降解菊粉担子菌的分离鉴定及其酶的产生

从菊芋地的腐木上分离到一株在以菊粉为唯一碳源和能源的培养基上生长良好,具有较高菊粉酶活性的担子菌菌株,经鉴定为采绒革盖菌（Ｃｏｒｉｏｌｕｓｖｅｒｓｉｏｌｏｒ）。该菌的菊粉酶大部分是胞外酶,此酶对菊粉的专一性高,其Ｉ／Ｓ比值在发酵过程中不断变化。菊粉酶活性平行地随菌体生长而增加。该酶的合成受菊粉诱导,受果糖抑制。当果糖浓度大于２．７ｍｇ／ｍｌ时,菊粉酶活性为零。菌体的匀质化可使生长加快从而获得大量菊粉酶。     

Bioconversion of inulin to 2,3-butanediol by a newly isolated Klebsiella pneumoniae producing inulinase

Inulin could be converted to bio-based chemicals by an inulinase producer without external inulinase, and the production of 2,3-butanediol was less than 50 g/L. In this work, a novel inulinase producer of Klebsiella pneumoniae H3 was isolated, and inulinase catalytic properties as well as 2,3-butanediol fermentation were investigated. The enzyme was an intracellular inulinase with an optimal pH of 6 ∼ 7 and a temperature of 30 °C. The use of inulin by H3 was dependent on the degree of polymerization (DP), and the average DP of inulin in fermentation broth increased from 2.82 to 8.08 in 24-h culture of batch fermentation. Acidic pretreatment was developed to increase inulin utilization by adjusting medium pH to 3.0 prior to sterilization. In batch fermentation with optimized medium and fermentation conditions, the concentration of target product (2,3-butanediol and acetoin) was 80.4 g/L with a productivity of 2.23 g/(L⋅h), and a yield of 0.426 g/g inulin.     

Studies on a beta-fructosidase (inulinase) produced bySaccharomyces fragilis

Summary A study was made of a β-fructosidase, which is produced extracellularly and intracellularly bySaccharomyces fragilis. The enzyme catalyzes the hydrolysis of inulin, bacterial levans, sucrose, and the fructose portion of raffinose, by splitting off terminal fructosyl units. It attacks β-2,1 as well as β-2,6 linkages. The enzyme content of inulin-grown cells is sufficient to allow fermentation of inulin at the same rate as glucose. The ratio of hydrolysis rates with sucrose and inulin was about 25 for the β-fructosidase ofS. fragilis and about 14,000 for invertase.S. fragilis does not contain significant amounts of invertase and it ferments inulin, sucrose and raffinose with the aid of a related, but different enzyme, inulinase. Conditions of growth were established which favor inulinase synthesis. Highest yields were obtained with inulin as the carbon source, and somewhat lower yields with raffinose. Glucose, fructose and sucrose were poor inducers of inulinase. The pH of the medium during growth on inulin had to be in the range where inulinase could act, otherwise growth was tardy and poor. In an inulin containing medium aeration favored enzyme production as a result of stimulation of growth. The inulinase content of the cells in a unit volume was generally greater than that in the culture medium. The intracellular inulinase could be solubilized quantitatively by autolysis. The intra-and extracellular inulinases were concentrated and purified to the same extent. Comparison of the two preparations with respect to substrate specificity, rate of inactivation by heat, pH optima with sucrose (4.2) and with inulin (5.0), and elution patterns from a column of diethylaminoethyl cellulose, indicated that the intra-and extracellular enzymes were identical.     

The state of the art in the production of fructose from inulin enzymatic hydrolysis

The present work reviews the main advancements achieved in the last decades in the study of the fructose production process by inulin enzymatic hydrolysis. With the aim of collecting and clarifying the majority of the knowledge in this area, the research on this subject has been divided in three main parts: a) the characteristics of inulin (the process reactant); b) the properties of the enzyme inulinase and its hydrolytic action; c) the advances in the study of the applications of inulinases in bioreactors for fructose production. Many vegetable sources of inulin are reported, including information about their yields in terms of inulin. The properties of inulin that appear relevant for the process are also summarized, with reference to their vegetable origin. The characteristics of the inulinase enzyme that catalyzes inulin hydrolysis, together with the most relevant information for a correct process design and implementation, are described in the paper. An extended collection of data on microorganisms capable of producing inulinase is reported. The following characteristics and properties of inulinase are highlighted: molecular weight, mode of action, activity and stability with respect to changes in temperature and pH, kinetic behavior and effect of inhibitors. The paper describes in detail the main aspects of the enzyme hydrolysis reaction; in particular, how enzyme and reactant properties can affect process performance. The properties of inulinase immobilized on various supports are shown and compared to those of the enzyme in its native state. Finally, a number of applications of free and immobilized inulinases and whole cells in bioreactors are reported, showing the different operating procedures and reactor types adopted for fructose production from inulin on a laboratory scale.     

