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PROJECT SEMESTER REPORT B.

Tech Biotechnology (January-June, 2011)

ISOLATION AND SCREENING OF INULINASE PRODUCING F UNGI FROM RHIZOSPHERE SOIL OF INULIN CONTAINING PLANTS

Submitted by Danish goyal Roll No. 700900036 Under the Guidance of Dr.Sumit jain

Department of Biotechnology & Environmental Sciences Thapar University, Patiala June, 2011

TABLE OF CONTENTS S.NO CHAPTERS ABSTAC T 1. 2. 2.1 2.1.1 2.1.2 2.1.3 2.2 2.2.1 2.2.2 2.2.3 INTRODUCTION REVIEW OF LITREATURE Inulinase-expressing microorganisms Moulds Yeast Bacteria Characteristics of inulinases Molecular Weight of inulinases Optimum pH and temperature activity of inulinases Effect of metals ions and protein inhibitors on inulinase activity Applications of inulinases High fructose syrup Inulo-oligosaccharide production Bioethanol production Other applications of inulinases MATERIALS AND METHODS Materials used Collections of samples Isolation of inulinase producing fungi Purification and maintaince of fungal isolates Characterization and Identification of fungal isolates PAGE NO.

2.3 2.3.1 2.3.2 2.3.3 2.3.4 3. 3.1 3.2 3.3 3.4 3.5

3.6 3.6.1 3.6.2 3.6.2.1 3.6.2.2 3.6.2.3 3.6.2.4 3.7

Screening and enzymatic estimation Enzyme production media used Enzymatic assay Reagents required Substrates Stopping reagent (DNS reagent) Standard stock solution Effect of temperature, pH and metal ions on inulinase activity by potential producer A.niger G6

3.7.1 3.7.2 4. 4.1 4.2

Effect of pH and temperature Effect of metal ions and detergents RESULTS AND DISSCUSSIONS Isolation and identification of fungi Screening and estimation of fungal isolates for inulinase activity

4.3

Effect of temperature, PH and metal ions on inulinase activity by potential producer A.niger G6

4.3.1 4.3.2 4.3.3 5. 6.

Effect of temperature Effect of pH Effect of metal ions and detergents SUMMARY AND CONCLUSIONS REFERENCES APPENDIX

TABLES S.NO 2.1 2.2 2.3 2.4 4.1 4.2 TABLES Substrates inulinase production used for 6 Inulin contents of some plants 8 Inulinase expressing microorganisms Properties of some microbial inulinase Distribution of samples in different habitats Fungal isolates with their morphological characteristics Fungi isolated from rhizospheric soil Inulinase activity of culture filtrates PAGE NO.

4.3 4.4

S. NO 2.1 2.2 Structure of inulin

LIST OF FIGURES

PAGE NO

Inulin being acted upon by microbial exo- and endo-inulinase enzymes. Standard curve of fructose Culture characteristics of selected fungi on SDA medium Effect of temperature on inulinase activity 34 Effect of pH on inulinase activity 35 Effect of metal ions on inulinase activity 36

3.1 4.1 4.2 4.3 4.4

ABSTRACT Inulin is present as a reserve carbohydrate in the roots and tubers of plants such as the dahlia, onion and garlic. Inulin has attracted considerable research attention because it is an abundant substrate for the production of fructose-rich syrups, as well as a source for the production of fructo-oligosaccharides (FOS) and inulooligosaccharides (IOS), FOS can be produced from inulin by microbial enzymes having hydrolytic and transiructosylatiiig activity. The aim of this work was to isolate inulinase producing moulds, identify their genera, the isolates were screened for high inulinase production through the enzyme activity procedure, and to test the characteristics of inulinase based on effect of temperature, pH and metal ions. A total of 18 strains of inulinase producing fungi were isolated from rhizosphere soils of inulin rich plants (onion, garlic and dahlia). The Aspergillus niger strains showed maximum inulinase activity as compared to other strains. Maximum inulinase activity by A.niger G6 was obtained as 220.36nkats/ml. The optimum pH and temperature for A.niger G6 inulin was found to be 5.0 and 60C respectively. The enzyme was inhibited by 10% T20 and SDS and activated by the presence of 1 mM Cu2+ and Mn2+. Results suggest that the awragus root powder induced exoinulinase synthesis in Aspergillus niger G6 and can be utrhded as a potential substrate for inulinase production.

Chapter 1 Introduction

Introduction l. INTRODUCTICN Inulin is a linear (0-2. l) linked fructose polymer that occur as a reserve carbohydrate in many plant families such as dahlia tubers, chicory roots, onion and garlic. Inulinase has received much more attention recently as it can be widely applied to hydrolyze inulin for the production of fuel ethanol, fructose and fructooligosaccahride, both of which are important ingredients in food and pharmaceutical industry. Such inulin has recently received a great interest as it represents a relatively inexpensive and abundant substrate for the production of high fructose syrup, for example, fructose syrup has beneficial effects in diabetic patients, increase the iron absorption in children, has high sweetening capacity so it can used in the diet of obese persons, stimulates growth of Bifidobacteria in large and small intestine, prevents colon cancer and is used as dietary fibers because of its fat like texture. (kumar et al., 2011). Chemically, inulin mainly consists of linear chains of [3-(2->1)-D-fructosyl-fructose links terminated by a sucrose residue (De Leenheer, 1996). Major sources of inulin for industrial scale production are chicory, Jerusalem artichoke (topinambur), and dahlia. The inulin %ent differs between the plant species. The world production of inulin is currently estimagto be about 350,000 tons. Main producers are Belgium, France, the Netherlands, and Chicory (Cichotium intybus) is a temperate climate biennial root crop. Crop requiregmts, harvesting, and processing are similar to sugar beet production. Jerusalem 3ItiChOHCli3HthUS tuberosus) is a perennial tuberous plant. The yield is higher if harvested annually dahlia is a tuberous plant mainly cultivated for its flowers. Dahlia tubers are used as inulgaource but the content is lower than that of chicory (Franck and De Leenheer, 2002; Peters, 2007).

