A Comparitive Study on Invertase Production, Characterization & Optimization by Penicillium Brevicompactum & Penicillium Chrysogenum on Pineapple Peel Waste.
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INTRODUCTION
Microorganisms are used extensively to provide a vast range of products and services. They have proved to be particularly useful because of the ease of their mass cultivation, speed of growth, use of cheap substrates (which in many cases are wastes) and the diversity of potential products. Their ability to readily undergo genetic manipulation has also opened up almost limitless further possibilities for new products and services from the fermentation industries (Trevor Palmor, 2004). Microorganisms are a large and disease group that exist as its single cell or cell cultures. These include bacteria, fungi, algae, protozoa and infectious agents at the borderline of life. Microbes are present most abundantly in soil, atmosphere and water plays a important role in the biochemical agent for the conversion of complex organic compounds into simple organic compounds (Glazer and Nikaido, 1995). Microorganisms are closely associated with health and welfare of human beings. Some Microorganisms are beneficial and others are detrimental. For example, Microorganisms are involved in the making of yoghurt, cheese and wine, in the production of Penicillin, interferons and alcohol, and the processing of industrial and domestic wastes. Microorganisms can cause disease, spoil food and deteriorate materials like iron, pipes, glass lenses and wood pilings (Glazer and Nikaido, 1995). Most of these Microorganisms irrespective of their origins, were subsequently modified by conventional strain improvement strategies, using mutagenesis or breeding programmes to improve their properties for industrial use (Trevor Palmor, 2004). In most cases regulatory considerations are of major importance when choosing microorganisms for industrial use. Fermentation industries often prefer to use established GRAS(Generally Regarded As Safe) microorganisms, particularly for the manufacture of food products and ingredients(Trevor Palmor, 2004). We mainly focus here on fungi and yeasts. It has recently become evident that some yeasts are also able to grow filamentously and vice versa, either n nutritional starvation or during certain stages of their development. There are some biotechnological fields in which fungi play an important role, namely the production of food, extra cellular enzymes, and secondary metabolites and of organic chemicals. In each case fungi compete with other biological systems such as bacteria or cell cultures or with the chemical synthesis if the respective compounds. Several extra cellular enzymes are used to produce food. They are either naturally synthesized or recombinantly introduced into foreign host (e.g., Devchand and Gwynne, 1991; Gelissen et al., 1992; Kinghorn and Unkles, 1994). For instance, the milk clotting enzyme chymosin from calves is recombinanly produced in Trichoderma and Aspergillus (Uusitalo et al. , 1991; Tsuchia et al. , 1993) and subsequently used for cheese production. The biological transformations in soil are catalyzed by soil enzymes. Soil enzymes are generated in the soil as a result of the activity of microorganisms and the activity of these enzymes are less influenced by the moisture balance, pH and also an optimum temperature conditions prevailing in soil. The most important as well as common enzymes present in the soil are urease, phosphatase, amylase, cellulase, invertase, protease etc., (Glazer and Nikaido, 1995). During the growth of microorganisms under ordinary culture conditions in a limited volume of medium, the growth rate continuously changes and the chemical composition of the medium varies as a result of the metabolism of the organisms (A. Davies, 1956). One way of achieving the culture in a steady state is by the use of continuous culture technique in which the growth rate is determined by the concentration of one particular constituents of the growth medium (A. Davies, 1956). The enzyme invertase β-D- fructofuranoside fructohydrolase E.C.3.2.1.26 catalyses the hydrolysis of any compound with an unsubstituted β-D- fructofuranosyl residue and the prime substrate in sucrose ( George Boguslawski, 1983) Sucrose + H2O invertase D- glucose + D fructose
A wide range of microorganisms produces an invertase and can thus utilize sucrose as a nutrient. Thus hydrolyzing sucrose to produce fructose and glucose in a equimolar mixture named inverted sugar. The inverted sugar is incorporated more easily in the industrial preparation and has more added value than sucrose (Chou and Jasovsky, 1993). Invertase is basically a yeast derived enzyme.But many microorganisms including fungi and bacteria produce this enzyme. Invertase production by microorganisms such as Aspergillus niger.ATC20611 (Hidaka etal., 1988), Acureobasid pullulans ITFCC 10524 (Yun etal., 1994), Aspergillus oryzae (Kuakake et.al., 1998), Zymomonas mobilis (Park etal., 1991) and Clavibaeter michiganensis (Baer etal,1998) are known (A.B.Bhake and M.B Patil,2005). The high cost of invertase production process, together with the low process extraction yield and purification have been limiting factors for the use of enzymatic hydrolysis in the industrial process. Reduction in the production cost could make if a more efficient and reliable technique to be used industrially for commercial production (Rodrigues et al., 2000).
Review of Literature
Fernandez.R.D et al., (2007), studied seventeen different strains of filamentous fungi and compared their abilities for the production of β- fructofuranosidase. Three of them Aspergillus oryzae IPT-301, Aspergillus niger ATCC 20611 and strain IPT-615, showed high production with total fructosyl transferase activity higher than 12,500 units 1(-1). In addition, β- fructofuranosidase of those strain have a high fructosyl transferase activity to hydrolytic activity ratio.
