Production, purification and characterization of invertase by Aspergillus niger using fruit peel waste as substrate.

A PROJECT REPORT

Submitted in partial fulfillment of theRequirements for the award of the degree of

Master of Science

In

Department Of BiotechnologyBy

JITESH S. PATEL

DEPARTMENT OF BIOTECHNOLOGYVEER NARMAD SOUTH GUJARAT UNIVERSITY

SURAT

2011

CERTIFICATE

DECLARATION

I hereby declared that the work presented in the Project entitled “Production, purification and characterization of invertase by Aspergillus niger using fruit peel waste as substrate” has been carried out by JITESH S. PATEL under the guidance of MISS. PRIYA BANDE, Project Guide, at MITCON, Pune. The entitled Work is original and no part of this work is either published or submitted in any university for the award of any degree or science.

Date:Place: Pune

JITESH S. PATEL

S.NO. CONTENTS PAGE.NO

1. Abstract 4.2. Introduction 5

3. Function and properties of invertase 7

4. Materials and Methods 8

5. Result and Discussion 13

6. Referance 16

7. Appendix 18

ABSTRACT

Three Aspergillus niger strains were grown in solid state fermentation systems with sucrose at100 g /l and also with the use of waste fruit peel (orange peel) as a substrate to produce invertase enzyme. Different parameter and growth conditions are studied with invertase and it’s production. The enzyme is purified with the use of chilled acetone and amonium sulphate. The molecular weight is determine by SDS-PAGE is 67Kda. The optimum temperature and optimum pH for the inveratse production is 37C and 4 respectively. The total protein concentration is invertase is 6.4mg/ml (sample 1) and 8.4 mg/ml (sample 2). The enzyme activity found highest at 25 minutes.

Objective:The objective of the present work was to production, partially characterize and determine optimum production parameter for the invertase production from isolated Aspergillus niger from soil semple using fruit peel waste as a subsrate.

Introduction:

Invertase (b-D-fructofuranoside-fructohydrolase, E.C. (3.2.1.26) catalyzes the hydrolysis of sucrose to glucose and fructose. Invertase is one of the most widely used enzymes in food industry, especially in the preparation of jams and candies.[1] The enzyme is a glycoprotein, with some residues of mannose being the major component of the carbohydrate moiety.[2,3] Invertase is mainly used in the food industry, where fructose is preferred over sucrose because it is sweeter and does not crystallize easily. However, the use of invertase is seriously limited because another enzyme, glucose isomerase,can be used to convert glucose to fructose at lower costs. A wide range of microorganisms produce invertase and can, thus, utilize sucrose as the only carbon source and as inducer of such enzyme. It has been extensively studied in yeast and some fungi[4]. Invertase is used for the inversion of sucrose in the preparation of invert sugar and high fructose syrup (HFS). It is one of the most widely used enzymes in food industry where fructose is preferred than sucrose especially in the preparation of jams and candies, because it is sweeter and does not crystallize easily [5]. The enzymatic activity of invertase has been characterized mainly in plants and microorganisms. Among microorganisms, Saccharomyces cerevisae[6], Candida utilis [7], Aspergillus flavus [8], Thermomyces lanuginosus [9] and Penicillium chrisogenum [10] has been widely studied.

Cultivation of micro-organisms by solid state fermentation (SSF) is an alternative to submerged fermentation (SmF) for the production of enzymes by moulds (Pandey et al. 1999). The main advantages of SSF over SmF are minor catabolic repression (Ramesh & Lonsane 1991, Solís-Pereira et al. 1993) higher enzyme productivity (Lekha & Lonsane 1994, Acuña- Argüelles et al. 1995) and enzyme titres (Pandey et al. 1999).

Invertase (_-D-fructo furanoside fructo hydrolase EC 3.2.1.26) seems to be an appropriate choice as a model system, since it has been studied in detail in Aspergillus niger (Boddy et al. 1993). The present communication compares SSF and SmF systems in relation to the production of biomass and secretion of invertase using three different strains of Aspergillus niger. Evidence is presented supporting higher titres of invertase by A. niger grown in SSF system are due to higher levels of biomass production, as compared to the lower invertase and biomass levels observed in SmF system.[11]

The present study trend is the utilization of waste material for production of byproducts which

boosts up high economic returns in many industries. In this study, the production, purification

and biochemical characterization of invertase produced by the filamentous fungus A. niger using

fruit peel waste as substrate has been carried out which has good potential for biotechnological

applications.