Introduction

Many inulin sources are being used as a renewable raw material in the production of inulinase. ie. ethanol, acetone, butanol, pullulan, gluconic acid, sorbitol, inulooligosaccharides and ultra-high-fructose syrup in pharmaceutical industries (Erdal et al., 2011). Microbial inulinases, the enzymes that hydrolyze inulin, have been proposed as the most promising approach to obtain fructose syrups from inulin rich feedstock. Although inulin-hydrolyzing activity has been reported from various microbial strains, yeast (Kluyveromyces spp.) and Aspergillus spp, have proved the best inulinase activity (Singh and Gill, 2006). Other inulinase producing microorganisms are Penicillium spp, Alternaria alternata, Rhizopus spp, and Bacillus spp, Clostridium spp, and Xanthomonas spp. (Singh and Gill, 2006). In the last decades a large number of fungal, yeast and bacterial strains were used for inulinase production. Among the various microbial strains Kluyveromyces marxianus and Aspergillus niger sources for inulinase production (Singh and Gill, 2006; Pandey et al., 1999; Chi et al., 2009). Inulinases have different catalytic properties, (molecular weight, optimum pH, optimum temperature, stability), depending especially upon their provenience. Generally, the inulinase activity (I) is accompanied by invertase activity (S) and the enz c complex is characterized by I/S ratio. When I/S ratio is higher than 10-2, the enzyme complex has a preponderate inulinase activity, while for invertase activity the I/S ratio lower than 104 (Sharma et al., 2006). Inulinases can be used in a wide range of industry applications: for ultra-high fructose syrup obtaining from inulin, bioethanol productio, inulooligosaccharide production, single-cell oil and single-cell protein production, some chemicals production, like citric acid, butanediol, alcohols and lactic acid (Chi et al., 2011; Chi et al., 2009; Pandey et al., 1999; Liu et al., 2010).

Introduction

Agro-industrial residues and vegeatable extracts appear to be a good source for inulinase production. Cassava flour, comcob, oat meal, rice straw, sugar cane bagasse, wheat bran, glucose and sucrose were used as carbon sources to establish the influence of carbon source on the production of inulinase by Aspergillus ochraceus (Guimaraes et al., 2007). The highest level of extracellular inulinase activity was obtained when sugar cane was used as carbon source (108 activity units). A. ochraceus inulinase activity was stimulated by the supplementation with glucose of the reaction medium (Guimaraes et al., 2007). Sharma et al. (2006) used also various substrates for inulinase production (rye, barley, banana, garlic, pure inulin, wheat, chicory, onion and dahlia). The highest inulinase activity was observed when garlic was used as carbon source. The major objective of this study was to isolate, identify and screen the inulinase producing fungi. However the overall study was conducted as follows: isolation of fungi from rhizospheric soil of inulin containing plants. Characterization and identification of recovered f\1ngi. Screening and estimation of inulinase from fungal isolates. Effect of temperature, pH, and metal ions on inulinase activity of potential producer.

Chapter 2
Review of literature

Review of Literature

2. REVIEW OF LITERATURE Inulin is present as a reserve carbohydrate in the roots and tubers of plants such as the Jerusalem artichoke, chicory, dahlia and in small amounts in garlic and onion. It consists of linear chains of [3-2, l-linked D-tructofuranose molecules terminated by a glucose (through a sucrose-type) linkage at the reducing end (Chi et al., 2009). (Fig 2.1)

Fig 3.1-structure of inulin (Vandamme and Derycke 1983)

Review of Literature

Inulin has attracted considerable research attention because it is an abundant substrate for the production of fructose-rich symps, as well as a source for the production of fructooligosaccharides (FOS) and inulooligosaccharides (IOS), both are low caloric saccharides, which plays an important role as a growth factor for beneficial microorganisms such as Bifidobacteria in large and small intestinal flora (Skowronek and Firedurek, 2004; Yuan et al., 2006). FOS can be produced from inulin by microbial enzymes having hydrolytic and transti'uctosylating activity. The two types of inulinase and invertase are hydrolytic enzymes. Inulinases have been produced using different substrates such as carbon sources, from pure inulin containing plant materials to agro-industrial residues, some of which are shown in (Table 2.1). Naturally occurring inulin rich materials are the preferred substrates that are used by researchers for inulinase obtaining but lately, agroindustrial residues have gained scientists attention. In nature, inulin can be found in many plant species from monoand dicotyledonous families, such are Liliaceae, Amaryllidaceae, Gramineae and Compositae (Chi et al., 2011). Excepting Gramineae plants, inulins are usually stored in bulbs, tubers and roots. Jerusalem artichoke and chicory, which belong to Compositae family, are the most commog used carbon sources used by researchers for inulinase production as they contain over 50% (dry matter) inulin (Chi et al., 2011, Pandey et al., 1999, Danilcenko et al., 2008)

Review of Literature

Table

Review of Literature

end endo-inulinase (2, l-B-fmctan fructanohydrolase, EC 3.2.1.7) hydrolyzes the internal linkages in inulin to yield inulo-oligosaccharides (Fernandez et al., 2004; Skowronek and Firedurek , 2004; Chi er al., 2009), and invertase breaks down sucrose to fructose and glucose

by catalyzing the hydrolysis of terminal non-reducing [3-fructofuranoside residues in |3tructofuranosides. While [3-b fructofuranosidase (bFFase) is a transfructosylatiiig enzyme, it can transfer the fiuctosyl residue to the sucrose molecule at a high concentration of sucrose, in which tructosyl residues are transferred to sucrose by B-2,1 glycosidic bonds (Rubio and Navarro, 2006; Kurakake et al., 2010). Most of these enzymes have been found in molds such as Aspergillus spp., Fusarium spp. and Aureobasidium spp. Microbes are known as the best source for commercial production of enzymes because of their easy cultivation and high yield of the enzymes (Siiisansaneeyakul et al., 2007a; Chi et al., 2009; Songpim et al., 2011). Since many enzymes of industrial significance are regulated by the composition of the medium, ge study of this regulation is important in the commercial production of such enzymes. the degree of polymerization ui mulm (MW 60,000) of plant origin IS < 200 and varies a<%`ding to plant species, weather conditions and age (Molina et al., 2005; Chi et al., 20% Some well known plant sources of inulin are listed in (Table 2.2).

Review of Literature

Table 3.1: Inulin content of some plants Modified from Van Loo et al.,(1995).