The regulation of invertase synthesis in Aspergillus niger with different carbohydrate was studied by M.C. Rubio and A.R. Navarro 2006. The synthesis was induced by β fructofuranoside substrate such as raffinose, sucrose and pyranose where as glucose and fructose which are their reaction product act as a repressor.
Cuitlahua C Aranda et al., (2006) was studied the invertase activity expressed by Aspergillus niger Aa-20 under different concentration of two substrates using solid state fermentation on poly-urethane glucose was used as repressor and sucrose act as the inducer. Induction repression ratio obtained using any glucose concentration was at least 2.5 times higher than that under basal condition
Ikram-ul-haq etal., (2005) studied invertase producing five strains of Saccharomyces cerevisiae was isolated form the dates. They were designated as GCA IV invertase producing capacity of the strains were analysed. The GCA II strain was found to be the better invertase yielding strain than the other cultures. The enzyme yield increased by 47.7% after the culture conditions were optimized.
A.B.Dhake and M.B.Patil (2005) studied with Penicillium purpurogenum, which produces both extracellular as well as intracellular acidic invertase. The effect of substrate feeding on the production of invertase. The addition of vitamins has no significant impact on the invertase production where as surfactants inhibit the growth of organism.
Anauclaudia Santana de Almeida et al., (2005) reported a comparative study on the sucrase hydrolyzing capacity of the soluble free invertase and auto immobilized invertase present in the intact cells of Cladosporium cladosporides. The immobilization sums to improve the invertase operational stability and gives a Km value higher than the soluble ones. So the immobilized cells can be sued in the industrial bioreactor instead of the soluble invertase, inorder to produce high quality product at low cost.
Anjali Hans and Arun Dev Sharma, (2005) reviewed the production and partial characterization of invertase from 9 Streptomyces strains designated as (ALKC 1-9) which was isolated from soil sample. When grown under the optimized conditions ALKC 1, 3, 4, and 5 showed high invertase activity therefore designated as the potential can dictate for the production of high fructose syrup and invert sugar production production at industrial scale.
K.Meena and T.K. Raja (2004) reported the immobilization of yeast invertase by gel entrapment. In their work they checked the feasibility of gel entrapment of yeast invertase using strontium alginate, barium alginate and calcium alginate. Calcium alginate entrapped invertase showed a considerable decrease in Km value after fifth day where as Barium and Strontium does not, because of their pore size and the permeability of the sucrose molecule. Thus strontium alginate and Barium alginate have displayed better properties such as mechanical strength of the beads, swelling characteristic and enzyme activity.
Mirza et al., (2003), studied the influenze of the carbon source during invertase production is well known, but little is understood about the nitrogen source influenze.
Ikram ul Haq et al., (2003) dealt with the time course study of three different varieties of Sacharomyces cereviseae (GCB-K5, GCA-II & KR-18) during invertase production by submerged fermentation.
Sikander Ali et al., (2003) studied the optimization of cultural conditions for the biosynthesis of β- fructofuranosidase by Saccharomyces cereviseae by submerged fermentation.
Skowronek et al., (2003) studied invertase activity of psychrotrophic fungi using 98 strains isolated from Artic tundra soil, among these strains Cladosporium herbarum1, was the most effective for invertase production which was selected by the cup plate method. Invertase showed a maximum activity at 500C and pH 6.0.
Mirza Ahsen Baig et al., (2003) studied the effect of carbon source on invertase production was well known but very little was understood about the effect of nitrogen source. Hence a study was carried out with three different nitrogen sources using Saccharomyces cerevisiae strain for the production of invertase. It was found that when a very little amount of urea was added to the fermentation medium there was an increase in invertase production. Kiran Shafiq et al., (2002), studied the effect of different mineral nutrients on the production of extra cellular invertases by Sacharomyces cereviseae GCB-K5.
J.C. Rossi Alva and M.H. Muguiz Rocha Leao (2002) made a comparative study on the invertase activity of free and entrapped cells of Saccharomyces cerevisiae mutant. The work concluded that the entrapped yeast cells had a weak ability to consume sugar.
P.L.Albertson, et al., (2001) determined both the soluble and cell wall invertase activity in sugarcane. The pH for the soluble and neutral invertase was found to be 4.5 and 7.3.They concluded that the soluble acid invertase (SAI) activity was greatly reduced in mature tissue and cell wall invertase (CWI) reduced in older leaves. Neutral invertase (N1) remained constant irrespective of tissue ages. The role of SA1 was in growth and differentiation CWI was intrinsically involved in these process where as NI plays a house keeping role in maintaining hexose concentrations with in the cytosol
Rodrigues et al.,(2000) characterized the invertase from Pichia anomala. The utilization of fungal biomass for invertase production reduced the technological cost and provided a good result. Hence the production of invertase from fungal biomass can be implemented industrially which reduces the cost of production.
. Coutinho et al., (1999) studied the influence of reaction products in the inversion of sucrose by invertase and also stated that the enzyme has a great operational stability. Hence the enzyme can be reused significantly without any loss of activity.