Invertase was one of the enzymes isolated from yeast more than a century ago [12]. The enzymeoccurs widely in many plants, microorganisms and animal sources [13,14,15]. The expressionand distribution of plant invertase have been especially well documented, because it plays animportant role in sugar metabolism [16,17,18,19]. Strong invertase activity has been detected inripe grape berries and the accumulation of sugar in the fruit during maturation has been shownto correlate with high level of invertase activity [20]. Grape invertase isolated from certain whitegrapes has been shown to be present in both soluble and bound forms [21,22]. Soluble invertasepurifi ed from Semillon [23] is stable unde acidic conditions.

Protein is an important part of life and nutrition; it is the substance that composes a large portion

of your body’s structure. Proteins are made up of amino acids arranged in differentcombinations. Next to water, protein is the most abundant substance in the human body. It ispart of all body cells and is a vital building block in the growth, maintenance and repair of thebody tissue.

Of the 20 amino acids that make up the proteins of the body, 9 are known as essential aminoacids. These cannot be made by the body fast enough to meet its needs for growth andmaintenance, so they must be obtained through your diet. Therefore, it is important for you toknow how much protein you need..

Proteins are composed of small units. These units are the amino acids which are calledthe building blocks of protein. There are about 20 different amino acids which are commonlyknown. Each different protein is composed of various amino acids put together in varyingorder with almost limitless combinations. Most proteins are large molecules that may containseveral hundred amino acids arranged in branches and chains.

Function and properties of invertase:

Invertase is the common name of the enzyme that catalyzes the hydrolysis of table sugar (i.e.sucrose) into a much sweeter, equimolar mixture of glucose and fructose called “invert” sugar. Because invert sugar is a key ingredient in a number of sweets and confectionary products, the bakery industry provides one of the most important commercial applications of this enzyme reaction. For this reason, the enzyme has been extensively characterized and commercial sources of pure invertase are readily available.

invertaseSucrose (aq) + H2O Fructose (aq) + glucose (aq) (“invert” sugar)

While aqueous solutions of either pure sucrose or glucose display weakly dextrorotatory behavior, meaning they cause a slight right-handed rotation of plane polarized light, solutions of pure fructose are strongly levorotatory and cause a much greater left-handed rotation of the light.

The enzyme reaction, therefore, catalyzes the inversion of the righthanded rotation of polarized light observed through sucrose solutions to the left-handed rotation observed for solutions of “invert” sugar, hence the enzyme’s common name of “invertase”. For similar reasons, the common monosaccharides glucose and fructose are also known as dextrose and levulose, respectively. Because enzymes are systematically named and classified by the substrate and subclass of reaction that they catalyze, the systematic name of invertase is “sucrose glycosidase”implying that it is a member of the subclass of enzymes that hydrolyze glycosidic (or acetal) linkages with a substrate specificity for sucrose. The yeast form of the enzyme has been assigned the unique four digit enzyme classification code (EC) number of 3.2.1.26 and it is also commonly called β-fructofuranosidase or sucrase. The intestinal enzyme lactose glycosidase (or lactase, EC 3.2.1.108), which hydrolyzes milk sugar into an equimolar mixture of galactose and glucose, is a related member of this enzyme subclass that may be more familiar because a deficiency of this enzyme is associated with symptoms related to lactose intolerance. In yeast cells, invertase is classified as an extra-cellular, glycoprotein which is localized to the thin volume of space that exists between the yeast’s plasma membrane and its outer cell wall (this peripheral volume is often called the periplasmic space). The enzyme serves the important biological function of cleaving sucrose on the outside of the cell into monosaccharides that can be transported (and subsequently metabolized) in the cytoplasm. That is, in the absence of invertase, yeast would have a difficult time utilizing table sugar as an energy source. Kinetic studies indicate that this extracellular form of invertase has a pH and temperature optima of about 4.8 and 40° C, respectively, and the Km for its substrate is about 5 mM sucrose. The enzyme’s native mass of about 270 kiloDaltons is constructed from two identical and heavily glycosylated subunits with a molecular weight of about 135 kiloDaltons (Neumann & Lampen, 1967). Because extracellular proteins are typically conjugated with oligosaccharide chains (i.e. glycosides) by post-translational modification before they are exported from eukaryotic cells, it is not surprising that the periplasmic form of yeast invertase is indeed a glycoprotein. However, invertase is unusual in that the numerous oligosaccharide chains attached to the two identical subunits account for nearly 50% of enzyme’s native mass (Lampen, 1971).These cellular and structural features of yeast invertase offer several advantages in this purification project: (i) first, the enzyme can be gently and selectively extracted from yeast cells by using conditions that disrupt the cell wall while leaving the plasma membrane intact; (ii) the high oligosaccharide content increases the stability of the extracted enzyme (either by preventing protein aggregation or reducing its susceptibility to attack by proteases and other undesirable reactions) (Schulke & Schmid, 1988); and(iii) variations in the sugar content of each subunit causes them to migrate as a smeared band