SOURCES

Plant part

Inulin content (% of fresh weight)

Onion Jerusalem artichoke Dahlia Chicory Leek Garlic Artichoke Banana Rye Barley Dandelion Burdock Camas Murnong Yacon Salsify

Bulb Tuber Tuber Root Bulb Bulb Leaves-heart Fruit Cereal Cereal Leaves Root Bulb Root Root Root

2-6 14-19 9-12.5 15-20 3-10 9-16 3-10 0.3-0.7 0.5-1 0.5-1.5 12-15 3.5-4.0 12-22 8-13 3-19 4-11

Review of Literature

lnulinases are fructofuranosyl hydrolases produced by a wide range of organisms including plants, bacteria, molds and yeasts. The general reaction mainly involves action of

two enzymes: (i) Exoinulinase (E.C. 3.8.l.80) which splits the terminal fructose units from inulin and (ii) Endoinulinase inulooligosaccharides (E.C. 3.2.1 .7) that breaks down inulin into

(IOS). Former can be used for production of high fructose syrup from natural inulins (saccharification) while the latter are used for producing inulooligosaccharides of varying lengths (Figure 2.2).
, 2-1 fructosyl linkage F G Inulin

Exo-inulinase

Endo-inulinase

Hi-fructose syrup

Inulo-oligosaccharides

Low calorie sweetener

Neutraceutical (Dietary fiber)

Fermentable substrate

Functional food ingredient

Figure 2.2: Inulin being acted upon by microbial exo- and endo-inulinase enzymes. Action of exoinulinase liberates fructose from the macromolecule while endoinulinase produces inulooligosaccharides. F- Fructose, G- Glucose. Review of Literature

2.1 Inulinase - expressing microorganisms A huge number of fnmgi, bacteria, and yeasts have been used for inulinase production

(Table 2.3). Among them, the strains belong to Aspergillus and Kluyveromyces genus were the most common and preferred choice for inulinase production. Table 2.3 Inulinase expressing microorganisms

Microorganisms

Maximal activities

References

Moulds 1.75 g/L Aspergillus niger 100 Ua/mL Aspergillus niger 52.5 IUa/mL Aspergillus niger 176 U/mL Aspergillus niger Aspergillus fumigatus Not available Aspergillus awamori Not available Aspergillus ochraceus 108 Total U Aspergillus ficuum 193.6 U/gdsb Aspergillus parasiticus 2.9 U/mL Geotrichum candidum 45.65 IU/mL Rhizoctonia solani 1.792 U/mL Chrysosporium pannorum 115 U/mL Bacteria 2.48 g/L Paenibacillus spp. 524 IU/L Streptomyces spp. 89 U/gds Streptomyces spp. Bacillus spp. 42.36 U/mL Pseudomonas spp. Not available Arthrobacter spp. Not available Yeasts Pichia guilliermondii 39.56 U/mL Pichia guilliermondii 61.5 U/mL Pichia guilliermondii 130.38 U/mL Pichia guilliermondii 60.1 U/mL Cryptococcus aureus 52.37 U/mL Cryptococcus aureus 85 U/mL Cryptococcus aureus 436.2 U/gds

Gern et al., 2001 Ge and Zhag, 2005 Kango, 2008 Kumar et al., 2005 Gill et al., 2006 Nagem et al., 2004 Guimaraes et al., 2007 Chen et al., 2011 Ertan et al., 2003b Mughal et al., 2009 Ertan et al., 2003a Xiao et al., 1988 Gern et al., 2001 Sharma et al., 2006 Dilipkumar et al., 2011 Zherebtsov et al., 2002 Kim et al., 1997 Kang et al., 1998 Gao et al., 2007 Chi et al., 2009 Yu et al., 2009 Gong et al., 2007 Gao et al., 2007 Chi et al., 2009 Chi et al., 2009

Yarrowia lipolitica Yarrowia lipolitica

Debaryomyces hansenii Candida kefyr Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces marxianus

62.85 U/mL 22.5 U/mg 52.53 U/mL 40 U/mL 194.1 U/mL 127 U/mL 176 IU/mL 208 IU/mL 262.9 U/mg 1294 U/mL 18743 U/mL 50.2 IU/mL 47.1 IU/mL 250 U/gds 47.2 U/mL 1317 U/mL 1139 U/mL

Gao et al., 2007 Liu et al., 2010 Gao et al., 2007 Pessoa and Vitolo, 1998 Kalil et al., 2010 Kalil et al., 2001 Santisteban et al., 2005 Santisteban et al., 2009 Golunski et al., 2011 Treichel et al., 2009 Kushi et al., 2000 Singh and Bhermi, 2008 Singh et al., 2006 Mazutti et al., 2007 Mazutti et al., 2010 Treichel et al., 2009 Sguarezi et al., 2009

2.1.1 .Moulds Inulinase activity has been obtained using different mould strains, out of which Aspergillus spp_ being the favourite specie for inulinase production. As per results, sixteen ftmgal strains and three bacterial strains, reported as inulinase producers, were investigated by Gem et al. (2001) for endo-inulinase production. The best endo-inulinase producer was the strain coded CDB 003, identified as Paenibacillus spp. From the fungal strains, Aspergillus niger DSM 2466 was selected as the best endo-inulinase producer by Gem et al. (2001). Ge and Zhang (2005) used also an Aspergillus niger strain and obtained a maximum inulinase activity of 100 U/mL in the presence of S-770 sucrose ester as nutritive substrate added into the fermentative medium at a concentration of 6 g/L. Using an infusion prepared of tap roots of dandelion, Kango (2008) obtained 52.5 IU/mL inulinase activity after 96h of cultivation with A.niger which was a selected strain. (Kumar et al. 2005) obtained a maximum inulinase activity of 176 U/mL at a 5% (W/v) inulin concentration in the medium, using a fungal strain isolated from soil and identified as A. niger .Other strains of Aspergillus species were reported in literature for inulinase production and characterization, such are A. fumigatus (Gill et al., 2006), A. awamori (Nagem et al., 2004), 3106), A. awamori (Nagem et al., 2004), A. ochraceus (Guimaraes et al., 2007), A. ficuum gen et al., 2011) and A. parasiticus (Ertan et al., 2003b). In the research of Mughal . (2009), soil samples of eight strains of Geotrichum candidum were isolated and itested fcliulinase activity. The activities of these strains ranged hom 0.12 to 1.38 IU/mL. After improvement through induced mutagenesis using methyl methane sulphonate, ethyl methane sulphonate and UV exposure, culture gave a 50-fold improved inulinase activity (45.65 IU/mL) compared to wild isolate (Mughal et al., 2009). Rhizoctonia salani, strain

