PROJECT SEMESTER REPORT

PROJECT SEMESTER REPORTB.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. 700900036Under the Guidance ofDr.Sumit jain

Department of Biotechnology & Environmental SciencesThapar University, PatialaJune, 2011

TABLE OF CONTENTSS.NO CHAPTERSPAGE NO.

ABSTAC T

1. INTRODUCTION

2. REVIEW OF LITREATURE

2.1 Inulinase-expressing microorganisms

2.1.1 Moulds

2.1.2 Yeast

2.1.3 Bacteria

2.2 Characteristics of inulinases

2.2.1 Molecular Weight of inulinases

2.2.2 Optimum pH and temperature activity of inulinases

2.2.3 Effect of metals ions and protein inhibitors on inulinase activity

2.3 Applications of inulinases

2.3.1 High fructose syrup

2.3.2 Inulo-oligosaccharide production

2.3.3 Bioethanol production

2.3.4 Other applications of inulinases

3. MATERIALS AND METHODS

3.1 Materials used

3.2 Collections of samples

3.3 Isolation of inulinase producing fungi

3.4 Purification and maintaince of fungal isolates

3.5 Characterization and Identification of fungal isolates

3.6 Screening and enzymatic estimation

3.6.1 Enzyme production media used

3.6.2 Enzymatic assay

3.6.2.1 Reagents required

3.6.2.2 Substrates

3.6.2.3 Stopping reagent (DNS reagent)

3.6.2.4 Standard stock solution

3.7 Effect of temperature, pH and metal ions on

inulinase activity by potential producer A.niger G6

3.7.1 Effect of pH and temperature

3.7.2 Effect of metal ions and detergents

4. RESULTS AND DISSCUSSIONS

4.1 Isolation and identification of fungi

4.2 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 Effect of temperature

4.3.2 Effect of pH

4.3.3 Effect of metal ions and detergents

5. SUMMARY AND CONCLUSIONS

6. REFERENCES

APPENDIX

TABLESS.NO TABLESPAGE NO.

2.1Substrates inulinase production used for 6

2.2 Inulin contents of some plants 8

2.3Inulinase expressing microorganisms

2.4Properties of some microbial inulinase

4.1Distribution of samples in different habitats

4.2Fungal isolates with their morphological characteristics

4.3Fungi isolated from rhizospheric soil

4.4Inulinase activity of culture filtrates

S. NOLIST OF FIGURESPAGE NO

2.1Structure of inulin

2.2Inulin being acted upon by microbial exo- and endo-inulinase enzymes.

3.1Standard curve of fructose

4.1Culture characteristics of selected fungi on SDA medium

4.2Effect of temperature on inulinase activity 34

4.3Effect of pH on inulinase activity 35

4.4Effect of metal ions on inulinase activity 36

.

ABSTRACTInulin 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 1Introduction

Introductionl. INTRODUCTICNInulin 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, 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).

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 2Review of literature

Review of Literature

2. REVIEW OF LITERATUREInulin 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 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 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 areshown in (Table 2.1). Naturally occurring inulin rich materials are the preferred substratesthat are used by researchers for inulinase obtaining but lately, agroindustrial residues havegained 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 androots. Jerusalem artichoke and chicory, which belong to Compositae family, are the mostcommog used carbon sources used by researchers for inulinase production as they containover 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 internallinkages in inulin to yield inulo-oligosaccharides (Fernandez et al., 2004; Skowronek andFiredurek , 2004; Chi er al., 2009), and invertase breaks down sucrose to fructose and glucoseby catalyzing the hydrolysis of terminal non-reducing [3-fructofuranoside residues in |3-tructofuranosides.While [3-b fructofuranosidase (bFFase) is a transfructosylatiiig enzyme, it can transferthe fiuctosyl residue to the sucrose molecule at a high concentration of sucrose, in whichtructosyl 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 asAspergillus spp., Fusarium spp. and Aureobasidium spp. Microbes are known as the bestsource for commercial production of enzymes because of their easy cultivation and highyield 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 themedium, ge study of this regulation is important in the commercial production of suchenzymes. the degree of polymerization ui mulm (MW 60,000) of plant origin IS < 200 andvaries a