Inulinase Producer Research Articles

Virulence, inulinolytic potency and biological control of fungi associated with banana leaf blight disease

A leaf blight disease is the widespread infection of bananas in tropical and subtropical regions. Early symptoms included irregular, tiny, necrotic lesions ranging in colour from pale grey to black-brown, mainly affecting the top three to five leaves. Necrotic and withering leaves indicated severe infection. Thirty-one fungal isolates were isolated from 30 samples of symptomatic banana leaves, collected from the field, on Potato Dextrose Agar (PDA) medium. The initial morphological identification of the isolates was confirmed using a molecular technique that involved amplifying the genes of β-tubulin for all collected isolates and cytochrome b for AspergillusNigri group. The pathogenicity test and re-isolation of the pathogen were done to confirm Koch's postulates. Light microscope and scanning electron microscopy were applied to notice the mycological infections in tested banana leaves. All collected isolates were evaluated to inulinase activity. Phytopathogenic fungi were biologically controlled by using four antagonistic fungal strains, Aspergillus flavipes, Phoma sp., Epicoccumpurpurascens and Verticilliumalbo-atrum. The results indicated that eighteen species represented by thirty-one isolates were recorded by phylogenetic analysis. Achroiostachys levigata (AEMC2 and 3) and Colletotrichum musae (AEMC16) exhibited pathogenic activity with highly virulent range (+++) and produced leaf lesions with 15–18.66 mm. Eighteen strains of banana leaf blight fungi were inulinase producers with variable proficiencies. The highest activity, 148.02667 U/ml was verified by Epicoccum nigrum (AEMC20). Phytopathogenic fungal growth was significantly suppressed by Epicoccum purpurascens and Aspergillus flavipes with maximum repression percentage of 50%. Therefore, biological control can be used to reduce the usage of chemical fungicides against leaf blight disease-caused fungi.

Aspergillus welwitschiae inulinase enzyme cocktails obtained on agro-material inducers for the purpose of fructooligosaccharides production

Production of fructooligosaccharides (FOS) is a trending topic due to their prebiotic effect becoming increasingly important for the modern human diet. The most suitable process for FOS production is the one using fungal inulinases. Introduction of new fungal inulinase producers and their implementation in production of inulinase enzymes is therefore gaining interest. This study provides a new approach to FOS synthesis by fungal enzyme complex without prior separation of any specific enzyme. Inulinase enzyme complexes could be used for the synthesis of FOS in two possible ways – hydrolysis of inulin (FOSh) and transfructosylation process of sucrose (FOSs), as demonstrated here. Depending on the fungal growth inducing substrate, a variety of inulinase enzyme complexes was obtained – one of which was most successful in production of FOSh and another one of FOSs. Substrates derived from crops: triticale, wheat bran, Jerusalem artichoke and Aspergillus welwitschiae isolate, previously proven as safe for use in food, were utilized for production of inulinase enzyme cocktails. The highest FOSs production was obtained by enzyme complex rich in β-fructofuranosidase, while the highest FOSh production was obtained by enzyme complex rich in endoinulinase. Both FOSh and FOSs showed antioxidant potential according to ABTS and ORAC, which classifies them as a suitable additive in functional food. Simultaneous zymographic detection of inulinase enzymes, which could contribute to expansion of the knowledge on fungal enzymes, was developed and applied here. It demonstrated the presence of different inulinase isoforms depending on fungal growth substrate. These findings, which rely on the innate ability of fungi to co-produce all inulinases from a cocktail, could be useful as a new, easy approach to FOS production by fungal enzymes without their separation and purification, contributing to cheaper and faster production processes.

Selection of Non-Mycotoxigenic Inulinase Producers in the Group of Black Aspergilli for Use in Food Processing.

Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production. Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production. The most common species were Aspergillus tubingensis (51.2%), followed by A. niger (23.1%), A. welwitschiae (23.1%) and A. uvarum (2.6%). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production. The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally.

