WO2008117301A1 - A process for the production of multienzyme system using fermentation - Google Patents

A process for the production of multienzyme system using fermentation Download PDF

Info

Publication number
WO2008117301A1
WO2008117301A1 PCT/IN2007/000617 IN2007000617W WO2008117301A1 WO 2008117301 A1 WO2008117301 A1 WO 2008117301A1 IN 2007000617 W IN2007000617 W IN 2007000617W WO 2008117301 A1 WO2008117301 A1 WO 2008117301A1
Authority
WO
WIPO (PCT)
Prior art keywords
range
fermentation
multienzyme system
production
multienzyme
Prior art date
Application number
PCT/IN2007/000617
Other languages
French (fr)
Inventor
Siddalingaiya Gurudutt Prapulla
Jolly Paul
Original Assignee
Council Of Scientific & Industrial Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Council Of Scientific & Industrial Research filed Critical Council Of Scientific & Industrial Research
Publication of WO2008117301A1 publication Critical patent/WO2008117301A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae

Definitions

  • Multienzyme system is defined as multifunctional system of more than two kinds of enzymes potentially acting with functional integration and cooperation effect.
  • the development of Multienzyme system is an interesting research field with great industrial potential. It finds wide applications in the food, feed, detergent industries and also for bioremediation. Multienzyme systems are used for the degradation of the lignocellulosic waste material and also for sewage treatment.
  • Aspergillus orzyae is used for producing protease, alpha- amylase and lactase enzyme (through fermentation process). It is available on: http://www.lifeenergytics.com/products/ pnutrizyme. Htm.
  • the Nutrizyme is a blend of enzymes from different sources including plants and fungi. Out of the thirteen enzymes present in the product only three enzymes are of Aspergillus ory ⁇ ae.
  • the other enzymes including amylase, amyloglucosidase, cellulase and beta galactosidase are from varying sources like plants, yeast and other fungi. So to make an effective consortium, different enzymes from varying sources have been mixed.
  • the main object of the present invention is to provide a process for the production of multienzyme system by fermentation.
  • the present invention provides a process for the production of multienzyme system using fermentation which comprises the steps of:
  • step (a) adding the inoculum, obtained in step (a), to the fermentation medium at the concentration in the range of 10 - 25 percent(v/v) followed by fermenting for a period in the range of 48-120 hours under shaking condition having angular speed in the range of 200 - 250 rpm for a period in the range of 48- 120 hours in order to obtain the fermentation culture;
  • step (b) centrifuging by a known method the fermentation culture, obtained in step (b), followed by separation of culture fluid from pellets using filter paper (Whatman no.l) in order to obtain the said multienzyme system in form of culture fluids.
  • the fermentation medium used in the step (b) has the following composition:
  • the fermentation period used in step (b) is preferably 72 hours in order to obtain multienzyme system having ⁇ -amylase and amyloglucosidase components of the multienzyme system capable of hydrolyzing starch in to glucose at the concentration in the range of 103.20- 530.12 mg.
  • the fermentation period used in step (b) is preferably 96 hours in order to obtain multienzyme system having ⁇ -amylase and amyloglucosidase capable of hydrolyzing starch in to glucose at the concentration in the range 149.68 - 552.26 mg.
  • the fermentation period used in step (b) is preferably 120' hours in order to obtain multienzyme system having ⁇ -amylase and amyloglucosidase capable of hydrolyzing starch in to glucose at the concentration in the range 160.00-614.19 mg.
  • the fermentation period used in step (b) is preferably 96 hours in order to obtain multienzyme system having pectinase capable of reducing banana pulp from the viscosity in the range of 980 - 890 cps to the viscosity in the range of 880 - 520 cps.
  • the fermentation period used in step (b) was preferably 96 hours in order to obtain multienzyme system having carboxymethyl cellulose (CMCase) capable of hydrolyzing polysaccharides, selected from the group consisting of cellulose, carboxy methyl cellulose and sugar cane bagasse, in to glucose at the concentration in the range 0.23-2.28 rng.
  • CMCase carboxymethyl cellulose
  • Fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast culture fluid, 0.9% KH 2 PO 4 , 2% NaNO 3 , 0.03% MgSO 4 JH 2 O, 0.4% K 2 HPO 4 , 0.6% NaCl and 1% NH 4 Cl: pH 5.0. Incubated for 48-120 h at 30 0 C at 250 rpm
  • Alpha-amylase hydrolyzes carbohydrates; starch in plants and glycogen in animals. Its major industrial applications are gelatinization of starch and production of maltose syrup and maltooligosaccharides.
  • AMG/Glucoamylases " produce glucose by successive cleavage at the non reducing terminals of starch, glycogen, dextrin and maltose molecules. This enzyme has been widely used for glucose syrup production.
  • Cellulases are the extra cellular cellulose degrading enzymes produced by various fungi, bacteria, insects and lower animals. The economic potential of this enzyme arises due to its application for the hydrolysis of lignocellulosic waste materials to glucose. It is widely used for bioremediation.
  • pectinase The transformation of pectin into sugar and galacturonic acid is catalyzed by pectinase.
  • Pectinase is a group of multiple enzymes required for the degradation of pectic substances ⁇ uu it ⁇ ses oi esterases ⁇ ectin esterase) and glycosidases (pectinase). This is used for bioremediation and also in fruit industry to reduce the fruit juice viscosity.
  • ⁇ -galactosidase cleaves lactose to glucose and galactose and is one of the recommended digestive enzymes for combating lactose intolerance.
  • Xylanases hydrolyse xylan into xylose monomers. They are industrially useful for the production of xylitol, xylose, Xylooligosaccharides etc.
  • The. novelty of the process is the simultaneous production of six different classes of enzymes with significant activity. The action of any of these individual enzymes is not hindered by the presence or action of other enzymes of the system.
  • the individual enzyme component of the multienzyme system produced can either be used in isolation for the production of a few products like glucose syrup or as a combination for bioremediation.
  • This multienzyme system produced by fermentation in the present invention comprises of ⁇ -amylase, amyloglucosidase, CMCase, pectinase, ⁇ -galactosidase, and traces of xylanase.
  • the advantage of the present invention over that of others sited in the prior art is the production of a multiple enzyme system comprising of ⁇ -amylase, amyloglucosidase, CMCase, pectinase, and traces of xylanases which can be used for the degradation of cellulose, starch, pectin and xylan components of industrial waste materials resulting in effective waste treatment protocol.
  • a seed culture of Aspergillus ory ⁇ ae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0 C with 250 rpm shaking for 24h.
  • the specifically designed fermentation media containing 4% starch, 1% glucose, 0.8% yeast extract, 0.9% KH 2 PO 4 , 2% NaNO 3 , 0.03% MgSO 4 JH 2 O, 0.4% K 2 HPO 4 , 0.6% NaCl and 1% NH 4 Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks. The flasks were kept for incubation at 30 + 1 0 C under shaking condition of 250 rpm. On completion of the fermentation, the fungal pellets were separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 4 0 C and was used as a source of enzymes.
  • Amylase activity was estimated by incubating an equal volume of appropriately diluted culture fluid with 1% starch solution in acetate buffer 0.05M, pH 4.8 at 30 0 C for 45 minutes. The reaction was terminated by keeping the reaction mixture in a boiling water bath for 5 minutes; the amount of reducing sugars liberated was estimated using Dinitro salicylic acid (DNS) method (Miller G.L., Anal Chem., 31:426-431, 1959).
  • DMS Dinitro salicylic acid
  • One unit of amylase activity is defined as the ⁇ mols of reducing sugar liberated per minute under the specified assay conditions.
  • Amyloglucosidase activity in the culture fluid was estimated by incubating 200 ⁇ l of appropriately diluted culture fluid with 5 ml of 4% starch solution in acetate buffer 0.2 M, pH 4.2 at 60 0 C for lhr.
