Enzymes

Enzymes are proteins that function as organic catalysts in cells. They speed up the metabolic reactions over a million fold, allowing the biochemical reactions that are essential for life. Many enzymes are produced commercially for use in the biotechnology industry. By the end of the 1990s the annual world wide sale of industrial enzymes stood in the region of US 1.5, approximately double the figure recorded a decade earlier Protease accounted for almost 50% of the market share, while carbohydrate degrading enzymes accounted for much of the rest (Davis,J,.2001). (Table 1) TABLE 1- ESTIMATED ANNUAL GLOBAL SALES VALUE OF INDUSTRIAL ENZYMES | ENZYME |MARKET VALUE (USS, MILLION) | |PROTEASE |700 | |CELLULASE |180 | |α-AMYLASE |135 | |LIPASE |90 | |HEMICELLULASE |75 | |GLUCOAMYLASE |60 | |PECTINASES |60 | |LACTASE |15 | |GLUCOSE ISOMERASE |15 | |OTHERS |100 | REHM ET AL (1999) THE MAJORITY OF THE INDUSTRIAL ENZYMES ARE OBTAINED FROM MICROORGANISMS. THE PRODUCER STRAINS ARE USUALLY MEMBERS OF THE FAMILY OF THE MICROBES CLASSIFIED AS GRAS. THE BACTERIA AND FUNGI, MOST NOTABLY BY THE MEMBERS OF THE GENERA BACILLUS AND ASPERGILLUS, PRODUCE SUCH BULK ENZYMES. TRADITIONALLY YIELDS WERE INCREASED BY STRAIN IMPROVEMENT SUCH AS MUTAGENESIS FOLLOWED BY THE IDENTIFICATION OF HYPER PRODUCING MICROBIAL STRAINS. POLYSACCHARIDE DEGRADING ENZYMES REPRESENT ONE OF THE MOST SIGNIFICANT GROUPS OF INDUSTRIALLY IMPORTANT BULK ENZYMES. SUCH ENZYMES INCLUDE AMYLASES, PECTINASES AND CELLULASES. IN ADDITION, SEVERAL OTHER CARBOHYDRATE TRANSFORMING ENZYMES SUCH AS GLUCOSE ISOMERASE, INVERTASE AND LACTASE ALSO ENJOY SIGNIFICANT COMMERCIAL NICHE MARKETS. AMYLOLYTIC ENZYMES ENZYMES THAT PARTICIPATE IN THE HYDROLYTIC DEGRADATION OF STARCH ARE REFERRED TO AS AMYLOLYTIC ENZYMES OR AMYLASES AND BELONG TO THE FAMILY OF HYDROLASES ALSO KNOWN AS DIASTASE AS THEIR POWER OF SEPARATING SOLUBLE DEXTRINS FROM THE INSOLUBLE ENVELOPS OF STARCH GRAINS. SPECIFIC ENZYMES CLASSIFIED WITHIN THIS GROUP INCLUDE α-AMYLASE β-AMYLASE, GLUCOAMYLASE ALSO KNOWN AS AMYLOGLUCOSIDASE PULLULANASE AND ISO AMYLASE. ALPHA AMYLASE IS AN ENZYME THAT IS INDUSTRIALLY IMPORTANT FOR THE PRODUCTION OF CORN SYRUP FROM CORNSTARCH, AS AN ADDITIVE IN LAUNDRY, FOR THE SIZING IN TEXTILE INDUSTRY, IN THE BREWING OF BEER, AND IN THE PRODUCTION OF BREAD. ENZYMATIC DEGRADATION OF STARCH YIELDS GLUCOSE, MALTOSE, AND OTHER LOW MOLECULAR MASS SUGARS. FURTHER MORE, ENZYMATIC MEDIATED ISOMERIZATION OF THE GLUCOSE YIELDS HIGH FRUCTOSE SYRUPS. Table 2: Sources and characteristics of some industrially significant amylolytic enzymes. |Enzyme |Endo- or exo acting|Glycosidic |SOURCE |PH OPTIMUM |TEMP OPT(C0) | | | |BOND CLEAVED | | | | |α- AMYLASE |ENDO |α ,1-4 |BACILLUS SUBSTILUS |6.0 |650 | |GLUCOAMYLASE |EXO |α ,1-4 |ASPERGILLUS NIGER |6.0 |600 | |β-AMYLASE |EXO |α ,1-4 |BACILLUS SP |5.5 |600 | | | | |CLOSTRIDIUM SP | | | |PULLULANASE |ENDO |α ,1-6 |KLEBSIELLA SP |4.5 |800 | |ISO AMYLASE |ENDO |α ,1-6 |PSEUDOMONAS SP |4.5 |550 | (GRUGER,1990) Starch and glycogen are the storage polymers of glucose made via dehydration synthesis reactions. Starch is abundant in plants whereas glycogen is abundant in animal tissues. Abundant supplies of starch may be obtained from corn, wheat, rice, tapioca, and potato. The wide spread availability of starch from such inexpensive sources, coupled with large scale production of amylolytic enzymes, facilities production of syrups containing glucose, fructose or maltose, which are of considerable importance in food and confectionary industry. Furthermore, they may be produced quite competitively when compared to the production of sucrose, which is obtained directly from traditional sources such as sugar beet or sugar cane. THE STARCH SUBSTRATE STARCH REPRESENTS THE MOST ABUNDANT STORAGE FORM OF POLYSACCHARIDE IN PLANTS, AND NEXT TO CELLULOSE IT IS THE MOST ABUNDANT POLYSACCHARIDE FOUND ON EARTH. IT IS ABUNDANT IN SEEDS SUCH AS CORN AND IN A WIDE VARIETY OF TUBERS. IT IS STORED IN GRANULAR FORM IN PLANT CELLS. THE STARCH POLYMER CONSISTS OF EXCLUSIVELY OF GLUCOSE UNITS. TWO FORMS EXISTS NAMELY AMYLOSE AND AMYLOPECTIN. AMYLOSE WHICH IS LINEAR CHAIN OF GLUCOSE UNITS JOINED BY α-1, 4-GLYCOSIDIC LINKAGE HAVING 500 TO 2000 GLUCOSE UNITS IN IT AND THE OTHER IS, AMYLOPECTIN, A BRANCHED POLYMER THAT IS SIMILAR TO AMYLOSE EXCEPT ITS BRANCHES ARE ATTACHED TO THE BACKBONE VIA α-1, 6 LINKAGES. THE DEGREE OF BRANCHING IN AMYLOPECTIN IS APPROXIMATELY ONE PER TWENTY-FIVE GLUCOSE UNITS IN THE UNBRANCHED SEGMENTS. AGRICULTURAL CROPS THAT ARE NOT USED FOR FOOD ARE USED TO PRODUCE LARGE AMOUNTS OF STARCH FOR THE MANUFACTURE OF ADHESIVES AND SUGAR SYRUPS. SUGAR SYRUPS THAT ARE MADE FROM STARCH INCLUDE GLUCOSE MALTOSE, FRUCTOSE AND SORBITOL. WHILE CONSIDERING GLYCOGEN IT IS STORAGE POLYMER IN THE ANIMAL TISSUE ITS STRUCTURE RESEMBLES TO AMYLOPECTIN EXCEPT IT IS MORE HIGHLY BRANCHED. GLYCOGEN HAS BRANCHING PER 12 UNITS .THE DEGREE OF BRANCHING AND THE CHAIN LENGTH VARY FROM SOURCE TO SOURCE, BUT IN GENERAL THE MORE THE CHAINS ARE BRANCHED, THE MORE THE STARCH IS SOLUBLE. MODE OF ENZYME ACTION IN ORDER TO MAKE USE OF THE CARBON AND THE ENERGY STORED IN STARCH, IT MUST BREAKDOWN INTO SMALLER ASSIMILABLE SUGARS, WHICH IS EVENTUALLY CONVERTED TO THE INDIVIDUAL BASIC GLUCOSE UNITS. THIS BREAK DOWN IS DONE THROUGH THE USE OF BIOLOGICAL CATALYSTS, WHICH IS AMYLASE HERE. ALL CELLS SYNTHESIZE ENZYMES THAT ARE USED FOR METABOLIC PROCESSES WITHIN THE CELLS .IN MAMMALS α-AMYLASE IS MADE IN THE SALIVARY GLANDS AND IS SECRETED INTO THE SALIVA. ALTHOUGH STOMACH ACIDS DESTROY SALIVARY α-AMYLASES, THE PANCREAS MAKES MORE α-AMYLASE AND SECRETES IT INTO THE INTESTINES, WHERE IT PARTICIPATES WITH OTHER DIGESTIVE ENZYMES IN THE BREAKDOWN OF STARCH AND GLYCOGEN TO GLUCOSE. MANY BACTERIA AND FUNGI ALSO PRODUCE α-AMYLASE, WHICH THEY TYPICALLY SECRETE INTO THE ENVIRONMENT TO BREAKDOWN STARCH SO THAT SMALLER SUGARS CAN ENTER THE MICROBIAL CELLS. α-AMYLASE IS STORED IN THE SEEDS OF PLANTS AND IS ACTIVATED UPON GERMINATION TO BREAK DOWN THE STARCH IN THE SEED THAT LEADS TO THE PRODUCTION OF GLUCOSE FOR THE PLANT EMBRYO. The enzyme α-amylase is an endoacting enzyme catalyzes the hydrolysis of α-1,4 linkages in starch (or glycogen) to yield maltose, maltotriose, and α-dextrin, depending upon the relative position of the bond under attack as counted from the end of the chain. Alpha amylase is incapable of hydrolyzing α-1, 6 glycosidic linkages present on the branch points of amylopectin chains. One exception to this is the α-amylase produced by Thermoactinomyces vulgaris, which can hydrolyze both α-1,4 and α-1, 6 glycosidic linkages. [pic] Figure-1: Hydrolysis of starch by α-amylase and β-amylase Products formed by starch hydrolysis . DEPENDING UPON THE RELATIVE POSITION OF THE BOND UNDER ATTACK AS COUNTED FROM THE REDUCING END OF THE CHAIN, THE PRODUCTS OF THIS DIGESTIVE PROCESS ARE DEXTRINS, MALTOTRIOSE, MALTOSE AND GLUCOSE ETC. MALTOSE HAS TWO GLUCOSE RESIDUES IN AN ALPHA1, 4 LINKAGES; IT IS FORMED IF THE SECOND MOLECULE FROM THE REDUCING END IS UNDER ATTACK. . MALTOTRIOSE HAS THREE GLUCOSE RESIDUES LINKED ALPHA1, 4. IT IS FORMED IF THE THIRD BOND FROM THE REDUCING END OF STARCH IS UNDER ATTACK. ALPHA DEXTRIN CONSISTS OF A α-1,4 BACKBONE AND ASSOCIATED α-1-6 BRANCH POINTS; THESE ARE SHORTER BROKEN STARCH SEGMENTS THAT FORM AS THE RESULT OF RANDOM HYDROLYSIS OF INTERNAL GLUCOSIDIC BONDS. GLUCOSE, A MOLECULE OF GLUCOSE IS OBTAINED IF THE BOND BEING CLEAVED IS THE TERMINAL ONE. THE ENZYME β-AMYLASE, FOUND IN BARLEY SEEDS ALSO HYDROLYZES STARCH BY CLEAVING SUCCESSIVE MALTOSE UNITS FROM THE ENDS OF THE CHAINS. THE BREAKDOWN OF LARGE POLYMERS DRASTICALLY REDUCES THE VISCOSITY OF GELATINIZED STARCH SOLUTION, RESULTING IN A PROCESS CALLED LIQUEFACTION BECAUSE OF THE THINNING OF THE SOLUTION. THE FINAL STAGE OF DEPOLYMERIZATION IS MAINLY THE FORMATION OF MONO-, DI-, AND TRI-SACCHARIDES. THIS PROCESS IS CALLED SACCHARIFICATION, DUE TO THE FORMATION OF SACCHARIDES. THE SPECIFICITY OF THE BOND ATTACKED BY ALPHA AMYLASE COMMONLY DEPENDS ON THE SOURCE OF THE ENZYME. CURRENTLY, MAJOR CLASSES OF ALPHA AMYLASE ARE COMMERCIALLY PRODUCED THROUGH MICROBIAL FERMENTATION. THEY ARE CLASSIFIED ACCORDING TO THEIR STARCH LIQUEFYING AND/OR SACCHAROGENIC EFFECT, PH OPTIMUM, TEMPERATURE RANGE, AND STABILITY. SACCHAROGENIC AMYLASES PRODUCE FREE SUGARS, WHEREAS STARCH-LIQUEFYING AMYLASES BREAK DOWN THE STARCH POLYMER BUT DO NOT PRODUCE FREE SUGARS. MANY ORGANISMS PRODUCE SEVERAL AMYLASES. [pic] FIG -2: PRODUCTS OF STARCH HYDROLYSIS BY α-AMYLASE IMPORTANT BACTERIAL SPECIES PRODUCING α-AMYLASE BACTERIA, WHICH PRODUCE α -AMYLASE, ARE BACILLUS SUBTILIS, B.CEREUS, B.AMYLOLIQUEFACIENS, B.COAGULANS, B.POLYMYXA, B.STEAROTHERMOPHILUS, B.LICHENOFORMIS, LACTOBACILLUS, MICROCOCCUS, PSEUDOMONAS, ESHERICIA, PROTEUS, THERMOMONOSPORA, AND SERRATIA. THREE VERY SIMILAR STRAINS PRODUCING SACCHROGENIC α-AMYLASE ARE B.SUBTILIS VAR. B. SUBTILIS MARBURG, B.NATTO, AND AMYLOSACCHARATICUS. THE STRAIN AMYLOLIQUEFACIENS DIFFERS FROM THESE IN THAT IT PRODUCES A LIQUEFYING α - AMYLASE. SOME α-AMYLASE PRODUCING FUNGI ARE FROM THE GENRA ASPERGILLUS, PECILLIUM, CANDIDA, NEUROSPORA, AND RHIZOPUS.