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Journal of Bioscience and BioengineeringVOL. 119 No. 4, 421e426, 2015

Comparative study of fungal strains for thermostable inulinase production

Adriana C. Flores-Gallegos,1 Juan C. Contreras-Esquivel,1 Jesús A. Morlett-Chávez,2 Cristóbal N. Aguilar,1

and Raúl Rodríguez-Herrera1,*

Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Boulevard Venustiano Carranza and José Cárdenas s/n, República Oriente, Saltillo 25280,Coahuila, Mexico1 and Clinical and Molecular Diagnosis Department, Universidad Autónoma de Coahuila, Boulevard Venustiano Carranza and José Cárdenas s/n, República Oriente,

Saltillo 25280, Coahuila, Mexico2

Received 19 December 2013; accepted 23 September 2014Available online 6 November 2014

* Correspond4390511.

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1389-1723/$http://dx.doi

Fructose and fructo-oligosaccharides (FOS) are important ingredients in the food industry. Fructose is considered analternative sweetener to sucrose because it has higher sweetening capacity and increases iron absorption in children,and FOS’s are a source of dietary fiber with a bifidogenic effect. Both compounds can be obtained by enzymatic hy-drolysis of inulin. However, inulin presents limited solubility at room temperature, thus, fructose and FOS production iscarried out at 60�C. Therefore, there is a growing interest to isolate and characterize thermostable inulinases. The aim ofthis work was to evaluate the capacity of different fungal strains to produce potential thermostable inulinases. A total of27 fungal strains belonging to the genera Aspergillus, Penicillium, Rhizopus, Rhizomucor and Thermomyces were eval-uated for production of inulinase under submerged culture using Czapek Dox medium with inulin as a sole carbonsource. Strains were incubated at 37�C and 200 rpm for 96 h. Crude enzyme extract was obtained to evaluate inulinaseand invertase activity. In order to select the fungal strain with the highest thermostable inulinase production, a se-lection criterion was established. It was possible to determine the highest inulinase activity for Rhizopus microsporus13aIV (10.71 U/mL) at 36 h with an optimum temperature of inulinase of 70�C. After 6 h at 60�C, the enzyme did not showany significant loss of activity and retained about 87% activity, while it only retains 57% activity at 70�C. According tohydrolysis products, R. microsporus produced endo and exo-inulinase.

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[Key words: Inulin; Rhizopus microsporus; Exoinulinase; Endoinulinase; Thermal stability]

Inulin is a widespread, naturally occurring reserve of poly-fructan found in plants. It consists of linear b-2,1-linked poly-fructose chains, containing a terminal glucose unit. It can behydrolyzed by two types of enzymes: exo-inulinase (b-D-fructanfructanohydrolase, EC 3.2.1.80) which split-off the terminal b-(2,1)fructofuranosidic bonds yielding fructose, and endo-inulinase (2,1-b-D-fructan fructanohydrolase, EC 3.2.1.7), which hydrolyze theinternal linkages of inulin and release oligosaccharides (1). Theseoligosaccharides, called FOS, are made of 1e3 fructose units bondedto one molecule of sucrose, and they are classified as 1-kestose(GF2), nystose (GF3) and 1F fructofuranosyl nystose (GF4) (2).

FOS are widely used in many countries as food ingredients sincethey havemany health benefits such as promoting a good balance inthe intestinal flora, inducing proliferation of intestinal bifidobac-teria (probiotics). Currently, oligosaccharides have received GRASstatus (generally recognized as safe) by the FDA (Food and DrugAdministration) and have an approximated value of 200 US dollarper kg (3). Inulin has been proposed as a feedstock for production ofinulo-oligosaccharides (IOS) through action of endo-inulinases;however, inulin is insoluble in cold water and only slightly (5%)soluble in water at 55�C. Unfortunately, only a few inulinases

ing author. Tel.: þ52 844 4169213, þ52 844 4161238; fax: þ52 844

ress: raul.rodriguez@uadec.edu.mx (R. Rodríguez-Herrera).

e see front matter � 2014, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2014.09.020

required for industrial applications have an optimum temperatureof 50�C or higher (4e6). Therefore, the search for thermostableinulinase producers has received increasing attention.