that is easy to detect during SDS-PAGE analysis (Moreno et al., 1980). On the other hand, this unusually high sugar content also reduces the ability of the protein to bind to Coomassie brilliant blue, the key component of the Bradford dye-binding protein assay. For this reason, solutions of pure commercial invertase prepared by dissolving a weighed mass of the solid enzyme to a final concentration of 1 mg per mL are observed to have a relative or equivalent concentration of only 0.10 mg per mL when compared to bovine serum albumin as the standard, reference protein in the Bradford assay.

MATERIALS AND METHODS:

Organism and inoculum preparation method:

Culture was screened for invertase enzyme production and fungal strain A. flavus

selected for the production of invertase was prepared from 4 days old slant culture. Fungal

strains were isolated from soil.Spores of Aspergillus niger used in this work. Inoculum was

prepared by transferring the spores to potato dextroseagar medium, incubated at 30 °C for 5

days. Spores were scraped into 0.01 % Tween 80 solution. Composition of culture medium for

invertase production was (in g/L): sucrose 20, yeast extract 10, ammonium sulphate 1.0,

magnesium sulphate 0.75, potassium dihydrogen phosphate 3.5, pH 5.0.The sucrose in the media

was substituted with fruit peel waste (orange peel) as substrate.Cultivation was carried out in 250

ml Erlenmeyer flasks each containing 50 ml of sterile medium. After inoculation (106 spores/ml),

the flasks were incubated at 30 °C for seven days in a incubator shaker at 125rpm. At the end of

fermentation, the supernatant was harvested by centrifugation at 10,000 rpm for 10 min (4 oC)

and was used as crude enzyme extract.

Processing of the substrate:The fruit peel waste (Orange peel) were obtained from the fruit market, washed and then sliced.

The sliced pieces were spread on the trays and put it in to oven for completely dry and then crush

it which was used as substrate. They were autoclaved at 15 lbs for 20 minutes before use.

Solid-state fermentation (SSF):

Cultivation was carried out in 250 ml Erlenmeyer flasks containing 50 ml of sterile medium.

Culture conditions were: 30 °C, The medium used for enzyme production under submerged

fermentation comprised of (gm/L): sucrose 20, yeast extract 10, ammonium sulphate 1.0,

magnesium sulphate 0.75, potassium dihydrogen phosphate 3.5, pH 5.0, initial moisture content

of 65 % and incubation time of 7 days.

Extraction Of Enzyme:

For enzyme extraction, the content of each reactor was mixed with distilled water (1:10, mass

per volume) and vortexed for 1 min. Solids were filtered with filter paper and then with

Whatman no.1filter paper and the clear filtrate was assayed for extracellular invertase activity.

The mycelial mass was collected by filtration and its dry weight was determined. The mycelia

were disrupted in a porcelain mortar with acid-washed sea sand at 4ºC, extracted in distilled

water and centrifuged (23000g) for 10 minutes. The supernatant was called intracellular crude

extract and was used to determine intracellular invertase activity.

Enzymatic assay:

The β-D-fructofuranosidase activity was determined using 1% sucrose as substrate in sodium acetate buffe. Invertase activity was determined using the method of Sumner and Howells[24] by incubating 1.0 ml of enzyme solution with 9.0 ml of sucrose in 0.03 M acetate buffer (pH 5.0). To stop the reaction, 1 ml of dinitrosalicylic acid reagent was added and heated for 5 min in a boiling water bath. Finally the absorbance was read at 540 nm in spectrophotometer. One unit of enzyme activity (U) was defined as amount of enzyme that releases 1 µmol of glucose per min under the assay conditions. The values of enzymatic activity were expressed as U/ml for SbmF or U/g of substrate for SSF.