Response Surface Methodology-Based optimization of Inulinase Production from New Bacillus Isolates

This study aimed to investigate new bacterial sources with the ability to produce inulinase and to optimize fermentation conditions for inulinase production. The inulinase production was carried out using newly isolated Bacillus licheniformis and Bacillus velezensis among identified sixteen bacterial strains. The four essential variables incubation time (1-3 days), temperature (25-40°C), pH (5-7), and Wheat Bran (WB) level (1-5%) determined by the Plackett–Burman design (PB) were selected for optimization studies with Box-Benhken Design (BBD). These parameters were found to be very effective on inulinase production. The maximum inulinase activities for B. licheniformis and B. velezensis were 401.18 EU/mL and 344.61 EU/mL. Both inulinases sustained 50% of their initial activity at 30°C for 9 days.. The results point out that bacteria are an important source as inulinase producer. The new isolates can be used in production of inulinase for industrial processes.

The power of two: An artificial microbial consortium for the conversion of inulin into Polyhydroxyalkanoates

One of the major issues for the microbial production of polyhydroxyalkanoates (PHA) is to secure renewable, non-food biomass feedstocks to feed the fermentation process. Inulin, a polydisperse fructan that accumulates as reserve polysaccharide in the roots of several low-requirement crops, has the potential to face this challenge.In this work, a “substrate facilitator” microbial consortium was designed to address PHA production using inulin as feedstock. A microbial collection of Bacillus species was screened for efficient inulinase producer and the genome of the selected strain, RHF15, identified as Bacillus gibsonii, was analysed unravelling its wide catabolic potential. RHF15 was co-cultured with Cupriavidus necator, an established PHA producer, lacking the ability to metabolize inulin. A Central Composite Rotary Design (CCRD) was applied to optimise PHA synthesis from inulin by the designed artificial microbial consortium, assessing the impact of species inoculum ratio and inulin and N-source concentrations. In the optimized conditions, a maximum of 1.9 g L−1 of Polyhydroxybutyrate (PHB), corresponding to ~80% (gpolymer/gCDW) polymer content was achieved. The investigated approach represents an effective process optimization method, potentially applicable to the production of PHA from other complex C- sources.

Molecular diversity and hydrolytic enzymes production abilities of soil bacteria.

Soil is an integral part of ecosystem which is niche for varieties of microflora. The present study was investigated to isolate varied strains of bacteria from soil samples of three different geographical regions of Tamil Nadu (India) and evaluate their hydrolytic enzymes (amylase, cellulase, and inulinase) producing potentialities. Among 72 bacterial cultures isolated from Ambattur Industrial Estate, Neyveli Lignite Corporation, and Arignar Anna Zoological Park regions, 41.66, 38.88, and 36.11% of isolates were observed amylase, cellulase, and inulinase producers, respectively. On the other hand, 20.83% of total bacteria isolated from all three regions exhibited concurrent production of amylase, cellulase, and inulinase. Potent isolates depicting maximum enzyme activities were identified as Bacillus anthracis strain ALA1, Bacillus cereus strain ALA3, Glutamicibacter arilaitensis strain ALA4, and Bacillus thuringiensis strain ALA5 based on molecular characterization tools. Further, the thermodynamics parameters, open reading frames (ORFs) regions, and guanine-cytosine (GC) content were determined by distinct bioinformatics tools using 16S rRNA sequences of strains. Minimum free energy values for strain ALA1, strain ALA3, strain ALA4, and strain ALA5 were calculated as −480.73, −478.76, −496.63, and −479.03 kcal/mol, respectively. Mountain plot and entropy predicted the hierarchical representation of RNA secondary structure. The GC content of sequence for strain ALA1, strain ALA3, strain ALA4, and strain ALA5 was calculated as 53.06, 52.94, 56.78, and 53.06%, respectively. Nine ORFs were obtained for strain ALA1, strain ALA3, and strain ALA5 while 10 ORFs were observed for strain ALA4. Additionally, bootstrap tree demonstrated close resemblance of strains with existing bacteria of similar genus. Findings showed higher variability of bacterial diversity as hydrolytic enzymes producers in the investigated geographical regions.