Arruda et al., (1999) did a comparative their study on the characterization of free soluble invertase and invertase that are entrapped into calcium alginate beads. The study revealed that the activity of the entrapped invertase was higher than the soluble free invertase.
B.K. Gogoi et al., (1998) isolated and characterized the extracellular α-amylase and invertase from amylolytic yeast Saccharomycopsis fibuligera and showed the Saccharomyces strain as an excellent producer of extracellular α-amylase and invertase under the cultivation conditions. Therefore this strain can be used industrially for the dual enzyme fermentation.
Baer et al.,(1998) reported that the maximum extracellular invertase was produced after 4 days and intracellular invertase level reached to its peak on 5 days after inoculation. They also reported that the optimum production of invertase by Clavibacter michiganensis after 4 days of incubation.
The effect of pH, temperature and dissolved oxygen concentration on the production of glucose-6-phosphate dehydrogenase and invertase using Saccharomyces cerevisiae. The study explained that the G6PDH was highly sensitive to pH, temperature and dissolved oxygen (DO) variation. Its production was increase three times when the temperature, pH and dissolved oxygen was increased where as the invertase activity decreased at least 50% when dissolved oxygen, temperature, pH were increased as reported by J. Abrahao Neto et al., (1997).
Kurkura et al., (1996) studied the effect of pH on the transfructosylation and hydrolysis by β-fructofuranosidase from Aspergillus oryzae showed that the maximum activity was observed in pH 4.5. Hence the pH 4.5 was considered as the optimum pH for invertase production.
Chou C.C and Jasovsky.,(1993) studied the advantages of Ecosorb TM precoats in the production of liquid sugar. The study concluded that the inverted sugar can be more easily incorporated in the industrial preparation because it is sweeter like sucrose and does not crystallize easily.
The most effective strain for the production of invertase was selected using the method of test tube micro culture. It was then subjected to mutagenesis and the product was analysed for invertase activity. The mutant strain showed an 2- fold increase in the invertase activity when compared with the parental strain as reviewed by J.Fiedwrek etal.,(1990).
A sucrose hydrolyzing enzyme was isolated from Zymomonas mobilis and characterized by Lawrence etal., 1990. The extracellular protein isolated was dialysed by two dimensional gel electrophoresis. The molecular weight of the protein purified was found to be 46 kDa. The partially purified protein also catalysed transfructosylation reactions.
G.E.Bracho and J.R.Whitaker (1989) studied the purification and partial characterization of potato (Solanum tuberosum) invertase and its endogenous proteinaceous inhibitor. An purified acid invertase contains 10.9% carbohydrate by DEAE sephadex chromatography. At pH 4.7, the enzyme was found to be most stable and had a maximum activity around pH 5. An inhibitor which was purified contains single polypeptide without glycol group stable over the pH 2-7 and temperature 370C for 1 hour.
Moussa et al., (1987), studied the purification, properties and comparison of one invertase, 5 exoinulinases and 3 endoinulinases from Aspergillus ficuum and showed that invertase was more active on sucrose than on inulin.
K.C.Aker et al., (1987) study was undertaken as an initial step in the development of a full-scale process for the bioconversion of the fermentable sugars in waste whole banana by the nutritionally well accepted yeast Candida utilis. And also reported growth kinetics, biomass yield, protein content and the specific oxygen respiration rate of C.utilis for the various fermentations investigated.
Palanivelu et al.,(1984) studied the co-induction of sucrose transport and invertase activities in the Thermophilic fungus, Thermomyces lamuginosus. The incorporation of sucrose into the fungus occurred only in the mycelia which were previously exposed to sucrose or raffinose. And they confirmed that the transport of sucrose does not dependent on the ATP but by a proton gradient across the plasma membrane.
Marian Carlston et al., (1984), studied that the SNF1 gene product of Saccharomyces cereviseae is required to derepress expression of many glucose-repressible genes, including the SUC2 structural gene for invertase.
Ramesh Maheshwari et al., (1983) reported that the invertase activity appeared in the culture of Thermomyces lamuginosus only when supplement with sucrose or raffinose as the carbon source. The behavior of the T. lamuginosus differs from invertase of yeasts and molds in many features such as invertase activity depends on the carbon source used, activity was also related to the growth of the fungus (i.e) the activity was seen during the fungal growth but disappeared after maximal growth. The enzyme was very unstable in the presence of cycloheximide and the invertase was readily secreted into the medium.
George Boguslawski (1983) reported that the invertase catalyses the hydrolysis of any compound with an unsubstituted β-D-fructofuranosyl residue and the prime substrate was sucrose. A simplified method for measuring the activity of invertase. In this method, the fructose produced from sucrose by invertase action was converted to D-sorbitol by sorbitol dehydrogenase in the presence of NADH, the product formed was measured at 340 nm. In the same year D.B. Lee and S.J. Free studied the isolation and characterization of invertase from Neurospora mutants. The mutants showed an increase in invertase activity when the strain was transferred to a carbon free medium.
F. Parra et al., (1980) studied the effect of media constituents on invertase formation. The study used the strain of Saccharomyces carlbergensis in order to reveal the role of the internal invertase in synthesis and secretion. And they concluded that the intermediate forms of invertase were secreted by cells when grown in 2-deoxy-D-glucose, showing that the complete glycosylation of the enzyme was not essential for its release from the cell.