Microbial inulinases have been reported from filamentous fungiAspergillus fumigatus, Aspergillus niger NK 126 (7), Penicillium sp.TN-88 (8), Rhizopus sp. TN 96 (9), yeast Kluyveromyces marxianus(10), Cryptococcus aureus G7a (11) and bacteria Streptomyces sp. (5),Bacillus sp. (12), Staphylococcus sp. (10). However, among thediverse microbial strains, K. marxianus and A. niger are reported asthe most common and preferred microorganisms for inulinaseproduction (13e15). The aim of this work was to evaluate the po-tential of twenty seven Mexican fungal strains to produce ther-mostable inulinases.

MATERIALS AND METHODS

Microorganisms Twenty one fungal strains isolated from plants and soil andbelonging to the Food Research Department-UAC collection (16) and sixthermotolerant and thermophilic strains from the Chemical EngineeringDepartment-CUCEI (17) culture collection were tested for inulinase production(Table 1). The strains were maintained on potato dextrose agar, while spores weremixed with a solution of skinned milk (9%) and glycerol (1%) and kept at �80�C.

Evaluation for inulinase production Screening experiments were carriedout in triplicate using a rotary shaker and a 250 mL Erlenmeyer flask with 30 mL ofculture medium, with the following composition (gL�1): inulin 10.0, NaNO3 7.65,KH2PO4 3.04, MgSO4 1.52, and KCl 1.52. The pH of culture medium was adjusted to5.0 before sterilization. Flasks were inoculated with a spore suspension containing107 spores mL�1 and incubated in a rotary shaker at 37�C and 200 rpm for 96 h.

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TABLE 1. Fungal strains tested for inulinase production.

Code Strain Collection

MesophiliAa20 Aspergillus niger UACGH1 Aspergillus niger UACPSH Aspergillus niger UACGS Aspergillus fumigatus UACESS Penicillium citrinum UACNH4 Aspergillus oryzae UACAN1 Aspergillus niger UACAN5 Aspergillus aculeatinus UACAN9 Aspergillus niger UACAN13 Aspergillus niger UACAN15 Aspergillus niger UACAN22 Aspergillus aculeatus UACAN45 Aspergillus niger UACAN49 Aspergillus homomorphus UACAN63 Aspergillus niger UACAN103 Aspergillus aculeatus UAC

Thermotolerant4a Rhizomucor pusillus UAC4b Rhizomucor pusillus UAC4c Rhizomucor pusillus UAC4d Rhizomucor pusillus UAC5a Aspergillus fumigatus UAC19aIV Rhizopus microsporus var. tuberosus CUCEI23aIV Rhizomucor pusillus CUCEI13aIV Rhizopus microsporus var. rhizopodiformis CUCEI43aIV Rhizopus microsporus var. chinensis CUCEI

ThermophilicT1.10 Thermomyces lanuginosus CUCEI5S-2 Thermomyces lanuginosus CUCEI

UAC, Universidad Autónoma de Coahuila; CUCEI, Centro Universitario de CienciasExactas e Ingenierías.

422 FLORES-GALLEGOS ET AL. J. BIOSCI. BIOENG.,

Samples for analysis were filtered using filter paper (Whatman no. 1) and thencentrifuged at 3000 �g for 5 min. Supernatants were used as crude enzymethroughout the experiments for enzyme assays.

Enzymatic assay Enzyme activities were assayed by measuring the con-centration of reducing sugars released from inulin and sucrose. The reactionmixturecontaining 0.1 mL of diluted crude enzyme and 0.1 mL of inulin or sucrose (1% w/v in0.1 M acetate buffer pH 5.0) was incubated for 15 min at 50�C. Reactionwas stoppedby boiling the sample for 5 min (18). Reducing sugars were subsequently analyzedby 3,5-dinitrosalicylic acid-DNS reagent (19). One unit of inulinase/invertase (U)was defined as the amount of enzyme which produced 1 mmol min�1 of fructose/glucose under the assay conditions mentioned above. The ratio between thesetwo activities was expressed as inulin/sucrose (I/S) (20).

Protein determination Protein content was determined as previously re-ported (21), using bovine serum albumin as a standard.