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.

Optimization of fermentation parameters for high-activity inulinase production and purification from Rhizopus oryzae by Plackett-Burman and Box-Behnken.

The aim of study was to optimize fermentation parameters for inulinase production from Rhizopus oryzae by a statistical approach and to carry out purification of inulinase. Five isolated fungal strains were screen out inulin degradation by using Lugol's iodine solution. R. oryzae exhibited maximum zone of clearance around the colony and was used as an inulinase producer. The effect of carbon sources (inulin, glucose, maltose, sucrose, lactose, onion peel, stevia root, wheat bran) as medium component and fermentation parameters (temperature (25-45°C), initial pH (4-7), time (3-7days)) on inulinase production was investigated by Plackett-Burman Design. Wheat Bran (WB), temperature, pH, and incubation time were found to be significant for the production of inulinase (P < 0.05). Furthermore, Box-Behnken Design was employed to optimize fermentation conditions. The maximum experimental results for inulinase activity and specific activity were 348.36EU/mL and 3621.78EU/mg, respectively. The results were obtained at 5days of incubation time, 35°C of incubation temperature, initial pH of 5.5, and 2% (w/v) WB. Also, inulinase was purified by using ammonium sulfate precipitation, gel filtration chromatography with 2.19-fold and its molecular weight was found as 89.12kDa. The optimal pH and temperature of the purified enzyme were 4.0 and 60°C, respectively. Furthermore, the purified enzyme showed excellent stability at 60°C. In conclusion, the present study offers cost-effective method to produce inulinase from Rhizopus oryzae. Also, it can be suggested that the purified inulinase has strong potential for usage in production of fructose syrup and other industrial areas.

Inulinase hyperproduction by Kluyveromyces marxianus through codon optimization, selection of the promoter, and high-cell-density fermentation for efficient inulin hydrolysis

This study aimed to overexpress an inulinase gene in Kluyveromyces marxianus to achieve the inulinase overproduction and preparation of ultra-high-fructose syrup. First, the inulinase gene (INU1 gene) was overexpressed through codon optimization and selection of a suitable promoter. Then, the inulinase was overproduced by high-cell-density fermentation. Finally, ultra-high-fructose syrup was prepared. It was found that optimization of the codons of the native INU1 gene encoding inulinase from Kluyveromyces marxianus KM-0 made a recombinant strain KM-N70 carrying the optimized INU1N gene produce 251.4 U/mL of the inulinase activity. Furthermore, inulinase activity produced by a recombinant KM-KN16 strain carrying the optimized INU1N gene directed by the native TPS1 promoter from K. marxianus KM-0 reached 338.5 U/mL and expression level of the optimized INU1N gene in the recombinant KM-KN16 strain was also greatly enhanced. During a 10-L fermentation, the recombinant KM-KN16 strain could produce 374.3 U/mL of inulinase activity within 24 h, while during a high-cell-density fed-batch fermentation, the recombinant KM-KN16 strain could produce 896.1 U/mL of inulinase activity and OD600nm value of its culture reached 108. The crude inulinase preparation obtained in this study had an inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation. It was found that 90.3% of 332.4 g/L of inulin was hydrolyzed to produce 41.0 g/L of glucose and 256.0 g/L of fructose and 91.1% of 328.2 g/L of inulin in the extract of the tubers of Jerusalem artichoke was hydrolyzed to produce 48.3 g/L of glucose and 250.5 g/L of fructose by the crude inulinase preparation (75 U/g of the substrate) within 8 h. The hydrolysates contained major monosaccharides and a trace amount of trisaccharides and the monosaccharides were composed of around 85% fructose and 15% glucose. So far, any other yeasts available have produced only up to 120 U/mL of inulinase activity. Together, this made the recombinant KM-KN16 strain be the best inulinase producer at this moment. The inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation and the ultra-high-fructose syrup with 41.0 g/L of glucose and 256.0 g/L of fructose were obtained. The inulinase activity obtained in this study was the highest among all the inulinase activities produced by yeast, fungal, and bacterial strains obtained so far.