isolated from soil, had the maximum inulinase activity of 1.792 U/mL in the second day of cultivation using Jerusalem artichoke powder as carbon source (Extan et al., 2003a). 2.1.2 Yeasts Yeasts have been used in enzyme production for years, as they are easier to grow and handle in comparison with bacteria. Among the yeasts which are able to produce inulinases, Kluyveromyces spp., Pichia spp. and Candida spp, shows a huge potential for producing high yields of inulinase activity. Gao et al. (2007) screened over 400 marine yeasts and found that some of his isolated marine yeasts strains have the ability to secrete large quantities of inulinase. Among them, Pichia guilliermandii, C13/ptocaccus aureus, Yarrowia Iipalitica and Debarjyomyces hansenii can secret over 40 U/mL of extracellular inulinase (Gao et al., 2007, Chi et al., 2009, Gong et al., 2008, Gong et al., 2007, Sheng et al., 2008). Using mutagenesis, ultraviolet exposure combined with LiC1 treatment of the cells of Pichia guilliermondii, Yu et al. (2009) obtained a maximal inulinase activity of 130.38 U/mL in the medium optimization studies. Surface-engineered Yarrowia Iipolitica yeast can produce 22.5 U/mg inulinase activities within 96 h (Liu et al., 2010). Kluyveromyces spp. is, by far, the most widely used yeast for inulinase production. (Kalil et al. 2010) obtained a maximum inulina tivity of 194.1 U/mL when investigated the main parameters affecting the inulinase production obtained from Kluyveromyces marxianus var. bulgaricus, using an ion exchangxed bed column. In his hirious study, the maximum inulinase activity obtained was 127 U/mL in the optimized medium for the optimization of inulinase production (Kalil et al., 2001). Santist& et al.(2005) investigated during his work about some key factors in inulinase production (agitation, aeration and shear stress) by Kluyveromyces marxianus and found the

best fermentation conditions of 1 vvm aeration, 450 rpm agitation, with pitched blade up impeller in a stirred reactor, with a production of 176 TU/mL. Santisteban er al. (2009) investigated the effects of carbon and nitrogen sources and oxygenation on the inulinase production in his later work and 208 IU/mL inulinase activity was obtained when 20 g/L sucrose was used as carbon source. In their trials for ethanol ultra filtration and precipitation of inulinases from Kluyveromyces marxianus, Golunski et al. (2011) obtained a maximum inulinase specific activity of 262.9 U/mg. In their studies on the production, partial purification and characterization of inulinase from K. marxianus (Treichel et al. 2009) obtained a maximum inulinase activity of 1294 U/ml, using agro-industrial residues as substrate, while from K. marxianus Kushi et al. (2000) obtained 18743 U/mL after dialysis and lyophilisation of extracellular inulinase. Singh and Bhermi (2008) and Singh et al. (2006) concerning their study on inulinase production using roots of Aspergillus spp. and Kluyveromyces marxianus obtained 50.2 IU/mL and 47.1 IU/mL respectively, showing that inulinase yield is six times higher when produced in bioreactor as compare to shake flask trials. Kluyveromyces marxianus was used in many optimization studies for inulinase production, when different yields of inulinase activity were obtained. Mazutti et al. (2007) obtained a maximum inulinase activity of 250 U/gds isolid-state fermentation of sugar cane bagasse and 47.2 U/mL in submerged liquid fermentation (Mmm 61 az., 2010), Bender et az. (2006) obtained 444.8 U/g inulinase activity from agro-industrial residues using solid-state fermentation, (Treichel et al. 2009) obtained 1317 using agro-industrial residues as substrate using agro-industrial residues as substrate.

2.1.3 Bacteria Bacterial strains are also used for the production of inulinase, mainly because of their thermostability. Bacterial strains mainly concem biosynthesis of endo-inulinases. Streptomyces spp. was found good producer of inulinases; using garlic as substrate, Sharma et al. (2006) obtained 524 IU/L inulinase activity with Streptomyces spp. and Dilipkumar et al. (2011) had a maximrun inulinase activity of 89 U/gds by Streptomyces spp, using pressmud as carbon source. Bacteria of genus Bacillus are also active producers of extracellular inulinase 42.36 U/mL inulinase activities on sucrose as substrate (Zherebtsov et al., 2002). Pseudomonas spp. and Arthrobacter spp. were also tested for the ability to produce inulinases; data concerning their inulinase activities are not available. (Kim et al., 1997, Kang et al., 1998). 2.2 Characteristics of inulinases 2.2.1 Molecular weight of inulinases It was reported form previous studies that inulinases produce from microbes have over 50kDa molecular weight (Chi et al., 2009). For example, Cryptococcus aureus purified and characterized from inulinase and its molecular weight was estimated to 60.0 kDa (Sheng et al. 2008). For Pichia guilliermondii derived inulinases, molecular weight of 50 kDa was reported by Gong et al. (2008) and 54 kDa by (Chi et al. 2009). Inulinases with molecular weight of 250 kDa were produced by Kluyveromyces fragilis (Pandey et al., 1999). Bacterial strains had similar molecular weight as yeasts for production of inulinases. (Kang et al. 1998) charecterized the endoinulinases produced by Arthrobacter spp. and estimated its molecular weight at 75 kDa. Inulinases produced by moulds strains molecular weights between 50 kDa and 200 kDa. A. ochraceus - 79 kDa (Guimaraes et al., 2007), Penicillium spp. - 68 kDa (Qt al., 2009), F.oxysporum - 300 kDa (Pandey et al., 1999).

