ISOLATION AND SCREENING OF INULINASE1.doc

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 60°C 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.

1

Chapter 1

Introduction

2

Introduction

l. INTRODUCTICN

Inulin is a linear (β-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 β-(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 3ItiChO§HCli3HthUS

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).

3

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 10-4 (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, inulo-

oligosaccharide 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).

4

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 fungi.

Screening and estimation of inulinase from fungal isolates.

Effect of temperature, pH, and metal ions on inulinase activity of potential

producer.

5

Chapter 2

Review of literature

6

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 β-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)

7


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 fructo-

oligosaccharides (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

trans’ti'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 substratesthat 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 mono-and 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)

8


Substrates used for inulinase production

Substrates Microorganisms ReferencesPure substratesInulin Pichia guilliermondii Yu et al., 2009

K. lactis, K. marxianus spp.

Guerrero et al., 2006

Streptomyces spp. Sharma et al., 2006

Sucrose Kluyveromyces marxianus Kalil et al., 2001, Santisteban et al., 2009

A. niger, A. oryzae, A. ficuum

Ge and Zhang, 2005

Santisteban et al., 2009Guerrero et al., 2006Santisteban et al., 2009

Fructose Kluyveromyces marxianus Guerrero et al., 2006K. lactis, K. marxianus spp.

Glucose Kluyveromyces marxianus

Inulin containing plant materials (rye, barley, banana, garlic, onion,wheat, chicory, dahlia, dandelion, Asparagus roots, Jerusalem artichoke)

Streptomyces spp. Sharma et al., 2006A. niger Kango, 2008Kluyveromyces marxianus YS-1

Singh et al., 2006

Rhizoctonia solanis Singh and Bhermi, 2008

Ertan et al., 2003a

Agro-industrial residues (cassava flour, corncob, oat meal, rice straw, sugar cane bagasse, wheat bran, pressmud)

Aspergillus ochraceus Guimaraes et al., 2007Streptomyces spp. Dilipkumar et al., 2011

9


The exo-inulinase (β Dfructanohydrolase, 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 |3-

tructofuranosides.

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 β-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).

10


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

SOURCES Plant part Inulin content

(% of fresh weight)

Onion Bulb 2-6

Jerusalem artichoke Tuber 14-19

Dahlia Tuber 9-12.5

Chicory Root 15-20

Leek Bulb 3-10

Garlic Bulb 9-16

Artichoke Leaves-heart 3-10

Banana Fruit 0.3-0.7

Rye Cereal 0.5-1

Barley Cereal 0.5-1.5

Dandelion Leaves 12-15

Burdock Root 3.5-4.0

Camas Bulb 12-22

Murnong Root 8-13

Yacon Root 3-19

Salsify Root 4-11

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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 (E.C. 3.2.1 .7) that breaks down inulin into inulooligosaccharides

(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).

12

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 inulo-oligosaccharides. F- Fructose, G- Glucose.

Endo-inulinase

Neutraceutical (Dietary fiber)

