Title of Invention

"AN IMPROVED PROCESS FOR THE IMMOBILIZATION OF BETA-FRUCTOFURANOSIDASES"

Abstract The present invention relates to an improved process for the immobilization of beta-fructofuranosidases by microencapsulation in inert matrices such as mesoporous silica. The immobilized enzyme produced by the process of the present invention has improved catalytic activity and enhanced stability. Further, the enzyme can be reused, making the catalysis cheaper.
Full Text AN IMPROVED PROCESS FOR IMMOBILIZATION ENZYMES.
This invention relates to an improved process for immobilization of hydrolyzing. enzymes. More particularly it relates to the said process of immobilization of hydrolyzing enzymes by microencapsulation enzymes in inert matrices such as silica.
Microencapsulation of proteins in inert matrices is important for their use as biocatalysts. The matrics should protect the proteins from degradation, aggregation, or denaturation and it should not perturb the native properties of the proteins. Such immobilization can prolong the shelf life of the protein while preserving its activity. The protective environment of the host can inhibit microbial degradation, hydrolysis and extend their useful lifetime. Recently Tetsuji Itoh et al have reported stabilization of chlorophyll a in mesoporous silica (J.mater.chem.2002,12,3275-3277). Horseradish peroxidase enzyme immobilization in folded sheet mesoporous materials are obtained by Takahashi group. They have studied its oxidative activity in toluene (R and D review of Toyoto CRDL vol.35 No.4 , 2000.12). Kuroda et al have reported the effective inclusion of chlorophyllous pigments into mesoporous silica modified with α,w Diols. (Chem.mater.2001,13,2722) Acording to Eric J. Ackerman Entrapping organophosphorous enzymes in a functionalized nanoporous support show double activity than plain enzyme(JACS 124(38) 11242) G.D.Stucky et al have carried out catalytic halogenation reactions using Vanadium bromoperoxidase in titanium grafted MCM41(JACS 1997,199,6921) Same author has investigated the size
exclusion properties of the small pore and large pore ARTS silylated SBA15
and MCF using three proteins of varying size vis; conalbumin, chicken egg
ovalbumin and soyabean trypsin inhibitor protein.(JACS 1999,121,9897)
There are two references by Humphrey Hp.yiu.et al. They have selectively
adsorbed trypsin to MCM 48 and MCM41 and studied the hydrolysis of N a-
benzoyl -DL arginine -4 nitro anilide. They have found that amount of
enzyme adsorbed is directly proportional to the pore size of the molecular
sieve, (microporous and mesoporous mater. 2001,44-45 763)
In another reference same people have immobilized trypsin on thiol chloride
and acid functionalized silica and did the hydrolysis of BAPNA. They claim
that reuse of the catalyst is possible. (J.Mol.Cat.B enzyme 2001,15,81-92),
However there are scanty reports on immobilization of p-fructofuranosidases
on mesoporous silica.
The main object of the invention is to provide an improved process for
immobilization of hydrolyzing enzymes exemplified by immobilization of p
Fructofuranosidases on mesoporous silica.
Another object is to reuse the catalyst.
Yet another object is to reduce the cost of the catalyst and make the process
cheaper.
Still another object is to have improvements in catalytic activity and stability
upon immobilization.
The process of immobilization provided by the present invention is illustrated
by taking a representative example of ßFructo- furanosidases enzymes which
catalys the hydrolysis of sucrose and related glycosides. The immobilized
enzyme is useful in hydrolyzing the sucrose into invert sugars. Though there
is ubiquitoes distribution, the enzyme of commercial interest originates from
yeasts sp. Hydrolysis of sucrose results in a mixture of sugars, (invert syrup) which is sweeter than sucrose due to the high degree of sweetness of fructose. Hence one of the most important applications of invertase lies in production of invert syrup from sucrose. The product is a 1: 1 mixture of glucose and fructose, which is more soluble than sucrose hence the sugar content can be increased considerably without the crystallization of sugar. Due to hygroscopic nature of invert syrup, it is used as humectants in the manufacture of chocolate-coated sweeteners and fondants. Invertase is also involved wherever sucrose containing substrates are subjected to fermentation, namely alcoholic beverages, lactic acid, glycerol etc.
Invertases are known to have insulinase activity, which can be used for the hydrolysis of insulin (polyfructose) to fructose.(1978- A Wiseman). D-fructose which is one of the product obtained by sucrose hydrolysis by invertase is 1.8 times sweeter than sucrose, well tolerated by diabetics, improves iron absorption and favours the removal of ethanol from the blood of alcoholics. (Brazilien Journal of Chemical Engineering -Vol.16 No.3 pp237-245 -1999 A.Pessoa and M.Vitolo)