Source

K. marxianus K. marxianus K. marxianus K. fragilis Yarrowia lipolitica Pichia guilliermondi i Cryptococcus aureus Arthrobacter spp. Bacillus spp. Streptomyce s spp. A. niger A. niger Penicillium janczewskii A. ochraceus F. oxysporum

Molecula r weight kDa 250 50

optimu m ph 3.5 4.4 4.75 4.5 6

optimum temperatur e (c) 60 50 55 55 50 60

ph stabilit y range 03-Jul 06-Jul

temperatur e stability range (c) 40 50 60

References Mazutti et al., 2007 Mazutti et al., 2010 Kushi, 2000 Pandey et al., 1999 Liu et al., 2010 Gong et al., 2008

60 75 70 79 300

5 7.5 7 6 4.4 5 4.8-5.0 4.5 5.8-6.2

50 50 60 40 3545 60 3037

4.0-6.5 5-10.5 06-Aug -

65 3040 2540 6070 60 -

Sheng et al., 2008 Kang et al., 1998 Zherebtsov, 2002 Sharma et al., 2006 Sharma et al., 2006 Pandey et al., 1999 Sharma et al., 2006 Guimaraes et al., 2007 Pandey et al., 1999

2.2.2 Optimum pH and temperature activity of inulinases From moulds and yeasts the optimum pH activity of inulinases varies, in general, in the range of 4.5-6.0 (Table 2.4). A. niger were found to have an optimum pH at 4.4 for the production of inulinases, inulinases from A. versicolor have the optimal pH at 5.5 and Penicillium janczewskii produces inulinases with optimal pH at 4.8-5.0 (Sharma et al., 2006). Inulinases produced by A. ochraceus are optimal at pH 4.5 (Guimaraes et al., 2007). There are also few inulinases with lower optimal pH: K. marxianus produces inulinases which have the highest activity at 3.5 (Mazutti et al., 2007) and Pichia guilliermondii at 3.4 (Gao et al., 2007). In contrast, the inulinases produced by some bacterial strains hydrolyze inulin at the optimrun pH of 7.0-7.5, like Arthrobacter spp. (Kang et al., 1998) and Bacillus polymyxa (Zherebtsov et al., 2002, Chi et al., 2009). Usually, the purified inulinases are optimal activity at the temperature range of 50-60C. Kluyveromyces spp. strains produce inulinases with maximum activity at 50-55C (Mazutti et al., 2010, Pandey et al., 1999, Kushi, 2000), while inulinase derived from Pichia guilliermondii (Gong et al., 2008, Chi et al., 2009), A. ochraceds (Guimaraes et al., 2007) and Streptomyces spp. (Sharma et al., 2006) have optimum activity at 60C. F . oxysporum (Pandey et al., 1999), Penicillium janczewskii (Sharmalgaal, 2006) and A. niger (Pandey et al., 1999) produce inulinases optimal at 30~ 40 C. lndicating that the optimal temperatures of inulinases tram different species of microorganisms are significantly different.

2.2.3 Effect of metal ions and protein inhibitors on inulinase activity Some metal ions added to the reaction medium can enhance the inulinase activity, while others have inhibitory effect on the enzyme. Inulinase activity of Kluyveromyces marxianus is promoted by Co+2, , Mg" and low concentrations of SDS (0.00l%) and is inhibited by Cu+2, Fe+3, Zn+2, Tween 20, Tween so and Brij-35 (Singh and Bnenni, zoos) _ and also by Ca+2, Ba+2, Zn+2, Na+ (Kushi, 2000). Inulinase derived from Cryptococcus aureus is activated by low concentrations of Ca+2, K+, Na+, Zn and Cu+2 and inhibited by Mg+2, Hg+2 and Ag+ (Sheng et al., 2008). Similar results were observed for Pichia guilliermondii inulinase also (Gong et al., 2008). Both yeast strains were isolated from marine enviromnent. Hg+2 and Ag+ have inhibitory effect on inulinase activity produced by moulds and bacteria also, suggesting the importance of thiol-containing amino acid residues in the enzymes function. Arthrobacter spp. (Kang et al., 1998), Streptomyces spp. (Sharma et al., 2006) and Aspergillus ochraceus (Guimaraes et al., 2007) inulinases are inhibited by the presence of these metals into the reaction medium. 2.3 Applications of inulinases Inulinases can be used in a large spectrum of applications, ranging from pharmcology to food industry and bioethanol production. 2.3.1 high fructose syrup in the last years researchers attention has been directed toward producing natural polysacharides using microbial fermentation that was used in industry on a large scale. Fructose the sweetest of all naturally occurring carbohydrates and is often produced by enzymgprocess from starch. Conversion of starch to fructose involves the use of three different enzymes and the maximum yield is 45% (Pandey et al., 1999). A simple and high

productivity method to obtain high fructose syrup is the enzymatic hydrolysis of inulin, a single step process that uses inulinases and yields 95% pure fructose (Chi et al., 2009, Ricca or al., 2009). Fructose has beneficial effects in diabetics, obesity, stimulates calcium absorption, stimulates grow of bitidobacteria, increases the iron absorption in children and prevents colon cancer (Chi et al., 2009). Furthermore, fructose metabolism bypasses the known metabolic pathway of glucose and does not require insulin (Rocha et al., 2006, Gong et al., 2007). It is widely used in food industry, pharmaceutics and beverages (Chi et al., 2009, Rocha et al., 2006). 2.3.2 Inulo-oligosaccharide production The endo-inulinases are used for the production of inulooligosaccharides (IOS). Many microorganisms have been reported as endoinulinases producers: Yarrowia Iipolitica, Cryptococcus aureus (Gao et al., 2007), Arthrobacter spp. (Kang et al., 1998), Pseudomonas spp. (Chi et al., 2009), Paenibacillus spp. (Gem et al., 2001). IOS have found many applications in food industry: confectionery, milk desserts, yoghurt and cheese production, bakery, chocolate, ice-cream and sauces (Chi et al., 2011). It was found that the major IOS obtained after inulin hydrolysis with endo-inulinases has oligosaccharides are prebiotics; their positivgfect on human health has been widely acknowledged (Rocha et al., 2006, Chi et al., 2009) 2.3.3 Bioethanol production biochemical and thermo-chemical conversion technologies can convert biomass into carbon containing biofuels such as biodiesel and other liquids. The primary feedstock for ethanol production worldwide remains sugar or starch from agricultural crops, and its primary use is as a blend with gasoline (at 5-90% blend). Nowadays, studies concerning bioethanol production from various unconventional feedstock, such are lignocelluloses