Functional food ingredient

Inulo-oligosaccharides

Exo-inulinase

Hi-fructose syrup

Low calorie sweetener

Fermentable substrate

InulinF G

β, 2-1 fructosyl linkage


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

MICRO ORGANISMS MAXIMAL ACTIVITY REFRENCES

Moulds

Aspergillus niger 1.75 g/L Gern et al., 2001

Aspergillus niger 100 Ua/mL Ge and Zhag, 2005

Aspergillus niger 52.5 IUa/mL Kango, 2008

Aspergillus niger 176 U/mL Kumar et al., 2005

Aspergillus fumigatus Not available Gill et al., 2006

Aspergillus awamori Not available Nagem et al., 2004

Aspergillus ochraceus 108 Total U Guimaraes et al., 2007

Aspergillus ficuum 193.6 U/gdsb Chen et al., 2011

Aspergillus parasiticus 2.9 U/mL Ertan et al., 2003b

Geotrichum candidum 45.65 IU/mL Mughal et al., 2009

Rhizoctonia solani 1.792 U/mL Ertan et al., 2003a

Chrysosporium pannorum 115 U/mL Xiao et al., 1988

Bacteria

Paenibacillus spp. 2.48 g/L Gern et al., 2001

Streptomyces spp. 524 IU/L Sharma et al., 2006

Streptomyces spp. 89 U/gds Dilipkumar et al., 2011

Bacillus spp. 42.36 U/mL Zherebtsov et al., 2002

Pseudomonas spp. Not available Kim et al., 1997

Arthrobacter spp. Not available Kang et al., 1998

Yeasts

Pichia guilliermondii 39.56 U/mL Gao et al., 2007

Pichia guilliermondii 61.5 U/mL Chi et al., 2009

Pichia guilliermondii 130.38 U/mL Yu et al., 2009

Pichia guilliermondii 60.1 U/mL Gong et al., 2007

13

Cryptococcus aureus 52.37 U/mL Gao et al., 2007

Cryptococcus aureus 85 U/mL Chi et al., 2009

Cryptococcus aureus 436.2 U/gds Chi et al., 2009

Yarrowia lipolitica 62.85 U/mL Gao et al., 2007

Yarrowia lipolitica 22.5 U/mg Liu et al., 2010

Debaryomyces hansenii 52.53 U/mL Gao et al., 2007

Candida kefyr 40 U/mL Pessoa and Vitolo, 1998

Kluyveromyces

marxianus

194.1 U/mL Kalil et al., 2010

Kluyveromyces

marxianus

127 U/mL Kalil et al., 2001

Kluyveromyces

marxianus

176 IU/mL Santisteban et al., 2005

Kluyveromyces

marxianus

208 IU/mL Santisteban et al., 2009

Kluyveromyces

marxianus

262.9 U/mg Golunski et al., 2011

Kluyveromyces

marxianus

1294 U/mL Treichel et al., 2009

Kluyveromyces

marxianus

18743 U/mL Kushi et al., 2000

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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

15


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

16


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).

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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).

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Source

Molecular

weight

kDa

optimum

ph

optimum

temperature

(c)

ph

stability

range

temperature

stability

range (c)

References

K. marxianus - 3.5 60 - - Mazutti et al., 2007

K. marxianus - 4.4 50 - - Mazutti et al., 2010

K. marxianus - 4.75 55 - 40 Kushi, 2000

K. fragilis 250 - 55 - - Pandey et al., 1999

Yarrowia

lipolitica

- 4.5 50 03-Jul 50 Liu et al., 2010

Pichia

guilliermondii

50 6 60 06-Jul 60 Gong et al., 2008

Cryptococcus

aureus

60 5 50 4.0-6.5 65 Sheng et al., 2008

Arthrobacter

spp.

75 7.5 50 5-10.5 30…40 Kang et al., 1998

Bacillus spp. - 7 - 06-Aug 25…40 Zherebtsov, 2002

Streptomyces

spp.

- 6 60 - 60…70 Sharma et al., 2006

A. niger - 4.4 - - - Sharma et al., 2006

A. niger 70 5 40 - - Pandey et al., 1999

Penicillium

janczewskii

- 4.8-5.0 35…45 - - Sharma et al., 2006

A. ochraceus 79 4.5 60 - 60 Guimaraes et al.,

2007

F. oxysporum 300 5.8-6.2 30…37 - - Pandey et al., 1999

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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 50-60°C. Kluyveromyces spp. strains produce inulinases with

maximum activity at 50-55°C (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 60°C. 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.

20


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+2 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).

21


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

22

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 single- cell 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)

23

Chapter 3

Materials and methods

24

Materials & 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 4°C 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 28°C 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 4°C as

25

Materials & Methods

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 28°C 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 50°C, and pH 5.0 from 1% (w/v) inulin

(Chicory, Sigma Chemical Co., USA).

26

Materials & Methods

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.

27

Materials & Methods

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 50°C, add (in this order)

3.0 mL DNS, 0.2 mL enzyme.

1. Add 1.8 mL substrate solution to a test tube and temperature to 50°C.

2. Add 0.2mL enzyme diluted appropriately (dilution in buffer), mix by vortexing.

3. Incubate exactly 15 min. at 50°C.

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).

8. 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 Effect of pH and temperature

28

Materials & Methods

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

50°C. 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 30°c, 40°C, 50°C, 6o"c, 70°C, 80°Cand 90°c.

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 30°C for 1 h. The

residual activity of inulinase was then detennined as compared to control i.e. untreated

enzyme sample.

29

Chapter 4

Result and discussions

30

Result & Discussions

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 different habitat

S. NO. SOURCE NO. OF ISOLATES

1 ONION 3

2 ONION 2

3 DAHLIA 2

4 GARLIC 4

5 GARLIC 5

6 GARLIC 2

TOTAL 6 18

31


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)

32


Table: Fungal isolate with their morphological characteristics

S. No. Sample No. Fungus Colonial Morphology Microscopic Appearance

1 O5,G5,G6 Aspergillus niger

Colonis start white to pale yellow but quickly form jet black condidia. The reverse is buff or yellow grey

Conidial heads are large, condidiophores are smooth walled hyaline or turning towards the vesicle. Conidia are dark brown to black and roughly walled.

2 O1,O3, O4,G1,G4,G8,G11

Aspergillus

Fumigatus

Colonies are cottony or powdery, white at first then darkening to green, greenish grey or greenish brown with a white apron at the margin.

Condidia are arising in chains, conidiophores are long and have club-shaped vesicles, and vesicles are uniseriate.

3 D1, D2, G2,G3,G10

Aspergillus flavus

Colonies are flat, often with radical grooves, yellow at first becoming white to dark yellow green.

Conidial heads are typically radiate, latter spilt to loose columns, conidiophores are hyaline and coarsely roughened.

4 O2 Aspergillus terreus

Colonies are velvety, brown to orange brown, the reverse is white to brown

Smooth elliptical condia from long chains, condidiophores are hyaline and smooth walled, vesicles are small, dome shaped.

