber extract by immobilized cells in 3 h. The same immobilized cells were recycled and used repeatedly for 10 batch cycles starting with fresh juice at the beginning of each cycle. It was found that immobilized cells were extremely stable and the percent hydrolysis was almost constant for all 10 batch cycles. In a continuous reactor using an immobilized cell concentration of 65.7 g (dry wt) l^?1 of total working bioreactor volume, the percent hydrolysis was found to remain constant at ～100% at dilution rates <1.26 h^?1, but beyond that it decreased. Volumetric productivity attained its maximum value at D = 2.08 h^?1 and was found to be 100 g l^?1 h^?1. This was achieved at a feed sugar conversion of 80%. At 90% conversion and D = 1.66 h^?1, the productivity was found to be 90 g l^?1 h^?1. Continuous operation of the immobilized cell bioreactor at a constant dilution rate of 1.65 h^?1 for 240 h resulted in only 2% loss of original activity.     

Fructose production from Jerusalem artichoke by inulinase immobilized on chitin

Summary Inulinase fromAspergillus ficuum was immobilized by cross-linking with glutaraldehyde on chitin. Batch and continuous production of fructose from Jerusalem artichoke tuber was studied using this immobililized inulinase. In a batch reactor, the extent of hydrolysis attained 90% (D-fructose/D-glucose :86/14) in 10h and 77.5g/L of D-fructose was produced from the Jerusalem artichoke tuber juice. In a continuous packed bed column reactor, the maximum volumetric productivity of 61 g/L, h was obtained at residence time of 0.9h and conversion yield of 55%. At a fixed residence time of 2.6 h and 40° C, this could be operated for over two weeks with only a slight loss of activity (4.8%).     

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater

Aims: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. Methods and Results: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38°C for growth and inulinase production, and 35°C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l^?1, and the highest ethanol yield of 0·467, or 91·5% of the theoretical value of 0·511, was achieved for the slurry with a solid concentration of 200 g l^?1. Conclusions: Halophilic Jerusalem artichoke can be used for fuel ethanol production. Significance and Impact of the Study: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.     

Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells

In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2 g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2 l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6 g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (50.6%).     

Continuous production of fructose-syrups from inulin by immobilized inulinase from recombinantSaccharomyces cerevisiae

Recombinant exoinulinase was partially purified from the culture supernatant ofS. cerevisiae by (NH₄)₂SO₄ precipitation and PEG treatment. The purified inulinase was immobilized onto Amino-cellulofine with glutaraldehyde as a cross-linking agent. Immobilization yield based on the enzyme activity was about 15%. Optimal pH and temperature of immobilized enzyme were found to be 5.0 and 60°C, respectively. The enzyme activity was stably maintained in the pH ranges of 4.5 to 6.0 at 60°C. 100% of enzyme activity was observed even after incubation for 24 hr at 60°C. In the operation of a packed-bed reactor containing 412 U inulinase, dahalia inulin of 7.5%(w/v) concentration was completely hydrolyzed at flow rate of 2.0 mL/min at 60°C, resulting in a volumetric productivity of 693 g-reducing sugars/L/h. Under the reaction conditions of 1.0 mL/min flow rate with 2.5% inulin at 60°C, the reactor was successfully operated over 30 days without loss of inulinase activity.     

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 × His tag was found to be 22.6 U mg^?1 of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 °C, respectively and the inulinase was stable in the pH range of 3–8 and in the temperature range of 0–50 °C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g^?1 of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10 h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L^?1 citric acid and 5.3 g L^?1 iso-citric acid from inulin while 68.9 g L^?1 of citric acid and 4.1 g L^?1 iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.     

Immobilization of thermostable exo-inulinase from mutant thermophilic Aspergillus tamarii-U4 using kaolin clay and its application in inulin hydrolysis

In this study, attempts were made to immobilize purified exo-inulinase from mutant thermophic Aspergillus tamarii-U4 onto Kaolinite clay by covalent bonding cross-linked with glutaraldehyde with an immobilization yield of 66% achieved. The free and immobilized inulinases were then characterized and characterization of the enzymes revealed that temperature and pH optima for the activity of the free and immobilized enzymes were both 65?°C and pH 4.5 respectively. The free inulinase completely lost its activity after incubation at 65?°C for 6 h while the immobilized inulinase retained 16.4% of its activity under the same condition of temperature and incubation time. The estimated kinetic parameters K_m and V_max for the free inulinase as estimated from Lineweaver-Burk plots were 0.39?mM and 4.21?µmol/min for the free inulinase and 0.37?mM and 4.01?µmol/min for the immobilized inulinase respectively. Inulin at 2.5% (w/v) and a flow rate of 0.1?mL was completely hydrolysed for 10?days at 60?°C in a continuous packed bed column and the operational stability of the system revealed that the half-life of the immobilized inulinase was 51?days. These properties make the immobilized exo-inulinase from Aspergillus tamarii-U4 a potential candidate for the production of fructose from inulin hydrolysis.     