materials or kitchen refuse, are increasing. Alcohol production from inulin rich feedstock has been studied since the end of 19th century. Although more widely recognized now, the dramatic enviromnental, economic, strategic and infrastructure advantages offered by the production of ethanol were not appreciated in the past. Lnulin rich raw materials gained researchers attention for bioethanol production. The microbial exo-inulinases can remove the terminal H'uctose residues from the non-reducing end of inulin molecule, producing fructose and glucose, which can easily be fermented to ethanol by Saccharomyces spp. yeast strains (Chi et al., 2011). Some yeast strains can perform simultaneous hydrolysis and fermentation of the inulin: Kluyveromyces marxianus and some Saccharomyces spp. yeasts can produce both active inulinase and ethanol (Chi et al., 2011, Rosa et al., 1986, Kim et al., 1998, Lim et aL,2011) 2.3.4 Other applications of inulinases Inulinases have also found their application for inulin substrates hydrolysis for singlecell oil and single-cell protein production (Chi et al., 2011). The marine yeast Cryptococcus aureus can be used for single cell protein production by cultivation on inulin hydrolysates from Jerusalem artichoke tubers. The same applications are important to produce citric acid, 2,3 butanediol, lactic acid and sugar alcohols, like mannitol (Chi et al., 2011; Saha, 2006; Liu et al.,2010)

Chapter 3
Materials and methods

3. MATERIALS AND METHODS 3.1 Materials used The media and chemicals used in present investigation were procured from Hi Media Laboratories and Loba Chemicals, Mumbai. All the glassware used was of borosil. 3.2 Collections of samples A total 10 rhizosphere soil samples of inulin containing plants (dahlia, garlic, onion) were collected from different sites. The samples of rhizosphere soil (soil adjacent to the roots) were collected in sterile polythene bags with the help of sterile spatula and all these samples were brought to the laboratory under aseptic conditions and stored at 4C till further use. 3.3 Isolation of inulinase producing fungi Sabouraud dextrose agar plates were prepared. All the constituents of medium was accurately weighed and mixed in distilled water before autoclaving at 121 C for 20 minutes. The medium was adjusted to pH 5.6. 10ug/ml ampicillin was added to suppress the growth of unwanted bacteria and encourage the growth of desired fungi. The samples were suspended in sterile water blanks, vortex for few minutes and serially diluted. The dilutions of l0`3, l0`4 and 10 5 were plated on Sabouraud dextrose agar plates and incubated at 28C for five to seven days. The plates were monitored at regular time intervals for appearance of any fungal forms.the appeared colonies were picked up and transferred on sterile Sabouraud Dextrose Agar plate for their purification (Amare Gessesse et al., 2003).

3.4 Purification and maintaince of fungal isolates The fungi were purified by repeated point inoculation. The purity of the isolated fungi was confirmed by microscopic examination of the culture at 40X magnification using light microscope. After ensuring purity, the cultures were sub-cultured on Sabouraud Dextrose Agar slants and allowed to grow for a period of 5-7 days and subsequently stored at 4C as stock cultures in slant. Usually, working as well as stock cultures are maintained and the working cultures were transferred to fresh Sabouraud dextrose agar slants at regular intervals of three months. 3. 5 Characterization and Identification of fungal isolates For the identification of the isolated fungi, the petri plates were further used for microscopic examination by using lactophenol-cotton blue staining as per the standard procedure. On the basis of their colonial and morphological characteristics, the fungi were identified. 3.6 Screening and enzymatic estimation 3.6.1 Enzm production media used erlenmeyer flasks (150 mL) containing 50 mL of asparagus medium were autoclaved (20 min, 121 c) and inoculated with two discs of all the fungal isolates for the enzymatic production submerged condition. Flasks were incubated at 28C on a rotary shaker (150 rpm) for 4 days. The contents were then flittered with Whatman filter paper and filtrates were used for enzymatic estimation.

Materials and Methods 3.6.2 Enqvmatic' assay Inulinases hydrolyze inulin into fructose and inulooligosaccharides. The reducing sugars released by the action of inulinase were determined by 3, 5-dinitrosalicyclic acid (DNS) method using fructose as standard (Miller, 1959). The 3, 5-dinitrosalicyclic acid was reduced to 3-amino-5-nitrosalicyclic acid under alkaline condition resulting in colour changes which was analyzed at 540nm. One nkat of inulinase was defmed as the amount of enzyme which produced lnano mol of fructose per mL per sec at 50C, and pH 5.0 from 1% (w/v) inulin (Chicory, Sigma Chemical Co., USA). 3.6.2.1 Reagents required 1. Sodium acetate buffer (0.2M, pH 5.0) Stock solution 0.2M sodium acetate and 0.2M acetic acid Dissolve 8.2 g sodium acetate in 500 mL distilled water (solution A). Dissolve 5.8 mL acetic acid in water (final volume 500 mL) (solution B) Sodium acetate buyer (0.2M pH 5. 0) Add 350 mL solution A and 150 mL solution B mix properly and fmal volume 500 mL. 3.6.2.2. Substrates 1% inulin in 0.2M sodium acetate buffer, pH 5.0 Homogenize 1 g inulin in 80 mL sodium acetate buffer for 1 hour. Make up final volume up to 100 mL with buffer and continue stirring for l hour. 3.6.2.3 stopping reagent (DNS reagent) Dissolve 8.0 g of NaOH to 350.0 mL water. add in small portions (with continuous stirring) the mixture containing: 150 g K-Na tartrate (Himedia)

5.0 g 3, 5- dinitrosalicyclic acid > Mix by stirring until all the crystals have dissolved. > Make up to 500.0 mL with water in a volumetric flask. Stored in a dark tightly closed bottle at room temperature. In case, the reagents after storage contain particles or insoluble precipitates filter it using filter paper. 3.6.2.4 Standard stock solution (0. 01M fructose): Dissolve 180 mg of fructose (Hi-media) in 0.2M sodium acetate buffer and make the volume up to 100 mL in a voltunetric flask. Procedure: Enzyme blank: Add 1.8 mL substrate, incubate 15 min. at 50C, add (in this order) 3.0 mL DNS, 0.2 mL enzyme. l. Add 1.8 mL substrate solution to a test tube and temperature to 50C. 2. Add 0.2mL enzyme diluted appropriately (dilution in buffer), mix by vortexing. 3. Incubate exactly 15 min. at 50C. 4. Add 3.0 mL stopping reagent (DNS), mix and remove the tube from the water bath. 5. Place the tube (all samples, enzyme blanks, fiuctose standards and reagent blank) in a boiling wgbath all in one time. After boiling for exactly Smin., remove the tubes and cool in cold water bath. 6. Measure the colour formed at 540 nm against the reagent blank. 7. Using the standard curve, convert the corrected value to enzyme activity units (nkat/mL). Multiply by dilution factor of the enzyme sample to calculate the activity in the original (undilluted)sample dilution. (Figure 3.1).