The State of the Art in the Production of Fructose from Inulin Enzymatic Hydrolysis

ABSTRACT

The present work reviews the main advancements achieved in the last decades in the study of the fructose production process by inulin enzymatic hydrolysis. With the aim of collecting and clarifying the majority of the knowledge in this area, the research on this subject has been divided in three main parts: a) the characteristics of inulin (the process reactant); b) the properties of the enzyme inulinase and its hydrolytic action; c) the advances in the study of the applications of inulinases in bioreactors for fructose production.

Many vegetable sources of inulin are reported, including information about their yields in terms of inulin. The properties of inulin that appear relevant for the process are also summarized, with reference to their vegetable origin.

The characteristics of the inulinase enzyme that catalyzes inulin hydrolysis, together with the most relevant information for a correct process design and implementation, are described in the paper. An extended collection of data on microorganisms capable of producing inulinase is reported. The following characteristics and properties of inulinase are highlighted: molecular weight, mode of action, activity and stability with respect to changes in temperature and pH, kinetic behavior and effect of inhibitors. The paper describes in detail the main aspects of the enzyme hydrolysis reaction; in particular, how enzyme and reactant properties can affect process performance. The properties of inulinase immobilized on various supports are shown and compared to those of the enzyme in its native state.

Finally, a number of applications of free and immobilized inulinases and whole cells in bioreactors are reported, showing the different operating procedures and reactor types adopted for fructose production from inulin on a laboratory scale.     

Localization of inulinase and invertase in Kluyveromyces species

In vivo hydrolysis of inulin and sucrose was examined in selected yeasts of the genus Kluyveromyces. Cells, grown in sucrose-limited chemostat cultures, were subjected to treatments for the removal of inulinase, the enzyme responsible for the hydrolysis of both inulin and sucrose. The effects of these treatments were studied by measurement of inulin-dependent and sucrose-dependent oxygen consumption by cell suspensions. In Kluyveromyces marxianus var. marxianus, inulinase was partially secreted into the culture fluid. Removal of culture fluid inulinase by washing had no effect on sucrose-dependent oxygen consumption by this yeast. However, this treatment drastically reduced inulin-dependent oxygen consumption. Treatment of washed cells with sulfhydryls removed part of the cell wall-retained inulinase and reduced inulin-dependent oxygen consumption by another 80%. Sucrose-dependent oxygen consumption was less affected, decreasing by 40%. Cell suspensions of K. marxianus var. drosophilarum, K. marxianus var. vanudenii, and Saccharomyces kluyveri rapidly utilized sucrose but not inulin. This is in accordance with the classification of these yeasts as inulin negative. Supernatants of cultures grown at pH 5.5 did not catalyze the hydrolysis of inulin and sucrose. This suggested that these yeasts contained a strictly cell-bound invertase, an enzyme not capable of inulin hydrolysis. However, upon washing, cells became able to utilize inulin. The inulin-dependent oxygen consumption further increased after treatment of the cells with sulfhydryls. These treatments did not affect the sucrose-dependent oxygen consumption of the cells. Apparently, these treatments removed a permeability barrier for inulin that does not exist for sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous saccharification of inulin and ethanol fermentation by recombinantSaccharomyces cerevisiae secreting inulinase

VariousSaccharomyces cerevisiae strains were transformed with a 2 μ-based multicopy expression plasmid, pYIGP, carryingKluyveromyces marxianus inulinase gene under the control ofGAPDH promoter. Among them two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinantS. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.     