Penicillium subrubescens, a new species efficiently producing inulinase

Inulin is a reserve carbohydrate in about 15 % of the flowering plants and is accumulated in underground tubers of e.g. chicory, dahlia and Jerusalem artichoke. This carbohydrate consists of linear chains of β-(2,1)-linked fructose attached to a sucrose molecule. Inulinases hydrolyse inulin into fructose and glucose. To find efficient inulin degrading fungi, 126 fungal strains from the Fungal Biotechnology Culture Collection (FBCC) at University of Helsinki and 74 freshly isolated strains from soil around Jerusalem artichoke tubers were screened in liquid cultures with inulin as a sole source of carbon or ground Jerusalem artichoke tubers, which contains up to 19 % (fresh weight) inulin. Inulinase and invertase activities were assayed by the dinitrosalicylic acid (DNS) method and a freshly isolated Penicillium strain originating from agricultural soil (FBCC 1632) was the most efficient inulinase producer. When it was cultivated at pH 6 and 28 °C in 2 litre bioreactors using inulin and Jerusalem artichoke as a carbon source, inulinase and invertase activities were on day 4 7.7 and 3.1 U mL^?1, respectively. The released sugars analysed by TLC and HPLC showed that considerable amounts of fructose were released while the levels of oligofructans were low, indicating an exoinulinase type of activity. Taxonomic study of the inulinase producing strain showed that this isolate represents a new species belonging in Penicillium section Lanata-divaricata. This new species produces a unique combination of extrolites and is phenotypically and phylogenetically closely related to Penicillium pulvillorum. We propose the name Penicillium subrubescens sp. nov. (CBS 132785^T = FBCC 1632^T) for this new species.     

Production and properties of inulinase from Penicillium sp. NFCC 2768 grown on inulin-rich vegetal infusions

Inulinase production by Penicillium sp. NFCC 2768 isolated from the rhizosphere soil of dahlia was studied on media containing inulin-rich plant extracts. The maximum inulinase activity (64.54 nkat/ml) was observed with the tuber extract of dahlia (Dahlia pinnata). The fungus produced substantial inulinase activity on asparagus root powder (45.23 nkat/ml) and garlic extracts (41.32 nkat/ml). The apparent molecular weight of the purified inulinase was 68 kDa. The optimum pH and temperature for enzyme activity were 5.0 and 50°C, respectively. Mn²⁺ and Ca²⁺ were found to enhance the inulinase activity, while Hg²⁺ was found to be a strong inhibitor. Inulinase liberated fructose, glucose, sucrose, kestose (GF₂), nystose (GF₃), and inulooligosaccharides (IOS). This study suggested the use of dahlia tuber extract and asparagus root powder as suitable substrates for inulinase production by the newly isolated Penicillium sp. NFCC 2768, and its application in the generation of fructose and IOS.     

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol^?1, respectively. K_m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.     

鹰嘴豆孢克鲁维酵母利用菊芋原料同步糖化与发酵生产乙醇

菊芋含有大量的菊粉多糖,且种植简单、产量高,是极具开发价值的替代玉米等粮食作物生产燃料乙醇的原料。文中研究了鹰嘴豆孢克鲁维酵母Y179利用菊芋原料同步糖化与发酵生产乙醇。鹰嘴豆孢克鲁维酵母Y179具有高效分泌菊粉酶的能力,摇瓶试验显示Y179酵母能够利用完全由菊芋原料配制而成的培养基良好生长并发酵产生乙醇。通气及温度对乙醇产量影响明显,相对厌氧环境对Y179酵母发酵产乙醇具有促进作用,30℃发酵温度相对37℃和42℃更有利于乙醇产量提高。种子液培养时间及接种量对乙醇产量影响较小。在5 L发酵罐中以10%(V/V)量接入预培养36 h的Y179种子液,发酵液完全由菊芋干粉配制而成,总糖含量22%(W/V),30℃不通气,300 r/min搅拌,发酵144 h时,乙醇浓度达到12.3%(V/V),糖醇转化效率86.9%,糖利用率大于93.6%。初步研究结果显示鹰嘴豆孢克鲁维酵母Y179在利用菊芋原料生产乙醇方面具有良好应用前景。     