(PATRICIA.,1995) PHYSIOCHEMICAL PROPERTIES OF AMYLASES. α-AMYLASES OCCURS WIDELY IN BACTERIA AND FUNGI AND IS AN ENDO ACTING ENZYME, UNABLE TO HYDROLYZE THE α-1,6 LINKAGES IN AMYLOPECTIN BUT ARE ABLE TO BY PASS THEM. THE MOLECULAR WEIGHTS OF VARIOUS α-AMYLASES DO NOT DIFFER CONSIDERABLY. TABLE 3: MOLECULAR WEIGHTS OF DIFFERENT α-AMYLASES |SOURCES OF α-AMYLASE |MOLECULAR WEIGHTS | |α-AMYLASE (3.2.1.1)(MALT) |59000 | | α- AMYLASE (3.2.1.1)(B SUBTILIS) |48,700 | | α-AMYLASE(3.2.1.1) (PANCREAS) |45,000 | |α- AMYLASE (3.2.1.1) (B STEAROTHERMOPHILUS) |15,500 | |α-AMYLASE (3.2.1.1)(B. AMYLOLIQUEFICIENS) |49,000 | |α- AMYLASE (3.2.1.1) (A. NIGER) |58,000 | (GRUGER, 1990) VERY SENSITIVE STUDIES ON THESE ENZYMES SHOWED THAT THESE HAVE ALMOST 15% NITROGEN AND ARE METALLOPROTEINS. THEY ALL CONTAIN A LARGE PROPORTION OF TYROSINE AND TRYPTOPHAN IN THE ENZYME PROTEIN AND MOST REQUIRE CALCIUM AS STABILIZER, AND THESE ENZYMES ARE INACTIVATED BY THE IODIDE AND FLUORIDE IONS. AMYLASES ARE THERMOLABILE AND THEIR HEAT STABILITY VARIES MARKEDLY WITH THE TYPE OF AMYLASE AND WITH THE DEGREE OF PURITY OF THE ENZYME PREPARATIONS. SO, OPTIMUM TEMPERATURE HAS LITTLE MEANING. THE VELOCITY OF SUBSTRATE DEGRADATION INCREASES WITH TEMPERATURE IN USUAL WAY. AMYLASE ACTIVITY IS REGULATED BY DEFINITE RANGE OF PH. FOR EACH AMYLASE THERE IS A HYDROGEN IONS ACTIVITY OVER WHICH IT IS MORE STABLE AND MOST ACTIVE, BUT EXACT VALUES DEPEND UPON A NUMBER OF FACTORS, AMONG WHICH THE KIND AND CONCENTRATION OF ELECTROLYTE, TEMPERATURE AND TIME OF EXPOSURE ARE IMPORTANT. FOR EXAMPLE MALT α- AMYLASE IS MOST STABLE AT A PH CLOSE TO 7 BUT THE OPTIMUM PH WAS NOT ABOVE 5 FOR ACTIVITY AT 300C. THE MOST IMPORTANT SUBSTRATES FOR AMYLASE ACTION ARE STARCHES AND GLYCOGENS IN THE FORM OF PASTES AND SOLUTIONS. CLASSIFICATION OF AMYLASES SIMULTANEOUS QUANTITIVE OBSERVATIONS ACCOMPLISHED DURING AMYLASE ACTION REVEAL THE EXISTENCE OF DIFFERENT KINDS OF AMYLASES. THEY ARE CLASSIFIED ACCORDING TO THERE ➢ SACCHROGENIC EFFECTS ➢ PH OPTIMUM ➢ TEMPERATURE RANGE [pic] [pic][pic] FIG- 3: THE SUMMARY OF STARCH DEGRADING ENZYMES AND THEIR INTER RELATIONSHIP. EXOSPLITTING AMYLASES BETA (β) AMYLASE:[EC3.2.1.2] THE MAIN TYPE OF AMYLASES RECOGNIZED AT PRESENT, IS SACCHAROGENIC AMYLASE ALSO KNOWN AS β-1-4-GLUCANHYDROLASE. THESE AMYLASES ARE CHARACTERIZED BY THEIR ABILITY TO REMOVE SUCCESSIVE MALTOSE UNITS FROM THE REDUCING ENDS OF POLYSACCHARIDE CHAINS OF STARCHES, WITHOUT MARKEDLY DISRUPTING THE REST OF STARCH MOLECULE. THE DEXTRIN WHICH REMAIN AFTER THE ACTION OF SACCHROGENIC AMYLASES HAVE RELATIVELY HIGH MOLECULAR WEIGHTS, ARE NON REDUCING, AND RETAIN THE PROPERTY OF GIVING WITH IODINE. THE MALTOSE FORMED EXHIBITS RISING (DEXTROROTATORY) OR β MUTA ROTATION , HENCE THE NAME β AMYLASE. GLUCO- AMYLASE [EC 3.2.1.3] GLUCOAMYLASE IS ALSO KNOWN AS α,1-4GLUCAN GLUCANOHYDROLASE. IT REMOVES SUCCESSIVE GLUCOSE UNITS FROM THE NON-REDUCING ENDS OF THE SUBSTRATE CHAIN. THE ACTION OF THE ENZYME ON A CHAIN DECREASES MARKEDLY WHEN AN α-1,6-LINKAGE IS ENCOUNTERED AS IN AMYLOPECTIN AND GLYCOGEN. Endosplitting amylase ALPHA-(α) AMYLASE [EC 3.2.1.1] ENDO SPLITTING AMYLASES HAVE CHARACTERISTIC NAMES LIKE DEXTRINOGENIC OR LIQUEFYING AMYLASES. THESE ENZYMES HYDROLYZE THE α -1-4-GLUCOSIDIC BONDS, THOUGH A α-1-6- BONDS CONSTITUTING THE BRANCH POINTS OF AMYLOPECTIN AND GLYCOGEN REMAIN UNATTACKED. RAPID HYDROLYSIS RESULTS IN MALTOSE OR GLUCOSE AND REDUCING DEXTRIN, WHICH GIVE NO COLOR WITH IODINE (DEXTRINOGENIC ACTION). THESE CHANGES ARE USUALLY ACCOMPANIED BY CORRESPONDING RAPID DECREASE IN THE VISCOSITIES OF THE STARCH PASTES (LIQUEFYING ACTION). IN SEVERAL CASES THE PRODUCTS FORMED BY DEXTRINOGENIC AMYLASES HAVE BEEN FOUND TO EXHIBIT FALLING OR A MUTAROTATION; HENCE THE NAME α- AMYLASE IS OFTEN APPLIED TO THEM . THEY ARE ALSO NAMED AS α-1,4 GLUCANO HYDROLASE. Types of Amylases CLASSIFICATION BASED ON THE SOURCE OF ENZYME IS OF MORE VALUE FOR THE AMYLASE PRODUCER THAN TO THE USER. THE VISUAL CLASSIFICATION ACCORDING TO SOURCE CONSISTS OF A DIVISION INTO SOME MAJOR GROUPS DISUSED AS UNDER; ANIMAL α -AMYLASE IT INCLUDES THE SALIVARY, PANCREATIC, BLOOD, URINE, MUSCLE AND LIVER AMYLASE SECRETED FROM THE RESPECTIVE ORGANS OF ANIMALS. THEY ALL PERFORM THE SAME FUNCTION AND QUITE STABLE IN PURE SOLUTION FORMS PLANT α -AMYLASE RIPE OR NON-GERMINATED CEREALS, GENERALLY CONTAIN ONLY TRACES OF AMYLASES, BUT DURING THE GERMINATION OF THE GRAINS LARGE AMOUNTS OF THE ENZYME ARE CONVERTED FROM INACTIVE INTO AN ACTIVE STATE. THESE AMYLASES ARE KNOWN AS MALTA AMYLASES. MALTED GRAINS INCLUDE OATS, RYES, RICE AND WHEAT. SMALL AMOUNTS OF CALCIUM SALTS STABILIZE THESE ENZYMES AND THESE ARE NOT ACTIVATED BY CHLORIDE IONS. THEIR OPTIMUM PH IS ABOUT 5. YEAST α -AMYLASE THE YEAST ENZYME IS IDENTICAL WITH ISOPHOSPHORYLASE, CAPABLE OF BREAKING AND SYNTHESIZING 1-6-GLUCOSIDIC LINKAGES AND IS ALSO CAPABLE OF REMOVING ANOMALOUS END GROUPS OF DEXTRINS. MOLD α-AMYLASE MANY MOLDS ARE EXTREMELY RICH IN AMYLASE AND ARE IMPORTANT COMMERCIALLY. THESE AMYLASES ARE EXECRATED PARTICULARLY, FROM THE MEMBERS OF THE GENERA ASPERGILLUS, RHIZOPUS, AND ENDOMYCETES. NATURALLY, THE FUNGAL AMYLASES ARE REQUIRED IN LARGE QUANTITIES FOR THEIR ACTION TO COMPENSATE FOR THEIR LOSS IN THE PROCESS. BACTERIAL α- AMYLASE MANY AMYLASES OF BACTERIAL ORIGIN ARE KNOWN WHICH APPEARS TO BE DEXTRINOGENIC AMYLASE SIMILAR TO THOSE ALREADY DISUSED. BACTERIAL AMYLASES HAVE FOUND WIDE APPLICATION IN INDUSTRY, BIOTECHNOLOGY AND HAVE BEEN INVESTIGATED IN CONSIDERABLE DETAILS. MOSTLY BACTERIAL AMYLASES ARE ORIGINATED FROM THE GENUS BACILLUS OF THE FAMILY BACILLACEAE. BACTERIAL AMYLASES ARE GENERALLY THERMOSTABLE AND ARE QUITE OFTEN THERMOPHILLIC AND COULD BE USED AT LOW LEVELS OF ENZYMES CONCENTRATION. THEREFORE, THERE IS AN INCREASING DEMAND FOR BACTERIAL AMYLASES. (DAVIS,1992) INDUSTRIAL APPLICATIONS OF AMYLASES AMYLOLYTIC ENZYMES MAINLY OF MICROBIAL ORIGIN ARE USED EXTENSIVELY IN A WIDE VARIETY OF INDUSTRIAL PROCESSES. ONE OF THE MORE IMPORTANT USES AND CERTAINLY ONE, WHICH HAS BEEN ACCORDED MOST, IS THE HYDROLYSIS OF CEREAL MASHES PRIOR TO ALCOHOLIC FERMENTATION. THE FOOD INDUSTRY ALSO USES LARGE AMOUNTS OF AMYLASE, PARTICULARLY FOR PRODUCING GLUCOSE SYRUPS FROM STARCHY MATERIALS, THE TYPE OF AMYLOLYTIC ENZYME CHOSEN DEPENDING UPON THE RAW MATERIAL USED AND THE PRODUCT REQUIRED. The textile and paper industries too find uses for amylase. Preparations in removing starchy seizing from fabrics as well as for coating paper. (Takagi, 1971). Other uses to which amylases are put include fortification of flour in baking (West, Todd, and Mason, 1953) in the manufacture of pre cooked baby foods, treatment of chocolate and liquorices syrups to prevent them from coagulation, and removal of wallpaper. Somewhat small amounts are also used as digestive aids (Zoltowoska, 2001) A FEW INDUSTRIAL APPLICATIONS OF α-AMYLASES ARE LISTED BELOW. THESE CAN SHOW THE SIGNIFICANCE OF THE ENZYME IN OUR COUNTRY. TEXTILE INDUSTRY; AS DESIZING AGENT IN THE TEXTILE INDUSTRY FOR REMOVING STARCH FROM THE GRAY CLOTHE BEFORE IT’S FURTHER PROCESSING IN BLEACHING AND DYEING. PAPER INDUSTRY: RAW STARCH HYDROLYZED WITH ALPHA AMYLASE IS USED FOR SIZING AND COATING PAPER INSTEAD OF EXPENSIVE CHEMICALLY MODIFIED STARCHES. GLUCOSE INDUSTRY: MANUFACTURE OF GLUCOSE FROM STARCH THROUGH ENZYME IS CHEAPER AND EASIER PROCESS THUS ELIMINATING ACID REACTION THAT LOWERS THE YIELD OF GLUCOSE. SUGAR INDUSTRY: THE PRESENCE OF STARCH AND OTHER POLYSACCHARIDES IN CANE SUGAR JUICE GIVES PROBLEMS THROUGHOUT THE SUGAR MANUFACTURING PROCESS, FOR INSTANCE BY CAUSING LOWER FILTRATION RATES, INCREASED VISCOSITIES AND INFERIOR CRYSTALLIZATION CHARACTERISTICS. THESE POLYSACCHARIDES CAN BE REMOVED BY THIS ENZYME. FEED INDUSTRY: NUTRITIONAL VALUE OF FEEDS CONTAINING BARLEY OR OTHER MATERIALS CAN BE IMPROVED BY THE ADDITION OF α-AMYLASE. THE SUPPLEMENTATION OF FEEDS WITH ENZYMES IS ACTIVELY BEING STUDIED ABROAD. LIVESTOCK RAISING EXPERIMENTS HAS PROVED ITS SUITABILITY FOR THIS PURPOSE. ALCOHOL INDUSTRY: IN THE PRODUCTION OF ETHYL ALCOHOL FROM GRAIN, POTATOES, AND OTHER STARCH CONTAINING RAW MATERIALS THE STARCH IS FIRST CONCERTED TO FERMENTABLE SUGARS. THE USE OF BACTERIAL ENZYME PARTLY REPLACES MALT IN BREWING INDUSTRY, THUS MAKING THE PROCESS MORE ECONOMICAL. BREAD INDUSTRY: THE ADDITION OF ENZYME TO FLOUR IMPROVES THE KEEPING QUALITIES, TASTE, AROMA AND POROSITY OF THE BREAD. MORE THAN 70% BREAD IN USA, RUSSIA, AND EUROPEAN COUNTRIES CONTAIN THIS ENZYME. FIRST CLEAR RECOGNITION OF AN ENZYME WAS MADE BY PAYAN AND PERSOZ IN 1883, WHEN THEY FOUND THAT AN ALCOHOL PRECIPITATE OF MALT EXTRACT CONTAINED A THERMOLABILE SUBSTANCEWHICH CONVERTED STARCH INTO SUGAR. THIS SUBSTANCE, WHICH IS KNOWN AS AMYLASE, WAS NAMED BY THEM AS DIASTASE, FROM SEPARATION BECAUSE OF ITS POWER OF SEPARATING SOLUBLE DEXTRIN FROM THE INSOLUBLE ENVELOPS OF THE STARCH GRAINS. THE NAME DIASTASE LATER CAME TO BE USED AS A GENERAL TERM FOR ENZYMES.(ENCARTA 2002) Amylases are found in saliva, pancreatic juice, blood cells, blood serum, liver, in many plant seeds, and grains, also found in molds and bacteria. Extensive work on the production, characterization and purification techniques of α-amylase by various sources is reported such as, • MICROBIAL SOURCE • ANIMAL SOURCE • PLANT SOURCE MICROBIAL α-AMYLASE Davis et al ,(1980)studied the production of α-amylase (α-1,4-glucan 4-glucanohydrolase; EC 3.2.1.1) by a strain of Bacillus stearothermophilus isolated from leaf litter was investigated in a tryptone-maltose medium at 55 0C in batch and chemostat culture. Amylase production was growth-limited and restricted to the exponential phase in batch culture. The enzyme yield was reduced by 40% when the culture pH was maintained at pH 7.2. Amylase production in chemostat culture was influenced by the growth rate throughout the dilution rate range used. AUGUSTIN ET AL, (1981) STUDIED THE PRODUCTION OF α-AMYLASE BY MICROSCOPIC FUNGI. THE AMYLOLYTIC ACTIVITY AND ESPECIALLY THE PRODUCTION OF α-AMYLASE (EC 3.2.1.1) AND α-GLUCOSIDASE (EC 3.2.1.20) WAS SCREENED IN IMPERFECT FUNGI, MUCORAL FUNGI AND SOME ASCOMYCETES. THE AMYLOLYTIC ACTIVITY WAS FOUND IN 29 STRAINS OUT OF THE 49 TESTED. GHOSH ET AL, ( 1984) WORKED ON THE PURIFICATION AND EVALUATED SOME PROPERTIES OF A THERMOSTABLE α--AMYLASE FROM BACILLUS APIARIUS CBML 152 BY ETHANOL PRECIPITATION, STARCH AMYLASE COMPLEX FORMATION AND BY ION-EXCHANGE CHROMATOGRAPHY USING DEAE-CELLULOSE AT PH 8.6, ELUTED WITH 0.2 TO 0.3 M NACL IN THE STARTING BUFFER (0.5 M TRIS-HCL, PH 8.6). SRIVASTAVA ET AL,(1986) WORKED ON THE PARTIAL PURIFICATION AND PROPERTIES OF THERMOSTABLE INTRACELLULAR AMYLASES FROM A THERMOPHILIC BACILLUS SP. THE CRUDE ENZYME, HAVING PH OPTIMUM AT 6.5. AND TEMPERATURE OPTIMUM AT 680C WAS PURIFIED BY DEAE-CELLULOSE COLUMN CHROMATOGRAPHY. THREE SEPARABLE ENZYME FRACTIONS HAVING STARCH HYDROLYZING PROPERTY WERE ELUTED BY LOWERING THE PH FROM 8.5 TO 7.0. LIN ET AL, (1987) STUDIED THE EXTRACELLULAR THERMOSTABLE α-AMYLASE FROM BACILLUS STEAROTHERMOPHILUS Q8, IT WAS PURIFIED BY AMMONIUM SULFATE PRECIPITATION AND CM-SEPHADEX CHROMATOGRAPHY. THE ACTIVITY THE OF PARTIAL PURIFIED α-AMYLASE WAS TO BE PROTECTED BY BOVINE SERUM ALBUMIN CA2+ AND MG2+. THE ENZYME SHOWED 100% ACTIVITY AT PH 9.0; 98%, AT PH 8.0 AND 41%; AT PH 10.0. IT EXPRESSED OPTIMAL REACTION TEMPERATURE AT 900C, 81% OF THE ACTIVITIES REMAINED AT 1000 C. AFTER 15 MIN INCUBATION AT 1000C WITH THE ADDITION OF 10 MM CA2+, THE ENZYME ONLY RETAINED 67% ACTIVITY. THE ENZYME, HOWEVER, RETAINED 10% OF THE MAXIMAL ACTIVITY AFTER 2 H INCUBATION AT 900C, IN THE ABSENCE OF SUBSTRATE AND WITH THE ADDITION OF CA2+. OF CATIONS, NA+ AT 0.1 AND 1 MM, MN2+ AT 0.1 MM SHOWED STIMULATORY EFFECT; OF ANIONS OH-CL-I-HCO3-NO2-N3- AT 10 MM SHOWED STIMULATORY EFFECT. ALI ET AL ,(1989)STUDIED THE EFFECT OF 8 GROWTH REGULATORS AT CONCENTRATIONS OF 1,000, 5,000 AND 10,000 PPM ON THE ACTIVITY OF FUNGAL (ASPERGILLUS FLAVUS VAR. COLUMNARIS) α-AMYLASE WAS STUDIED. INDOL ACETIC ACID (IAA) AND NAPHTHALENE ACETIC ACID (NAA) INHIBITED α -AMYLASE ACTIVITY BY 2% AND 7% AT 1,000 PPM. THE OTHER 6 GROWTH REGULATORS, INDOL BUTYRIC ACID (IBA), GIBBERELLIC ACID, CUMARIN, CYCOCEL (CCC), ATONIK-G AND KYLAR, DID NOT INHIBIT BUT STIMULATED α -AMYLASE ACTIVITY (0 TO 9%) AT 1,000 PPM. ALL GROWTH REGULATORS STUDIED INHIBITED α -AMYLASE ACTIVITY AT 5,000 AND 10,000 PPM CONCENTRATION EXCEPT KYLAR. MORANELLI ET AL ,(1990)PURIFIED AND CHARACTERIZED THE EXTRACELLULAR α-AMYLASE ACTIVITY OF THE YEAST SCHWANNIOMYCES ALLUVIUS HAD BEEN PURIFIED BY ANION-EXCHANGE CHROMATOGRAPHY ON DEAE-CELLULOSE AND GEL-FILTRATION CHROMATOGRAPHY ON SEPHADEX G-100. SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) AND N-TERMINAL AMINO ACID ANALYSIS OF THE PURIFIED SAMPLE INDICATED THAT THE ENZYME PREPARATION WAS HOMOGENEOUS. THE ENZYME WAS A GLYCOPROTEIN HAVING A MOLECULAR MASS OF 52 KILODALTONS (KDA) ESTIMATED BY SDS-PAGE AND 39 KDA BY GEL FILTRATION ON SEPHADEX G-100. CHROMATOFOCUSING SHOWED THAT IT WAS AN ACIDIC PROTEIN. IT WAS RESISTANT TO TRYPSIN BUT SENSITIVE TO PROTEINASE K. ITS ACTIVITY WAS INHIBITED BY THE DIVALENT CATION CHELATORS EDTA AND EGTA AND IT WAS INSENSITIVE TO SULFHYDRYL-BLOCKING AGENTS. EXOGENOUS DIVALENT CATIONS WERE INHIBITORY AS WERE HIGH CONCENTRATIONS OF MONOVALENT SALTS. THE ENZYME HAD A PH OPTIMUM BETWEEN 3.75 AND 5.5 AND DISPLAYED MAXIMUM STABILITY IN THE PH RANGE OF 4.0-7.0. UNDER THE CONDITIONS TESTED, THE ACTIVITY IS MAXIMAL BETWEEN 450 AND 500 C AND WAS VERY THERMOLABILE. VIHINEN ETAL,(1990) CHARACTERIZED A LIQUEFYING TYPE BACILLUS STEAROTHERMOPHILUS α-AMYLASE . THE CODING GENE WAS CLONED IN BACILLUS SUBTILIS AND THE ENZYME WAS PRODUCED IN THREE DIFFERENT HOST ORGANISMS: B. STEAROTHERMOPHILUS, B. SUBTILIS, AND ESCHERICHIA COLI. PROPERTIES OF THE PURIFIED ENZYME WERE SIMILAR IRRESPECTIVE OF THE HOST. TEMPERATURE OPTIMUM WAS AT 700-800C AND PH OPTIMUM AT 5.0-6.0. THE ENZYME WAS STABLE FOR 1 H IN THE PH RANGE 6.0-7.5 AT 800C. THE ENZYME WAS STABILIZED BY CA2+, NA+, AND BOVINE SERUM. PAQUET ET AL, (1991) CHARACTERIZED THE EXTRACELLULAR α-AMYLASE FROM CLOSTRIDIUM ACETOBUTYLICUM ATCC 824. IT WAS PURIFIED TO HOMOGENEITY BY ANION-EXCHANGE CHROMATOGRAPHY (MONO Q) AND GEL FILTRATION (SEPHADEX). THE ENZYME HAD AN ISOELECTRIC POINT OF 4.7 AND A MOLECULAR WEIGHT OF 84,000, AS ESTIMATED BY SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS. BAJPAI ET AL ,(1991)WORKED ON THE INCREASED PRODUCTION OF THERMOSTABLE α-AMYLASE ENZYME BY BACILLUS SP TCRDC-25A WITH MALTODEXTRINS. MALTODEXTRINS AND HYDROLYSATES OF RICE AND CORN FLOUR OF VARYING DEXTROSE EQUIVALENTS (DE) HAVE BEEN USED AS A CARBON SOURCE FOR α-AMYLASE ENZYME PRODUCTION BY BACILLUS SP TCRDC-25A. THE RATE AND TOTAL ENZYME PRODUCTION WAS HIGHER IN MALTODEXTRIN MEDIA THAN IN CORNSTARCH. THE ENZYME PRODUCTION INCREASED WITH INCREASE IN DE UP TO 45%. ARAKAWA ET AL, (1992) STUDIED THE STABILITY OF FUNGAL α-AMYLASE IN SODIUM DODECYLSULFATE.UNFOLDING OF A FUNGAL α-AMYLASE IN AQUEOUS SODIUM DODECYLSULFATE (SDS) SOLUTION WAS EXAMINED BY SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE). WHEN THE α-AMYLASE WAS INCUBATED WITH 1% SDS AT ROOM TEMPERATURE. THE OBSERVED SMALL SHIFT OF THE MELTING TEMPERATURES BY SDS SUGGESTS THAT THE DESTABILIZING ACTION OF SDS ON THE α-AMYLASE WAS WEAK. THIS INDICATED THAT THE SDS-INDUCED UNFOLDING OF THE α-AMYLASE WAS A SLOW PROCESS. Khoo et al, (1994)purified and characterized α-amylase from Aspergillus flavus. Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate. Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an α -amylase as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. ABE ET AL, (1994) PURIFIED AND CHARACTERIZED PERIPLASMIC α-AMYLASE FROM XANTHOMONAS CAMPESTRIS K-11151.IT WAS ISOLATED FROM SOIL, PRODUCED A PERIPLASMIC α -AMYLASE OF A NEW TYPE. THE ENZYME WAS PURIFIED TO HOMOGENEITY, AS SHOWN BY SEVERAL CRITERIA. THE PURIFIED ENZYME SHOWED ALMOST THE SAME ACTIVITIES ON α -, β-, AND γ-CYCLODEXTRINS, SOLUBLE STARCH, AND AMYLOSE. CHEN ET AL,(1995).AN α-AMYLASE GENE FROM STREPTOMYCES SP WL6 WAS CLONED ON A 3.1KB DNA FRAGMENT, WHICH WAS COMPLETELY SEQUENCED. THE 3088 NUCLEOTIDE SEQUENCE OBTAINED CONTAINS THREE PUTATIVE CODING REGIONS IN THE SAME ORIENTATION. THE ONE CORRESPONDING TO THE STRUCTURAL REGION OF THE ALPHA-AMYLASE GENE HAS A DEDUCED AMINO ACID SEQUENCE OF 459 RESIDUES, SHOWING UP TO 71% IDENTITY TO OTHER ALPHA-AMYLASES. AN INCOMPLETE ORF WAS IDENTIFIED UPSTREAM THE α-AMYLASE GENE, AND THE DEDUCED PRODUCT PRESENTS SOME HOMOLOGY TO PROTEINS INVOLVED IN CATABOLIC REGULATION MARCO ET AL,(1996) PURIFIED AND CHARACTERIZED BACILLUS SUBTILIS α-AMYLASE PRODUCED BY ESCHERICHIA COLI.BACIILLS AMYLASE GENE WAS INSERTED INTO A PLASMID WHICH WAS TRANSFERRED TO ESCHERICHIA COLI. DURING CLONING, A 3' REGION ENCODING 171 CARBOXY-TERMINAL AMINO ACIDS WAS REPLACED BY A NUCLEOTIDE SEQUENCE THAT ENCODED 33 AMINO ACID RESIDUES NOT PRESENT IN THE INDIGENOUS PROTEIN. BOSE ET AL ,(1996)STUDIED THERMOSTABLE α -AMYLASE PRODUCTION USING BACILLUS LICHENIFORMIS NRRL B14368. MAXIMUM AMOUNT OF EXTRACELLULAR α -AMYLASE OF B. LICHENIFORMIS NRRL B14368 WAS OBTAINED DURING THE STATIONARY PHASE. HIGHEST YIELD OF α -AMYLASE WAS ACHIEVED WITH HIGH LEVEL OF CRUDE PROTEIN AND LOW CARBOHYDRATE LEVEL. THERE WAS A CATABOLITE REPRESSION IN THE ORGANISM. PROTEASE WAS PRODUCED CONCURRENTLY WITH α -AMYLASE. IT WAS ALSO OBSERVED THAT SOYABEAN ACTS AS AN INHIBITOR OF THE PROTEASE. IT WAS ALSO OBSERVED THAT α -AMYLASE PRODUCTION WAS A NON-GROWTH ASSOCIATED PRODUCT. MALTOSE WAS AN EXCELLENT INDUCER FOR α -AMYLASE PRODUCTION. CA2+ (0.01 M) INCREASED THE THERMOSTABILITY OF THE ENZYME. ALPHA-AMYLASE PURIFICATION STUDIES WERE CARRIED OUT BY USING ISOPROPANOL, ACETONE, AMMONIUM SULPHATE SOLUTION, ION EXCHANGE CHROMATOGRAPHY. ACETONE WAS FOUND MOST SUITABLE FOR THE SEPARATION OF α -AMYLASE. PROTEIN RECOVERY AND RELATIVE ENZYME ACTIVITY (AS COMPARED TO THAT OF THE MAXIMUM ACTIVITY OF THE CRUDE ENZYME) WERE 30.77% AND 3.03 RESPECTIVELY ABOU-ZEID ET AL,(1997) ISOLATED FILAMENTOUS FUNGI FROM CEREALS AND SCREENED FOR THEIR ABILITY TO PRODUCE α -AMYLASE (1,4-α -GLUCAN GLUCANOHYDROLASE, EC 3.2.1.1). A SELECTED STRAIN IDENTIFIED AS ASPERGILLUS FLAVUS SHOWED HIGH ENZYMATIC ACTIVITY. A SINGLE EXTRACELLULAR α -AMYLASE WAS PURIFIED TO HOMOGENEITY BY A STARCH ADSORPTION METHOD. AGHAJARI ET AL, (2002)INVESTIGATED THE MECHANISM AND FUNCTION OF ALLOSTERIC ACTIVATION BY CHLORIDE IN SOME ALPHA-AMYLASES, THE STRUCTURE OF THE BACTERIAL α-AMYLASE FROM THE PSYCHROPHILIC MICRO-ORGANISM PSEUDOALTEROMONAS HALOPLANKTIS IN COMPLEX WITH NITRATE HAD BEEN SOLVED AT 2.1A0, AS WELL AS THE STRUCTURE OF THE MUTANTS LYS300GLN (2.5A0)ANDLYS300ARG(2.25A0). RICHARDSON ET AL,(2002) USED HIGH THROUGHPUT SCREENING OF MICROBIAL DNA LIBRARIES TO IDENTIFY α-AMYLASES WITH PHENOTYPIC CHARACTERISTICS COMPATIBLE WITH LARGE SCALE CORN WET MILLING PROCESS CONDITIONS. SINGLE AND MULTIORGANISM DNA LIBRARIES ORIGINATING FROM VARIOUS ENVIRONMENTS WERE TARGETED FOR ACTIVITY AND SEQUENCE-BASED SCREENING APPROACHES. ANIMAL AMYLASE SUTHERLAND ET AL,( 1976)ANALYZED TWELVE PANCREASES FROM HUMAN INFANTS ONE YEAR OLD OR LESS , FOR TISSUE INSULIN AND AMYLASE CONTENT BEFORE AND AFTER DISPERSAL OF PANCREATIC FRAGMENTS BY MINCING AND COLLAGENASE DIGESTION. TISSUE INSULIN AND AMYLASE CONTENT PROVIDED AN INDEX OF PANCREATIC ISLET MASS AND EXOCRINE DIGESTIVE ENZYME CONTENT, RESPECTIVELY. THE RESULTS WERE COMPARED WITH SIMILAR ANALYSES PERFORMED ON JUVENILE AND ADULT HUMAN PANCREASES BEFORE AND AFTER ISLET ISOLATION AND ON INTACT AND DISPERSED NEONATAL RAT AND ADULT RAT PANCREAS. INFANT HUMAN PANCREAS HAD AN AVERAGE TISSUE INSULIN CONCENTRATION OF 1,128 μG/L. OF TISSUE AND A TOTAL INSULIN CONTENT OF 1,718 μG/L PANCREAS, AS AGAINST VALUES OF 140 μG./L OF TISSUE AND 7,209 μG./LPANCREAS FOR ADULT HUMAN PANCREAS. Abdullah et al , (1977) derived the action pattern of human salivary α-amylase in the vicinity of the branch points of amylopectin .Salivary α-amylase hydrolysed amylopectin in stages. Six such dextrins were found. Of these, two were capable of being further hydrolysed by α-amylase, whereas the remaining four were true, amylase-resistant α-limit dextrins. AGGETT ET AL,(1980) NORMAL PAEDIATRIC RANGE OF PLASMA α-AMYLASE ACTIVITY WAS DETERMINED USING THE PHADEBAS BLUE STARCH METHOD. THE RANGE FOR CHILDREN OVER ONE YEAR WAS 98-405 IU/L. PLASMA AMYLASE ACTIVITY INCREASED THROUGHOUT INFANCY. MATURE LEVELS OF ACTIVITY WERE OBSERVED IN SOME CHILDREN BY AGE 2 MONTHS AND IN MOST OF THEM BY 9 MONTHS. Bretaudiere et al,(1981)studied the suitability of control materials for determination of α-amylase activity and it was assessed in comparison with reference groups of authentic human serum specimens containing α-amylase of either pancreatic or salivary origin, specimens from patients with no pancreatic pathology, and normal specimens to which porcine pancreatic α-amylase was added. They concluded that porcine enzyme should not be used for interlaboratory quality-control surveys or intermethod comparison studies. CHAN Y, ET AL,(1984). STUDIED THE HYDROLYSIS OF PARTIALLY HYDROXYETHYLATED AMYLOSE BY PORCINE PANCREATIC α-AMYLASE GIVES RISE TO A NUMBER OF HYDROXYETHYLATED DI-, TRI-, AND TETRASACCHARIDES, AS WELL AS LARGER PRODUCTS. NO MODIFIED MONOSACCHARIDES WERE DETECTED. Ameliushkina et al,(1991)Alpha-Amylase activity was measured with the use of a new chromogenic insoluble substrate, Testamyl, developed and manufactured by Kemotex, Tallinn, Estonia. Reproducibility trials of the new method demonstrated a low coefficient of variations within a lot (4.4%) and among the lots (3.5%). alpha-Amylase activity values obtained with the use of Testamyl and by other technique were in good correlation. CHATTERTON ET AL,(1996)THIS INVESTIGATION WAS DESIGNED TO EVALUATE THE PRODUCTION RATES AND CONCENTRATIONS OF SALIVARY α-AMYLASE AS A MEASURE OF ADRENERGIC ACTIVITY UNDER SEVERAL CONDITIONS OF STRESS IN HUMAN SUBJECTS. SALIVA AND BLOOD SAMPLES WERE SIMULTANEOUSLY COLLECTED FROM MEN AT FOUR 15 MIN INTERVALS BOTH BEFORE AND AFTER REGIMENS FOR EXERCISE, A WRITTEN EXAMINATION, OR A REST PERIOD. LEVELS OF α-AMYLASE AND NE RETURNED TO CONTROL LEVELS WITHIN 30-45 MIN AFTER EXERCISE, BUT EP REMAINED ELEVATED BY APPROXIMATELY 2-FOLD DURING THE REMAINING HOUR OF OBSERVATION, THEY CONCLUDED THAT SALIVARY α-AMYLASE CONCENTRATIONS ARE PREDICTIVE OF PLASMA CATECHOLAMINE LEVELS, PARTICULARLY NE, UNDER A VARIETY OF STRESSFUL CONDITIONS, AND MAY BE A MORE DIRECT AND SIMPLE END POINT OF CATECHOLAMINE ACTIVITY THAN ARE CHANGES IN HEART RATE. Chopra et al,(1993)studied the secretion of α-amylase in human parotid gland epithelial cell culture. The secretions of the salivary gland system are essential for the maintenance of oral health. The nature of cell-specific secretions of the various glands and their regulation is not completely understood. The objective of this study was to establish epithelial cell cultures from the human parotid gland that exhibit the tissue-specific function of α-amylase secretion.. They established a long-term epithelial cell culture model of human parotid gland epithelial cells that exhibited differentiated function and retained the intact β-adrenergic receptor. Zoltowska .