  • the amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD) (Pazur J.H., Cepure A. and Knull H.R. Carbohyd. Res. 21, 1, 1971), after stopping the reaction by adding 0.8 ml 4 N NaOH.
  • One unit of amyloglucosidase activity is defined as the ⁇ mols of glucose liberated per minute under the specified assay conditions.
  • CMCase activity in the culture fluid was estimated by incubating 500 ⁇ l of culture fluid with ImI of 1% Carboxy methyl cellulose solution in citrate buffer 0.05 M, pH 4.8 at 50 0 C for 30 minutes. The reaction was terminated by keeping the reaction mixture in a boiling water bath for 5 minutes; the amount of reducing sugars liberated was estimated using DNS method (Miller G.L., Anal Chem., 31:426-431, 1959). One unit of CMCase activity is defined as the ⁇ mols of reducing sugar liberated per minute under the specified assay conditions.
  • Fermentation was carried out for 48h.
  • the enzyme activities of ⁇ -amylase, amyloglucosidase and CMCase were found to be 1 U, 0.55XlO 5 U and 6.67U respectively.
  • Fermentation was carried out for 72h.
  • the enzyme activities of ⁇ -amylase, amyloglucosidase and CMCase were found to be 1.1U, 0.73XlO 5 U and 6.19U respectively.
  • Fermentation was carried out for 96h.
  • the enzyme activities of ⁇ -amylase, • amyloglucosidase and CMCase were found to be LlU, 0.72XlO 5 U and 5.84U respectively.
  • Fermentation was carried out for 12Oh.
  • the enzyme activities of ⁇ -amylase, amyloglucosidase and CMCase were found to be 1.06U, 1.17XlO 5 U and 5.63U respectively.
  • a seed culture of Aspergillus oryzae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0 C with 250 rpm shaking for 24h.
  • the specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH 2 PO 4 , 2% NaNO 3 , 0.03% MgSO 4 .7H 2 O, 0.4% K 2 HPO 4 , 0.6% NaCl and 1% NH 4 Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks.
  • the flasks were kept for incubation at 30 + 1 0 C under shaking condition of 250 rpm and fermentation was carried out for 48h.
  • the fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 4 0 C and was used as a source of enzymes.
  • Pectinase activity in the culture fluid was estimated by incubating 200 ⁇ l of culture fluid with 2ml of 0.5% pectin solution in acetate buffer 0.1 M, pH 4.3 at 50 0 C for 10 minutes.
  • the amount of reducing sugar liberated was estimated using Nelson — Somogyi method (Nelson N. J. Biol. Chem. 153, 375-6, 1944: Shaffer P.A., Somogyi M. J. Bio. Chem. 100, 695, 1933).
  • One unit of Pectinase activity is defined as the ⁇ mols of galacturonic acid liberated per minute under the specified assay conditions.
  • ⁇ -galactosidase activity in the culture fluid was estimated by incubating 0.5 ml of culture fluid with 50 ⁇ l of 2OmM o- nitro phenyl - ⁇ - D- galactopyranoside prepared in acetate buffer 0.1 M, pH 5.0 at 60 0 C for 10 minutes. The amount of o-nitro phenol liberated was estimated (Norimasa Onishi, Takashi Tanaka, Appli. Enviro. Micro. Nov. 1995, 4026 - 30).
  • One unit of ⁇ -galactosidase activity is defined as the ⁇ grams of o-nitro phenol liberated per minute under the specified assay conditions.
  • Xylanase activity in the culture fluid was estimated by incubating 50 ⁇ l of culture fluid with 0.5 ml 1% w/v birch wood xylan prepared in 0.2M NaOH at 28 0 C for 15minutes. The amount of reducing sugars liberated was estimated using DNS method (Miller G.L., Anal Chem., 31:426-431, 1959).
  • DNS method iller G.L., Anal Chem., 31:426-431, 1959.
  • One unit of xylanase activity is defined as the ⁇ mols of xylose liberated per minute under the specified assay conditions.
  • Fermentation was carried out for 48h.
  • the enzyme activities of ⁇ -amylase, amyloglucosidase CMCase, pectinase, ⁇ -galactosidase and xylanase were found to be 6.57 U 5 LI lXlO 5 U,
  • Fermentation was carried out for 72h.
  • the enzyme activities of ⁇ -amylase, amyloglucosidase CMCase, pectinase, ⁇ -galactosidase and xylanase were found to be 10.34U, 1.21XlO 5 U,
  • Fermentation was carried out for 12Oh.
  • the enzyme activities of ⁇ -amylase, amyloglucosidase CMCase, pectinase, ⁇ -galactosidase and xylanase were found to be 15.47 U, 0.83XlO 5 U,
  • a seed culture of Aspergillus oryzae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0 C with 250 rpm shaking for 24h.
  • the specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH 2 PO 4 , 2% NaNO 3 , 0.03% MgSO 4 .7H 2 O, 0.4% K 2 HPO 4 , 0.6% NaCl and 1% NH 4 Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks.
  • the flasks were kept for incubation at 30 + 1 0 C under shaking condition of 250 rpm and fermentation was carried out for 72h.
  • the fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 4 0 C and was used as a source of enzymes.
  • amylase and amyloglucosidase activity in the culture fluid was estimated and was used for the production of glucose syrup.
  • the amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 103.20 mg.
  • a seed culture of Aspergillus ory ⁇ ae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0 C with 250 rpm shaking for 24h.
  • the specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH 2 PO 4 , 2% NaNO 3 , 0.03% MgSO 4 .7H 2 O, 0.4% K 2 HPO 4 , 0.6% NaCl and 1% NH 4 Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks.
  • the flasks were kept for incubation at 30 + 1 0 C under shaking condition of 250 rpm and fermentation was carried out for 96h.
  • the fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 4 0 C and was used as a source of enzymes.
  • amylase and amyloglucosidase activity in the culture fluid was estimated and was used for the production of glucose syrup.
  • the amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 149.68 mg.
  • a seed culture of Aspergillus oryzae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 C with 250 rpm shaking for 24h.
  • the specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH 2 PO 4 , 2% NaNO 3 , 0.03% MgSO 4 JH 2 O, 0.4% K 2 HPO 4 , 0.6% NaCl and 1% NH 4 Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks.
  • the flasks were kept for incubation at 30 + 1 0 C under shaking condition of 250 rpm and fermentation was carried out for 12Oh.
  • the fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 4 0 C and was used as a source of enzymes.
  • amylase and amyloglucosidase activity in the culture fluid was estimated and was used for the production of glucose syrup.
  • a seed culture of Aspergillus ory ⁇ ae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 ⁇ 1 0 C with 250 rpm shaking for 24h.
  • the specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH 2 PO 4 , 2% NaNO 3 , 0.03% MgSO 4 JH 2 O, 0.4% K 2 HPO 4 , 0.6% NaCl and 1% NH 4 Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks.
  • the flasks were kept for incubation at 30 + 1 0 C under shaking condition of 250 rpm and fermentation was carried out for 96h.
  • the fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 4 0 C and was used as a source of enzymes.
  • the pectinase activity in the culture fluid was estimated and was used for thinning of banana pulp.
  • a) 18O g of banana pulp was incubated with 5U of pectinase for 30 minutes to 330 minutes and the viscosity was measured every 60minutes using Brookfield Synchro-lectric viscometer. The viscosity has been found to reduce steadily from 980 to 720 cps.
  • a seed culture of Aspergillus ory ⁇ ae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0 C with 250 rpm shaking for 24h.
  • the specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl- cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH 2 PO 4 , 2% NaNO 3 , 0.03% MgSO 4 .7H 2 O, 0.4% K 2 HPO 4 , 0.6% NaCl and 1% NH 4 Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks.
  • the flasks were kept for incubation at 30 + 1 0 C under shaking condition of 250 rpm and fermentation was carried out for 96h.
  • the fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 4 0 C and was used as a source of enzymes.
  • the carboxy methyl cellulase activity in the culture fluid was estimated and was used for the production of glucose syrup from cellulose, carboxy methyl cellulose, corn cob and sugar cane bagasse.
  • b) 5 ml of 0.5 g of carboxy methyl cellulose dissolved in 0.1M acetate buffer pH 5.0 was incubated with IU of CMCase for 60 minutes. The amount of glucose liberated was estimated by DNS method. The glucose concentration was found to be 13.28 mg.
  • Thinning of banana pulp during the time course of reaction (example 6) is given in Table 4.