The light and intermediate molecular forms of yeast invertase act as the precursors of the heavy enzyme. The level of the intermediate molecular forms of invertase depends on the metabolic state of the yeast Sacchromyces cerevisiae. The light invertase was not affected by the changes in the glucose concentration where as the heavy and intermediate molecular forms undergo changes when the glucose level in the medium was decreased was reported by C.F. Iglesias et al., (1980). RA.Leigh et al.,(1979) studied the presence of sucrose and acid invertase in the vacuole .And isolated from the freshly cut slices of the storage root of beet root and the slices washed with aerated water was determined using correlative method and concluded that beet root have most of the sucrose and acid invertase in the vacuoles.
E.J.Laishley, (1975) studied the regulation and properties of Chlostridium pastaeuranium utilizing sucrose as its carbon sourcefor the growth and concluded that the enzyme synthesized could be repressed by addition of fructose to a sucrose growing culture.
Kazuhiro et al., (1974) worked on the purification and properties of Dextransucrase and invertase from Streptococcus mutans, and concluded that the dextransucrase had both dextran synthesizing and invertase like activity.
Howard.K (1973), studied the characterization of invertase from carcinogenic Streptococcus mutans GS-5. And it showed that none of the common glycolytic intermediate or adenine nucleotides had any significant effect on enzyme activity. The enzyme doesnot appear to be catabolically repressed by glucose nor induced by sucrose.
A comparative study of dextransucrase and invertase from Streptococcus mutans was studied by Kazuhiro et al., (1973), which showed that the antigenic determinant of invertase was different from dextransucrase on immunodiffusion.
Cynthia.H.Bigger et al., (1972), showed that the slime mutant of Neurospora crassa produced invertase and was serologically identical to wild type Neurospora crassa invertase. The mutant secreted 95 % of the invertase into the medium whereas cells of the wild type retained almost all the invertase.
The biochemical and histochemical localization of invertase in Neurospora crassa during conidial germination and hyphal growth was studied by Patricia L.Y. Chung et al., (1970), which showed (1) conidial invertase was uniformly distributed along the cell periphery, (2) growing hyphal tips of germinating conidia showed pronounced invertase activity. J.R. Trevithik and R.Z. Metzenberg (1966) were reviewed. The secretion of invertase by young mycelia of Neurospora, which indicated that the fractionation of light invertase from heavy invertase occurs at the cell wall. The findings have shown that invertase can undergo a reversible transition between an active monomer and an aggregated active form. It was expected that the light invertase escapes rapidly across the cell wall than the heavy form. Hence the extracellular invertase constitutes mainly the light form.
A study was made of Penicillium chrysogenum and some other fungi to determine the relative distribution of intra and extra cellular invertase produced by them in submerged fermentation. The proportion of each type of enzyme varied with the organism and the period of fermentation. Most of the enzyme initially boumd to the mycelium was released into the medium with the progress of fermentation. Difference was observed in effect of cultural conditions of enzyme production in P. chrysogenum & S. cereviseae. This was done by Poonawalla et al. , 1965.
E.P. Hill and A.S. Sussman (1964) studied the levels of trehalase and invertase formed during the development of Neurospora. Invertase activity was highest after the mycelial growth has been completed where as trehalase was found in ungerminated. The result confirmed that their activity was coordinately controlled. A comparative study on the production of extracellular and total invertase by Candida utilis, Saccharomyces cerevisiae and other yeasts are carried out by R.G. Dworschack and L.J. Wickerham (1960), Candida utilis was found to be superior when compared with the Saccharomyces cerevisiae and Saccharomyces carlbergensis in the production of extracellular and total invertase.
Invertase formation in Saccharomyces fragilis was studied by A. Davies, (1956), using continuous culture technique. The invertase formation was inhibited by glucose at concentration greater than 0.001 % (w/v). The mean generation time, the concentration of ammonia and growth factors had no significant effect on invertase activity.
The effect of the generation time and the concentration of ammonia and growth factors on the formation of Invertase in Saccharomyces fragilis were studied using continuous culture technique by R. Davies, 1953. The study revealed that the addition of sucrose and raffinose to the medium aerobically stimulated the production of invertase.
AIM AND OBJECTIVES
The main aim of the study is the production and characterization of the invertase enzyme from an isolated species using pineapple peel waste as the carbon source .The objective are as follows:
1 Isolation and enumeration of soil microorganisms.
2 Selection of organism based on the utilization of sucrose.
3 Optimization of culture conditions.
4 Determiniation of pH, Temperature, Biomass, Reducing sugar, invertase activity, Protein, and Substrate concentration, Standardization of enzyme activity.