(2001) purified and characterized α -amylases from the intestine and muscle of Ascaris suum (Nematoda).Alpha-Amylase (EC 3.2.1.1) was purified from the muscle and intestine of the parasitic helminth of pigs Ascaris suum. The enzymes from the two sources differed in their properties. Isoelectric focusing revealed one form of amylase from muscles with pl of 5.0, and two forms of amylase from intestine with pI of 4.7 and 4.5. SDS/PAGE suggested a molecular mass of 83 kDa and 73 kDa . CHANDRAN ET AL,(2002)  WORKED ON THE EXPRESSION, CHARACTERIZATION, AND BIOCHEMICAL PROPERTIES OF RECOMBINANT HUMAN SALIVARY AMYLASE. HUMAN SALIVARY AMYLASE, A MAJOR COMPONENT OF HUMAN SALIVARY SECRETIONS, POSSESSES MULTIPLE FUNCTIONS IN THE ORAL CAVITY. IT IS THE ONLY ENZYME IN SALIVA CAPABLE OF DEGRADING OLIGOSACCHARIDES, WHICH ARE USED BY THE ORAL MICROFLORA FOR NUTRITIONAL PURPOSES. PLANT AMYLASES BALASUBRAMANIAN ET AL,(1989)STUDIED THE CHANGES IN CARBOHYDRATE AND NITROGENOUS COMPONENTS AND AMYLASE ACTIVITIES DURING GERMINATION OF GRAIN AMARANTH .GRAIN AMARANTH (AMARANTHUS HYPOCHONDRIACUS), YERCAUD LOCAL VARIETY, WAS SOAKED OVERNIGHT AND GERMINATED FOR 192 H TAKING THE SOAKED GRAINS AS THE ZERO TIME (0 H) SAMPLE. THE CHANGES IN THE ACTIVITIES OF ALPHA- AND β-AMYLASES, STARCH, SUGAR, PROTEIN AND LYSINE CONTENTS DURING GERMINATION ARE REPORTED. AN INCREASE OF WATER SOLUBLE PROTEIN CONTENT WAS NOTICED IN 24 H GERMINATED GRAINS. CHANDLER ET AL,(1991) USED A PRIMER EXTENSION TO CHARACTERIZE α-AMYLASE MRNAS FROM ALEURONE TISSUE OF BARLEY (HORDEUM VULGARE L. CV. HIMALAYA) GRAINS. TWO SYNTHETIC OLIGONUCLEOTIDES, SPECIFIC FOR THE LOW-PI AND HIGH-PI α-AMYLASE GROUPS, WERE USED AS PRIMERS FOR SYNTHESIS OF CDNA FROM TOTAL RNA PREPARATIONS. AJANDOUZ ET AL,(1992)STUDIED ISOFORMS AMY1, AMY2-1 AND AMY2-2 OF BARLEY ALPHA-AMYLASE WHICH WERE PURIFIED FROM MALT. AMY2-1 AND AMY2-2 ARE BOTH SUSCEPTIBLE TO BARLEY ALPHA-AMYLASE/SUBTILISIN INHIBITOR. THE ACTION OF THESE ISOFORMS WAS COMPARED USING SUBSTRATES RANGING FROM P-NITROPHENYLMALTOSIDE THROUGH P-NITROPHENYLMALTOHEPTAOSIDE CHI ET AL,(1995) DEVISED AN IMPROVED PROCESS FOR HIGH-CONCENTRATION ETHANOL PRODUCTION FROM COOKED CORN STARCH BY SACCHAROMYCES SP. THE MASH WAS SACCHARIFIED AT 600C BY USING HIGH-EFFICIENCY GLUCOAMYLASE. WHEN 0.9 G OF AMMONIUM SULFATE WAS ADDED TO 280 ML OF THE FERMENTATION MEDIA, 18.9% (V/V) ETHANOL COULD BE REACHED IN THE MASH AFTER 50 HRS OF FERMENTATION, LEAVING 0.27% REDUCING SUGAR AND 3.1% TOTAL SUGAR IN THE FERMENTED MEDIA. CHOUDHURY ET AL,(1996) STUDIED THE CHARACTER OF A WHEAT AMYLASE INHIBITOR PREPARATION AND EFFECTS ON FASTING HUMAN PANCREATICOBILIARY SECRETIONS AND HORMONES. AMYLASE INHIBITION INDUCES CARBOHYDRATE TOLERANCE, SATIETY, AND WEIGHT LOSS AND PROLONGS GASTRIC EMPTYING, EFFECTS THAT MAY BE USEFUL IN THE TREATMENT OF OBESITY AND NON-INSULIN-DEPENDENT DIABETES MELLITUS. THEIR RESULTS SHOWED THAT A HIGH PROTEIN PURITY AND A HIGH SPECIFIC ACTIVITY AGAINST α-AMYLASE ACTIVITY AND EFFECTIVELY INHIBITS HUMAN PANCREATIC AMYLASE ACTIVITY SECRETED INTO THE DUODENUM. COLLADO ET AL(1999) ESTIMATED SWEETPOTATO AMYLASE ACTIVITY BY FLOUR VISCOSITY ANALYSIS. SWEETPOTATO FLOUR (SPF), PREPARED FROM 44 GENOTYPES ADAPTED TO PHILIPPINE CONDITIONS, SHOWED WIDE VARIATION IN RAPID VISCO-ANALYZER (RVA) PASTING CHARACTERISTICS DUE TO ITS VARIATION IN COMPOSITION AND ENDOGENOUS AMYLASE ACTIVITY. THE RVA PASTING PARAMETERS OF PEAK VISCOSITY DETERMINED IN WATER (PV1) AND THAT DETERMINED IN 0.05 MM ABE ET AL(1999).STUDIED THE CULTURED CELLS OF RICE (ORYZA SATIVA CV SASANISHIKI) PRODUCE TWO α-AMYLASE ISOZYMES, AMY-I AND AMY-III. USING A BACTERIAL EXPRESSION SYSTEM, EIGHT CHIMERIC GENES CONSTRUCTED WITH VARIOUS COMBINATION OF AMY-I AND AMY-III CDNA FRAGMENTS WERE EXPRESSED, AND EACH RECOMBINANT CHIMERIC PROTEIN WAS CHARACTERIZED. CHEN ET AL,(2002 ) STUDIED THE EXPRESSION OF α-AMYLASE GENES IN CEREALS WHICH WAS INDUCED BY BOTH GIBBERELLIN (GA) AND SUGAR STARVATION .THESE ENHANCERS MAY FACILITATE THE DESIGN OF HIGHLY ACTIVE AND TIGHTLY REGULATED COMPOSITE PROMOTERS FOR MONOCOT TRANSFORMATION AND GENE EXPRESSION.THIS STUDY ALSO REVEALS THE EXISTENCE OF CROSS-TALK BETWEEN THE SUGAR AND GA SIGNALING PATHWAYS IN CEREALS AND PROVIDES A SYSTEM FOR ANALYZING THE UNDERLYING MOLECULAR MECHANISMS INVOLVED. IMMOBILIZATION OF α-AMYLASE GALICH ET AL,(1976) THEY WORKED ON THE PRODUCTION OF IMMOBILIZED α-AMYLASE. ACTIVE IMMOBILIZED α-AMYLASE WAS OBTAINED WHEN APPLYING AE-CELLULOSE CHROMATOGRAPHY AND GLUTARIC DIALDEHYDE. THE TIME OF THE ENZYME INTERACTION WITH THE CARRIER, AMOUNT OF GLUTARIC DIALDEHYDE NECESSARY FOR BINDING, OPTIMAL ENZYME: CARRIER RATIO AS WELL AS THE METHODS FOR DESICCATION OF THE IMMOBILIZED AMYLASE PREPARATIONS WERE SPECIFIED. CONDITIONS WERE SELECTED FOR α-AMYLASE STABILIZATION WITH THE PRESENCE OF GLUTARIC ALDEHYDE. IMMOBILIZED AMYLASE AS COMPARED TO FREE ENZYME WAS SHOWN TO BE MORE PH-STABLE IN THE ACID AND ALKALINE ZONES OF PH (2.0-3.5 AND 10.5-12.0), THERMOSTABLE (WITHIN A TEMPERATURE RANGE OF 20-600C) AND RESISTANT TO THE EFFECT OF 5.5 M UREA. CARVALHO ET AL,(1987)STUDIED THE ACTIVITY OF IMMOBILIZED α-AMYLASE. ALPHA-AMYLASE IMMOBILIZED ON POLYAMIDE 11 SHOWED HIGHER SPECIFIC ACTIVITY AND RETENTION OF ACTIVITY THAN THE DERIVATIVES EMPLOYING POLYACRYLAMIDE AND POLYETHYLENETEREPHTHALATE AS SUPPORTS. POLYAMIDE 11 AND POLYETHYLENETEREPHTHALATE α-AMYLASE DERIVATIVES EXHIBITED A HIGHER EXTENT OF MULTIPLE ATTACKS ON STARCH THAN THE WATER-SOLUBLE ENZYME WHEREAS THE POLYACRYLAMIDE DERIVATIVE PRESENTED LESS. THE POLYAMIDE 11α-AMYLASE DERIVATIVE ACTED ON AMYLOSE-AZURE IN THE SAME WAY AS THE WATER-SOLUBLE α -AMYLASE. BAYRAMOGLU ET AL,(1992) IMMOBILIZED α-AMYLASE INTO PHOTOGRAPHIC GELATIN BY CHEMICAL CROSS-LINKING WITH CHROMIUM (III) ACETATE AND CHROMIUM (III) SULPHATE. CELLULOSE TRIACETATE FILM STRIPS, ENABLED SIMPLE HANDLING WHEN COATED WITH AN ALPHA-AMYLASE-GELATIN MIXTURE, ACCOMPLISHING A HIGH DEGREE OF DURABILITY DURING CONSECUTIVE IMMERSIONS INTO REACTION MEDIA. PHOTOGRAPHIC GELATIN WAS FOUND TO BE A VERY EFFICIENT NATURAL POLYMER, DUE TO ITS EXTRAORDINARY DIFFUSION CHARACTERISTICS FOR IMMOBILIZATION AS A CARRIER. CHEN ET AL,(1998) MADE A COMPOSITE MEMBRANE BY CASTING HYDROGEL ONTO A NONWOVEN POLYESTER SUPPORT AND USED FOR ENZYME IMMOBILIZATION. THE HYDROGEL CONSISTS OF N-ISOPROPYLACRYLAMIDE, CROSS-LINKER N, N'-METHYLENEBIS(ACRYLAMIDE), 2-HYDROXYETHYL METHACRYLATE, SOLUBLE STARCH, AND N-(ACRYLOXY)SUCCINIMIDE (NAS). THE COMPOSITE MEMBRANE WAS TEMPERATURE SENSITIVE WITH A LOWER CRITICAL SOLUTION TEMPERATURE (LCST) AROUND 350C. ALPHA-AMYLASE WAS IMMOBILIZED TO THE MEMBRANE BY COVALENT BONDS THROUGH REACTING WITH THE HIGH REACTIVE ESTER GROUPS IN THE BEST OPERATING CONDITION WAS CYCLING THE TEMPERATURE BETWEEN 50 AND 200C EVERY 5 MIN. THE MEMBRANE REACTOR WAS OPERATED UP TO EIGHT TIMES FOR SUCCESSIVE STARCH HYDROLYSIS. AKSOY ET AL,(1998) STABILITY OF α-AMYLASE IMMOBILIZED ON POLY(METHYL METHACRYLATE-ACRYLIC ACID) MICROSPHERES. THEY PREPARED POLY(METHYL METHACRYLATE-ACRYLIC ACID) MICROSPHERES AND THE ACID GROUPS WERE ACTIVATED BY USING EITHER CARBODIIMIDE (CDI) OR THIONYL CHLORIDE (SOCL2). ALPHA-AMYLASE WAS COVALENTLY BOUND ON THESE ACTIVATED MICROSPHERES. THE PROPERTIES OF THE IMMOBILIZED ENZYME WERE INVESTIGATED AND COMPARED WITH THOSE OF THE FREE ENZYME. THE RELATIVE ACTIVITIES WERE FOUND TO BE 80.4 AND 67.5% FOR CARBODIIMIDE AND THIONYL CHLORIDE BOUND ENZYMES, RESPECTIVELY. MAXIMUM ACTIVITIES WERE OBTAINED AT LOWER PHS AND HIGHER TEMPERATURES UPON IMMOBILIZATION COMPARED TO FREE ENZYME. NO