Abstract

The present invention relates to a process for the production of multienzyme system by fermentation. The process in particular uses Aspergillus oryzae MTCC 5154 under optimized conditions for the production of multienzyme system by submerged fermentation. The product in particular is a mixture of different enzymes such as α-amylase, amyloglucosidase (AMG), carboxy methyl cellulase (CMCase), pectinase β-galactosidase and xylanase.

Description

"A Process for the Production of Multienzyme system using
Fermentation"
Multienzyme system is defined as multifunctional system of more than two kinds of enzymes potentially acting with functional integration and cooperation effect. The development of Multienzyme system is an interesting research field with great industrial potential. It finds wide applications in the food, feed, detergent industries and also for bioremediation. Multienzyme systems are used for the degradation of the lignocellulosic waste material and also for sewage treatment.
References may be made to Bruno, Mark (Raleigh, NC), Enzyme Research and Development Corporation, United States Patent application no. 222108 wherein multienzymes powdered composition containing bacteria has been used for the waste water treatment. A consortium of degradative enzymes and bacterial cultures was used to digest and liquefy the organic wastes flushed into onsite waste disposal systems. Individual enzymes from different commercial sources were mixed to 'get the multienzyme composition.
References may be made to Triantafyllou, Angelika Oste (Lund, SE), Cereal Base (CEBA AB), United States Patent application no. 302127 wherein an enzyme suspension comprising of different hydrolytic enzymes was used for the hydrolysis of cereal starch for the production of the cereal suspensions. Individual enzymes from different commercial sources were mixed together to obtain the multienzyme consortium.
Environmental stress due to the accumulation of the waste materials has necessitated more sudies on the waste degradation by multienzyme system. References may be made to Abdel-Mohsen, Saber Ismail, Process Biochemistry, 31, 7, 645-650, 1996 wherein the orange peel, an accumulating waste of orange-based industries, has been utilized for the production of a multienzyme system of pectinases, cellulases and xylanases. But lack of some of the other hydrolytic enzymes such as amylases, amyloglucosidase etc. limits the use of this multienzyme system.
References may be made to F. O. Omogbenigun, C. M. Nyachoti and B. A. Slominski, J. Animal Science, 82, 1053-1061, 2004 wherein the use of multienzyme system in the diet of the poultry and farm animals has been studied. The effect of multienzyme preparations on nutritional digestibility and growth performance in weaned pigs has been investigated. The study suggests a cost effective formulation and environmentally friendly diet for weaned pigs containing multienzyme systems.
References may be made to Seung - Goo Lee, Seung - Pyo Hong and Moon-Hee Sung, Enz. and Microb. Tech, 25, 298-302, 1999 wherein biologically important intracellular multienzyme complexes from Streptococcus faecalis for the production of dopamine, a mammalian neurotransmitter, was developed invitro.
References may be made to Shigeyuki Imamura, Shiguru Ueda, Michmao Mizugaki and Akihiko Kawaguchi, J. Biochem., 107, 184-189, 1990 wherein a multienzyme complex from Pseudomonas fragi grown on oleic acid as the sole source of carbon for fatty acid oxidation was characterized and purified.
References may be made to Diomi Mamma, Emmanuel Kalogeris, Dimitris G. Hatzinikolaou, Afroditi Lekanidou, Dimitris kekos, Basil J. Macris and Paul Christakopoulos, Food Biotechnology, 18, 1, 1-18, 2004, wherein attempts have been made to produce useful extracellular multienzyme system. PeniciIIium decumbens grown on rutin was shown to produce a multienzyme system of monooxygenase and glycosidases that can degrade rutin, a non toxic flavanoid glycoside, to water soluble products. The use of this system is limited to rutin degradation.
It has been reported in Applied Microbiology and Biotechnology, vol. 73, No.3, December 2006, pp 533 -540 that Aspergillus niger is used for production of multi-enzymes preparation containing cellulose, hemicellulase, glucoamylase, pectinase and proteinase. However, the microbial source and the consortium components are different between the said report and the present invention.
Apart from this, one industrially produced nutrizyme is available in the market, in this multi- enzyme blend (nutrizyme), Aspergillus orzyae is used for producing protease, alpha- amylase and lactase enzyme (through fermentation process). It is available on: http://www.lifeenergytics.com/products/ pnutrizyme. Htm. However, the Nutrizyme is a blend of enzymes from different sources including plants and fungi. Out of the thirteen enzymes present in the product only three enzymes are of Aspergillus ory∑ae. The other enzymes, including amylase, amyloglucosidase, cellulase and beta galactosidase are from varying sources like plants, yeast and other fungi. So to make an effective consortium, different enzymes from varying sources have been mixed.
Bruno et α/ U.S. patent application no. 222108 claims a consortium of degradative enzymes and bacterial cultures which was used to digest and liquefy the organic wastes flushed into onsite waste disposal systems. In the Triantafyllou et α/ U.S. patent application no. 302127 an enzyme suspension comprising of different hydrolytic enzymes used for the hydrolysis of cereal starch for the production of the cereal suspensions is claimed. In all the above stated references of Nutrizyme, Bruno et al consortium and Triantafyllou et al consortium, individual enzymes from different origins or commercial sources were mixed to get the final composition of the multienzyme consortium. Whereas, in the present patent, we claim a multienzyme consortium produced by fermentation using Aspergillus oryzae. The components - α-amylase, amyloglucosidase (AMG), carboxy methyl cellulase (CMCase), pectinase, β -galactosidase and xylanase, are obtained at the end of single fermentation process using a single microbe, A. oryzae. Thus, the novelty of the present patent resides in the production of nutrizyme at a single stage through fermentation and hence cost-effective. As well as there has been no blending of enzymes from different sources, hence better quality control.
References may also be made to E. Emtiazi and I. Nahvi, Biomass and Bioenergy, 19, 1, 31- 37, 2000 wherein Cellulomonas sp. isolated from silk worm and rabbit waste was used for the production of multienzymes, comprising of CMCase, cellobiase, filter paperase (Fpase), xylanase, amylase and manganese peroxidase by the fermentation of wheat straw, a major lignocellulosic waste. The multienzyme thus produced has been investigated for improving the digestibility of straw for animal feed. No food grade applications have been show using this system.