Statistical analysis All experiments were established under a randomizedblock design with three replications. The data were analyzed using an analyses ofvariance (ANOVA) procedure and when needed means comparison was performedusing the Tukey’s range procedure (p � 0.05) employing the SAS software version6.1.7600.

Effect of temperature and thermal stability The effect of temperature oninulinase activity was determined by incubating 0.1 mL of enzyme and 0.1 mL ofinulin (1% w/v in 0.1 M acetate buffer, pH 5.0) for 15 min at different temperatures.To determine the thermal stability, enzymes were incubated at different times(0e6 h). Residual activity was estimated under a standard assay conditions aftereach incubation level, and expressed as relative activity (%).

Determination of hydrolysis products In order to analyze the end productsfrom inulin hydrolysis, a mixture of 100 uL of enzyme and 100 uL of inulin (1%w/v in0.1 M acetate buffer pH 5.0) were incubated at 50�C. Samples were withdrawn atdifferent time intervals. Products were analyzed using thin layer chromatography(TLC) on silica gel 60 plates. Plates were developed at room temperature with asolvent system containing propanol: water: butanol (12:4:3, v/v/v) (22). Sugar spotswere revealed by spraying the plates with diphenylamine-aniline-phosphoric acidreagent and by heating them at 100�C for 5 min (23,24) . High purity 1-kestose,nystose and 1-fructofuranosylnystose (SigmaeAldrich) were used as standards foridentification of low molecular weight oligomers, as well as, fructose and glucose.

RESULTS AND DISCUSSION

Fungal production of inulinase Atotalof twentyseven fungalstrains were screened for inulinase production. Although inulin-

hydrolyzing activity has been reported fromvariousmicrobial strains,yeast (Kluyveromyces spp.) and (Aspergillus spp.) have showed thebestinulinase activity (14). However, in the present study it was observedthat thermotolerant and thermophilic strains belonging toRhizomucor, Rhizopus and Thermomyces genera showed higherinulinase activity than mesophilic strains belonging to Aspergillusand Penicillium (except for A. fumigatus). Inulinase activity rangedfrom a minimum of 1.52 U/mL (Penicillium citrinum ESS) to amaximum of 4.90 U/mL (A. fumigatus 5a) (Fig. 1A). A. fumigatus 5aalso showed high amounts of specific activity (1.37 mmol of fructosereleased per minute per milligram of protein), followed byRhizomucor pusillus 4a (1.24) and Thermomyces lanuginosus 5S-2(1.23) (Fig. 1B).

Moreover, studies by Fawsi (25) tested the capacity of differentfungal genera (Aspergillus, Clodobotryum, Curvularia, Fusarium,Mucor, Penicillium, Rhizomucor, Rhizopus, Talaromyces and Thielavia)for inulinase production finding that Thielavia terrestris NRRL 8126(thermophile), and Aspergillus foetidus NRRL 337 (mesophile) werethe best extracellular inulinase producers (8.42 and 8.36 U/mLrespectively), after being grown on a basal medium containingchicory root extract 2% (w/v) as a sole carbon source for six days.Nandogopal and Kumari (26) mentioned that Aspergillus niveus andPenicillium purpurogenum when grown on chicory root producedthe highest inulinase activity at levels of 7.0 and 9.0 U/mL respec-tively. Gern et al. (27) tested 16 fungal strains reported as endo-inulinase producers. The strain identified as A. niger DSM 2466 wasselected as the best endoinulinase producer. Ge and Zhang (28) alsoused an A. niger strain and obtained a maximum inulinase activityof 100 U/mL in the presence of S-770 sucrose ester as nutritivesubstrate added into the fermentative medium. Kumar et al. (29)obtained a maximum inulinase activity of 176 U/mL at a 5% (w/v)inulin concentration in the medium, using a soil isolate fungalstrain identified as A. niger.