Determination of protein concentration:

Protein concentration was determined colorimetrically by Bradford method and Lowery method.

Bradford protein assay:

The method described below is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg protein.

Assay:

1. Warm up the spectrophotometer before use. 2. Dilute unknowns if necessary to obtain between 5 and 100 µg protein in at least one

assay tube containing 100 µl sample 3. If desirred, add an equal volume of 1 M NaOH to each sample and vortex. Add NaOH to

standards as well if this option is used. 4. Prepare standards containing a range of 5 to 100 micrograms protein (albumin or gamma

globulin are recommended) in 100 µl volume.

5. Add 5 ml dye reagent and incubate 5 min. 6. Measure the absorbance at 595 nm.

Protein Estimation by Lowry’s Method:

Procedure:

1) Different dilutions of BSA solutions are prepared by mixing stock BSA solution (1 mg/ ml) and water in the test tube as given in the table. The final volume in each of the test tubes is 5 ml. The BSA range is 0.05 to 1 mg/ ml.2) From these different dilutions, pipette out 0.2 ml protein solution to different test tubes and add 2 ml of alkaline copper sulphate reagent (analytical reagent). Mix the solutions well.3) This solution is incubated at room temperature for 10 mins.4) Then add 0.2 ml of reagent Folin Ciocalteau solution (reagent solutions) to each tube and incubate for

30 min. Zero the colorimeter with blank and take the optical density (measure the absorbance) at 660 nm

5) Plot the absorbance against protein concentration to get a standard calibration curve.6) Check the absorbance of unknown sample and determine the concentration of the unknown sample.

Purification and characterization of invertase:

Crude extract was precipitated by 70% saturation with ammonium sulphate or chilled acetone

and then resuspend in 0.2 M acetate buffer.

Ammonium sulfate fractionation for purification:

Solid ammonium sulfate was added, over ice, into the crude extract to 30% saturation; aftercentrifugation (10 000_g, 20 min), ammonium sulfate was added to bring the supernatant to 100% saturation. The latter was stored overnight at 4°C and then centrifuged. The precipitate was redissolved and dialyzed against several changes of 0.01 M phosphate buffer (pH 6.5).

SDS-PAGE electrophoresis was carried out and molecular weight was

determined.

SDS-PAGE:

Protocol1) Prepare polyacrylamide gel according to standard protocol.2) Load samples and run gel at 25 mA in 1x SDS Running Buffer.3) At this point, the gel can either be transferred to a membrane or stained with Coomassive.4) Place gel in a plastic container. Cover with isopropanol fixing solution and shake at room temperature. For 0.75 mm-thick gels, shake 10 to 15 min; for 1.5 mm thick gels, shake 30 to 60 min.5) Pour off fixing solution. Cover with Coomassie blue staining solution and shake at RT for 2 hr.6) Pour off staining solution. Wash gel with 10% acetic acid to destain, shaking at RT ON.

The kinetic parameter of the purified invertase enzyme was determined and the optimum pH and temperature on the activity of the enzyme was also assayed.

Determination of Optimum temperature:

The different flask of fermentation medium are kept at different tempetature like 4C,25C,37C, and 65C. then the concentration of the all flask are carry out by Bradford method or Lowery method, the highest concentration gives the optimum tempetature. The graph of temperature v/s O.D. is prepared and the peek indicates the optimum temparature.

Determination of Optimum pH:

The different flask of fermentation medium are kept at different pH like 4,7 and 9. then the concentration of the all flask are carry out by Bradford method or Lowery method, the highest concentration gives the optimum pH. The graph of pH v/s O.D. is prepared and the peek indicates the optimum pH.

RESULTS AND DISCUSSION:

Production of invertase by fungi in shaken flask culture:

Invertase production by A. niger was studied in shaken flask culture technique by inoculating 106 spores/ml of fermentation medium containing the fruit peel waste as substrate. The C: N ratio in CHNS analyzer was estimated which shows the carbon content in orange and pomegranate was similar and comparatively more than pineapple peel whereas in the case of nitrogen, orange peel showed high value than other two substrates.