Inulin hydrolysis by the Debaryomyces phaffii inulinase immobilized on Deae cellulose

Debaryomyces phaffii inulinase was immobilized by adsorption on DEAE-cellulose. The immobilization caused a drop in optimum pH, a slight increase in optimum temperature and an important increase in the thermal stability of the system. The activity yield of immobilized inulinase was 75%.A continuous reactor operation was carried out. The utilization of this system permited the production of fructose syrup from inulin.     

一步法发酵菊芋产乙醇菌种的筛选及产酶条件、酶学性质的研究

从腐烂的菊芋及实验室保存的菌种中,选育到一株发酵菊芋产乙醇的菌株克鲁维酵母Kluyveromyces marxianus Y1。利用正交实验法对克鲁维酵母产菊粉酶的培养基组成及培养条件进行优化,确定培养基组成（g/L）为：菊粉40,酵母粉4,蛋白胨4,尿素1;初始pH5．0,温度30℃,150r／min条件下培养达到最佳产酶效果（57U／mL）。该菌株所产菊粉酶的性质测定结果表明：以菊粉为底物,该菊粉酶最适反应温度为55℃,在60℃以下稳定性很好,高于60℃时酶迅速失活;最适pH为5．0,pH4．6—5．2范围内酶稳定性很好;该酶属于外切型菊粉酶,体积分数为8％的乙醇对酶活力基本没有影响。     

Alkaline inulinase production by a newly isolated bacterium Marinimicrobium sp. LS–A18 and inulin hydrolysis by the enzyme

To date, all of microbial inulinases reported showed optimal activity at pH values ranging from 3.5 to 7.0. A bacterial strain, Marinimicrobium sp. LS-A18, showing high extracellular inulinolytic activity was isolated from a marine solar saltern of the Yellow Sea in China. Maximum enzyme activity was obtained at 55°C and pH 9.0, respectively. The inulinase activity was induced by inulin, but not by the other carbon sources employed. Under the optimal medium and culture condition, the highest inulinase activity, 14.6 U/ml, was obtained after 96 h of incubation at shake flask level. The optimal medium for inulinase production was MHI medium containing 4% inulin, 1% peptone and 5% NaCl, while the optimal culture condition for inulinase production were pH 7.5, temperature 37°C, agitation speed 210 rpm, medium volume 40 ml in 250 ml shake flask, and incubation time 96 h. A large amount of monosaccharides was released after inulin hydrolysis by the inulinase from strain LS-A18. This is the first report on alkaline inulinase production from microorganism.     

Considerazioni su Gelsi Ingialliti per Fame

Jerusalem artichoke (Helianthus tuberosus L.), an important crop, containing over 50% inulin in its tubers on a dry weight basis is an agricultural and industrial crop with a great potential for production of ethanol and industrial products. Inulin is a good substrate for bioethanol production. Saccharomyces cerevisiae 6525 can produce high concentrations of ethanol, but it cannot synthesize inulinase. In this study, a new integration vector carrying inuA1 gene encoding exoinulinase was constructed and transformed into 18SrDNA site of industrial strain S. cerevisiae 6525. The obtained transformant, BR8, produced 1.1 U mL^? 1 inulinase activity within 72 h and the dry cell weight reached 12.3 g L^? 1 within 48 h. In a small-scale fermentation, BR8 produced 9.5% (v/v) ethanol, with a productivity rate of 0.385 g ethanol per gram inulin, while wild-type S. cerevisiae 6525 produced only 3.3% (v/v) ethanol in the same conditions. In a 5-L fermentation, BR8 produced 14.0% (v/v) ethanol in fermentation medium containing inulin and 1% (w/v) (NH4)₂SO₄. The engineered S. cerevisiae 6525 carrying inuA1 converted pure nonhydrolyzed inulin directly into high concentrations of ethanol.     