The traditional methods for the preparation of multienzyme systems are:
1. Mixing of the desired enzymes from different sources. 2. Using genetically engineered microbial cultures capable of producing the desired multienzyme systems.
3. By mixed culture fermentation, using different well defined microbes.
No reports are available on a bioprocess for the production of multienzyme system by submerged fermentation using Aspergillus oryzae.
The main object of the present invention is to provide a process for the production of multienzyme system by fermentation.
Accordingly the present invention provides a process for the production of multienzyme system using fermentation which comprises the steps of:
a) growing the culture Aspergillus oryzae having MTCC 5154 on a medium having sucrose at the concentration of about 1% and Yeast extract at the concentration of about 0.2% at a temperature in the range of 25 - 35 deg C and pH in the range of 5 — 6 for a period in the range of 48-120 hours under shaking condition having angular speed in the range of 200 - 250 rpm in order to obtain an inoculum;
b) adding the inoculum, obtained in step (a), to the fermentation medium at the concentration in the range of 10 - 25 percent(v/v) followed by fermenting for a period in the range of 48-120 hours under shaking condition having angular speed in the range of 200 - 250 rpm for a period in the range of 48- 120 hours in order to obtain the fermentation culture; AND
c) centrifuging by a known method the fermentation culture, obtained in step (b), followed by separation of culture fluid from pellets using filter paper (Whatman no.l) in order to obtain the said multienzyme system in form of culture fluids. In an embodiment of the present invention the fermentation medium used in the step (b) has the following composition:
Figure imgf000006_0001
In an embodiment of the present invention the multienzyme system obtained is comprised of the following composition:
Figure imgf000006_0002
In an embodiment of the present invention the fermentation period used in step (b) is preferably 72 hours in order to obtain multienzyme system having α-amylase and amyloglucosidase components of the multienzyme system capable of hydrolyzing starch in to glucose at the concentration in the range of 103.20- 530.12 mg.
In an embodiment of the present invention the fermentation period used in step (b) is preferably 96 hours in order to obtain multienzyme system having α-amylase and amyloglucosidase capable of hydrolyzing starch in to glucose at the concentration in the range 149.68 - 552.26 mg.
In an embodiment of the present invention the fermentation period used in step (b) is preferably 120' hours in order to obtain multienzyme system having α-amylase and amyloglucosidase capable of hydrolyzing starch in to glucose at the concentration in the range 160.00-614.19 mg.
"In an embodiment of the present invention the fermentation period used in step (b) is preferably 96 hours in order to obtain multienzyme system having pectinase capable of reducing banana pulp from the viscosity in the range of 980 - 890 cps to the viscosity in the range of 880 - 520 cps.
In an embodiment of the present invention the fermentation period used in step (b) was preferably 96 hours in order to obtain multienzyme system having carboxymethyl cellulose (CMCase) capable of hydrolyzing polysaccharides, selected from the group consisting of cellulose, carboxy methyl cellulose and sugar cane bagasse, in to glucose at the concentration in the range 0.23-2.28 rng.
The process for the production of multienzyme system by fermentation is illustrated in the following flow chart.
One loop full of spores from 5 day old slants of Aspergillus oryzae MTCC 5154
Medium containing 1% sucrose an 1d 0.2% yeast extract at a pH of 5.5 Incubation for 24 h at 300C at about 250 rpm
10% (v/v) of inoculum
!
Fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast culture fluid, 0.9% KH2PO4, 2% NaNO3, 0.03% MgSO4JH2O, 0.4% K2HPO4, 0.6% NaCl and 1% NH4Cl: pH 5.0. Incubated for 48-120 h at 300C at 250 rpm
Cultu 1re broth Filtered through filterr ppasper (Whatman no 1)
Culture fluid (source of enzymes)
Estimation of e Inzyme activities Alpha-amylase hydrolyzes carbohydrates; starch in plants and glycogen in animals. Its major industrial applications are gelatinization of starch and production of maltose syrup and maltooligosaccharides.
AMG/Glucoamylases" produce glucose by successive cleavage at the non reducing terminals of starch, glycogen, dextrin and maltose molecules. This enzyme has been widely used for glucose syrup production. Cellulases are the extra cellular cellulose degrading enzymes produced by various fungi, bacteria, insects and lower animals. The economic potential of this enzyme arises due to its application for the hydrolysis of lignocellulosic waste materials to glucose. It is widely used for bioremediation.
The transformation of pectin into sugar and galacturonic acid is catalyzed by pectinase. Pectinase is a group of multiple enzymes required for the degradation of pectic substances αuu it υυπψπses oi esterases φectin esterase) and glycosidases (pectinase). This is used for bioremediation and also in fruit industry to reduce the fruit juice viscosity.
β-galactosidase cleaves lactose to glucose and galactose and is one of the recommended digestive enzymes for combating lactose intolerance. Xylanases hydrolyse xylan into xylose monomers. They are industrially useful for the production of xylitol, xylose, Xylooligosaccharides etc.
NOVELTY
The. novelty of the process is the simultaneous production of six different classes of enzymes with significant activity. The action of any of these individual enzymes is not hindered by the presence or action of other enzymes of the system. The individual enzyme component of the multienzyme system produced can either be used in isolation for the production of a few products like glucose syrup or as a combination for bioremediation. This multienzyme system produced by fermentation in the present invention, comprises of α-amylase, amyloglucosidase, CMCase, pectinase, β-galactosidase, and traces of xylanase.
ADVANTAGES
The advantage of the present invention over that of others sited in the prior art is the production of a multiple enzyme system comprising of α-amylase, amyloglucosidase, CMCase, pectinase, and traces of xylanases which can be used for the degradation of cellulose, starch, pectin and xylan components of industrial waste materials resulting in effective waste treatment protocol.
A few reports are available on the production and use of multienzyme systems for a variety of applications like lignocellulosic waste degradation, sewage treatment, food supplements etc as referred to in the above prior art. However there are no reports on the concomitant production of multiple enzymes consisting of a few important hydrolytic enzymes and a transferase for food applications and in particular, no reports are available on a bioprocess for the production of multienzyme system comprising α-amylase, amyloglucosidase (AMG), carboxy methyl cellulase (CMCase), pectinase and β-galactosidase by submerged fermentation using Aspergillus ory∑ae.