Isolation of an inulinase derepressed mutant of Pichia polymorpha for the production of fructose

Pichia polymorpha has inulinase activity and could be used for the production of fructose syrup from inulin. The application of immobilized P. polymorpha whole cells for the continuous hydrolysis of inulin is, however, limited since the biosynthesis of this enzyme system is repressed by the reaction products, dextrose and fructose. A derepressed mutant hyperproducer of inulinase was isolated after treatment with EMS followed by a selection step with deoxyglucose.     

Inulinase-producing Marine Yeasts: Evaluation of their Diversity and Inulin Hydrolysis by Their Crude Enzymes

Total 427 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish, and marine algae were obtained. After inulinase activity of the yeast cultures was estimated, we found that four strains (OUC1, G7a, OUC2, and G7a1) of the marine yeasts grown in the medium with inulin could secrete a large amount of inulinase into the medium. The results of routine identification and molecular methods show that they belong to Pichia guilliermondii OUC1, Cryptococcus aureus G7a, Yarrowia lipolytica OUC2, and Debaryomyces hansenii G7a1, respectively. The optimal pHs of inulinase activity produced by them were 6.0, 5.0, 5.0, and 5.0, respectively, while the optimal temperatures of inulinase activity produced by them were 60°, 50°, 60°, and 50°C, respectively. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis by the crude inulinase produced by P. guilliermondii OUC1, indicating that the crude inulinase had a high exoinulinase activity while a large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase produced both by C. aureus G7a and D. hansenii G7a1. However, no monosaccharides and disaccharides were detected after inulin hydrolysis by the crude inulinase produced by Y. lipolytica OUC2, suggesting that the crude inulinase had no exoinulinase activity.     

Production of inulinase by Xanthomonas campestris pv phaseoli using onion (Allium cepa) and garlic (Allium sativum) peels in solid state cultivation

AIMS: To access inulinase production by Xanthomonas campestris pv phaseoli using the submerged and solid state cultivation (SSC) methods. METHODS AND RESULTS: Various carbon sources, inulin-rich solid substrates and pure synthetic inulin were tested for their efficiency in inulinase induction. The highest inulinase production (17.42 IU ml(-1)) in submerged cultures of X. campestris was observed with inulin as a carbon source with an initial pH, temperature and agitation of 7.0, 37 degrees C and 150 rev min(-1) respectively. Among the various substrates, garlic peels (117 IU gds(-1)) and onion peels (101 IU gds(-1)) were found to be the best for inulinase production. CONCLUSION: The inulinase production level of X. campestris was 6.7-fold higher in garlic and 5.8-fold in onion, under optimized SSC conditions compared with the submerged culture. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on inulinase production from garlic and onion peels by X. campestris using SSC. SSC is an efficient method for inulinase production by X. campestris for commercial applications.     

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.     

Comparative analysis of thermostability of extracellular inulinase activity from Aspergillus fumigatus with commercially available (Novozyme) inulinase

Thermostable inulinase activity was identified in the extracellular extract of Aspergillus fumigatus. At its optimum temperature of 60 degrees C, the ammonium sulphate fraction retained approximately 70% of its maximum activity after 72 h incubation in the absence of inulin. The two isoforms of A. fumigatus inulinase were purified and their thermostability was studied. In the presence of inulin, isoform II was more thermostable when compared to other two fractions and retained approximately 54% activity after 3h at 60 degrees C. The higher thermostability of inulinase of A. fumigatus makes it a potential candidate for commercial production of fructose.     

The β-fructofuranosidase activities of a strain of Clostridium acetobutylicum grown on inulin

Summary The -fructofuranosidase activities of a strain of Clostridium acetobutylicum, selected for its capacity to grow on inulinic substrates, were investigated. When grown on inulin, this strain produced extracellular and intracellular -fructofuranosidases, both of which hydrolysed inulin (inulinase activity) and sucrose (invertase activity). Inulinase activity was higher than invertase activity in the extracellular preparation, the opposite being observed for the cellular preparation. The effects of pH and temperature, substrate specificity and the kinetic constants for inulin and sucrose were studied on both preparations, as well as induction by inulin and repression by glucose and fructose of inulinase and invertase activities. The overall results were consistent with the existence of a least one inulinase, (EC 3.2.1.7), mainly but not entirely released in the extracellular medium, and an invertase (3.2.1.26) localized within the cell.Time course hydrolysis experiments of dalhia inulin and Jerusalem artichoke inulofructans by extracellular inulinase showed that this preparation had a remarkably high specificity for hydrolysis of long chain inulofructans.     