As many microbial preparations of inulinase possess remarkableinvertase (S) activity accompanying the inulinase activity (IN), theircatalytic activity is described in terms of I/S or S/I ratios (30). When I/S ratio is higher than 10�2, the enzyme complex has a preponderantinulinase activity, while for invertase activity the I/S ratio is lowerthan 10�4 (31). In this study, I/S ratios varied from 0.57 to 2.49, thus,concluding that inulinase activity is dominant in all strains. How-ever, higher I/S ratios were observed for Aspergillus strains con-firming that they have a stronger preponderant inulinase activity(Fig. 1C). A range of I/S ratio between 0.02 and 7.9 for various mi-crobial inulinases has been previously reported by different authors(32). Ertan and Ekinci (33) also reported I/S ratios of 1.22 for Alter-naria alternata and A. niger, and 1.04 for Trichoderma harzanium.

With the aim to select the fungal strains with the highestpotential for inulinase production, a selection criterion was estab-lished. The response variables [specific activity (SA), inulinase ac-tivity (IN) and I/S ratio (IS)] were weighted according to theirimportance in the process [S ¼ 4.0 (SA) þ 2.0 (IN) þ 1.0 (IS)]. Thehighest value was given to the specific activity (U/mg protein) as anindicator of purity of the obtained enzyme, and the lowest value tothe preponderance of inulinase over invertase activity.

Through the use of ANOVA and later Tukey’s multiple rangetests for treatment means, it was possible to select A. fumigatus 5a,Rhizopus microsporus13aIV, Thermomyces lanoginosus T1.10 and5S-2 and encouraged further investigation (Fig. 2).

Kinetic evaluation of inulinase production The fourselected strains were evaluated for kinetic production of inulinaseusing the same medium mentioned above and incubating at 37�Cfor 96 h at 200 rpm. R. microsporus 13aIV showed a maximuminulinase activity of 10.71 U/mL at 36 h of fermentation whileA. fumigatus SOC-5A showed its maximal enzymatic activity(7.97 U/mL) at 60 h (Fig. 3). On the other hand, T. lanoginosus T1.10

FIG. 1. Inulinase production (A), specific activity (B) and I/S ratio (C) of 27 Mexican fungal strains belonging to Aspergillus, Penicillium, Rhizomucor, Rhizopus and Thermomyces.

VOL. 119, 2015 FUNGAL THERMOSTABLE INULINASE PRODUCTION 423

FIG. 2. Selection index for fungal strains with major potential to produce thermostable inulinase.

424 FLORES-GALLEGOS ET AL. J. BIOSCI. BIOENG.,

and 5S-2 showed its maximum inulinase activity at 72 and 60 h,respectively (6.28 and 7.23 U/mL). In all cases, after maximumactivity was reached, a gradual and steady decline of activityoccurred. A potential explanation for this may be due to secretionof proteolytic enzymes by the microbial strains; these enzymesare known to cause denaturation of inulinase (34). Time course ofinulinase production by A. niger NK-126 using dandelion extractshowed that maximum inulinase production (52.5 U/mL) at 96 h(7). Cruz et al. (35) reported that A. niger-245 reached themaximum inulinase activity (2 U/mL) at 48e60 h. Thus, thefastest inulinase production is generated by R. microsporus 13aIVis an advantage over other microorganisms already proposedtherefore making it a suitable property for industrial application.

Effect of temperature and thermal stability Inulinases withan optimum temperature higher than 50�C, is an extremelyimportant factor for commercial production of fructooligo-saccharides and fructose from inulin, since high temperaturesensure proper solubility of inulin and also prevent microbialcontamination (20). Likewise a high thermostability also bringsdown production cost of some industrially used enzymes,because lower amounts of enzyme are required to produce thedesired product. To determine the optimum temperature ofinulinases produced in the present study, enzyme reactions wereperformed at different temperatures (50e80�C) in acetate buffer(pH 5.0) using inulin as substrate. As shown in Table 2, theoptimum temperature of inulinase from R. microsporus was 70�C.This temperature is higher than that reported for inulinase fromdifferent fungal species, such as Fusarium oxysprum (13),Penicillium janczewskii (31) and A. niger (7) which produceinulinases with optimal temperatures between 30�C and 40�C.Inulinase preparation from Rhizopus sp. TN-96 has its optimalalso at 40�C (9). Other microorganisms such as Kluyveromycesspp. produce inulinases with maximum activity at 50e55�C(36,37) while inulinases derived from Pichia guilliermondii (15,38),Aspergillus ochraceus (39) and Streptomyces spp. (31) haveoptimum activity at 60�C.