The following examples are given by a way of illustration of the present invention and should not be constructed to limit the scope of the present invention.
EXAMPLE 1
A seed culture of Aspergillus ory∑ae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0C with 250 rpm shaking for 24h. The specifically designed fermentation media containing 4% starch, 1% glucose, 0.8% yeast extract, 0.9% KH2PO4, 2% NaNO3, 0.03% MgSO4JH2O, 0.4% K2HPO4, 0.6% NaCl and 1% NH4Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks. The flasks were kept for incubation at 30 + 1 0C under shaking condition of 250 rpm. On completion of the fermentation, the fungal pellets were separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 40C and was used as a source of enzymes.
Amylase activity was estimated by incubating an equal volume of appropriately diluted culture fluid with 1% starch solution in acetate buffer 0.05M, pH 4.8 at 300C for 45 minutes. The reaction was terminated by keeping the reaction mixture in a boiling water bath for 5 minutes; the amount of reducing sugars liberated was estimated using Dinitro salicylic acid (DNS) method (Miller G.L., Anal Chem., 31:426-431, 1959). One unit of amylase activity is defined as the μmols of reducing sugar liberated per minute under the specified assay conditions.
Amyloglucosidase activity in the culture fluid was estimated by incubating 200μl of appropriately diluted culture fluid with 5 ml of 4% starch solution in acetate buffer 0.2 M, pH 4.2 at 600C for lhr. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD) (Pazur J.H., Cepure A. and Knull H.R. Carbohyd. Res. 21, 1, 1971), after stopping the reaction by adding 0.8 ml 4 N NaOH. One unit of amyloglucosidase activity is defined as the μmols of glucose liberated per minute under the specified assay conditions.
CMCase activity in the culture fluid was estimated by incubating 500μl of culture fluid with ImI of 1% Carboxy methyl cellulose solution in citrate buffer 0.05 M, pH 4.8 at 500C for 30 minutes. The reaction was terminated by keeping the reaction mixture in a boiling water bath for 5 minutes; the amount of reducing sugars liberated was estimated using DNS method (Miller G.L., Anal Chem., 31:426-431, 1959). One unit of CMCase activity is defined as the μmols of reducing sugar liberated per minute under the specified assay conditions.
a) Fermentation was carried out for 48h. The enzyme activities of α-amylase, amyloglucosidase and CMCase were found to be 1 U, 0.55XlO5U and 6.67U respectively.
b) Fermentation was carried out for 72h. The enzyme activities of α-amylase, amyloglucosidase and CMCase were found to be 1.1U, 0.73XlO5U and 6.19U respectively. c) Fermentation was carried out for 96h. The enzyme activities of α-amylase, • amyloglucosidase and CMCase were found to be LlU, 0.72XlO5U and 5.84U respectively. d) Fermentation was carried out for 12Oh. The enzyme activities of α-amylase, amyloglucosidase and CMCase were found to be 1.06U, 1.17XlO5U and 5.63U respectively.
EXAMPLE 2
A seed culture of Aspergillus oryzae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0C with 250 rpm shaking for 24h. The specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH2PO4, 2% NaNO3, 0.03% MgSO4.7H2O, 0.4% K2HPO4, 0.6% NaCl and 1% NH4Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks. The flasks were kept for incubation at 30 + 1 0C under shaking condition of 250 rpm and fermentation was carried out for 48h. The fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 40C and was used as a source of enzymes.
Pectinase activity in the culture fluid was estimated by incubating 200μl of culture fluid with 2ml of 0.5% pectin solution in acetate buffer 0.1 M, pH 4.3 at 500C for 10 minutes. The amount of reducing sugar liberated was estimated using Nelson — Somogyi method (Nelson N. J. Biol. Chem. 153, 375-6, 1944: Shaffer P.A., Somogyi M. J. Bio. Chem. 100, 695, 1933). One unit of Pectinase activity is defined as the μmols of galacturonic acid liberated per minute under the specified assay conditions.
β-galactosidase activity in the culture fluid was estimated by incubating 0.5 ml of culture fluid with 50μl of 2OmM o- nitro phenyl - β- D- galactopyranoside prepared in acetate buffer 0.1 M, pH 5.0 at 600C for 10 minutes. The amount of o-nitro phenol liberated was estimated (Norimasa Onishi, Takashi Tanaka, Appli. Enviro. Micro. Nov. 1995, 4026 - 30). One unit of β-galactosidase activity is defined as the μgrams of o-nitro phenol liberated per minute under the specified assay conditions.
Xylanase activity in the culture fluid was estimated by incubating 50μl of culture fluid with 0.5 ml 1% w/v birch wood xylan prepared in 0.2M NaOH at 280C for 15minutes. The amount of reducing sugars liberated was estimated using DNS method (Miller G.L., Anal Chem., 31:426-431, 1959). One unit of xylanase activity is defined as the μmols of xylose liberated per minute under the specified assay conditions.
a) Fermentation was carried out for 48h. The enzyme activities of α-amylase, amyloglucosidase CMCase, pectinase, β-galactosidase and xylanase were found to be 6.57 U5 LI lXlO5 U,
15.34 U, 0.32 U, 0.76 U and 0.75 U respectively. b) Fermentation was carried out for 72h. The enzyme activities of α-amylase, amyloglucosidase CMCase, pectinase, β-galactosidase and xylanase were found to be 10.34U, 1.21XlO5U,
41.26 U, 0.45U, 0.59U and 0.5 IU respectively.
I l c) fermentation was carried out for 96h. The enzyme activities of α-amylase, amyloglucosidase CMCase, pectinase, β-galactosidase and xylanase were found to be 14.34U, 1.94XlO5U,
44.26 U, 0.76 U, 1.29 U and 0.41 U respectively. d) Fermentation was carried out for 12Oh. The enzyme activities of α-amylase, amyloglucosidase CMCase, pectinase, β-galactosidase and xylanase were found to be 15.47 U, 0.83XlO5U,
29.12 U, 0.04 U, 0.34 U and 0.43 U respectively.
EXAMPLE 3
A seed culture of Aspergillus oryzae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0C with 250 rpm shaking for 24h. The specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH2PO4, 2% NaNO3, 0.03% MgSO4.7H2O, 0.4% K2HPO4, 0.6% NaCl and 1% NH4Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks. The flasks were kept for incubation at 30 + 1 0C under shaking condition of 250 rpm and fermentation was carried out for 72h. The fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 40C and was used as a source of enzymes.
The amylase and amyloglucosidase activity in the culture fluid was estimated and was used for the production of glucose syrup. , a) 1000 U of amyloglucosidase was incubated with 25 ml of 5% starch solution in citrate phosphate buffer 0.1M, pH 5.5 in the presence of ImM CaCl2 at 500C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 103.20 mg. b) 1000 U of amyloglucosidase enzyme was incubated with 25 ml of 10% starch solution in citrate phosphate buffer 0.1 M, pH 5.5 in the presence of ImM CaCl2 at 500C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 408.00 mg. c) 1000 U of amyloglucosidase enzyme was incubated with 25 ml of 15% starch solution in citrate phosphate buffer 0.1M, pH 5.5 in the presence of ImM CaCl2 at 500C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 530.12 mg.