5 G7,G9 Aspergillus oxysporum

Colonies growing rapidly, arieal mycelium white then becoming purple, reverse hyaline to dark purple

Conidiophores are short, single, and latter arranged in densely branched cluster, chalmydospores are hyaline or rough walled

33


Table 4.3: Fungi isolated from rhizospheric soil

Sr. No. Sample Isolate Name Fungus

1 ONION

O1 Apergillus fumigatus

O2 Apergillus terreus

O3 Apergillus fumigatus

2 ONIONO4 Apergillus fumigatus

O5 Apergillus niger

3 DAHLIAD1 Apergillus flavus

D2 Apergillus flavus

4 GARLIC

G1 Apergillus fumigatus

G2 Apergillus flavus

G3 Apergillus flavus


5 GARLIC

G5 Apergillus niger

G6 Apergillus niger

G7 Fusarium oxysporum


G9 Fusarium oxysporum

6 GARLICG10 Apergillus flavus


34


35


Figure 4.1 Cultural characterstics of selected fungi on sabouraud’s dextrose agar medium

A: Fusarium Oxysporum B: Aspergillus terreus C: Aspergillus Niger

D: Aspergillus Fumigatatus E: Aspergillus Flavus

36


4.2 Screening and estimation of fungal isolates for inulinase activity

in 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.

37


Table 4.4: Inulinase activity of culture filtrates

S.NO. FUNGUS ISOLATE NO INSULINASE ACTIVITY

(nkat/mL)

1 Apergillus niger G6 220.36

2 Apergillus niger G5 124.23

3 Apergillus flavus D1 86.03

4 Fusarium oxysporum G7 72.42

5 Apergillus fumigatus O1 68.94

6 Apergillus niger O5 66.13

7 Aspergillus Terrus O2 44.48

8 Apergillus fumigatus G11 41.26

9 Apergillus flavus G10 37.53



12 Fusarium oxysporum G9 12.04





17 Apergillus flavus D2 4.04


38


4.3 Effect of temperature. pH. and metal ions on inulinase activity produced by potential

producer A. niger G6

4.3.1 Effect of temperature

The effect of different temperatures on the enzyme activity was studied. From the results

obtained maximal activity was observed at 60°C (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 50°C and 30°C 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

40°C. Cruz-Guerrero et ci/.,(l999) and Wenling ef al.,( 1999) have also been reported

temperature Optima of 50°C for K. marxainus.

Temperature range for maximum growth and inulinase production by iimgi has been reported

to be 28-30°C (Vandamme and Derycke, 1983).

Figure 4.2: Effect of temperature on inulinase activity

39

Chart Title

29.25

51.64

81.97

100

79.5

16.4911.940

20

40

60

80

100

120

0 20 40 60 80 100

Temperature (c)

Rel

ative

acti

vity

(%)

Series1


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).

40

76.81

100

51.93

32.6122.86

3.50

20

40

60

80

100

120

0 2 4 6 8 10

pH

Rela

tive

acti

vity

(%)

Series1


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.

0

20

40

60

80

100

120

140

Cacl2 MnSo4 CuSo4 Bacl2 Mgso4 Tween20 SDS

Effect of Metal ion & sufactants

Rel

ativ

e A

citv

ity

%

Cacl2

MnSo4

CuSo4

Bacl2

Mgso4

Tween20

SDS

41

Chapter 5

Summary and conclusion

42

Summary & 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.

In this study, isolation of fungi producing inulinase was conducted by serial dilution

method of the samples collected from different places.

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.

Out of 18 isolates, all isolates showed positive results and G6 gave the highest alinase

activity i.e. 220.36nkats/ml for quantitative screening.

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.

43

Chapter 6

References

44

Refrences

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54

Appendix

55

Appendix

(A) PREPARTION OF CULTURE MEDIA

(a) Sabouraud dextrose agar (SDA)

Yeast extract 10 g

Dextrose 40 g

Agar 20 g

Distilled water 1000 ml

(b) Asparagus medium

Asparagus root powder 5 g

Yeast extract 3 g


(B) PREPARATION OF REAGENTS AND BUFFERS

(a) Sodium acetate buffer (0.2 M,,pH 5.0)

0.2 M Sodium acetate(Stock solution A)

Sodium acetate 8.203 g


0.2 M Acetic acid (Stock solution B)

Acetic acid 5.72 ml


Appendix

56

Sodium acetate buffer(0.2M.pH 5.0)

Stock solution A 350 ml

Stock solution B 150 ml

(b) Substrates

1% inulin in 0.2 M sodium acetate buffer,pH 5.0

Inulin 1 g

Sodium acetate buffer 100 ml

(c) standard stock solution (0.01 M fructose)

Fructose 90 mg

Sodium acetate buffer 100 ml

(d) Stopping reagent (DNS reagent)

NaOH 10 g

Na-K tartrate 40 g

DNS 20 g


Dilutions (standard solutions)

1:1 undiluted =10.0 umol/ml = 11.1 nkat/ml

1:2 (1 ml stock+1 ml buffer) =5.0 umol/ml = 5.7 nkat/ml

1:3 (1 ml stock+2 ml buffer) =3.3 umol/ml = 3.7 nkat/ml

1:5 (1ml stock+4 ml buffer) =2.0 umol/ml = 2.23nkat/ml

57

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