For determining the thermostability, the enzyme solution washeated at 60e80�C for up to 6 h at pH 5.0. After treatment, theresidual activities were measured at 50�C (Fig. 4). Incubation ofinulinase at 60�C did not show any significant loss of activity and itretained about 87% activity after 6 h. At 70�C, it resulted in a gradualloss of activity but still retained the 80% of its activity after 2 h and57% after 6 h. Incubation of inulinase at 80�C for 4 h showed a re-sidual activity of 72%. However, prolonging incubation at 80�Cresulted in rapid loss of activity (61%). Thermal stability of inulinaseproduced by R. microsporus 13aIV at different temperatures isconsiderably higher than that reported for inulinases of other mi-croorganisms such as that from A. fumigatus which retained 77.3%of its original activity after incubation at 60�C for 3 h (4),K. marxianuswith a residual activity of 70% at 60�C for 140min (40),Fusarium oxysporum with 50 % of its original activity at 50�C after45 min (41), A. niger which is stable for 30 min at 60�C andAspergillus candidus for 60min at 55�C (42). Thus, it can be deducedthat the higher optimum temperature and greater thermostabilityof inulinase produced by R. microsporus 13aIV makes it a moresuitable and desirable strain for industrial and commercialapplications.

Determination of hydrolysis products To determine theexo- or endo-nature of the crude inulinase, TLC analysis was con-ducted using different sugars as standards. It was observed pro-duction of nystose, kestose, 1-fructofuranosyl nystose and otheroligosaccharides with higher polymerization degree. While at 96 h,was observed that fructose was the major sugar produced duringhydrolysis (Fig. 5). Therefore, it was concluded that crude enzymepreparation contained exo- and endo-inulinases. The release ofinulobiose (F2) and other oligosaccharides by Rhizopus TN-96 hasbeen reported by Ohta et al. (9) while fructose was confirmed asthe only reaction product by TLC for A. fumigatus (MTCC No.3009) inulinase (4).

In the present study, using a basal medium with inulin as solecarbon source, it was possible to identify R. microsporus 13aIV froma total of 27 fungal strains as the best inulinase producer. On the

FIG. 3. Kinetic evaluation for inulinase production (A), specific activity (B) and I/S ratio(C) for four fungal strains. Diamonds, Aspergillus fumigatus SOC-5A; squares, Thermo-myces lanuginosus T1.10; triangles, T. lanuginosus 5S-2; circles, R. microsporus 13aIV.

TABLE 2. Effect of different temperatures on inulinase activity ofR. microsporus.

Temperature (�C) Residual activity (%)

50 43.99 � 0.9660 89.18 � 3.2070 100 � 0.0280 73.71 � 0.88

FIG. 4. Thermostability of inulinase produced by R. microsporus in a range of 60e80�Cfor 6 h. Diamonds, 60�C; squares, 70�C; triangles, 80�C.

FIG. 5. Analysis of hydrolysis profile of inulin by inulinases produced fromR. microsporus 13aIV at different time intervals. G, glucose; F, fructose; S, sucrose; K,kestose; N, nystose; 1F, 1-fructofuranosyl nystose.

VOL. 119, 2015 FUNGAL THERMOSTABLE INULINASE PRODUCTION 425

other hand, according to the generated hydrolysis products, it canbe concluded that the enzyme preparation has endo and exo-actingactivity with its optimum activity at 70�C and with stability at 60�Cretaining 87% of its original activity after 6 h. Thermophilic en-zymes have gained a great interest as biocatalyst for application at

large scale, thus R. microsporus 13aIV could be a good candidate forfructose and FOS production at industrial levels. Further analysisconcerning characterization of the enzymes produced by thismicroorganism is needed.

426 FLORES-GALLEGOS ET AL. J. BIOSCI. BIOENG.,

ACKNOWLEDGMENTS

Adriana C. Flores-Gallegos wants to thank The National Councilfor Science and Technology (CONACYT) Mexico, for the financialsupport during her Ph. D. studies, grant number 322162.

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