EXAMPLE 4
A seed culture of Aspergillus ory∑ae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0C with 250 rpm shaking for 24h. The specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH2PO4, 2% NaNO3, 0.03% MgSO4.7H2O, 0.4% K2HPO4, 0.6% NaCl and 1% NH4Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks. The flasks were kept for incubation at 30 + 1 0C under shaking condition of 250 rpm and fermentation was carried out for 96h. The fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 40C and was used as a source of enzymes.
The amylase and amyloglucosidase activity in the culture fluid was estimated and was used for the production of glucose syrup. a) 1000 U of amyloglucosidase was incubated with 25 ml of 5% starch solution in citrate phosphate buffer 0.1M, pH 5.5 in the presence of ImM CaCl2 at 500C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 149.68 mg. b) 1000 U of amyloglucosidase enzyme was incubated with 25 ml of 10% starch solution in citrate phosphate buffer 0.1M, pH 5.5 in the presence of ImM CaCl2 at 500C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 452.40 mg. c) 1000 U of amyloglucosidase enzyme was incubated with 25 ml of 15% starch solution in citrate phosphate buffer 0.1M, pH 5.5 in the presence of ImM CaCl2 at 500C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 552.26 mg.
EXAMPLE 5
A seed culture of Aspergillus oryzae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 C with 250 rpm shaking for 24h. The specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH2PO4, 2% NaNO3, 0.03% MgSO4JH2O, 0.4% K2HPO4, 0.6% NaCl and 1% NH4Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks. The flasks were kept for incubation at 30 + 1 0C under shaking condition of 250 rpm and fermentation was carried out for 12Oh. The fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 40C and was used as a source of enzymes.
The amylase and amyloglucosidase activity in the culture fluid was estimated and was used for the production of glucose syrup. a) 1000 U of amyloglucosidase was incubated with 25 ml of 5% starch solution in citrate phosphate buffer 0.1M, pH 5.5 in the presence of ImM CaCl2 at 500C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD — POD Kit, Autospan). The glucose concentration was found to be 160 mg. b) 1000 U of amyloglucosidase enzyme was incubated with 25 ml of 10% starch solution in citrate phosphate buffer 0. IM, pH 5.5 in the presence of ImM CaCl2 at 50 C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD — POD Kit, Autospan). The glucose concentration was found to be 388.60 mg. c) 1000 U of amyloglucosidase enzyme was incubated with 25 ml of 15% starch solution in citrate phosphate buffer 0.1M, pH 5.5 in the presence of ImM CaCl2 at 500C for 96 hours and samples were collected. The amount of glucose liberated was estimated using Glucose oxidase - peroxidase method (GOD - POD Kit, Autospan). The glucose concentration was found to be 614.19 mg. EXAMPLE 6
A seed culture of Aspergillus ory∑ae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 ± 1 0C with 250 rpm shaking for 24h. The specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH2PO4, 2% NaNO3, 0.03% MgSO4JH2O, 0.4% K2HPO4, 0.6% NaCl and 1% NH4Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks. The flasks were kept for incubation at 30 + 1 0C under shaking condition of 250 rpm and fermentation was carried out for 96h. The fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 40C and was used as a source of enzymes.
The pectinase activity in the culture fluid was estimated and was used for thinning of banana pulp. a) 18O g of banana pulp was incubated with 5U of pectinase for 30 minutes to 330 minutes and the viscosity was measured every 60minutes using Brookfield Synchro-lectric viscometer. The viscosity has been found to reduce steadily from 980 to 720 cps. b) 180 g of banana pulp was incubated with 1OU of pectinase for 30 minutes to 330 minutes and the viscosity was measured every 60minutes using Brookfield Synchro-lectric viscometer. The viscosity has been found to reduce steadily from 960 to 655 cps. c) 18O g of banana pulp was incubated with 15U of pectinase for 30 minutes to 330 minutes and the viscosity was measured every βOminutes using Brookfield Synchro-lectric viscometer. The viscosity has been found to reduce steadily from 920 to 600 cps. d) 18O g of banana pulp was incubated with 2OU of pectinase for 30 minutes to 330 minutes and the viscosity was measured every 60minutes using Brookfield Synchro-lectric viscometer. The viscosity has been found to reduce steadily from 890 to 520 cps. EXAMPLE 7
A seed culture of Aspergillus ory∑ae MTCC 5154 was prepared by inoculating the organism to the inoculation medium and incubating at 30 + 1 0C with 250 rpm shaking for 24h. The specifically designed fermentation media containing 4% starch, 1% glucose, 1% carboxy methyl- cellulose, 0.5% pectin, 0.8% yeast extract, 0.9% KH2PO4, 2% NaNO3, 0.03% MgSO4.7H2O, 0.4% K2HPO4, 0.6% NaCl and 1% NH4Cl, of pH 5.0 was inoculated with the seed culture (10% v/v) in 500 ml conical flasks. The flasks were kept for incubation at 30 + 1 0C under shaking condition of 250 rpm and fermentation was carried out for 96h. The fungal pellets were then separated from the culture broth using a filter paper (Whatman no.l) and the culture fluid was stored at 40C and was used as a source of enzymes.
The carboxy methyl cellulase activity in the culture fluid was estimated and was used for the production of glucose syrup from cellulose, carboxy methyl cellulose, corn cob and sugar cane bagasse. a) 5 ml of 0.5 g of cellulose in 0.1M acetate buffer pH 5.0 was incubated with IU of CMCase for 60 minutes. The amount of glucose liberated was estimated by DNS method. The glucose concentration was found to be 0.23 mg. b) 5 ml of 0.5 g of carboxy methyl cellulose dissolved in 0.1M acetate buffer pH 5.0 was incubated with IU of CMCase for 60 minutes. The amount of glucose liberated was estimated by DNS method. The glucose concentration was found to be 13.28 mg. c) 5 ml of 0.5 g of sugar cane bagasse in 0.1 M acetate buffer pH 5.0 was incubated with IU of CMCase for 60 minutes. The amount of glucose liberated was estimated by DNS method. The glucose concentration was found to be 2.28 mg.
A summary of enzyme activities during the course of fermentation (example 1: a-d) is given in Table 1
Figure imgf000017_0001
A summary of enzyme activities during the course of fermentation (examples 2: a-d) is given in Table 2
Figure imgf000018_0001
A summary of glucose concentration during the course of fermentation (examples 3-5) is given in Table 3.
Figure imgf000018_0002
Thinning of banana pulp during the time course of reaction (example 6) is given in Table 4.
Figure imgf000019_0001