Inulinase activity ofDebaromyces cantarellii

Debaromyces cantarellii Capriotti contains an inulinase activity which is inducible by growth on inulin but not on other β-fructosides. The induction is inhibited by glucose and fructose. The system is situated in the cell wall and can be best extracted with a 20 mm phosphate buffer at pH 8.5. The inulinase activity shows pH optima at 4 and 6, suggesting the presence of two enzymes, the latter being more tightly bound to the cell wall. Both enzymes degrade inulin from the nonreducin end. The cells also contain a constitutive β-fructofuranosidase with a specificity partly overlapping with that of the inulinase(s).     

Studies on enzymic methods for extraction of inulin from Jerusalem artichokes

The release of inulin, d-fructose and protein from Jerusalem artichokes has been studied under diffusion and maceration conditions. The effects on release of added inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7), protease and cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] enzymes have been investigated. The results indicate that added enzymes do not improve the efficiency of inulin or d-fructose release and that mechanical methods represent the most efficient means of carbohydrate solubilization. Treatment of plant extracts with inulinase is shown to have the disadvantage of increasing peptide solubilization. The potential for improved inulin solubilization by use of endo-inulinases is discussed.     

Optimization of simultaneously enzymatic fructo- and inulo-oligosaccharide production using co-substrates of sucrose and inulin from Jerusalem artichoke

Prebiotic substances are extracted from various plant materials or enzymatic hydrolysis of different substrates. The production of fructo-oligosaccharide (FOS) and inulo-oligosaccharide (IOS) was performed by applying two substrates, sucrose and inulin; oligosaccharide yields were maximized using central composite design to evaluate the parameters influencing oligosaccharide production. Inulin from Jerusalem artichoke (5–15% w/v), sucrose (50–70% w/v), and inulinase from Aspergillus niger (2–7 U/g) were used as variable parameters for optimization. Based on our results, the application of sucrose and inulin as co-substrates for oligosaccharide production through inulinase hydrolysis and synthesis is viable in comparative to a method using a single substrate. Maximum yields (674.82?mg/g substrate) were obtained with 5.95% of inulin, 59.87% of sucrose, and 5.68 U/g of inulinase, with an incubation period of 9?hr. The use of sucrose and inulin as co-substrates in the reaction simultaneously produced FOS and IOS from sucrose and inulin. Total conversion yield was approximately 67%. Our results support the high value-added production of oligosaccharides using Jerusalem artichoke, which is generally used as a substrate in prebiotics and/or bioethanol production.     

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。     

Effect of media supplements and culture conditions on inulinase production by an actinomycete strain

Streptomyces sp. GNDU 1 produced high levels of extra-cellular inulinase (0.552 IU/ml) after 24 h at pH 7.5, temperature 46 degrees C in the presence of 1% inulin. The optimum temperature and pH for enzyme activity were 60 degrees C and 5.5 respectively. Yeast extract as a nitrogen source was found to be most suitable one for inulinase production whereas ammonium ion was inhibitory to the enzymatic production. All these conditions make Streptomyces sp. GNDU 1, a potential candidate for industrial enzymatic production of fructose from inulin.     

Trends in inulinase production – a review

This article highlights the research work carried out in the production of inulinases from various inulin substrates using strains of bacteria, yeast and fungi. Inulin is one of the numerous polysaccharides of plant origin that contains glucose or fructose. It is used as a substrate in industrial fermentation processes and in food industries due to its relatively cheap and abundant source for the microbiological production of high-fructose syrups, ethanol and acetone–butanol. The various oligosaccharides derived from inulin also find their application in the medical and dietary sector. The inulinase acts on the β-(2,1)-D-fructoside links in inulin releasing D-fructose. Hence, this article illustrates the capability of various microbes in hydrolyzing the carbon at its optimum nutrient concentration and operating condition towards inulinase production.     

Inulinase (β-fructofuranosidase) production by Kluyeromyces marxianus in batch culture

Summary Production of inulinase in batch fermentation using various carbon sources with Kluyermoyces marxianus was examined. Inulinase synthesis in the culture proceeded parallel to cell growth. Glucose, fructose and sucrose were inferior carbon sources for inulinase broduction. Highest production (212 U/mL) was achieved on inulin based media.