Determination of the sugar concentration:To determine the reducing sugar DNSA method of Sumner and Howells perform which indicates the reduction of sugar after fermentation process. Initial sugar concentration is higher then final sugar concentration.. For Standerd Graph (DNSA)

Sr. No. Std. solution(ml)

Dist. Water(ml)

DNSA solution(ml)

O.D(nm)

1. 1 9 1 0.122. 2 8 1 0.233. 3 7 1 0.374. 4 6 1 0.505. 5 5 1 0.586. 6 4 1 0.707. 7 3 1 0.838. 8 2 1 0.859. 9 1 1 0.9010. 10 0 1 0.92

For Samle1. 1 9 1 0.442. 3 7 1 0.533. 5 5 1 0.71

Protein Estimation:The concentration of total protein content is determine by Bradford method.. The protein concentration of flask at different temperature and pH are also measured by Bradford method which gives the optimum temperature and pH for the invertase production. Accordin to that the optimum tempetature and pH is 37C and 4 respectively.

For standerd graph (Bradford method):

Sr. No.

Std BSA(ml)

Dist. Water(ml)

Bradford solution(ml)

O.D. at430nm

Blank 0 8 1 0.001 1 7 1 0.652 2 6 1 0.773 3 5 1 0.904 4 4 1 1.115 5 3 1 1.136 6 2 1 1.257 7 1 1 1.338 8 0 1 1.50

For sampleA.niger 1 7 1 1.28

Optimum pH and temperature on the basis of invertase produvtion:concentration of flask at different temperature and pH are also measured by Bradford method which gives the optimum temperature and pH for the invertase production. Accordin to that the optimum tempetature and pH is 37C and 4 respectively. For optimum temperature on the basis of invertase production:

Temp. Sample(ml)

Dist. Water(ml)

Bradford solution(ml)

Bradford solution(ml)

4C - - - -25C 1 7 1 0.40037C 1 7 1 0.43265C - - - -

For optimum pH on the basis of invertase production:pH Sample

(ml)Dist. Water(ml)

Bradford solution(ml)

Bradford solution(ml)

4 1 7 1 0.4967 1 7 1 0.3089 1 7 1 0.232

Enzyme Activity:

The enzyme activity of invertase is measured at different time interval of 5 min and the maximum activity was observed at 25 min at 37C and pH-4.0

Sr. No.

Enzyme solutiom(ml)

Sucrose solution(ml)

Time ofReaction(min)

DNSA(ml)

O.D. at430nm

1 1 9 5 1 0.0782 1 9 10 1 0.223 1 9 15 1 0.684 1 9 20 1 1.035 1 9 25 1 1.306 1 9 30 1 1.077 1 9 35 1 1.028 1 9 40 1 1.009 1 9 45 1 0.8610 1 9 50 1 0.71

Determination of the molecular weightThe active fractions were pooled separately and the purity was checked by SDS-PAGE. The fraction gave more than one band, indicating that it contained some other proteins.The single band obtained by SDS-PAGE indicated that the protein was a single polypeptide.By performing SDS-PAGE the molecular weight of invertase is about 67 Kda determine.

References

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Appendix:

For SDS-PAGE

30% acrylamide 10% SDS 10% APS (make fresh each time) TEMED 1.5 M Tris, (pH 8.8) (resolving gel)

M Tris, (pH 6.8) (stacking gel) Tris : 15 g Glycine : 72 g SDS : 5 g 10% (v/v) acetic acid 0.006% (w/v) Coomassie Blue dye 90% ddH2O 10% (v/v) acetic acid 25% (v/v) isopropanol 65% ddH2O ddH2O : 16 ml 0.5 M Tris, (pH 6.8) : 5 ml 50% Glycerol : 8 ml 10% SDS : 8 ml mercaptoethanol 2 ml (add immediately before use) bromophenol blue

Bradford reagent:

Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol

85% (w/v) phosphoric acid.

1 M NaOH

Lowery reagent:

BSA stock solution 2% sodium carbonate 0.1 N NaOH solution 1.56% copper sulphate solution 2.37% sodium potassium tartarate solution. Folin - Ciocalteau reagent solution (1N)