5 Effect of pH, temperature & substrate concentration on invertase activity.
6 Partial purification of invertase enzyme from Penicillium chrysogenum & Penicillium brevicompactum.
7 Determination of protein & specific activity of invertase.
MATERIALS & METHODS
ISOLATION AND PURIFICATION OF FUNGI FROM SOIL
A large number of fungi of different groups are found in soil. They constitute the major place among soil microorganisms .A small amount of soil sample was collected from four corners and centre of the sugarcane field at udumalapet by making a ‘V’ shaped pit .They were mixed to make one lot . 10g of the soil sample was weighed and then the sample was diluted with sterile distilled water .1.0 ml of the diluted soil suspensions was transferred aseptically into the CD Agar plates [Supplemented with aureomycin antibiotic each 30 mg /L] Gently rotate the plate so as to spread the suspensions on the medium .The plates were incubated at 37 0 C for 4-5 days (R.C Dubey and D .K Maheshwari 2007).
ISOLATION OF PURE CULTURE:
The fungi isolated from the soil contain a mixed population exhibiting diverse morphological and physiological characters. Single germinating spores were picked from the mixed culture containing several spores and sub-cultured (S.M Reddy and S.Ram reddy 2005) A pure culture was produced by repeated sub culturing. The purified cultures were then transferred to agar slant and sub cultured fortnightly which was then stored at 4 0 C until use. (Anjali Hans and Arun dev Sharma 2005)
SELECTION OF ORGANISM BASED ON THE UTILIZATION OF SUCROSE
The organism present in the pure culture were identified using “Lactophenol Cotton Blue” mounting method which was especially used for the identification of fungi .The organism used through out the experiment was Penicillium chrysogenum & Penicillium brevicompactum was selected and identified commonly using 2% glucose medium which was basically depends on their ability to utilize sucrose as their sole carbon source (R.C Dubey and D .K Maheshwari 2007 and Domcsh K M et al., 1980, Srinivasan etal., 1991 Nakamura etal., 1997).
GROWTH MEDIA AND CELL SUSPENSIONS
The basal medium in which the Penicillium chrysogenum & Penicillium brevicompactum was maintained in the Czapek Dox Agar medium .The composition of the CD medium was given below: Composition of the CD medium (g/L) Sucrose : 30 Sodium Nitrate : 2.0 Dipotassium hydrogen phosphate : 1.0 Magnesium sulphate : 0.5 Potassium Chloride : 0.5 Ferrous sulphate : 0.01 pH : 5.0-5.6
(The medium was supplemented with 30mg /L of aureiomycin antibiotic) (Anjali Hans and Arun dev Sharma, 2005 & Dhake and Patil, 2005)
The composition of fermentation medium was given below :
Composition of fermentation medium (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 medium substituted with pineapple peel waste which was used as the carbon source. (Marcin Skowronek et al., 2003).
PROCESSING OF THE SUBSTRATE
The pineapple peel waste was obtained from the local fruit stall at Coimbatore was washed several times in distilled water and then sliced .The sliced pieces were spread on the trays and then sieved .The substrate was stored in the polyethylene bags at room temperature .They were autoclaved at 15 lbs for 20 minutes before use.
CULTURE CONDITION
Erlenmeyer flask (500 ml ) containing 100 ml of the production medium was inoculated with the culture from 24 hours old stock culture slants (Gogoi et al.,1998) and incubated at 300 C . After 3 days incubation, Invertase activity, biomass, pH, and reducing sugar content of the medium were determined for 10 days with 24 hours interval. (Anjali Hans and Arun Dev Sharma, 2005).
CELL FREE EXTRACT PREPARATION:
The mycelial mass was collected by filtration after 3 days of incubation .The filtrate was centrifuged at 10,000 rpm for 20 minutes at 40 C .The supernatant was used as the source of Invertase enzyme mainly contains extracellular enzyme. The mycelial mass was washed several times with distilled water and used for dry weight determination .The cell growth (biomass) in the liquid culture was monitored by dry weight that was expressed as g-1 of the microbial culture.( Dhake and Patil ,2005 ; Anjali Hans and Arun Dev Sharma, 2005 ).
OPTIMIZATION OF THE CULTURE CONDITION:
In the industrial exploitation of microbes greater attention is always given to the culture design and standardization of the physiochemical parameter of the medium. Since microorganisms exhibit diverse pattern of nutritional and environmental requirement. The optimization of commercial processes but also for the meaningful determined by measuring the enzyme activity by varying the single parameter such as pH, temperature and substrate concentration of the medium keeping the remaining parameter unaltered (Peter F Stanbury et al., 1997).
1. EFFECT OF DIFFERENT pH ON THE INVERTASE PRODUCTION
The optimization pH for invertase production was determined by altering the pH of the fermentation media from pH 3.0 to 6.0 & 8.0. The 100ml of the pH adjusted media was distributed in Erlenmeyer flask under sterile condition and inoculated with 1.0 ml of the 24 hours old cultureof the two different species separately. It was then subjected to incubation at room temperature .The mycelial weight and invertase production was assayed .The effective pH for the maximal invertase production was determined. (Peter.F.Stanbury etal., 1997; Bull et al., 1990 and Savals et al., 1993).
2. EFFECT OF DIFFERENT TEMPERATURE ON INVERTASE PRODUCTION The effect of Temperature on Invertase production was studied by altering the incubation temperature .The temperature was adjusted in the range from 30-700C .The 100 ml of the media with standardized pH and substrate concentration was distributed in Erlenmeyer flask under sterile conditions. It was inoculated with 1.0 ml of 24 hours old culture of the two species separately and incubated at varying temperature .The biomass and invertase yield in the medium was assayed. The effective temperature for invertase production was determined. (Peter F Stanbury etal., 1997; Bull et al., 1990 and Savals et al.,1993).