Production, purification and characterization of Inulinase from fungus of dahlia rhizosphere

Inulinase and its studies are of immense importance in the present scenario. The use of microbial inulinases is suggested as a very potential approach, however, for obtaining inulinase in sufficient quantity from microbes, particularly fungii, optimization of the growth parameters is essential. The current study is focused over extraction of inulinase from fungal strains isolated from rhizospheric soil of dahlia plant and optimization of growth parameters. In a total of ten isolates, two organisms Fusarium oxysporum and Aspergillus sp. were identified as efficient inulinase producers. The optimum incubation time, temperature and pH of these organisms were found to be 40 min, 65°C at 6.5 pH and 30 min, 50°C at 6.5 pH respectively. Inulin was identified as appropriate carbon source for these microbes. Thin layer chromatography validated the presence of hydrolysis product via the action of inulinase on inulin into fructose unit. The highest enzyme activity and Specific activity was obtained from Aspergillus spp. for 20% fractionate was 1.81U/ml and 2.288U/mg, for 40% fractionate was 0.735U/ml and 1.047U/mg and for 60% fractionate was 3.74U/ml and 12.47U/mg respectively at the optimized condition. DEAE Sephadex A 50 Ion exchange column chromatography show that the fifth fraction of 20% fractionate had shown the maximum enzyme activity. The molecular weight of this enzyme was estimated at 66±2kDa and 43±2kDa using SDS-PAGE after ammonium sulphate precipitation, dialysis and chromatography analysis which showed exo-inulinase type.

Inulinase production from a new inulinase producer, Penicillium oxalicum BGPUP-4

Abstract The present study was carried out for the isolation and characterization of an efficient inulinase producing fungal strain. Out of 31 fungal isolates, only 16 strains showed the hydrolytic zone surrounding the fungal growth on an inulin-rich medium. Screening of 16 inulolytic fungal strains was carried out based on activity of enzyme on inulin and sucrose and their I/S ratio. Fungal isolate BGPUP-4 shown maximum inulinase activity (11.06 IU/mL) after 5 days of cultivation and the corresponding I/S ratio was 2.64. This I/S ratio confirmed the inulinase nature of the enzyme. Thin layer chromatography and densitometer scanning of the hydrolysate were obtained after hydrolysis of inulin by inulinases from 5 fungal strains having high enzyme activity. Micromorphological and macromorphological characteristics of the fungal isolate BGPUP-4 exhibiting maximum enzyme activity, revealed it to be Penicillium oxalicum . Furthermore, 18S rRNA gene sequencing and phylogenetic analysis confirmed the identification of the fungal strain. Out of various carbon sources tested, lactose (2%) in combination with inulin (1.5%, as an inducer) showed a stimulatory effect on inulinase production (19.14 IU/mL). Agitation mode of cultivation in shake-flask fermentations, supported maximum inulinase production (38.52 IU/mL) after 5 days of incubation. This is the first report on inulinase production from Penicillium oxalicum .

Biotechnological potential of microbial inulinases: Recent perspective

ABSTRACTAmong microbial enzymes, inulinases or fructo-furanosylhydrolases have received considerable attention in the past decade, and as a result, a variety of applications based on enzymatic hydrolysis of inulin have been documented. Inulinases are employed for generation of fructose and inulo-oligosaccharides (IOS) in a single-step reaction with specificity. The high fructose syrup can be biotransformed into value-added products such as ethanol, single cell protein, while IOS are indicated in nutraceutical industry as prebiotic. Myriad microorganisms produce inulinases, and a number of exo- and endo-inulinases have been characterized and expressed in heterologous hosts. Initially, predominated by Aspergilli, Penicillia, and some yeasts (Kluyveromyces spp.), the list of prominent inulinase producers has gradually expanded and now includes extremophilic prokaryotes and marine-derived microorganisms producing robust inulinases. The present paper summarizes important developments about microbial inulinases and their applications made in the last decade.