Claims

We claim:
1. A Process for the production of multienzyme system using fermentation which comprises the steps of:
(a) growing the culture Aspergillus oryzae having MTCC 5154 on a medium having sucrose at the concentration of about 1% and Yeast extract at the concentration of about 0.2% at a temperature in the range of 25 - 35 deg C and pH in the range of 5 - 6 for a period in the range of 48-120 hours under shaking condition having angular speed in the range of 200 - 250 rpm in order to obtain an inoculum;
(b) adding the inoculum, obtained in step (a), to the fermentation medium at the concentration in the range of 10 - 25 percent(v/v) followed by fermenting for a period in the range of 48-120 hours under shaking condition having angular speed in the range of 200 - 250 rpm for a period in the range of 48-120 hours in order to obtain the fermentation culture; AND
(c) centrifuging by a known method the fermentation culture, obtained in step (b), followed by separation of culture fluid from pellets using filter paper (Whatman no.l) in order to obtain the said multienzyme system in form of culture fluids.
2. The process for the production of multienzyme system according to claim 1, wherein the fermentation medium used in the step (b) has the following composition:
Figure imgf000020_0001
Figure imgf000021_0001
3. The process for the production of multienzyme system according to claims 1 and 2, wherein the multienzyme system obtained is comprised of the following composition:
Figure imgf000021_0002
4. The process for the production of multienzyme system according to claims 1-3, wherein the fermentation period used in step (b) is preferably 72 hours in order to obtain multienzyme system having α-amylase and amyloglucosidase components of the multienzyme system capable of hydrolyzing starch in to glucose at the concentration in the range of 103.20- 530.12 mg.
5. The process for the production of multienzyme system according to claims 1-3, wherein the fermentation period used in step (b) is preferably 96 hours in order to obtain multienzyme system having α-amylase and amyloglucosidase capable of hydrolyzing starch in to glucose at the concentration in the range 149.68 - 552.26 mg.
6. The process for the production of multienzyme system according to claims 1-3, wherein the fermentation period used in step (b) is preferably 120 hours in order to obtain multienzyme system having α-amylase and amyloglucosidase capable of hydrolyzing starch in to glucose at the concentration in the range 160.00-614.19 mg.
7. The process for the production of multienzyme system according to claims 1-3, wherein the fermentation period used in step (b) is preferably 96 hours in order to obtain multienzyme system having pectinase capable of reducing banana pulp from the viscosity in the range of 980 - 890 cps to the viscosity in the range of 880 - 520 cps.
8. The process for the production of multienzyme system according to claims 1-3, wherein the fermentation period used in step (b) was preferably 96 hours in order to obtain multienzyme system having carboxymethyl cellulose (CMCase) capable of hydrolyzing polysaccharides, selected from the group consisting of cellulose, carboxy methyl cellulose and sugar cane bagasse, in to glucose at the concentration in the range 0.23-2.28 mg.
PCT/IN2007/000617 2007-03-23 2007-12-31 A process for the production of multienzyme system using fermentation WO2008117301A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN656/DEL/2007 2007-03-23
IN656DE2007 2007-03-23

Publications (1)

Publication Number Publication Date
WO2008117301A1 true WO2008117301A1 (en) 2008-10-02

Family

ID=39323789

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2007/000617 WO2008117301A1 (en) 2007-03-23 2007-12-31 A process for the production of multienzyme system using fermentation