CHANGE IN VMAX AND APPROXIMATELY 12-FOLD INCREASE IN K(M) WERE OBSERVED FOR IMMOBILIZED ENZYMES. THE ENZYME ACTIVITIES, AFTER STORAGE FOR 1 MONTH, WERE FOUND TO BE 24.5 AND 52.5% OF THE INITIAL ACTIVITY VALUES FOR CDI AND SOCL2 ACTIVATED MATRICES, RESPECTIVELY. ON THE OTHER HAND THE FREE ENZYME LOST ITS ACTIVITY COMPLETELY IN 20 DAYS. IMMOBILIZATION, STORAGE STABILITY AND REPEATED USE CAPABILITY EXPERIMENTS CARRIED OUT IN THE PRESENCE OF CA2+ IONS DEMONSTRATED HIGHER STABILITY, SUCH AS SOCL2 IMMOBILIZED ENZYME RETAINED 83.7% AND CDI IMMOBILIZED ENZYME RETAINED 90.3% OF THE ORIGINAL ACTIVITY OF THE ENZYME. THE IMMOBILIZED ENZYMES THAT WERE USED 20 TIMES IN 3 DAYS IN REPEATED BATCH EXPERIMENTS DEMONSTRATED THAT, IN THE ABSENCE OF CA2+ IONS ABOUT 75% AND IN THE PRESENCE OF CA2+ IONS GREATER THAN 90% OF THE ORIGINAL ENZYME ACTIVITY WAS RETAINED. . . ISOLATION OF THE ORGANISM ACTIVE AMYLOLYTIC STRAINS WERE ISOLATED FROM THE SOIL SAMPLES NEAR THE KITCHEN WASTES, BY ENRICHMENT CULTURE AND SERIAL DILUTION METHODS. MEDIUM COMPOSITION THE COMPOSITION OF THE NUTRIENT AGAR MEDIUM (G/L) USED FOR THE CULTURING AND MAINTENANCE OF BACTERIA IS AS FOLLOWS: | COMPOSITION | G/L | |CASINE HYDROLYZATE |4.0 | |PEPTONE |6.0 | |YEAST EXTRACT |3.0 | |GLUCOSE.D |2.0 | |DISTILLED WATER |1000 ML | |PH |7.0 | ISOLATION OF THE ACTIVE AMYLOLYTIC STRAINS 1G OF SOIL SAMPLES WERE SUSPENDED IN DISTILLED AND STERILIZED WATER THEN ABOUT 50 ML OF THIS SUSPENSION WAS ADDED IN NUTRIENT BROTH IN 250 ML ERLENMEYER FLASKS .THE FLASKS WERE SHAKEN IN A GALLENKAMP ORBITAL INCUBATOR SHAKER AT 180 R.P.M. AND AT 370C FOR 24HOURS. THE GROWTH THUS OBTAINED WAS SUBCULTURE FOUR TIMES IN THE SAME MEDIUM. PREPARATION OF STARCH AGAR PLATES 100μL OF THE CULTURED BROTH WAS SERIALLY DILUTED IN STERILIZED CULTURE MEDIUM (1:10). 50μL OF SUITABLY DILUTED SAMPLES WERE THEN SPREAD ON NUTRIENT AGAR PLATES HAVING THE SAME COMPOSITION AS IN NUTRIENT BROTH CONTAINING 1.5% AGAR. THE PLATES WERE INCUBATED AT 37OC FOR 24 HOURS. THE GROWN COLONIES WERE FURTHER SUB CULTURED THREE TIMES AND STREAKED THEM TO GET SINGLE COLONIES. SCREENING OF AMYLOLYTIC BACTERIAL STRAINS BACTERIAL STRAINS WERE SCREENED FOR AMYLOLYTIC ACTIVITY ON STARCH AGAR PLATES HAVING THE SAME COMPOSITION AS FOR NUTRIENT MEDIUM EXCEPT REPLACING GLUCOSE WITH 0.2% STARCH. INOCULA FROM ISOLATED COLONIES WERE TRANSFERRED TO 0.2% STARCH AGAR PLATES UNDER STERILIZED CONDITIONS AND INCUBATED AT 37OC FOR 24 HOURS. A WHITE ZONE AROUND THE COLONIES INDICATED STARCH HYDROLYSIS ON 2% STARCH AGAR. THE BACTERIAL COLONIES WHICH PRODUCE WHITE ZONES WERE FURTHER STREAKED ON STARCH PLATES IN DUPLICATE. IODINE TEST THE COLONIES THAT PRODUCED WHITE ZONES WERE FURTHER CONFIRMED BY IODINE TEST. ABOUT 1 ML OF IODINE SOLUTION (2.5% IODINE, 1% POTASSIUM IODIDE, 32%ETHANOL IN WATER) WAS POURED ON THE PLATE. THE BACTERIAL COLONIES, WHICH PRODUCED WHITE ZONES ON STARCH AGAR PLATES, CLEAR WHITE ZONES AROUND THE COLONIES INDICATED THE STARCH HYDROLYSIS SO THE AMYLOLYTIC COLONIES WERE SCREENED. THE AMYLASE PRODUCING WERE PICKED AND PRESERVED IN CULTURE MEDIUM CONTAINING 14% GLYCEROL AT -80OC. IDENTIFICATION OF THE ORGANISM THE FOLLOWING TESTS WERE PERFORMED FOR THE IDENTIFICATION OF THE ORGANISMS. MOTILITY TEST MOTILITY TEST WAS PERFORMED BY HANGING DROP METHOD. TWO DROPS OF DISTILLED WATER WERE PLACED ON A COVER SLIP AND THEN A FRESH BACTERIAL COLONY WAS MIXED WITH WATER ON A COVER SLIP. SHAPE AND MOTILITY OF BACTERIAL CELLS WERE OBSERVED UNDER THE OIL IMMERSION LENS. CATALASE TEST FRESHLY GROWN BACTERIAL COLONY WAS PLACED ON A GLASS SLIDE AND TWO DROP OF 2% H2O2 WAS ADDED ON IT. IMMEDIATE BUBBLING IN BACTERIAL COLONY INDICATED GAS PRODUCTION WHICH WAS A POSITIVE TEST FOR CATALASE ENZYME. SPORE STAINING FOR SPORE STAINING, A SMEAR OF ONE-WEEK-OLD BACTERIAL CULTURE WAS MADE ON GLASS SLIDE, AIR-DRIED AND FIXED BY HEATING. THE SMEAR WAS THEN FLOODED WITH 0.76% MALACHITE GREEN STAIN AND INCUBATED IN A STEAM BATH FOR 10 MINUTES. AFTER STAINING, THE SLIDE WAS WASHED WITH WATER AND ADDED TWO DROPS OF SAFRANIN AND PLACED FOR 30 SECONDS. AFTER WASHING AND AIR-DRYING, THE SLIDE WAS OBSERVED UNDER OIL IMMERSION LENS. SPORES WERE STAINED GREEN WHILE VEGETATIVE CELLS REMAINED PINK IN COLOR. GRAM STAINING TWO DROPS OF WATER WERE PLACED ON A COVER SLIP AND BACTERIAL CULTURE WAS MIXED IN WATER AND SMEAR WAS MADE ON THE SLIDE AND AIR DRIED. BACTERIAL SMEAR WAS FIXED BY HEATING AND FLOODED WITH CRYSTAL VIOLET SOLUTION (2% CRYSTAL VIOLET AND 0.8% AMMONIUM OXALATE IN 2% ETHANOL) FOR TWO MINUTES AND WASHED WITH DISTILLED WATER. BACTERIAL SMEAR WAS THEN COVERED WITH IODINE SOLUTION (2.5% IODINE, 1% POTASSIUM IODIDE, 32%ETHANOL IN WATER) FOR ONE MINUTE AND WASHED WITH WATER. EXTRA STAIN WAS REMOVED BY DIPPING THE SLIDE IN ETHANOL. THE SMEAR WAS COUNTERSTAINED WITH SAFRANIN (2.5% SAFRANIN IN 95% ETHANOL) FOR ONE MINUTE AND WASHED WITH WATER. GRAM POSITIVE BACTERIA STAINED WITH VIOLET COLOR AND GRAM NEGATIVE WITH PINK COLOR. GELATIN LIQUEFACTION THE CULTURE WAS STABBED ON 12% GELATIN TUBE VERTICALLY, RIGHT THROUGH THE CENTER OF GELATIN. THE GELATIN TUBES WERE INCUBATED AT 37OC FOR 2 DAYS. TUBES WERE REFRIGERATED FOR 30 MINUTES TO DETECT LIQUEFACTION.. NITRATE REDUCTION TEST 5ML OF NITRATE CULTURE BROTH (0.5% PEPTONE, 0.3% BEEF EXTRACT AND 0.1% KNO3, PH 7.0) WERE TAKEN SEPARATELY IN FOUR TEST TUBES. TWO TEST TUBES WERE INCUBATED WITH FRESHLY GROWN BACTERIAL CULTURE AND OTHER TWO WERE UNDER AS CONTROL. THE CULTURE WAS INCUBATED AT 37OC FOR THREE DAYS. 0.5ML OF 0.5% (-NAPHTHYLAMINE AND 0.5ML OF 1% SULPHANILIC ACID WAS ADDED TO EACH TEST TUBE. PRODUCTION OF RED COLOR IN THE TEST TUBES INDICATED POSITIVE TEST. GROWTH IN 5% NACL NUTRIENT BROTH TUBES CONTAINING 5% NACL WERE INOCULATED WITH TEST STRAIN AND INCUBATED AT 37OC FOR 24 HOURS. TURBIDITY INDICATING GROWTH WAS APPEARED IN THE TUBES. INOCULUM PREPARATION 20ML OF THE BROTH WAS TAKEN HAVING THE SAME COMPOSITION AS FOR0.2% STARCH AGAR. STARCH WAS REPLACED BY 0.2% GLUCOSE AS A CARBON SOURCE IN 250ML ERLENMYER FLASKS IN DUPLICATE. THE MEDIUM WAS AUTOCLAVED AT 15 P.S.I. FOR 20 MINUTES. THE MEDIUM WAS INOCULATED WITH THE CELLS FROM PURE AMYLOLYTIC COLONIES FOR INOCULUM PREPARATION. THE FLASKS WERE INCUBATED IN GALLENKAMP ORBITAL INCUBATOR SHAKER AT 37OC AT 180 R.P.M. ON THE APPEARANCE OF TURBIDITY THE FLASKS WERE REMOVED FROM ORBITAL INCUBATOR SHAKER AND WHEN THE O.D WAS 0.6 AT 600NM THE INOCULUM WAS READY FOR FERMENTATION. FERMENTATION METHODS THE FERMENTATIONS WERE CARRIED OUT IN DUPLICATE IN 250ML ERLENMYER FLASKS CONTAINING 50ML OF THE NURIENT BROTH AT PH 7.0. THE MEDIUM WAS STERILIZED AT 15P.S.I. FOR 20 MINUTES. AFTER INOCULATING EACH FLASK WITH 1ML OF THE INOCULUM (OD 0.6 AT 600NM) WERE INCUBATED IN A GALLENKAMP ORBITAL INCUBATOR SHAKER AT 180R.P.M AND AT THE SPECIFIED TEMPERATURE. THE MEDIUM WAS INOCULATED ASEPTICALLY. AFTER SPECIFIC TIME INTERVALS SAMPLES WERE DRAWN FROM DUPLICATE FLASKS ASEPTICALLY. THESE WERE HARVESTED BY CENTRIFUGATION AT 6000 R.P.M. FOR 10 MINUTES. SUPERNATANT WAS USED AS THE EXTRA CELLULAR ENZYME SOURCE. PROTEIN ESTIMATION SOLUBLE PROTEINS IN THE CULTURE SUPERNATANT AND IN PURIFICATION EXPERIMENTS WERE ESTIMATED BY BRADFORD DYE-BINDING METHOD (BRADFORD, 1976). BOVINE SERUM ALBUMIN (SIGMA CHEMICAL CO., USA) WAS USED AS A STANDARD. DYE BINDING REAGENT WAS PREPARED BY DISSOLVING 100MG OF COOMASSIE BRILLIANT BLUE G250 IN 50ML ETHANOL AND 100ML ORTHOPHOSPHORIC ACID. VOLUME WAS MADE UP TO 1 LITER WITH DISTILLED WATER. THE REAGENT WAS FILTERED TWICE. FOR PROTEIN ESTIMATION 2.5ML OF THE ABOVE REAGENT WAS MIXED WITH 50μL SOLUTION. ABSORPTION WAS READ AT 280NM. ENZYME ASSAY THE REDUCING GROUPS RELEASED FROM STARCH WAS MEASURED BY THE REDUCTION OF 3,5 DINITROSALISYLIC ACID BERNFLED (1951) . ONE UNIT OF α -AMYLASE RELEASES FROM SOLUBLE STARCH ONE MICROMOLE OF REDUCING GROUPS, CALCULATED AS MALTOSE PER MINUTE UNDER THE SPECIFIED CONDITIONS. DNS, (DINITROSALISYLIC ACID), WAS PREPARED BY DISSOLVING 2.0 GRAMS OF 3,5-DINITROSALISYLIC ACID IN 30 ML REAGENT