F igure3.1: Standard curve of fructose 3.7 Effect of temperature, pH, and metal ions on inulinase activity produced by potential producer A. niger G6 3. 711 Efect of pH and temperature The effect of pH on inulinase activity was determined by incubating 0.2 mL of suitably diluted enzyme and 1.8 mL of inulin (dissolved 1% w/v in different buffers, 200 mM sodium acetate buffer: pH 4.0, 5.0, 6.0. 7.0, 8.0 and 9.0; 100 mM phosphate buffer for 15 min at 50C. The effect of temperature was determined by incubating 0.2 mL of suitably diluted enzyme and 1.8 ml of inulin (1% w/v in 200 mM sodium acetate buffer, pH 5.0) for 15 min at temperature range from 30c, 40C, 50C, 6o"c, 70C, 80Cand 90c.

3. 7.2 Effect of metal ions and detergents The effect of various metal ions (1mM BaCl2, 1mM CaCl2.2H2O, 1mM CuSO4.5H2O, 1mM MgSO4.7H;O, 1mM MnSO4) and detergents (10mM sodium dodecylsulphte, 10% (v/v) Tween 20) on inulinase activity was examined by incubation various metal salts and detergents with enzyme in 200 mM sodium acetate buffer (pH 5.0) at 30C for 1 h. The residual activity of inulinase was then detennined as compared to control i.e. untreated enzyme sample.

Chapter 4
Result and discussions

Results and Discussion 4. RESULTS AND DISCUSSION 4.1 Isolation and identification of fungi Filamentous fungi are used by industry for manufacture of a large variety of useful products. The products include metabolites, enzymes and food. Fungal cells can grow at different environmental conditions. The chemical and physical conditions used for fungal propagation will have a great impact on the capability of these cells to accumulate the desired products. And hence, in the present study an attempt has been made to isolate the inulinase producing fungi from decaying rhizosphere soils of inulin containing plants collected from different habitats. Rhizosphere soil samples of inulin containing plants (dahlia, garlic, onion) were collected from different sites of Chandigarh. A total of 10 samples collected from rhizosphere soil of different plants were examined for the presence of mycoflora. Samples used for isolation included 3 of rhizosphere soil of garlic , 2 of rhizosphere soils of onion, sample of 1 rhizosphere soil of dahlia, shown in (Table4.1). Table 4.1: Distribution of samples in diferent habitat s. No. soURCE No. or ISOLATES 1 oN1oN 3 2 oN1oN 2 3 DAI-ILIA 2 4 GARLIC 4 5 GARLIC 5 ' 6 GARLIC 2 TOTAL 6 18

A total of 18 fungal forms belonging to 2 genera and 5 species were isolated during the course of present study. The results of isolation are given in (Table 4.3, Figure4.l). In the present survey, rhizosphere soil of onion yielded Aspergillus terreus, Aspergillus niger, Aspergillus jiunigates The sample of rhizosphere soil of dahlia yielded 1 fungal form and was identified as Aspergillus flavus. A.niger is a fungus commonly found on grapes, apples and tomatoes (Yildz and Baysal, 2006). Bali et al., (2008) reported that black mold A. niger were caused post harvest spoilage in sweet orange and acid lime at field. Okereke et al., 2010) indicated that the fungi species isolated from the infected mangoes were A. niger, Alternaria sp. Bolryodiolodia theobromae and Colletotrichum gloeosporioides. Fusarium sp, A. flavus and Phoma sp. were also isolated but could not prove pathogenicity when inoculated into healthy mango huits. The soil moisture has a direct effect on the population of fungi positively hence, at higher moisture, the tolerance and colonization by fungi is badly affected (Adams et al., 1999). In the present survey, samples of rhizosphere soil of garlic support different fungal forms and were identified as Aspergillus fumigatus, Aspergillus flavus, Fusarium oxysporum, Aspergillus niger, The identification of fungi was based upon morphological and microscopic features showed by each fungus as shown in (table 4.2)

4.2 Screening and estimation of fungal isolates for inulinase activity ln the experiment, the inulinase activity in the culture filtrates of test fungi was quantified by estimating the amount of reducing sugar (fructose) liberated from dahlia inulin (Sigma, U.S.A.) and was expressed in nkat per milliliter (nkat/mL) of culture filtrate (Table 4.4). Among three strains of Aspergillus niger, A. niger (G6) was found to possess enzyme activity equivalent to 220.36 nkat/mL in its culture filtrate of asparagus root power media while the culture filtrates of other 2 strains of A. niger i.e. G5, O5 showed inulinase activity in the range of 66.l3nkat/mL to 124.23 nkat/mL in asparagus containing medium, respectively. Several strains of Aspergillus niger have been reported to produce high titers of inulinase (Derycke and Vandamme, 1984), (Viswanathan and Kulkarni , l995a)( Ji et al., 1998) (Skowronek and Fiedurek, 2004),( Kumar et al, 2005),( Ge and Zhang, 2005),(Mutanda et al., 2008),( Kango, 2008),(Cruz et al.,l998) have found A. niger-245 to produce maximum (9.9 U/mL) of inulinase on medium containing casein and dahlia extract. (Ongen-Baysal et al., 1994) observed higher inulinase activity of A. niger A42 (54 U/ mL) using crude Jerusalem artichoke extract and Kango (2008) have of 54 U/ mL of inulinase activity in culture filtrate of A. niger NK-126 grown on dandelion tap root extract (Ohta et al., 1993). The strain Aspergillus fumigatus (O5) showed inulinase activity equivalent to 68.94nkat/ml and in culture filtrate of asparagus medium, receptivity. While other test strains of A.fumigatus (Gl 1) including A. fumigatus (Gl), A. fumigatus (G8) and A.fumigatus (03), A. Qigatus (04), Afumigatus (G4) showed inulinase activity equivalent to 2.55 nkat/mL 51.26 nkat/mL in asparagus medium. The isolates of Aspergillus terreus (02) were able to produce inulinase range of 44.48 nkat/mL grown on asparagus medium. All test strains aspergillus flavus (DI, G10, D2, G2, and G3) were able to produce inulinase a range of 86.03 nkat/mL, 37.53 nkat/mL, 4.04 nkat/mL, 6.54 nkat/mL and 4.41 nkat/ml.