Direct conversion of chicory flour into l(+)-lactic acid by the highly effective inulinase producer Lactobacillus paracasei DSM 23505

Highly effective bio-process for lactic acid (LA) production by simultaneous saccharification and fermentation (SSF) of chicory flour was developed. The strain used, Lactobacillus paracasei DSM 23505 produced natural inulinase (EC 3.2.1.80) with molecular weight ∼130kDa, located in the cell wall fraction. In batch fermentation with optimized medium content and fermentation conditions, a complete conversion of 136g/L chicory flour (89.3% inulin and 10.7% mix of sucrose, fructose and glucose) into 123.7g/L LA was achieved. These yield and conversion rate are the highest obtained by SSF for LA production from inulin. The high efficiency, the cheap fermentation broth and the simple process performance disclose the promising use of the chicory flour in industrial biotechnology for LA production.

Production of inulinase from Rhizopusoryzae by solid state fermentation

Fifty Rhizopus spp. isolates were obtained from local natural habitat. The ability of inulinase production by these isolates was screened. The isolate RC-2 which isolated from composite plants was the highest inulinase producer on modified Czapek-Dox agar in primary screening. Secondary screening revealed that the same isolate was the highest production on Nakamura broth medium. Isolate was identified as Rhizopusoryzae. Measurements of reducing sugars in crude filtrate which represent the products of enzyme revealed that Rhizopusoryzae RC-2 produced inulinaseextracellular. The optimum culture conditions for inulinase production by solid state fermentation included a mixture of Jerusalem artichoke Helianthus tuberosus tuber with sugarcane Saccharum sp. bagasse with a ratio (1:1) as carbon source, 2.5% of corn steep liquor as nitrogen source, moisturizing ratio (3:1) (v:w) with tap water, the best inoculums rate was 2.5×105 spore/ml and incubation at (30)ºC for 4 days.

Gene encoding inulinase isolated from Penicillium citrinum ESS and its molecular phylogeny.

Inulinase is an enzyme produced by plants and several microorganisms, including fungi, to hydrolyze the β-2,1 glycosidic linkages present in some oligosaccharides to produce fructose and glucose. This enzyme, in conjunction with invertases, levanases, and two types of 1-fructosyl transferases have been described as members of the glycosyl hydrolases (family 32), the most diverse group of enzymes used by microbes for biomass degradation. As being part of the same clan, they have common evolutionary origin sharing the most important functional characteristics. Recently, a xerophylic fungi strain isolated from Mexican semi-desert, Penicillium citrinum ESS has been reported as inulinase producer, which could have greater stability than other enzymes due to a metabolic machinery adapted to typical temperature changes in this region. To continue the understanding of action mechanisms of these enzymes and to establish evolutionary relationships within this family, in the present study, phylogenetic analyses were used to analyze amino acid sequences coding fungal and yeast glycoside hydrolases of family 32, including the new sequenced inulinase of P. citrinum ESS. It was possible to elucidate the action mechanism of fungal glycoside hydrolases in present study and to classify inulinase from P. citrinum ESS as an exo-inulinase on the basis of their amino acid sequence phylogenetic affinities.

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.