GRADE WATER, ADDED TO IT 32 GRAMS OF NAOH IN 20 ML WATER (2M). AFTER THIS IT WAS STIRRED BY GENTLE BOILING UNTIL YELLOW ORANGE CLEAR SOLUTION WAS OBTAINED AFTER THIS POTASSIUM SODIUM TERTRATE 60 GRAMS WAS ADDED TO IT SLOWLY .A CLEAR SOLUTION OF DNS WAS OBTAINED VOLUME WAS MADE UP TO 200ML. THIS CAN BE STORED UP TO SIX MONTHS. IT WAS KEPT IN DARK AIRTIGHT BOTTLE AND NEEDS INCUBATION AT 250C PRIOR TO USE. 1% STARCH SOLUTION, IS PREPARED BY DISSOLVING 1.0 GRAMS OF STARCH IN 100 ML OF 0.02 M SODIUM PHOSPHATE BUFFER (3.561/100ML) PH 6.9WITH NAOH 1M. MIXED IT BY GENTLE BOILING AND MADE THE VOLUME100ML WITH WATER IF NECESSARY. INCUBATED IT PRIOR TO USE AT 350C FOR 4-5 MINUTES. MALTOSE STOCK SOLUTION, PREPARED BY DISSOLVING180 GMS MALTOSE (MWT 360.3) IN 100ML REAGENT GRADE WATER IN A VOLUMETRIC FLASK. PROCEDURE USING THE MALTOSE STOCK SOLUTION PREPARED THE STANDARD CURVE AS FOLLOWS; IN A NUMBERED TUBES PREPARED 10 MALTOSE DILUTIONS RANGING 0.3 TO5 MICROMOLE PER ML. ADDED 2ML OF THE DNS REAGENT TO EACH TUBE AND INCUBATED THEM IN BOILING WATER BATH FOR 5 TO 6 MINUTES. COOLED THEM TO ROOM TEMPERATURE THEN ADDED WATER TO MAKE VOLUME 10ML. THE ABSORBANCE WAS NOTED AT 550NM. PIPETTE OUT 0.5 ML OF THE CRUDE ENZYME IN A TEST TUBE, IT WAS WITH ADDED 0.5ML OF STARCH SOLUTION AND INCUBATED AT 350C FOR FIVE MINUTES THE ENZYME REACTION WAS INTERRUPTED BY THE ADDITION OF 2ML DNS AND TAKEN INTO THE BOILING WATER BATH FOR 3 MINUTES , THESE WERE COOLED TO ROOM TEMPERATURE IN RUNNING WATER. THE BLANK WAS PREPARED BY THE SAME MANNER WITHOUT ENZYME SOLUTION. CALIBRATION CURVE WAS ESTABLISHED WITH MALTOSE, USED TO CONVERT THE O.D INTO MICROGRAMS OF MALTOSE. EFFECT OF PH ON ENZYME ACTIVITY TO DETERMINE THE OPTIMUM PH FOR ENZYME ASSAY 0.02 M SODIUM PHOSPHATE BUFFER OF DIFFERENT PH VALUES RANGING FROM 4.0 TO 9.0 WERE USED, TO DISSOLVE THE SUBSTRATE STARCH IN IT AND THE ASSAY WAS PERFORMED EARLIER. EFFECT OF TEMPERATURE ON ENZYME ACTIVITY TO DETERMINE THE OPTIMUM TEMPERATURE FOR ENZYME ASSAY THE ENZYME SUBSTRATE MIXTURE WAS INCUBATED AT DIFFERENT TEMPERATURES IN THE RANGE OF 25OC TO 55OC IN A SHAKING WATER BATH AND ASSAY WAS PERFORMED. PURIFICATION PROCEDURES OF α-AMYLASE SALTING OUT (NH4)2SO4 PRECIPITATION CRUDE ENZYME SOLUTION WAS PRECIPITATED WITH 90 % SATURATION OF AMMONIUM SULFATE. THIS WAS ACHIEVED BY ADDING 55 G OF AMMONIUM SULFATE SLOWLY FOR ABOUT 10 MINS. THE ENZYME SOLUTION WAS PLACED ON A MAGNETIC STIR PLATE AT 40 C, WAS CENTRIFUGED AT 6000 RPM FOR 10 MINUTES AND THE PPT’S WERE SEPARATED. THESE WERE DISSOLVED IN 20 ML OF NA PO 4 BUFFER (0.02M) HAVING PH 7.0. DIALYSIS (REMOVAL OF SALT) THE PROTEIN OBTAINED AFTER SALTING OUT WAS POURED INTO THE DIALYSIS TUBES AND PLACED IN A BEAKER OF 1000 ML CONTAINING THE SAME BUFFER (NA –PO4). IT WADS PLACED AT 40C IN A MINI COLD LAB ON THE STIR PLATE. DIALYSIS WAS CARRIED OUT FOR 24 HRS AND DURING DIALYSIS BUFFER WAS CHANGED AT REGULAR INTERVALS I.E FIRST CHANGED AFTER 1 HRS, THEN AFTER 1.5, FOLLOWED BY 3HRS AND LAST BUFFER WAS CHANGED AFTER WHOLE NIGHT. NEXT DAY THE SALT FREE PROTEIN SOLUTION WAS OBTAINED, O.D WAS MEASURED AT 280NM FOR PROTEIN CONCENTRATION AND AT 550 NM FOR AMYLASE ACTIVITY. GEL FILTRATION THE CONCENTRATED ENZYME WAS SUBJECTED TO GEL FILTRATION ON SEPHADEX G-75 ON A COLUMN MEASURING (1.6 X 36 CM) AT ROOM TEMPERATURE. 4G OF SEPHADEX G-75 (PHARMACIA FINE CHEMICAL) WERE SWOLLEN IN 80ML OF 0.1M SODIUM PHOSPHATE BUFFER PH 7.5 IN A BOILING WATER BATH FOR 3 HOURS. THE GEL WAS COOLED AT ROOM TEMPERATURE. POURED THE PREPARED GEL SLURRY ALONG THE SIDE OF THE TILTED COLUMN, TAKING CARE THAT NO AIR BUBBLE WAS ENTRAPPED. THE COLUMN OUTLET WAS OPENED AND EQUILIBRATED WITH THREE COLUMN VOLUMES OF SAME BUFFER IN ORDER TO STABILIZE THE BED. VOID VOLUME OF THE COLUMN WAS MEASURED BY USING 0.1% BLUE DEXTRAN 2000 (PHARMACIA FINE CHEMICAL). 3 ML OF CONCENTRATED BROTH WAS LOADED ON THE GEL COLUMN. THE COLUMN WAS ELUTED WITH SODIUM PHOSPHATE BUFFER. TOTAL OF 120 FRACTIONS (3 ML EACH) WERE COLLECTED FOR THE LOADED SAMPLES OF ENZYME. PROTEIN ESTIMATION AND ENZYME ASSAY WAS PERFORMED FOR EACH ELUTED FRACTION. ACTIVE FRACTIONS WERE POOLED. ELUTION PROFILE SHOWED ONE PEAK FOR (-AMYLASE ACTIVITY AND TWO PEAKS FOR PROTEINS. AMYLASE PEAK WAS ELUTED IN FRACTION 19-30 WHILE SECOND PEAK FOR PROTEIN WAS ELUTED IN FRACTIONS 13- 17. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS ELECTROPHORESIS ON THE BASIS OF MOLECULAR MASS WAS CARRIED OUT ACCORDING TO LAEMMLI (1970). 10% RESOLVING GEL WAS PREPARED BY MIXING 6.67ML 30% ACRYLAMIDE BISACRYLAMIDE, 3.35ML OF 3M TRIS HCL (PH 8.8), 0.2ML OF 10% SDS, 9.77ML OF DISTILLED WATER, 9µL TEMED AND 90µL OF 10% AMMONIUM PERSULPHATE. 30% ACRYLAMIDE BISACRYLAMIDE WAS PREPARED BY DISSOLVING 29.2G OF ACRYLAMIDE AND 0.5G BISACRYLAMIDE IN 100ML OF DISTILLED WATER. SOLUTION WAS STORED AT 4OC. 3M TRIS HCL (PH8.8) WAS PREPARED BY DISSOLVING 39.3G OF TRIZMA BASE IN 80ML OF DISTILLED WATER. THEN PH WAS ADJUSTED TO 8.8 BY USING 1N HCL AND THE FINAL VOLUME WAS MADE UPTO 100ML. SOLUTION WAS STORED AT 4OC. FRESHLY PREPARED RESOLVING GEL WAS POURED INSIDE THE GEL ASSEMBLY LEAVING 0.5 INCHES VACANT SPACE AT THE TOP. ALMOST 100µL DISTILLED WATER WAS LAYERED AT THE TOP OF THE GEL TO GIVE A FLAT SURFACE AND TO REMOVE OXYGEN, WHICH INHIBITED POLYMERIZATION. THE GEL WAS ALLOWED TO POLYMERIZE FOR 30 MINUTES. THE STACKING GEL WAS PREPARED BY DISSOLVING 0.94ML OF 30% ACRYLAMIDE BISACRYLAMIDE, 55µL OF 10% SDS, 0.35ML OF IM TRIS HCL (PH6.8), 4.90ML OF DISTILLED WATER, 38µL OF 1% BROMOPHENOL BLUE, 10µL OF TEMED AND 80µL OF 10% AMMONIUM PERSULPHATE. 12.1G OF TRIZMA BASE DISSOLVED IN 80ML OF DISTILLED WATER. THEN PH WAS ADJUSTED TO 6.8 BY USING 1N HCL AND FINAL VOLUME WAS MADE UPTO 100ML. WATER WAS REMOVED AND STACKING GEL WAS POURED IN THE GEL ASSEMBLY. COMB WAS INSERTED. ALLOWED THE GEL TO BE POLYMERIZED AT ROOM TEMPERATURE FOR 45 MINUTES. COMB WAS TAKEN OUT OF THE POLYMERIZED GEL AND WELLS WERE WASHED WITH TRIS-GLYCINE BUFFER. TRIS-GLYCINE BUFFER WAS PREPARED BY DISSOLVING 15G OF TRIS, 72G OF GLYCINE AND 5G OF SDS IN 1000ML OF DISTILLED WATER. GEL ASSEMBLY WAS SETTLED ON THE GEL CHAMBER AFTER REMOVING THE BOTTOM SPACER AND SEALING THE CHAMBER WITH 1.5% AGAR. UPPER AND LOWER TANKS OF THE CHAMBER WERE FILLED WITH TRIS-GLYCINE BUFFER. 8µL OF ENZYME AND 2µL OF LOADING DYE CONTAINING DITHIOTHRIETOL (DDT), SDS AND BROMOPHENOL BLUE AT 7.7%, 10% AND 0.1% (W/V) AND 50% GLYCEROL (V/V) IN TRIS BUFFER (PH6.8) WAS DENATURED BY HEATING IN BOILING WATER FOR 1 MINUTE. SAMPLE WAS LOADED ON THE GEL AND ELECTROPHORESED AT A CONSTANT VOLTAGE OF 150 VOLTS FOR FOUR HOURS. WHEN THE GEL WAS RUN COMPLETELY, IT WAS PUT IN STAINING SOLUTION FOR 2 HOURS WITH CONSTANT AGITATION. AFTER STAINING, GEL WAS PLACED IN THE DESTAINING SOLUTION AGAIN WITH CONSTANT AGITATION UNTIL THE BACKGROUND BECAME TRANSPARENT AND PROTEIN FRACTIONS WERE VISIBLE IN THE FORM OF LIGHT AND DARK BANDS. ISOLATION AND IDENTIFICATION OF AMYLOLYTIC BACTERIA AMYLOLYTIC BACTERIA WERE ISOLATED FROM DECAYING MOIST SOIL SAMPLES. ABOUT 10 SOIL SAMPLES WERE USED FOR THIS PURPOSE. THE BACTERIA WERE ISOLATED BY ENRICHMENT CULTURE AND SERIAL DILUTION METHODS. SERIALLY DILUTED SAMPLES WERE SPREAD ON STARCH AGAR PLATES AND INCUBATED AT 37OC FOR 24 HOURS IN DUPLICATE. SINGLE COLONIES WERE PICKED AND SPOTTED ON STARCH AGAR PLATES IN DUPLICATE FOR SCREENING OF THE IMPOLITIC ACTIVITY OF THE ORGANISMS. AFTER INCUBATION AT 37OC FOR 24 HOURS, WHITE ZONES AROUND THE AMYLOLYTICALLY ACTIVE STRAINS APPEARED INDICATING THE HYDROLYSIS OF STARCH AS SHOWN IN FIG. 4A. SEVEN BACILLUS STRAINS WERE ISOLATED WHICH SHOWED POSITIVE AMYLASE ACTIVITY ON 0.2% STARCH AGAR PLATES, THESE WERE FURTHER CONFIRMED BY IODINE TEST. THE ISOLATES SELECTED FOR FURTHER STUDY WERE ASSIGNED AS A-1, A-2, A-3, ANDA-4. FIG.4B Morphological and Biochemical Characteristics of Amylolytically Active Organisms The active organisms were identified by their morphological and biochemical characteristics. Morphological features of amylolytically active strains (A-1 to A-4) are presented in Table 3. All strains are gram positive, catalase positive, motile and spore forming. Spores occupy a central location except in case of strain A-4. Shape of spore in all strains was cylindrical except in A-3 in which it was round. Different biochemical tests were performed to characterize and differentiate the strains. Results of these tests are shown in Table 4. All strains liquefied gelatin, hydrolyzed starch and casein. Nitrate was reduced by all of them except A-2. All of them could grow in 5% NaCl. Strain A-2, which showed maximum amylolytic activity, was selected for further studies. Fig. 4a: Zones of starch hydrolysis on 0. 