Review of inulinase production using solid-state fermentation

The purpose of the present study is to critically analyze the recent literature covering the production of inulinase enzyme from various sources by solid-state fermentation and discuss various approaches to increase its production in solid-state fermentation, purification, and its properties. The review deals with the solid-state fermentative production of inulinase production. Inulinases have many applications in industries, such as for the production of ultra-high fructose syrup, biofuels, lactic acid, citric acid, and single-cell oil. Solid-state fermentation (SSF) is more economic, requires smaller vessels, lowers water intake, reduces wastewater treatments, higher product yield, lesser chance of bacterial contamination, and lowers energy consumption. Furthermore, the crude products obtained from SSF can be directly used as the source of enzyme for biotransformation. Although many reports are available on a wide range of microbes which produces inulinases by SSF, it is important to isolate novel microbes for its production. Also, extensive research is going on to exploit unexplored sources for SSF. Higher yield of inulinases can be achieved by bioreactor modeling and proper monitoring of physical and chemical parameters in SSF.     

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.     

D201—GM大孔树脂吸附交联固定菊粉酶的研究

克鲁维酵母Ｙ－８５产生的胞内菊粉酶,以Ｄ２０１－ＧＭ大孔径阴离子交换树脂吸附交联法固定化,其制备固定化酶（ＩＥ）的适宜条件：树脂吸附酶时ｐＨ６．５、温度３０℃、时间３ｈ;交联时戊二醛浓度０．０３％、温度４℃、时间３ｈ。上述条件下制得ＩＥ的活性产率可达６２％,水解菊粉底物的最适温度５５℃,对热的稳定性和贮存稳定性均有明显提高,用ＩＥ填充床反应器连续降解菊粉抽提液（总糖４．５％）的实验结果表明,进料空     

Improvement of inulinase stability of calcium alginate immobilized Kluyveromyces marxianus cells by treatment with hardening agents

To improve the inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) stability of calcium alginate-immobilized Kluyveromyces marxianus cells, treatment with hardening agents has been investigated. Treatment of immobilized cells with some polycationic polymers resulted in little decrease in volumetric reactor productivity, but was most effective in increasing the inulinase stability of the immobilized cells. Inulinase stability of glutaraldehyde-hardened immobilized cells increased two-fold, and for hexamethylenediamine + glutaraldehyde and polyethyleneimine + glutaraldehyde-hardened cells increased six-fold compared with that of the unhardened cells.     

产菊糖酶克鲁维酶母Y-85的明胶固定化

克鲁维酵母Ｙ－８５的胞外菊糖酶占其总酶活的２８％,以１０％明胶包埋该酵母,酶活保留率为７３．９％。与游离细胞相比,固定化细胞菊糖酶的最适ＰＨ未改变,但当ＰＨ〈４和ＰＨ〉７时酶活稳定性更高;最适水解温度则升高了５℃,酶的热稳定性也有所提高。游离细胞的Ｋｍ值为９．３ｍｍｏｌ／Ｌ,固定化细胞则为１２．８ｍｍｏｌ／Ｌ。４℃贮存３０ｄ,固定化细胞酶活无损失,分批反应１０批次,固定化细胞酶活及机械强度保持良好     

Hydrolysis of inulin from Jerusalem artichoke by inulinase immobilized on aminoethylcellulose

Purified inulinase (inulase, 2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) of Kluyveromyces fragilis has been immobilized on 2-aminoethyl-cellulose by treatment with 2% glutaraldehyde in 0.05 m phosphate buffer, pH 7.0, for 2 h at room temperature. The immobilized enzyme preparation had 39.3 units inulinase activity per gram dried matrix, with 53.4% recovery yield of activity, and showed good operational stability in the presence of substrate, inulin or the tuber extract of Jerusalem artichoke. Optimum pH and temperature were 5.5 and 45°C, respectively. In a batch reactor, the conversion was 90% ($\text{d}\text{-fructose/}\text{d}\text{-glucose}\text{= 76/24}$) and 34 mg d-fructose per ml was produced from the artichoke tuber extract by the immobilized inulinase in 20 h. In column reactor packed with 28 ml immobilized enzyme, the following conditions were found to be optimal: height/diameter ratio of column, 10.3; space time, 3.8 h; temperature, 40°C. Operation under these conditions gave 90% conversion of a 7% inulin solution and the productivity was 102 mmol l^?1 h^?1.     