3. EFFECT OF DIFFERENT CONCENTRATION OF SUCROSE
(Carbon source) ON INVERTASE PRODUCTION
The effect of different concentration of sucrose on invertase production was studied by altering the concentration of sucrose of the fermentation media from 0.5 % - 5.0 %. The100 ml of the media with standardized pH and substrate concentration was distributed in Erlenmeyer flask under sterile condition. It was inoculated with 1ml of 24 hours old culture and incubated at the standard temperature. The biomass and invertase yield in the media was assayed. The effective sucrose concentration for the invertase production was determined. (Peter F Stanbury et al., 1997; Bull et al., 1990 and Savals et al., 1993).
4. EFFECT OF DIFFERENT CONCENTRATION OF YEAST
EXTRACT (Nitrogen source) ON INVERTASE PRODUCTION
The effect of different concentration of yeast extract on invertase production was studied by altering the concentration of year’s extract of the fermentation media from 0.5 % - 2.5 %. The100 ml of the media with standardized pH and substrate concentration was distributed in Erlenmeyer flask under sterile condition. It was inoculated with 1ml of 24 hours old culture and incubated at the standard temperature. The biomass and invertase yield in the media was assayed. The effective yeast extract concentration for the invertase production was determined. (Peter F Stanbury et al., 1997; Bull et al., 1990 and Savals et al., 1993).
PRODUCTION OF INVERTASE FROM PINEAPPLE PEEL WASTE (substituted carbon source) USING THE OPTIMUM CONDITION
The 100 ml of the fermentation media with optimum PH 5.5, substrate (pineapple peel waste) concentration 4g/100ml was transferred to the Erlenmeyer flask under sterile condition .It was then inoculated with 1.0 ml of 24 hours old culture of the two species separately and then incubated at the optimum temperature 400C. At the 6th day of incubation the biomass, invertase activity and protein content was estimated. (Anjali Hans and Arun Dev Sharma, 2005).
BIOMASS DETERMINATION
The mycelial mass was collected by filtration and its wet weight was determined by placing it on a filter paper .The dry weight of each mycelial mass was monitored by drying at 800C to constant weight (Anjali Hans and Arun Dev Sharma, 2005).
ENZYME ASSAY Fructofuranosidase assay was determined by measuring the reducing sugars released by the hydrolysis of sucrose. The reaction was carried out at 300C for 5 minutes. The reducing sugars released in the reaction mixture were assayed by DNSA method. The cell free extract obtained after centrifugation is used as the enzyme source for determining the crude enzyme activity.
ASSAY MIXTURE
The determination invertase activity was carried out of at 300C in a mixture of 1.0ml of 0.01M acetate buffer (pH 6),1.0 ml of 0.3 M sucrose solution and 0.1 ml of cell free extract (Cuitl;ahua C Aranda et al.,2006;Anaclaudia Santana et al.,2004;George Boguslawski .,1983,and 1959) The mixture was incubated at room temperature (~ 300C) for 5 minutes after making up the volume of the mixture to 4.0 ml with distilled water .The hydrolysis was stopped by adding 2.0 ml of DNSA reagents and then invertase activity was assayed as described using glucose or fructose as a standard(Sharma et al .,2002)
STANDARDIZATION
1 0.1 ml of cell free supernatant was taken in separate test tubes.
2 To that added 1.0 ml of sucrose solution and 1.0ml acetate buffer. Incubated at 300C for 5 minutes.
3 Pipette out 0.2-1.0 ml of the working standard solution into series of test tubes.
4 Make up the volume in all the test tubes to 4.0 ml with distilled water.
5 Added 2.0ml of DNSA reagent to all the test tubes.
6 Incubated in a boiling water bath for 15 minutes.
7 The tubes were cooled and the intensity of the colour developed was read at 540nm.
8 A standard graph was prepared with these data for computing enzyme activity of the sample (Srinivasa et al .,1990)
ENZYME UNITS
One units of invertase (IU) was defined as the amount of enzyme which liberated / mg of product / minute /ml under the assay condition. (Anjali Hans and Arun Dev Sharma 2005)
PURIFICATION OF INVERTASE The crude enzyme was treated with ammonium sulfate with continuous stirring and was precipitate into 30% saturation. The precipitate was collected by centrifugation 10,000rpm for 20 minutes at 40C.The precipitate obtained was dissolved in acetate buffer of pH 5.5.The enzyme solution was dialyzed against the same buffer for 12 hours at 40C with occasional change of buffer to remove the salts. The dialysate solution was then treated with 50% acetone .The invertase activity was determined for the ammonium sulfate treated sample the dialysate and acetone treated sample.
ESTIMATION OF PROTEIN The protein content present in the crude extract and partial purified sample were estimated according to the Lowry’s method using Bovine serum albumin as the standard (Lowry et al., 1951).
1 0. 1 ml of the extract was taken in the test tubes.