2% starch agar | I. A-1 |iv. A-4 vii- A-7 | | ii. A-2 |v. A-5 | | iii. A-3 |vi. A-6 | Fig. 4b: Selected amylolytically active strains. |A-1 |A-3 | |A-2 |A-4 | Table 4. Morphological characteristics of Bacillus strains showing amylolytic activity |No. |Gram |Catalase |Motility |Sporulat-ion |Location of spore |Shape of spore | | |Staining | | | | | | |A-1 |+ |+ |+ |+ |C |Cl | |A-2 |+ |+ |+ |+ |C |Cl | |A-3 |+ |+ |+ |+ |C |R | |A-4 |+ |+ |+ |+ |ST |Cl | Key: Positive (+): Subterminal (ST): Central (C): Cylindrical (Cl): Round (R) Table 5. Biochemical characteristics of Bacillus strains showing amylolytic activity |No. |Gelatin |Starch |NO3- to NO2- |Growth in 5% NaCl |Casein |Diameter of | | |Liquefica-tion |Hydrolysis | | |Hydrolysis |casein hydrolysis | | | | | | | |zones (mm) | |A-1 |+ |+ |+ |+ |+ |13 | |A-2 |+ |+ |+ |+ |+ |11 | |A-3 |+ |+ |- |+ |+ |14 | |A-4 |+ |+ |+ |+ |+ |14 | Key: Positive (+): Negative (-) Protein Estimation Soluble proteins in the culture supernatant and in purification procedures were estimated by comparing absorbance of the sample with the bovine serum albumin standard curve at 280nm as shown in Fig. 5. [pic] Fig. 5: Standard curve for protein estimation Maltose Standard Curve for Enzyme Assay The reducing groups released from starch hydrolysis by the action of amylase were determined by the use of maltose standard curve. This reducing sugar strongly absorbed at 550 nm. The absorbance of maltose follows Beer's Law between 0 to 1000µmoleml-1 as shown in (Fig. 6). [pic] Fig. 6: Standard graph for maltose concentration Culture Conditions for Amylase Production Culture conditions were optimized by studying the effect of different parameters of the culture medium at a time while keeping the other variables constant. Effect of fermentation period The effect of fermentation period on cell growth and enzyme production were also determined. Maximum amylolytic activity was attained after 48 hours of fermentation. Maximum O.D was obtained after 8 hours of fermentation. After this there was a decrease in absorbance. (Fig .7a). [pic] FIG. 7A: EFFECT OF FERMENTATION PERIOD ON AMYLASE PRODUCTION. EFFECT OF TEMPERATURE Effect of temperature on amylase production was studied by growing strain A-5 at different temperatures ranging from 27oC to 45oC in nutrient-agar-medium for 18 hours in duplicate flasks in an orbital incubator shaker. All samples were drawn from the flasks after 18 hours. As the fermentation temperature was increased from 27oC to 45oC amylase production in the culture supernatant gradually increased from 29 to 38.0 U/ml. At 37oC maximum amylase production was obtained (Fig. 7b). Above this temperature there was a gradual decrease in amylase activity. [pic] Fig. 7b: Effect of temperature on amylase production Effect of pH The effect of initial pH of culture medium on amylase production at 37oC showed that pH of the culture medium had a significant effect. The organism was cultivated in nutrient agar-medium with initial pH of 4.0 to 9.0 in different flasks in duplicate. The fermentations were carried out at 37oC for 18 hours. When the pH of the culture medium was increased from 4.0 to 7.0 the amylase production increased from 230 to 270.0 U/ml and at the pH 7.0 the amylase production was maximum 270.0 U/ml (Fig. 7c). [pic] FIG. 7C: EFFECT OF PH ON AMYLASE PRODUCTION Assay Conditions Effect of pH on enzyme activity To determine the optimum pH for enzyme assay 0.02M sodium phosphate buffer of different pH values ranging from 5.0 to 9.0 were used for enzyme assay. The pH optima noticed was 7.0 at which amylase exhibited maximum activity (250 U/ml) as shown in Fig. 8 .a [pic] FIG .8A: EFFECT OF PH ON ENZYME ACTIVITY Effect of temperature on enzyme activity The optimum temperature for enzyme assay was determined by incubating the assay mixture at different temperature ranging from 37oC to 55oC. At 37oC amylase exhibited maximum activity 107.5 U/ml (Fig.8.b). [pic] Fig. 8b : Effect of temperature on enzyme activity Purification of Amylase Concentrated enzyme obtained after freeze drying was purified on Sephadex G-75 column by the method described in materials and methods. Fractions were monitored for amylase activity by the standard maltose assay (Bernfled, 1951). The protein concentration was determined by measuring the absorbance at 280nm. Total amount of protein and active units remaining after dialysis and gel filtration were 10.5mg and 250 units respectively. After gel filtration 4.72mg protein and 120 units were left. Specific activity increased from 20.5 to 25.4U/mg. Total percentage recovery after gel filtration was 41.1%. As shown in Fig. 9 amylase activities appeared in fractions 19 to 30. Table 6: Fractionation table for amylase through gel filtration | |Total volume (ml) |Total protein |Total active units |Specific activity |Percentage | | | |(mg) | |(U/mg) |recoveries | |Crude enzyme |120 |18.5 |380 |20.5 |100 | | Dialysis |75 |10.5 |250 |23.8 |65.7 | |Gel filtration |60 |4.72 |120 |25.4 |31.57 | [pic] FIG. 9: FRACTIONATION OF A CULTURE FILTRATE OF AMYLASE ON A SEPHADEX G-75 COLUMN (1.6 X 36CM). PROTEIN ([pic]), ACTIVITY ([pic] ) FIG.10: SDS-PAGE ANALYSIS OF α-AMYLASE LANE A CRUDE BROTH LANE B PURIFIED ENZYME AFTER GEL-FILTRATION MICROBIAL AMYLASES ARE EXTENSIVELY USED FOR STARCH THINNING IN GLUCOSE MANUFACTURE, DOUGH FOR BREAD MAKING, DESIZING OF TEXTILES AND IN PHARMACEUTICAL PREPARATIONS. THESE ENZYMES ARE PRODUCED BY MANY SPECIES OF BACTERIA SUCH AS BACILLUS SUBTILIS, B.CEREUS, B.AMYLOLIQUEFACIENS, B.COAGULANS, B.POLYMYXA, B.STEAROTHERMOPHILUS, B.LICHENOFORMIS, LACTOBACILLUS, MICROCOCCUS, PSEUDOMONAS, ESHERICIA, PROTEUS, THERMOMONOSPORA, AND SERRATIA(GHOSH,1984;SRIVASTAVA,1986;BAJPAI ,1991;MARCO,1996 AND AGHAJARI ,2002). SOME α-AMYLASE PRODUCING FUNGI ARE FROM THE GENRA ASPERGILLUS, PECILLIUM, CANDIDA, NEUROSPORA, AND RHIZOPUS.(AUGUSTIN, 1981; ALI 1989; MORANELLI 1990 AND ABOU-ZEID 1997). MICROBIAL SOURCE OF (-ALPHA AMYLASES ARE PREFERRED ON INDUSTRIAL SCALE BECAUSE OF THEIR THERMO STABILITY AND AMYLASE PRODUCTION BY MICROBIAL SOURCES IS ECONOMICAL. THE OBJECT OF THIS INVESTIGATION WAS TO DETERMINE THE CONDITIONS MOST SUITABLE FOR MAXIMUM AMYLASE FORMATION. ALPHA AMYLASE BY BACILLUS SP. WAS MAXIMUM AT 8 HOURS AFTER INOCULATION WITH CULTURE MEDIUM. SEVEN AMYLASE PRODUCING ORGANISMS WERE ISOLATED. FOUR OUT OF THEM WERE SELECTED FOR FURTHER STUDIES. AMYLASE ACTIVITY WAS MEASURED BY DNS METHOD (BERNFELD, 1951). ISOLATED STRAIN A-2 HAD MAXIMUM ACTIVITY OF (380U/ML) IN THE PRESENCE OF 0.2% STARCH AFTER 48 HOURS OF FERMENTATION. THE ORGANISM FAILED TO GROW ON HIGHER TEMPERATURES. ENZYME WAS MOST ACTIVE AT NEUTRAL OR SLIGHTLY ACIDIC PH. AMYLASE FROM BACILLUS STEROTHERMOPHILLUS SHOWED 100% ACTIVITY AT PH 9.0, 98% AT PH 8.0 AND 41% AT PH 10.0. IT EXPRESSED OPTIMAL REACTION TEMPERATURE AT 900C.81% OF THE ACTIVITIES REMAINED AT 1000C.(LIN, ET AL 1987) TO DETERMINE THE EFFECT OF PH AND TEMPERATURE ASSAYS WERE PERFORMED. IT WAS A NEUTRAL AMYLASE WITH AN OPTIMAL PH 7.0 AND OPTIMAL TEMPERATURE 370C. IN GENERAL AMYLASE FROM BACILLUS SP. AND CLOSTRIDIUM SP. WERE PURIFIED TO HOMOGENEITY BY ANION EXCHANGE CHROMATOGRAPHY (MONOQ) AND GEL FILTRATION (SEPHAROSE) . THE ENZYME HAD AN ISOELECTRIC POINT OF 4.7 AND MOLECULAR WEIGHT OF 84KD. 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