菊粉酶基因在酿酒酵母中的表达及乙醇发酵

以乙醇耐受力较强的酿酒酵母为受体菌,构建了能够分泌菊粉酶的基因工程菌并进行了菊芋粉的生料发酵。首先,以马克斯克鲁维酵母Kluyveromyces marxianus中的基因组DNA为模板,PCR扩增菊粉酶编码基因inu,分别使用菊粉酶自身启动子和酵母磷酸甘油激酶 (Phosphoglycerate kinase,pgk) 启动子,构建重组表达质粒HO/p-inu和HO/pgk-inu。经NotⅠ线性化后,采用电击法转化酿酒酵母工业菌株Saccharomyces cerevisiae 6525,分别得到含菊     

Immobilized inulinase: a new horizon of paramount importance driving the production of sweetener and prebiotics

In recent times, inulinase has emerged as one the most prominent and industrially upcoming enzymes applied to meet the ever increasing demand of d-fructose and fructooligosaccharides (FOS) as sweetener and prebiotics in the food and pharmaceutical industry, respectively. This review deals with types of inulinase and the attempts made to modify it for better thermal stability and shelf life. The ease of immobilization of inulinase has led us to the path of experimenting with different methods of enzyme immobilization since 1979. Several modes of immobilization ranging from simple cross-linking of enzymes onto a polymer support to nanoparticles have been applied over the years. The approach and concept of this review provide a yet unexplored focus on pioneering advances for the development of white biotechnology, for instance production of immobilized inulinase-based reusable biocatalysts and bioreactors designed for their use and for the continuous production of fructose and FOS.     

Purification and characterization of extracellular inulinase from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the purified inulinase

The extracellular inulinase of the marine yeast Pichia guilliermondii strain 1 was purified to homogeneity resulting in a 7.2-fold increase in specific inulinase activity. The molecular mass of the purified enzyme was estimated to be 50.0 kDa. The optimal pH and temperature for the purified enzyme were 6.0 and 60°C, respectively. The enzyme was activated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Fe³⁺, Fe²⁺, Cu²⁺, and Co²⁺, but Mg²⁺, Hg²⁺, and Ag⁺ inhibited activity. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1, 10-phenanthroline. The K _m and V _max values of the purified inulinase for inulin were 21.1 mg/mL and 0.08 mg/min, respectively. A large number of monosaccharides were detected after the hydrolysis of inulin. The deduced protein sequence from the cloned P. guilliermondii strain 1 inulinase gene contained the consensus motifs R-D-P-K-V-F-W-H and W-M-N-D-P-N-G, which are conserved among the inulinases from other microorganisms.     

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.     

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.     

云南省部分地区产菊粉酶霉菌的初步调查

从昆明,石林,西双版纳,思茅等地区的菊科植物根系上群,昆明,石林地区市售洋姜表土,洋姜提取液中污染菌及洋姜表面霉烂处,分离纯化产菊粉酶菌株,共获得105株霉菌,对以上菌株进行了初步鉴定,对产酶情况进行了初步的研究。发现分属于拟青霉属（Paecilomyces sp.),头孢霉属（Cephalosporium sp.),阜孢霉属（Papularia sp.),丝衣霉属（Byssochlamys sp.),指轮枝孢霉属（Stachylidium sp.)。刚毛菌属（Lacellina sp.)交链孢属（Alternaria sp.),地霉属（Geotrichun sp.)毛霉属（Mucor sp.),枝孢霉属（Claddosporium sp.),金孢霉属（Chrysosporium sp.),镰刀菌属（Fusarium sp.),根霉属（Rhizopus sp．）等13个避的霉菌,是国内未见报道的新的菊粉酶产生菌。     

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.     

Operation of a fixed-bed bioreactor in batch and fed-batch modes for production of inulinase by solid-state fermentation

This work is focused on the inulinase production by solid-state fermentation (SSF) in a fixed-bed reactor (34 cm diameter and 50 cm height) with working capacity of 2-kg of dry substrate operated in batch and fed-batch modes. It was investigated different strategies for feeding the inlet air in the bioreactor (saturated and unsaturated air) as alternative to remove the metabolic heat generated during the microbial growth by evaporative cooling. The kinetic evaluation of the process carried out in batch mode using unsaturated air showed that the evaporative cooling decreasing the mean temperature of the solid-bed, although the enzyme production was lower than that obtained using saturated air. Results showed that maximum enzyme activity (586 ± 63 U gds⁻¹) was obtained in the fed-batch mode using saturated air after 24 h of fermentation. The enzymatic extract obtained by fed-batch mode was characterized and presented optimum temperature and pH in the range of 52–57 °C and 4.8–5.2, respectively. For a temperature range from 40 to 70 °C the enzyme presented decimal reduction time, D-value, ranging from 5748 to 47 h, respectively. For a pH range from 3.5 to 5.5 the enzyme showed good stability, presenting D-values higher than 2622 h. In terms of Michaelis–Mentem parameters were demonstrated that the crude inulinase activity presented higher affinity for substrate sucrose compared to inulin.     