2 Added 0.5ml of the acetate buffer to the enzyme extract.
3 Pipette out 0.2-2.0 ml of the working standard into a series of test tubes.
4 Make up the volume to 1.0 ml in all the test tubes by adding distilled water. A tube with 1.0ml of the water serves as the blank.
5 Added 5.0 ml of alkaline reagent D to all the tubes.
6 Then added 0.5 ml of the Folin’s phenol, reagent mixed well and incubated in dark at room temperature for 30 minutes.
7 The blue color developed was then read at 660nm.
8 A standard graph was drawn and the amount of protein in the sample was calculated (Sadasivam and Manickam, 1997).
SPECIFIC ACTIVITY
The specific activity was calculated using the formula (Trevor Palmer, 2004).
Invertase Activity Specific activity = ————————— Protein
RESULT
The comparative study between Penicillium chrysogenum & Penicillium brevicompactum was done and the optimum conditions for enzyme production by these two Penicillium Spp. were found out and the best strain was identified based on the invertase production. Enzyme source
The enzyme source was culture filtrate of Penicillium chrysogenum & Penicillium brevicompactum grown separately by submerged fermentation at room temperature (400C) for 6 days and 4 days respectively.
Optimum pH
The pH optimum of invertase from Penicillium brevicompactum was found to be 5.0 at 4th day of incubation, corresponding to 2.436 IU/ml of reducing sugar at room temperature for 5 minutes. (Table 1 & Figure 1)
The optimum pH of invertase from Penicillium chrysogenum was found to be pH 5.5 at 6th day of incubation, corresponding to 2.233 IU/ml of reducing sugar at room temperature for 5 minutes. (Table 2 & Figure 2)
Optimum Temperature
The optimum temperature of invertase was found to be 400C for both Penicillium chrysogenum & Penicillium brevicompactum and corresponding to 1.660 IU/ml and 1.710 IU/ml of reducing sugar at room temperature for 5 minutes respectively. (Table 3, 4 and Figure 3, 4).
Optimum Sucrose Concentration
The effect of various carbon sources on the production of invertase by Penicillium chrysogenum & Penicillium brevicompactum species were illustrated in table 5 and 6. The maximum enzyme activity was found out at 2 % sucrose concentration for both the Penicillium Sps.
Optimum Nitrogen Concentration
The effect of various nitrogen sources on the production of invertase by Penicillium chrysogenum & Penicillium brevicompactum species were illustrated in table 7 and 8. The maximum enzyme activity was found out at 1 % yeast extract concentration for both the Penicillium Sps.
Purification The result of purification studies of Penicillium brevicompactum & Penicillium chrysogenum has summarized in table 9(A) and 9(B) respectively. The specific activity of the purified enzyme after purification showed two fold increase in activity.
Crude Extract
The crude extract of the enzyme from Penicillium brevicompactum had specific activity of 2.764 IU/ml.
The crude extract of the enzyme from Penicillium chrysogenum had specific activity of 2.326 IU/ml.
Ammonium Sulphate precipitation
The crude extract was subjected to ammonium sulphate precipitation. The precipitate of Penicillium brevicompactum had the specific activity was 2.958 IU/ml.
The crude extract was subjected to ammonium sulphate precipitation. The precipitate of Penicillium chrysogenum had the specific activity was 2.607 IU/ml.
Acetone treatment
It is then subjected to 50% acetone treatment. The product of Penicillium brevicompactum had specific activity of 3.253 IU/ml. And the product of Penicillium chrysogenum had specific activity of 2.924 IU/ml.
TABLES
Table: 1
Effect of pH in invertase production by Penicillium brevicompactum.
Invertase is an enzyme that catalysis the hydrolysis of sucrose into glucose and fructose. It is also called sucrose or saccharase. The official name for invertase in β- fructofuranosidase, which implies that the reaction catalysed by this enzyme is the hydrolysis of the terminal non-reducing β-fructofuranosyl residue in β-fructofuranoside. Traditionally, invertase has been produced by liquid or submerged fermentation. In recent years, fermentation has gained renewed interest from researchers for the production of enzymes in view of several economic and engineering advantages. The selection of substrate for the fermentation media involves screening of several agricultural waste materials for microbial growth and product formation. An ideal media should provide all necessary nutrients to the microorganisms for their optimum functioning. Using various strains that were identified using Lacto phenol Cotton Blue mounting method carried out several studies on the production of invertase. The microorganism was isolated on the basis of its sucrose utilization capacity. The fungal strains used for the present study was Penicillium Spp., the biomass utilization of the fungus Penicillium brevicompactum& Penicillium chrysogenum as a natural support for invertase (auto immobilization), is a low cost technology with good results (Dhake, A.B., and Patil, M.B., (2005). The selection of substrate for the fermentation media involves screening of several agricultural materials for microbial growth and product formation. Several studies on the production of invertase were carried out by various fungal strains using different substrates under laboratory conditions. (Hidaka et al., 1988; Yun et al., 1995; Park et al., 1991; Baer et al., 1998; Dhake and Pattil, 2005). The substrate used for the present study was pineapple peel waste. Though invertase can be produced by the use of many substrates, the period of incubation for maximum production is still under question. In our study the maximum production was obtained on the 4th day of incubation for Penicillium brevicompactum and 6th day for Penicillium chrysogenum for pineapple peel waste. Baer et al., 1998, reported similar results. When Clavibacter mechiganesis was used for invertase production. The invertase level reached to its peak on the 5th day of inoculation. The optimum pH was determined by carrying out the study at varying pH within the range of 3.0 to 6.0 and 8.0. From this study the pH for maximum invertase production was found to be pH 5.0 for Penicillium brevicompactum and pH 5.5 for Penicillium chrysogenum for the substrate. Similar observation was obtained by John R.Trevithick and Robert L. Metzenberg (1966) using Neurospora, B.k.Gogoi etal ., 1998 using Saccharomycopsis fibuligera, Rubio. M.C and Navarro A.R (2006) using Aspergillus niger. By assaying the effect of temperature 200C, 30ºC, 40ºC, 50ºC, 60ºC and 70ºC, the optimum temperature was monitored. The optimum temperature for invertase production was found to be 40ºC for pineapple peel waste. Studies done by Ghosh. B.K et al., also showed that an optimum temperature of 30ºC is required for the maximal production of invertase by Saccharomyces mutant. This observation was also supported by Juan Carlos Rossi Alva et al., (2002) using Saccharomyces cerevisiae. At 70ºC, the enzyme activity will be high due to the evaporation of the media. The activity could be obtained only for the first five days and then the media got evaporated. The bioprocess production of inverted sugar as any other biotechnological process is very complex due to the number of variables involved. Hence a balanced environment with optimum temperature, pH and substrate concentration in necessity to reach a good productivity (Blancd etal., 1997) The enzyme activity was studied by growing the Penicillium Spp., under optimum culture condition using pineapple peel waste as the carbon source. The effect of various parameters on the crude enzyme obtained by centrifugation was studied. The data obtained reveals that the optimum temperature for the enzyme activity was 40ºC when incubated at varying temperature 30ºC to 70ºC. The result obtained was also supported by Dhake.A.B, and Patil, M.B., (2005) by using the strains Penicillium brevicompactum & Penicillium chrysogenum, the enzyme obtained from this species also showed an optimum temperature for the enzyme activity at 40ºC. Most purification protocols consist of 3±4 major steps. In this study, the invertase enzyme, from Penicillium brevicompactum & Penicillium chrysogenum, was purified in steps consisting of concentration of protein by ammonium sulphate precipitation and acetone treatment. The enzyme purification was done using invertase from Penicillium brevicompactum & Penicillium chrysogenum. The purified enzyme had low protein content when compared with the crude enzyme source. The activity of the enzyme source was increased after purification. Skowronek et al., 2003 by enzyme obtained from Cladosporium herbarum, also supported this.
The use of free enzymes in industrial applications has been limited, many due to high cost of enzymes, their instability and irrecoverability. Using immobilizing enzymes, which lead to greater product purity, cleaner processes and economic operational costs, circumvents these limitations of the enzymes. (Meena and Raja, 2003). The present study concludes that the pineapple peel waste can be more effectively used as a carbon source for the production of invertase using Penicillium Spp., under the optimized culture condition. Hence it can be substituted as a carbon source for the commercial production of invertase. Between the two species used, Penicillium brevicompactum shows more invertase production than Penicillium chrysogenum using pineapple peel waste as the substrate.
SUMMARY & CONCLUSION
Invertase production by Penicillium brevicompactum & Penicillium chrysogenum., at varying pH and period of incubation was studied using Czapek Dox agar as the basal medium. The sucrose source of the medium was substituted by pineapple peel waste. The result of the study reveals that the increase in the incubation time increases the enzyme production up to 4th day and 6th day of incubation for Penicillium brevicompactum & Penicillium chrysogenum respectively. Further increase in incubation period showed a gradual decrease in the enzyme production. The effect of pH on invertase production was studied using the carbon substituted media and it was inferred that the invertase production was maximum at pH 5 and pH 5.5 for Penicillium brevicompactum & Penicillium chrysogenum respectively. The incubation temperature had the significant effect on the production of invertase. The study reveals that the enzyme production by the strain is maximal at the temperature of 40ºC when grown in a carbon-substituted media. The concentration of the substrate at which enzyme production was maximum was determined. The result of the study reveals that the optimum substrate concentration for invertase production was 4gm/100ml of the production media. The result obtained reveals that the optimum culture condition for maximum production of invertase Penicillium brevicompactum was pH 5.0, temperature 40ºC, substrate concentration 4gm/100ml and for Penicillium chrysogenum was pH 5.5, temperature 40ºC, substrate concentration 4gm/100ml. The purification of invertase was carried out using ammonium sulphate, dialysis and acetone precipitation. The enzyme activity and protein content of the purified invertase was determined.. The partial purified enzyme showed a 2 fold increase in enzyme activity than the crude. The protein content decreased after partial purification. From the present study, we could see that parameters like pH, temperature had different effect on their respective activities. In conclusion, since Penicillium brevicompactum & Penicillium chrysogenum are producers of invertase under the same cultural conditions, with Penicillium brevicompactum as an excellent producer of invertase, this organism may find industrial application in enzyme fermentation.
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