Consolidated ethanol production from Jerusalem artichoke tubers at elevated temperature by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered with inulinase expression through cell surface display

Ethanol fermentation from Jerusalem artichoke tubers was performed at elevated temperatures by the consolidated bioprocessing strategy using Saccharomyces cerevisiae MK01 expressing inulinase through cell surface display. No significant difference was observed in yeast growth when temperature was controlled at 38 and 40 °C, respectively, but inulinase activity with yeast cells was substantially enhanced at 40 °C. As a result, enzymatic hydrolysis of inulin was facilitated and ethanol production was improved with 89.3 g/L ethanol produced within 72 h from 198.2 g/L total inulin sugars consumed. Similar results were also observed in ethanol production from Jerusalem artichoke tubers with 85.2 g/L ethanol produced within 72 h from 185.7 g/L total sugars consumed. On the other hand, capital investment on cooling facilities and energy consumption for running the facilities would be saved, since regular cooling water instead of chill water could be used to cool down the fermentation system.     

Expression of exo‐inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta‐gami B (DE3) and its characterization

Inulin is a linear carbohydrate polymer of fructose subunits (2‐60) with terminal glucose units, produced as carbon storage in selected plants. It cannot directly be taken up by most microorganisms due to its large size, unless prior hydrolysis through inulinase enzymes occurs. The hydrolyzed inulin can be taken up by microbes and/or recovered and used industrially for the production of high fructose syrup, inulo‐oligosaccharides, biofuel, and nutraceuticals. Cell‐free enzymatic hydrolysis would be desirable for industrial applications, hence the recombinant expression, purification and characterization of an Aspergillus niger derived exo‐inulinase was investigated in this study. The eukaroyototic exo‐inulinase of Aspergillus niger 12 has been expressed, for the first time, in an E. coli strain [Rosetta‐gami B (DE3)]. The molecular weight of recombinant exo‐inulinase was estimated to be ～81 kDa. The values of K_m and V_max of the recombinant exo‐inulinase toward inulin were 5.3 ± 1.1 mM and 402.1 ± 53.1 µmol min^?1 mg^?1 protein, respectively. Towards sucrose the corresponding values were 12.20 ± 1.6 mM and 902.8 ± 40.2 µmol min^?1 mg^?1 protein towards sucrose. The S/I ratio was 2.24 ± 0.7, which is in the range of native inulinase. The optimum temperature and pH of the recombinant exo‐inulinase towards inulin was 55°C and 5.0, while they were 50°C and 5.5 towards sucrose. The recombinant exo‐inulinase activity towards inulin was enhanced by Cu²⁺ and reduced by Fe²⁺, while its activity towards sucrose was enhanced by Co²⁺ and reduced by Zn²⁺. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:629–637, 2016     

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 (200?g?L(-1)) at 40?°C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2?g?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.     

A new approach for achievement of inulin accumulation in suspension cultures of Jerusalem artichoke (Helianthus tuberosus) using biotic elicitors

A promising protocol for achievement the accumulation rate of inulin compound in a suspension culture of Jerusalem artichoke (Helianthus tuberosus) was established. The effect of incorporated of cell cultures in combining with two type of biotic elicitors Aspergillus niger extract and Methyl-Jasmonate incorporation feeding medium on leaf cell growth patterns and production of inulin was investigated. The maximum value of cell growth parameters and highest content of inulinase activity (0.395 u/ml) were resulted from elicitation of augmented MS-medium with A. niger extract at the level of 0.2% in combination with Methyl-Jasmonate (150 μM) as compared with other concentrations after 2 weeks of cultivation. The chemical analyses of the different cell lines were spectro-photometerically performed. This study clearly indicates that combining of A. niger and Methyl-Jasmonate elicitors plays a critical role on inulin process and its accumulation in Jerusalem artichoke cell cultures.     

Immobilized thermophilic bacteria as a source of respiratory chain for the recycling of NAD+

Immobilization of thermophilic bacterium strain PS3 has been performed by crosslinking with 0.4% glutaraldehyde in the presence of 4.6% bovine serum albumin at ? 20°C. After immobilization of bacteria the plasma membrane became permeable to NADH. The yield of NADH respiration of the immobilized strain was ～10%. The apparent K_m for NADH with the immobilized thermophilic strain was $\text{6 × 10}{}^{\mathrm{?4}}\text{m}$. After immobilization of this strain no variations of activities were observed between pH 4.5 and 9.5. Recycling of NAD⁺ is at least 10 times better with the thermophilic strain compared to Escherichia coli. In a preliminary experiment at 45°C the half life obtained with Escherichia coli was 1 h and with PS3 was 12 h. This temperature increased the rate of respiration by a factor of ～4 (compared to 20°C) and may avoid most of bacterial contaminations (most bacteria are not able to grow above 42°C).