复合诱变在青霉高产菊粉酶菌株选育中的应用

以Penicillium sp．B01为出发菌株,经吖黄素或DES（硫酸二乙酯）分别与^60Co-γ射线对其孢子悬液进行复合诱变。经过初筛和复筛,在30μg／mL吖黄素诱变时间2h,剂量率为4．11Gy／min的^60Co-γ射线辐射使累计剂量为20．55Gy复合诱变的条件下,筛选出一株菊粉酶活比出发菌株高32％的突变菌株B01-A13-Co31。经同工酶电泳验证,变异株与出发菌株相比酶带有所变化。将此菌种连续传代6次进行产酶性能的稳定性测定,表明此菌株具有良好的遗传稳定性。     

Hydrolysis of inulin: a kinetic study of the reaction catalyzed by an inulinase from Aspergillus ficuum

A kinetic study of the hydrolysis of inulin was performed by using as catalyst a commercial inulinase from Aspergillus ficuum. The reaction was studied carrying out initial rate as well as time course measurements. Both inulinase and invertase activities of the enzyme were taken into account, and the corresponding kinetic parameters were determined in the temperature range 30-50 degrees C. The activation energies of the turnover constant for inulinase and invertase activities were found to be similar (56-57 kJ . mol(-1)). The ratio S/I of invertase to inulinase activity was 1.6 regardless of temperature. The thermal degradation of the enzyme was also investigated up to 70 degrees C, and an activation energy of 350-370 kJ . mol(-1) was evaluated.     

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

Aims: 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. Methods and results: 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. Conclusions: 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. Significance and impact of the study: 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.     

Production of inulinase by solid-state fermentation: effect of process parameters on production and preliminary characterization of enzyme preparations

This work was aimed at producing inulinase by solid-state fermentation of sugarcane bagasse, using factorial design to identify the effect of corn steep liquor (CSL) and soybean bran concentration, particle size of bagasse and size of inoculum. Maximum inulinase activity achieved was 250 U per g of dry substrate (gds) at 20% (w/w) of CSL, 5% (w/w) of soybean bran, 1 × 10¹⁰ cells mL⁻¹ and particle size of bagasse in the range 9/32 mesh. The use of soybean bran decreased the time to reach maximum activity from 96 to 24 h and the maximum productivity achieved was 8.87 U gds⁻¹ h⁻¹. The maximum activity was obtained at pH 5.0 and 55.0°C. Within the investigated range, the enzyme extract was more thermostable at 50.0°C, showing a D-value of 123.1 h and deactivation energy of 343.9 kJ gmol⁻¹. The extract showed highest stability from pH 4.5 to 4.8. Apparent K _m and V _max are 7.1 mM and 17.79 M min⁻¹, respectively.     

黑曲霉固态发酵及酶解玉米皮

以玉米提取淀粉后的玉米皮渣为主要原料,采用黑曲霉固态发酵法产酶再酶解的二步法降解玉米皮中纤维素类物质。经Plackett-Burman法及响应面设计优化发酵条件得:温度30℃,接种量10%,初始水分体积分数60%,物料厚度2.47 cm,初始pH 5.79,发酵时间6 d;滤纸比酶活可达11.01 U/g,较原始酶活提高了40.61%;产酶结束后加入pH 4.8醋酸-醋酸钠缓冲液,置于50℃下酶解144 h,中性洗涤纤维与酸性洗涤纤维降解率分别为46.09%、48.82%,还原糖质量分数达到9.02%。     

黑曲霉产菊粉酶菌株的选育及培养基优化

为获得高产菊粉酶的黑曲霉菌株,以Aspergillus niger YH-1为出发菌株,经过亚硝基胍(NTG)诱变,以高温高菊芋粉相结合的方式进行梯度驯化,选育出一株产菊粉酶菌株YH-3,并运用响应面实验方法对该菌株的培养基进行优化。确定了最佳培养基组成:菊芋粉25.2 g/L、豆饼粉40 g/L、蔗糖酯4.9 g/L、NaCl 5.5 g/L。发现内切菊粉酶活力(I)由60.9 U/mL提高到165.0 U/mL,比出发菌株提高了1.7倍。研究证明蔗糖酯对于黑曲霉YH-3发酵产菊粉酶是一种有效的促进剂。