Table

4.3 Effect of temperature. pH. and metal ions on inulinase activity produced by potential producer A. niger G6 4.3.1 Effect oftemperature The effect of different temperatures on the enzyme activity was studied. From the results obtained maximal activity was observed at 60C (Figure 4.2). Chen er al., (1997) and Passoni cr ul., (20()7) found the same results for A. niger 319 and Penicillium respectively.jain et al, (2012) and Kango (2008) found optimum temperature of 50C and 30C for K.marxanius and A. niger respectively. Similarly, Cho and Yun (2002) have shown that .xanthomonas oryzae produce an endoinulinase having optimum temperature of 50 C whereas Abeer (2004) indicated that StrepI0n1_i'c'es griseus produces an inulinase having optimum temperature of 40C. Cruz-Guerrero et ci/.,(l999) and Wenling ef al.,( 1999) have also been reported temperature Optima of 50C for K. marxainus. Temperature range for maximum growth and inulinase production by iimgi has been reported to be 28-30C (Vandamme and Derycke, 1983).

Figure 4.2: Effect of temperature on inulinase activity

4.3.2: Effect of pH The effect of different pH on the enzyme activity was studied and the maximal inulinase activity was observed at pH 5.0 (Figure 4.3). Jain et al., (2012) found optimum pH of 4.0 for K. mancainus. Xio et al., (1988) and Yokota et al., (1991) obtained the maximum activity at pH 5.5 with inulinase from Chrysosporium pannorum and Arthrobacter sp. H65- 7 respectively. This low pH value is advantageous for industrial preparation of sugar syrops because of reduced color formation at low pH values (Vandamme and Derycke, 1983). Many other authors observed that inulinase enzyme have an optimum pH in range of 4.5-5.0 (Snyder and phaff, 1960; Negoro and kito, 1973; Nakamura and Nakatsu, 1977).

4.33 Effect of metal ions and detergents In order to assess the practical utility of inulinase produced by present test organism, A. niger G6, effect of different metal ions and detergents on activity of inulinase was studied. The results of the experiments are given (Figure 4.4). The Mn2+ and Cu2+ ions were activating the enzyme by 31% and 13% respectively. Nakamura and Nakatsu (1977) reported the activation of inulinase by Mn2+. The other ions viz. Ba2+ and Mg2+ results in reduction of inulinase activity by 22% and 56% respectively. In the presence of 10% (v/v) concentration of Tween 20, only 49% residual activity of inulinase was observed in the test enzyme sample. Pretreated SDS (10mM) strongly inhibited activity of these enzymes. Enzyme sample treated with SDS showed to residual activity 36%. Sharma et al. (2006) and Gill et al. (2006) also found that SDS at 1M concentration results in almost total inactivated of enzymes.

Chapter 5
Summary and conclusion

5. SUMMARY AND CONCLUSIONS Inulin is a widespread plant polyfmctan that has linear chains of B- (2, 1)-linked fructose residues attached to a terminal sucrose residue. This polyfmctan serves as a storage polysacchaiide in the Compositae and Gramineae families, For example: Asparagus (Asparagus racemosus), Dahlia (Dahlia pinnata), Jerusalem artichoke (Helianthus tuberosus), Chicory(Cichorium intibus). These are widely studied in fungi e.g. Aspergillus niger, Aspergillus jiunigatus, Aspergillus jlavus, Aspergillus terreus. Applications of inulinase are high fructose syrup, inulo-oligosaccharide production, bioethanol production. 0 In this study, isolation of fungi producing inulinase was conducted by serial dilution method of the samples collected from different places. 0 18 different isolates from 3 different sources were obtained on SDA media and identified on the basis of their morphology. Production of fungal isolates was done using Asparagus medium in Erlenmeyer flask (150 ml) in shaking incubator set atl50 rpm and their filtrates were used for further analysis. 0 Out of 18 isolates, all isolates showed positive results and G6 gave the highest alinase activity i.e. 220.36nkats/ml for quantitative screening. 0 the optimum pH and temperature for A. niger G6 inulinase was found to be 5.0 and 20 c respectively. The enzyme was inhibited by 10% T20 and l0mM SDS and activated by the presence of lmM Cu and Mn2+. In the present study Aspergillus niger G6 was found to be a good producer of inulinase A. niger is potentially useful for producing commercial enzyme cocktail readily utilizable for making either sweeteners or prebiotics from inulin.

Chapter 6
References

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Appendix

(A) PREPARTION OF CULTURE MEDIA (a) Sabouraud dextrose agar (SDA) Yeast extract Dextrose Agar Distilled water 10 g 40 g 20 g 1000 ml

(b) Asparagus medium

Asparagus root powder Yeast extract Distilled water

5g 3g 1000 ml

(B) PREPARATION OF REAGENTS AND BUFFERS (a) Sodium acetate buffer (0.2 M,,pH 5.0) 0.2 M Sodium acetate(Stock solution A) 8.203 g 500 ml

Sodium acetate Distilled water

0.2 M Acetic acid (Stock solution B) 5.72 ml 500 ml

Acetic acid Distilled water

Sodium acetate buffer(0.2M.pH 5.0) 350 ml 150 ml

Stock solution A Stock solution B

(b) Substrates

1% inulin in 0.2 M sodium acetate buffer,pH 5.0 Inulin Sodium acetate buffer 1g 100 ml

(c) standard stock solution (0.01 M fructose) Fructose Sodium acetate buffer 90 mg 100 ml

(d) Stopping reagent (DNS reagent)

NaOH Na-K tartrate DNS Distilled water

10 g 40 g 20 g 500 ml

Dilutions (standard solutions) 1:1 undiluted 1:2 (1 ml stock+1 ml buffer) 1:3 (1 ml stock+2 ml buffer) 1:5 (1ml stock+4 ml buffer) =10.0 umol/ml = 11.1 nkat/ml =5.0 umol/ml = 5.7 nkat/ml =3.3 umol/ml = 3.7 nkat/ml =2.0 umol/ml = 2.23nkat/ml

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