Country Status (1)

Country Link
WO (1) WO2008117301A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010070146A1 (en) 2008-12-19 2010-06-24 Danisco A/S Process for production of an enzyme product
US20120003339A1 (en) * 2010-07-02 2012-01-05 Minacapelli Pompeo Compositions and methods for controlling blood glucose levels
US20120252102A1 (en) * 2011-03-31 2012-10-04 Bioremediation Specialists, LLC Bioremediation systems, compositions, and methods
US9056340B2 (en) 2012-03-30 2015-06-16 Bioremediation Specialists L.L.C. Bioremediation systems, compositions, and methods
US20160222367A1 (en) * 2013-09-06 2016-08-04 Council Of Scientific & Industrial Research Enzyme composition and process of preparation thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02203782A (en) * 1989-02-02 1990-08-13 Wakamoto Pharmaceut Co Ltd Production of galactooligosaccharide-containing koji
SU1641887A1 (en) * 1988-09-02 1991-04-15 Институт Ботаники Им.Н.Г.Холодного Strain of fungus aspergillus oryzae (ahlburg) cohn producing xylanase
WO2006103698A1 (en) * 2005-03-31 2006-10-05 Council Of Scientific & Industrial Research 'edible films and coatings based on fructooligosaccharides with probiotic properties'

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1641887A1 (en) * 1988-09-02 1991-04-15 Институт Ботаники Им.Н.Г.Холодного Strain of fungus aspergillus oryzae (ahlburg) cohn producing xylanase
JPH02203782A (en) * 1989-02-02 1990-08-13 Wakamoto Pharmaceut Co Ltd Production of galactooligosaccharide-containing koji
WO2006103698A1 (en) * 2005-03-31 2006-10-05 Council Of Scientific & Industrial Research 'edible films and coatings based on fructooligosaccharides with probiotic properties'

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 199038, Derwent World Patents Index; AN 1990-287564, XP002479141 *
DATABASE WPI Week 199211, Derwent World Patents Index; AN 1992-086957, XP002479140 *
MORKEBERG R ET AL: "Induction and repression of alpha-amylase production in batch and continuous cultures of Aspergillus oryzae", MICROBIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, GB, vol. 141, no. PART 10, 1 October 1995 (1995-10-01), pages 2449 - 2454, XP003010822, ISSN: 1350-0872 *
NAKADAI T ET AL: "CULTURE CONDITIONS OF ASPERGILLUS-ORYZAE FOR PRODUCTION OF ENZYME PREPARATION", JOURNAL OF FERMENTATION TECHNOLOGY, vol. 66, no. 5, 1988, pages 525 - 534, XP002479139, ISSN: 0385-6380 *
XIU-JUAN WANG ET AL: "Optimization of multienzyme production by two mixed strains in solid-state fermentation", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER-VERLAG, BE, vol. 73, no. 3, 27 June 2006 (2006-06-27), pages 533 - 540, XP019458726, ISSN: 1432-0614 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010070146A1 (en) 2008-12-19 2010-06-24 Danisco A/S Process for production of an enzyme product
US8765442B2 (en) 2008-12-19 2014-07-01 Dupont Nutrition Biosciences Aps Process for production of an enzyme product
US20120003339A1 (en) * 2010-07-02 2012-01-05 Minacapelli Pompeo Compositions and methods for controlling blood glucose levels
US20120252102A1 (en) * 2011-03-31 2012-10-04 Bioremediation Specialists, LLC Bioremediation systems, compositions, and methods
US9056340B2 (en) 2012-03-30 2015-06-16 Bioremediation Specialists L.L.C. Bioremediation systems, compositions, and methods
US20160222367A1 (en) * 2013-09-06 2016-08-04 Council Of Scientific & Industrial Research Enzyme composition and process of preparation thereof

Similar Documents

Publication Publication Date Title
Virupakshi et al. Production of a xylanolytic enzyme by a thermoalkaliphilic Bacillus sp. JB-99 in solid state fermentation
Irfan et al. One-factor-at-a-time (OFAT) optimization of xylanase production from Trichoderma viride-IR05 in solid-state fermentation
Kapoor et al. Cost-effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora
Divakaran et al. Comparative study on production of α-amylase from Bacillus licheniformis strains
US7727755B2 (en) Enzyme and methodology for the treatment of a biomass
Battan et al. High-level xylanase production by alkaliphilic Bacillus pumilus ASH under solid-state fermentation
Reda et al. Production of bacterial pectinase (s) from agro-industrial wastes under solid state fermentation conditions
KAPILAN et al. Paddy husk as support for solid state fermentation to produce xylanase from Bacillus pumilus
Dinarvand et al. Effect of extrinsic and intrinsic parameters on inulinase production by Aspergillus niger ATCC 20611
WO2008117301A1 (en) A process for the production of multienzyme system using fermentation
KR20120014813A (en) (bacillus pumilus with capacity of cellulose-decomposition and cultivation method thereof
KR101771960B1 (en) Paenibacillus jamilae BRC15-1 strain producing cellulase and use thereof
Singh et al. Microbial inulinases and pullulanases in the food industry
Mageshwaran et al. Growth kinetics of Bacillus subtilis in lignocellulosic carbon sources
Afifi Effective technological pectinase and cellulase by Saccharomyces cervisiae utilizing food wastes for citric acid production
Fadel et al. Cellulases and animal feed production by solid-state fermentation by Aspergillus fumigatus NRCF-122 mutant
Simoes et al. Optimization of xylanase biosynthesis by Aspergillus japonicus isolated from a Caatinga area in the Brazilian state of Bahia
Demirci et al. Xanthan gum production from hydrolyzed rice bran as a carbon source by Xanthomonas spp
Chesini et al. Aspergillus kawachii produces an inulinase in cultures with yacon (Smallanthus sonchifolius) as substrate
Youssef et al. Optimization of cultural conditions for β-mannanase production by a local Aspergillus niger isolate
Katapodis et al. Production of β-fructofuranosidase from Sporotrichum thermophile and its application in the synthesis of fructooligosaccharides
Chantorn et al. Suitable conditions for xylanases activities from Bacillus sp. GA2 (1) and Bacillus sp. GA1 (6) and their properties for agricultural residues hydrolysis.
JP5943326B2 (en) Glucose production method
Rao et al. Carbohydrases: A class of all-pervasive industrial biocatalysts
TWI418634B (en) Method for production of xylooligosaccharide with controllable composition by bacillus halodurans

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07859634

Country of ref document: EP

Kind code of ref document: A1

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07859634

Country of ref document: EP

Kind code of ref document: A1