Screening of novel yeast inulinases and further application to bioprocesses

Inulin is a carbohydrate composed of linear chains of β-2,1-linked D-fructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end. Jerusalem artichoke (JA) is one of the most interesting materials among unconventional and renewable raw materials, with levels of inulin reaching 50-80% of dry matter. Inulin or inulin-rich materials can be actively hydrolyzed by microbial inulinases to produce glucose and fructose syrups that can be used in bioprocesses. In this study, several microbial strains were isolated and their ability to inulinase biosynthesis was evaluated. The novel yeast strain Talf1, identified as Zygosaccharomyces bailii, was the best inulinase producer, attaining 8.67 U/ml of inulinase activity when JA juice was used as the inducer substrate. Z. bailii strain Talf1 and/or its enzymatic crude extract were further applied for bioethanol production and biodesulfurization (BDS) processes, using inulin and JA juice as carbon source. In a consolidated bioprocessing for ethanol production from 200 g/l inulin, Z. bailii strain Talf1 was able to produce 67 g/l of ethanol. This ethanol yield was improved in a simultaneous saccharification and fermentation (SSF) process, with the ethanologenic yeast Saccharomyces cerevisiae CCMI 885 and the Talf1 inulinases, achieving a production of 78 g/l ethanol. However, the highest ethanol yield (∼48%) was obtained in a SSF process from JA juice (∼130 g/l fermentable sugars), where the S. cerevisiae produced 63 g/l ethanol. Relatively to the dibenzothiophene BDS tests, the Gordonia alkanivorans strain 1B achieved a desulfurization rate of 4.8 μM/h within a SSF process using Talf1 inulinases and JA juice, highlighting the potential of JA as a less expensive alternative carbon source. These results showed the high potential of Z. bailii strain Talf1 inulinases as a versatile tool for bioprocesses using inulin-rich materials.

Production and Properties of Microbial Inulinases: Recent Advances

Microbial inulinases find application in enzymatic hydrolysis of inulin for production of fructose (sweetener) and inulooligosaccharides (prebiotics). Inulin is one of the most abundant nonstructural polysaccharides found widely dispersed in plants and awaits judicious utilization. Demands for an alternative healthy sweetener and multifunctional fructooligosaccharides have prompted investigators to explore microorganisms for inulinase production and to develop bioprocesses for production of high-fructose syrup and oligosaccharides based on enzymatic hydrolysis of raw inulin. Inulinases have been characterized in several molds, yeasts and a few bacteria. Recently, there has been a spurt of interest in finding novel inulinase producers, cloning and expression of inulinase gene in heterologous hosts and use of crude plant inulin and agro-industrial media in both submerged and solid state fermentation for inulinase production. The review discusses current knowledge on production, properties and applications of microbial inulinases and looks into the recent advances in the field.

Inulinase-expressing microorganisms and applications of inulinases

In this review article, inulinase-expressing microorganisms and its potential applications in transformation of inulin into very-high-fructose syrup, bioethanol, and inulooligosaccharides are overviewed. In the past 10 years, many new inulinase producers have been obtained and many genes encoding inulinases from different microorganisms have been cloned and characterized. Some novel processes for exoinulinase overproduction have been developed for bioethanol production and ultra-high-fructose syrup. The endoinulinases have also been used for production of inulooligosaccharides from inulin and inulin-containing materials.

Inulinase-hyperproducing strains of Kluyveromyces sp. isolated from aguamiel (Agave sap) and pulque

Two strains of Kluyveromycesmarxianus (A1 and A2) isolated from ‘aguamiel’ (agave sap) and one strain of K. lactis var. lactis (P7) isolated from ‘pulque’ (its fermented product), were studied to make a survey of inulinase production. The strains of K. marxianus A1 and A2 were the best producers of inulinase, giving up to 2.5 times more enzyme than the control hyperproducing strain K. marxianus CDBB-L-278, and showed lower catabolic repression than this. One strain isolated from pulque was identified as K. lactis var. lactis and was also an excellent inulinase producer, being the first strain of this species reported as such. These strains were very good inulinase producers and they had low susceptibility to catabolic repression probably because the source from which they were isolated was rich in sucrose and oligofructans. They can be used in the transformation of inulin to produce fructose and/or oligofructans.