Other uses of this enzyme include the manufacture of artifical honey, plasticizing agents in cosmetics, drugs and paper industry and enzyme electrodes in detecting sucrose.
Enzymatic hydrolysis of sucrose to invert sugar is preferential to acid hydrolysis, as it does not result in the production of furfural and other undesirable flavours as well as oligosaccharides.Though considerable attention has been paid to the production of high fructose syrup, very little importance has been given to invertase for the production of isomerase's syrup. Production of high fructose syrup requires starch hydrolysis as its starting material. In this respect, in countries like India where sucrose is readily available, utilization of sucrose will be advantageous. Additionally, molasses (a byproduct of sugar industry) can serve as source of sucrose because of its high sucrose content (approx. 50%). Invertase is also added in molasses to reduce osmolarity.
Beta fructo furanosidase when immobilized on functionalized mesoporous silica has been found to be very active for the production of invertase sugar syrup.
Accordingly, the present invention provides an improved process for the immobilization of beta-fructofuranosidases, wherein the steps comprising:
[a] soaking functionalized silica in a mixture of the enzyme beta-fructofuranosidases and buffer of pH 5.0 to 7.0 and adding a cross-linker which is glutaraldehyde;
[b] stirring the mixture of step [a] for a period of 10 to 18 hours at a temperature of not more than 10 degree C;
[c] separating the enzyme immobilized in silica by conventional methods;
[d] washing the immobilized enzyme of step [c] with buffer of pH 5.0 to 7.0 or with water to obtain the desired immobilized beta-fructofuranosidase.
In one of the embodiments of the present invention the functionalized silica designated as SBA-15 is obtained as per procedure given in science 1998, 278, 548. In another embodiment the cross linking agent may be an aldehyde such as glutaraldehyde. In still another embodiment the buffer may be a pH 5.0 to 7.0 buffer.
In another feature of the present invention the immobilized/crosslinked
enzyme may be used for the preparation of invert syrup comprising
The process of the present invention is described hereinbelow with reference to examples which are illustrative only and should not be construed to limit the scope of the present invention in any manner.
Example-1
This example illustrates the preparation of functionalized silica designated as Siliceous SBA-15 which was done as per literature procedure. (Science 1998, 279, 548)
4.0 g. portion of triblock copolymer Pluronic P -123 template was dissolved with stirring in a solution of 30 g of water and 120 g of 2N HCI at 313 K and 8.5 g of Tetra ethyl orthro silicate (TEOS) is then added. The resulting mixture was stirred at 40 °C for 20 hrs. and then was aged at 80 °C for 2 days under static condition. The as prepared sample was recovered by filtration and air dried at 80 °C overnight. The organic template was removed by calcinations in air at 540 °C for 6 hrs. The calcined SBA-15 was suspended in toluene, and refluxed to make it dry. Then 30% of aminopropyl triethoxy silane was added to it .The refluxing was continued for 6h It was then filtered, washed with toluene and dried at 100 °c .
Example 3
Immobilisation of invertase (p -fructofuranosidase) enzyme was done as follows. 1 gm of SBA-15 was suspended in 10 ml of 50 mM Sodium acetate buffer. 5 mg of invertase enzyme ( ß -fructofuranisidase) was added to this and stirred at 4°c - 10°c temperature overnight. Zeolite enzyme complex was separated by centrifugation. Buffer washings were given to the complex to
remove free enzyme. Activity of the supernatant and also of the complex was checked after 5 washes.
Example 4
This example illustrates the preparation of crosslinked enzyme. 1 gm of SBA-15 silica with functionalised NH2 group was mixed with sodium acetate buffer and 1 mi enzyme containing 5 ml of protein to the final concentration was stirred at 4°c-10°c for two hours and o.2 ml glutaraldehyde was added as cross linking reagent. This mixture was kept stirring further overnight. Zeolite enzyme complex was separated by centrifugation. Buffer washing were given to remove free enzyme. Activity of the supernatant and complex was checked by invertase assay described by Gascon and Lampen.(JBC 1968 vol 243 pi 573-1577.)
Example 5
Temperature stability was done with both complexes, plain as well crosslinked. Temperature selected were ranging from 30°c-80°c. After 1 hr incubation assay was done essentially as described above.
(Table Removed)
Example-6
This example illustrates the pH stability and profile (incubated at room temperature)
pH stability was done with both the complexes plain as well as crossed enzyme on zeolite. Buffers ranging from pH 4 to pH 10 were selected. 50 mm buffer of above pHs were used for suspending mesoporous silica complexes comprising approx. 30 L of an enzyme. After 1 hour incubation in respective buffer, the silicates were washed with 50 mm of acetate buffer and assayed as mentioned above.

(Table Removed)
Example-7
This example Illustrates the Both the complexes as
well as free enzyme were assayed at different temperatures ranging from 30°c-> 80 ° c. in 50mM sodium acetate buffer pH 4.5 . Substrate used was 2 g. % sucrose.

(Table Removed)
Example-8
This example illustrates pH stability of the enzyme. Both complexes and free enzyme were assayed using different pH buffers ranging from pH4—pH10. Sucrose concentration was 2 g.% and buffer concentration was 50mM. Incubation temperature was 50 ° c.

(Table Removed)
The main advantages of the present invention are as follows:
The process of this invention is to reuse the catalyst.
Another advantage of the present invention is to reduce the cost of the
catalyst and make the process cheaper.
Still another advantage of the present invention is to have improvements in
catalytic activity and stability upon immobilization.
















We claim:
1. An improved process for the immobilization of beta-fructofuranosidases
characterized in cross-linking functionalized silica with glutaraldehyde, wherein the
steps comprising:
[a] soaking functionalized silica in a mixture of the enzyme beta-fructofuranosidases and buffer of pH 5.0 to 7.0 and adding a cross-linker which is glutaraldehyde;
[b] stirring the mixture of step [a] for a period of 10 to 18 hours at a temperature of not more than 10 degree C;
[c] separating the enzyme immobilized in silica by conventional methods;
[d] washing the immobilized enzyme of step [c] with buffer of pH 5.0 to 7.0 or with water to obtain the desired immobilized beta-fructofuranosidase.
2. An improved process for the immobilization of beta-fructofuranosidases substantially as herein described with reference to the foregoing examples.

Documents:

1874-DEL-2004-Abstract-(05-08-2010).pdf

1874-DEL-2004-Claims (03-05-2011).pdf

1874-DEL-2004-Claims-(03-05-2011).pdf

1874-DEL-2004-Claims-(05-08-2010).pdf

1874-DEL-2004-Claims-(08-10-2010).pdf

1874-DEL-2004-Correspondence Others-(03-05-2011).pdf

1874-DEL-2004-Correspondence-others (03-05-2011).pdf

1874-DEL-2004-Correspondence-Others-(05-08-2010).pdf

1874-DEL-2004-Correspondence-Others-(08-10-2010).pdf

1874-DEL-2004-Description (Complete)-(08-10-2010).pdf

1874-DEL-2004-Form-1-(05-08-2010).pdf

1874-DEL-2004-Form-1-(08-10-2010).pdf

1874-DEL-2004-Form-2-(05-08-2010).pdf

1874-DEL-2004-Form-3-(05-08-2010).pdf

1874-DEL-2004-Form-3-(08-10-2010).pdf

1874-DEL-2004-Form-5-(05-08-2010).pdf

1874-DEL-2004-Form-5-(08-10-2010).pdf

1876-del-2004-abstract.pdf

1876-del-2004-claims.pdf

1876-del-2004-correspondence-others.pdf

1876-del-2004-description (complete).pdf

1876-del-2004-form-1.pdf

1876-del-2004-form-18.pdf

1876-del-2004-form-2.pdf

1876-del-2004-form-3.pdf

1876-del-2004-form-5.pdf


Patent Number 247991
Indian Patent Application Number 1874/DEL/2004
PG Journal Number 23/2011
Publication Date 10-Jun-2011
Grant Date 08-Jun-2011
Date of Filing 29-Sep-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 SANJEEVANI AMRIT PARDHY NATIONAL CHEMICAL LABORATORY, PUNE-411008, INDIA.
2 SHILPA SHIRISH DESHP ANDE NATIONAL CHEMICAL LABORATORY, PUNE-411008, INDIA.
3 ASMITA ASHUTOSH PRABHUNE NATIONAL CHEMICAL LABORATORY, PUNE-411008, INDIA.
4 ARCHANA VISHNU PUNDLE NATIONAL CHEMICAL LABORATORY, PUNE-411008, INDIA.
PCT International Classification Number C12N 11/14
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA