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Chapter - 8 Publications

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  • Chapter - 8

    Publications  

  • 579

    NSave Nature to Survive

    5(4) : 579-582, 2010

    SCREENING AND ISOLATION OF INTRACELLULAR ααααα-GALACTOSIDASE PRODUCING MICROORGANISMS FROM

    SUGARCANE WASTE SOIL

    E. SIRISHA, A. NAVEEN, V. BHARGHAV AND M. LAKSHMI NARASU*

    Centre for Biotechnology, Institute of Science and Technology,

    Jawaharlal Nehru Technological University, Hyderabad - 500 085, INDIA

    E-mail: [email protected]

    INTRODUCTION

    Microbial enzymes are widely used in different industries such

    as food, beverage, textile, leather, pharmaceutical and waste

    water treatment. Of these, glycosidases or carbohydrolases

    plays a pivotal role in hydrolysis of carbohydrates.

    Glycosidases are classified into two categories-

    exoglycosidases and endoglycosidases based on the nature

    of hydrolysis. The exoglycosidases (ex: galactosidase and

    glucosidases) break the glycoside bond at terminal

    monosaccharide residue whereas endoglycosidases (Ex:

    amylases, endoglycosidase H) cleave the polysaccharide

    chains in between the monosaccharide residues. (Henrissat,

    1991; Henrissat and Bairoch, 1993).

    Galactosides are glucosides containing D-galactose linked to

    the aglycone molecule by acetal linkage. The enzymes that

    hydrolyse the D-galactose linkage are called galactosidases.

    Depending on the plane of hydrolysis of D-galactose

    molecules, galactosidases are classified into-α-galactosidaseand β-galactosidases (Suzuki et al., 1970; Sinnott, 1990).α-galactosidases (EC 3.2.1.22) catalyze the hydrolysis of α-1-6linked terminal galactose residues from galactose

    oligosaccharides such as melibose, raffinose, stachyose and

    branched polysaccharides such as galactomannans and

    galactoglucomannans (Kumar and Mishra, 2010). α-galactosidases have variety of applications in medical and

    food industry. In beet sugar industry, this enzyme is used to

    remove raffinose from molasses, sugar syrups etc (Ohtakara

    and Mitsutomi, 1987). The enzyme is applied to increase the

    nutritional quality of legumes by hydrolysing

    galactooligosaccharides (Thippeswamy and Mulimani, 2002)

    and to increase gelling in guar gum (Buplin et al., 1990). In

    humans, mutations in gfA gene leads to a X-linked lysosomal

    storage disorder called Fabry’s disease. Enzyme replacement

    therapy with α-galactosidase is considered as potentialtreatment for Fabry’s patients (Fulle et al., 2004). In addition,

    the enzyme is applied for the conversion of type ‘B’

    erythrocytes to type ‘O’ erythrocytes (Yang et al., 2000).

    α- galactosidase has been isolated and purified from differentsources like plants, plant seeds, yeasts, fungi and bacteria.

    Because of its immense commercial applications, the present

    study is focused on isolation of bacteria capable of α-galactosidase production from soils.

    MATERIALS AND METHODS

    Sample source

    Soil samples were collected from sugar waste dumped soils

    from sugarcane industries located at Sangareddy (17º35' 07"

    N and 78º03' 50" E), Andhra Pradesh, India.

    Isolation of microorganisms

    The samples were serially diluted and plated on agar plates

    containing 1% (w/v) Sucrose, 1% (w/v) K2HPO

    4, 1% (w/v)

    Tryptone, 0.05% (w/v) Ascorbic acid, 0.01% (w/v) Thiamine

    HCl, 0.4% (w/v) MgSO4.7H

    2O, 0.2% (w/v) FeSO

    4.7H

    2O (pH

    7.0) hereby referred as growth media by spread plate

    technique. Plates were incubated at 37°C for 24 hr. Pure

    ABSTRACTIntracellular α-galactosidase (EC 3.2.1.22) producing bacteria were isolated from soil. The isolates weregrown on the growth media with sucrose as inducer. The isolate showing maximum activity was identified by

    following Bergey’s manual. Different media parameters were optimized for enhanced enzyme production. α-galactosidase activity was optimal at 36ºC and pH 7.0 and when raffinose and tryptone were used as carbon

    and nitrogen source.

    KEY WORDS

    α-GalactosidaseRaffinose

    Intracellular

    Acinetobacter

    Received on :

    21.09.2010

    Accepted on :

    13.12.2010

    *Corresponding

    author

  • 580

    E. SIRISHA et al.,

    cultures were obtained by using different pure culture

    techniques. The cultures were maintained on minimal medium

    agar slants containing sucrose, tryptone and K2HPO

    4 and

    subcultured every 15 days.

    Screening of the isolates for ααααα-galactosidase activity

    Isolated colonies were inoculated into sucrose media and

    incubated at 36ºC for 24 hr in shaking incubator at agitation

    speed of 170rpm. Cells were harvested from broth by

    centrifugation at 10,000 g and washed with 20mM Tris buffer

    (pH 7.0). The cells were suspended in the same buffer

    containing 0.3% (w/v) lysozyme, 0.1% (w/v) Triton X 100,

    1mM PMSF and incubated for 1 hr at 30ºC. The cells were

    further disrupted by sonication. Cell debris was removed by

    centrifugation (10,000 g, 20 minutes, 4ºC) and the supernatant

    was used for further study.

    ααααα-galactosidase assayα - galactosidase activity was assayed in a reaction systemcontaining 550μL of Tris buffer (pH 7.0), 100μL of enzyme

    preparation and 250μL of 2mm ρ-nitrophenyl-α-D-galactopyranoside (ρNPGal) or other synthetic substrates. Thereaction mixture was incubated at 60ºC for 10 minutes and

    stopped by addition of 0.2mM Na2CO

    3 and read at 405 nm

    (Dey et al., 1993). The activity was also measured by using

    raffinose as substrate in a reaction mixture containing 20mM

    tris buffer (pH 7.0) and enzyme preparation. The produced

    amount of reducing sugar was determined by adding 1 mL of

    3, 5-dinitrosalicylate reagent (Miller, 1956).

    One enzyme unit (U) was defined as the amount of enzyme

    required to produce one μmol of ρ-nitrophenol or reducingsugars (galactose) per min under the above assay conditions.

    Protein estimation

    Protein quantities in the enzymatic extract were determined

    by the (Lowry et al., 1951) method with bovine albumin serum

    as standard

    Culturing and characterization of the isolates

    The isolate showing α-galactosidase activity hereby referredto as G2 was cultivated in the medium at 36ºC at 170rpm for

    24 hr. Morphological and biochemical characteristics of the

    isolate have been studied for the identification of the isolate

    Optimization of different media parameters for increased

    enzyme activity

    Growth curve vs. Enzyme activity

    G2 was cultured in media broth at 36ºC in a shaker at 170rpm.

    The culture was harvested at 2 hr interval. The cells were

    disrupted by above mentioned procedure and the supernatant

    collected was used as a crude enzyme solution for enzyme

    assay.

    Effect of different carbon sources on ααααα-galactosidase activity

    The effect of different carbon sources on the growth of G2 has

    been studied by replacing the sole carbon source sucrose

    with glucose, raffinose, galactose, lactose, glucose + galactose

    at 1% (w/v) as final concentration whereas the other parameters

    were unaltered.

    Effect of different nitrogen sources on ααααα-galactosidase activity

    Growth of G2 has been studied by using different nitrogen

    sources like yeast extract, tryptone, peptone, soyabean meal,

    yeast extract+ tryptone and casein at 1% (w/v) as final

    concentration, the other parameters were same.

    Effect of pH on ααααα-galactosidase activityTo observe effect of pH on the growth media for the production

    of α-galactosidase, pH of the media was varied from 5-9, theother parameters were unaltered.

    Effect of temperature on ααααα-galactosidase activityFor the selection of optimum temperature for the production

    of α-galactosidase the temperature was varied from 28ºC to42ºC, by keeping the other parameters same.

    RESULTS AND DISCUSSION

    Microbiological analysis of soil samples revealed that sugar

    waste contaminated sites contain high bacterial count. 25

    colonies were isolated and tested for α-galactosidase activityand one of the isolate showing maximum α-galactosidaseactivity was selected for further studies and was designated as

    G2.

    Identification of the isolate

    Morphological and biochemical characteristics of the isolate

    (G2) have been studied (Table 1) and the properties have

    been compared with the standard characteristics described in

    Substrate, Test Result

    Gram Staining Negative

    Morphology Coccobacilli

    Citrate Positive

    Catalase Positive

    Gelatin liquefaction Negative

    Nitrate reduction Positive

    Oxidase Negative

    Fermentation of gas and acid

    Glucose Positive

    Sucrose Positive

    Mannose Positive

    Lactose Negative

    Indole Negative

    Methyl red Positive

    Voges-Proskauer Negative

    Table 1: Morphological and biochemical characterisation of G2

    isolate

    Carbon Source (1%) Activity (IU)

    Glucose 0.67

    Galactose 2.24

    Sucrose 3.85

    Lactose 1.28

    Raffinose 4.02

    Glucose + galactose 0.92

    Table 2: Effect of different carbon sources on ααααα-galactosidase activity

    Nitrogen Source (1%) Activiy (IU)

    Tryptone 3.54

    Peptone 0.84

    Soyabean meal 0.38

    Yeast extract 1.65

    Yeast extract + Tryptone 2.24

    Table 3: Effect of different nitrogen source

  • 581

    Bergey’s Manual of Determinative Bacteriology (8th Edition).

    The isolate studied to Gram- negative, coccobacilli, strictly

    aerobic and non-motile organism. The isolate has been

    tentatively identified to belong to the genus Acinetobacter.

    Growth vs. Enzyme activity

    The amount of alpha-galactosidase produced was observed

    after every 2 hr till 24 hr. Maximum activity was observed at

    12 hr of growth i. e., during the end of log phase as shown in

    figure (1). After that alpha-galactosidase yield got reduced due

    to the consumption of nutrients.

    Effect of carbon, nitrogen, pH and temperature on the

    enzyme activity

    Optimum media conditions for the maximum alpha-

    galactosidase activity were investigated by studying the effect

    of carbon source, nitrogen source, pH and temperature. From

    Figs. 2 and 3, activity was observed at wide pH range and

    temperature. However, maximum activity was reported at pH

    7 and temperature 36ºC. Maximum activity was recorded for

    both sucrose and raffinose as carbon sources and tryptone as

    the nitrogen source at the final concentration of 1% (w/v).

    (Tables 2 and 3)

    CONCLUSION

    Results present in the Tables (2, 3) and figures (2, 3) indicate

    that the various compositions in growth media influenced the

    enzyme production by the bacteria. It appears substrate

    specificity plays an important role in inducing the enzyme

    production. Raffinose and Sucrose are the inducers for alpha-

    galactosidase production. In future studies, intracellular α-galactosidase from Acinetobacter will be purified and

    characterised to determine its suitability as an industrial enzyme

    in different industries like food, medical and sugar industries

    for degradation of raffinose oligosaccharides.

    REFERENCES

    Buplin, P. V., Gidley, M. J., Jeffcoat, R. and Underwood, D. R.1990. Development of a biotechnological process for the modificationof galactomannan polymers with plant alpha-galactosidase. Carbohydr.Polym .12:155-168.

    Dey, P. M., Patel, S. and Brwonleader, M. D. 1993. Induction of α-galactosidase in Penicillum ochrochloron by guar (CyamopsistetragonalobusII) gum. Biotech. Appl. Biochem. 17: 361-71.

    Fulle, M., Lovejoy, M., Brooks, D. A., Harkin, M. L., Hopwood, J. J.and Meikle P. J. 2004. Immunoquantificatiion of alpha-galactosidase:evaluation for the diagnosis of Fabry disease. Clin. Chem. 50: 1979-1985.

    Henrissat, B. 1991. A classification of glycosyl hydrolases based onamino-acid sequence similarities. Biochem. J. 280:309-316.

    Henrissat, B. and Bairoch, A. 1993. New families in the classificationof glycosyl hydrolases based on amino- acid sequence similarities.Biochem. J. 293: 781-788.

    Kumar, S. and Mishra, S. K. 2010. Purification and characterizationof a thermostable alpha-galactosi dase with transglycosylation activityfrom Aspergillus parasiticus MTCC-2796. Process. Bio. chem. 45:1088-1093.

    Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951.Protein measurement with the Folin phenol reagent. J. Biol. Chem.193(1): 265–75

    Miller, G. L. 1956. Use of dinitrosalicylic acid reagent for determinationof reducing sugar. Anal.Chem. 31: 426-428

    Ohtakara, A. and Mitsutomi, M. 1987. Immobilization of thermostableα-galactosidase from Pycnoporus cinnabarinus on chitosan beads andits application to the hydrolysis of raffinose in beet sugar molasses. J.Ferment. Technol. 65: 493–498.

    Sinnott, M. L. 1990. Catalytic mechanisms of enzymatic glycosyltransfer. Chem. Rev. 90: 1171-1202.

    INTRACELLULAR α-GALACTOSIDASE PRODUCING MICROORGANISMS

    Figure 3: Effect of temperature on ααααα- Galactosidase activityTemperature ºC

    Acti

    vit

    y μ

    mL

    28 30 32 34 36 38 40 42

    3.5

    3

    2.5

    2

    1.5

    1

    0.5

    0

    Figure 1: Growth vs ααααα- Galactosidase activityTime in Hour

    Acti

    vit

    y μ

    mL

    OD

    valu

    e a

    t 6

    00

    nm

    0 8 16 24 32 40 48 56 64 72 80 88 96 104

    4

    3.5

    3

    2.5

    2

    1.5

    1

    0.5

    0

    3.5

    3

    2.5

    2

    1.5

    1

    0.5

    0

    Figure 2: Effect of pH on ααααα- Galactosidase activitypH

    Acti

    vit

    y μ

    mL

    5 5.5 6 6.5 7 7.5 8 8.5 9

    3.5

    3

    2.5

    2

    1.5

    1

    0.5

    0

  • 582

    Suzuki, H., Li, S. C. and Li, Y. T. 1970. α-Galactosidase fromMortierella vinacea. Crystallization and properties. J. Biol. Chem.

    245: 781–786.

    Thippeswamy, S. and Mulimani, V. H. 2002. Enzymic degradation of

    raffinose family oligosaccharides in soymilk by immobilized α-galactosidase

    E. SIRISHA et al.,

    from Gibberella fujikuroi. Process. Biochem. 38: 635-/640.

    Yang, J., Gong, F., Ji, S. P., Ren, H. M., Liu, Z. P. and Gao, X. 2000.

    Cloning and Expression of Alpha-galactosidase cDNA for

    seroconversion from Group B to O. Chin. J. Biochem. Mol. Biol.

    (Chin).16: 438-442

  • Advances in Biological Research 4 (5): 249-252, 2010ISSN 1992-0067© IDOSI Publications, 2010

    Corresponding Author: M. Lakshmi Narasu, Centre for Biotechnology, Institute of Science and Technology,Jawaharlal Nehru Technological University Hyderabad, Hyderabad-500085, India.E-mail: [email protected].

    249

    Isolation and Optimization of Lipase Producing Bacteria from Oil Contaminated Soils

    E. Sirisha, N. Rajasekar and M. Lakshmi Narasu

    Centre for Biotechnology, Institute of Science and Technology,Jawaharlal Nehru Technological University Hyderabad, Hyderabad-500085, India

    Abstract: Lipolytic bacteria were isolated from oil contaminated soils and grown on tributyrin mediacontaining 1% (w/v) olive oil. The isolate showing maximum activity was identified by followingBerger’s manual. Different media parameters were optimized for maximal enzyme production. Peak lipase activitywas observed for palm oil as carbon source, peptone as nitrogen source, at pH 7.0 and temperature at 37°C.

    Key words: Lipases % Tributyrin % Extracellular % Oil

    INTRODUCTION Isolation of Lipolytic Bacteria: Soil samples have

    Lipases (Triacylglycerol lipases, EC 3.1.1.3) are water base containing 0.5% (w/v) peptone, 0.3% (w/v) yeastsoluble enzymes which have the ability to hydrolyse extract, 1% (v/v) Tributyrin and 2% agar, pH 7.0) bytriacylglycerols to release free fatty acids and glycerol. spread plate method [13]. Plates were incubated atLipases constitute a major group of biocatalysts that 37°C for two days. Pure cultures of the isolates werehave immense biotechnology applications. Lipases have maintained on minimal media agar slants (yeast extract,been isolated and purified from fungi, yeast, bacteria, NaCl, Peptone and 2% agar, pH 7.0) and were subculturedplant and animal sources [1]. Of all these, bacterial lipases every 15 days.are more economical and stable [2].

    Bacterial lipases are used extensively in food and Screening of the Isolates for Lipase Activity: Lipolyticdairy industry for the hydrolysis of milk fat, cheese organisms were screened by qualitative plate assay.ripening, flavour enhancement and lipolysis of butter fat Isolates were grown on Tributyrin agar base plates andand cream [3]. Lipases are also used in detergent industry incubated at 36°C for 2 days. Zone of clearance wasas additive in washing powder [4], textile industry to observed due to hydrolysis of tributyrin.increase fabric absorbency [5], for synthesis ofbiodegradable polymers or compounds [6] and different Culturing and Characterization of the Isolates: Thetransesterification reactions [7]. In addition, the enzyme is isolate showing maximum zone of clearance herebyused as a catalyst for production of different products referred as L2 is selected for further analysis.used in cosmetic industry [8], in pulp and paper industry Morphological and biochemical characteristics of the[9], in synthesis of biodiesel [10], degreasing of leather isolate have been studied for the identification of the[11] and in pharmaceutical industry [12]. isolate.

    Currently bacterial lipases are of great demandbecause of potential industrial applications. The present Lipase Assay: Lipase activity was measured by titrimetricpaper focused on screening and isolation of method using olive oil as a substrate. Olive oil (10% v/v)microorganisms and optimisation of different parameters was emulsified with gum Arabic (5% w/v) in 100mMfor maximal enzyme activity. potassium phosphate buffer pH 7.0. 100 µl of enzyme

    MATERIALS AND METHODS at 37°C. The reaction was stopped and fatty acids were

    Sample Collection: Samples were collected from oil and (1:1). The amount of fatty acids liberated were estimatedfat contaminated soils of diary and oil refinery industries by titrating with 0.05M NaOH until pH 10.5 using asituated in and around Hyderabad andhra Pradesh, India. phenophathelin indicator [14].

    been serially diluted and plated on to tributyrin agar

    was added to the emulsion and incubated for 15 minutes

    extracted by addition of 1.0ml of acetone: ethanol solution

  • Advan. Biol. Res., 4 (5): 249-252, 2010

    250

    One unit of enzyme is defined as the amount of plated on tributyrin agar base. Morphological andenzyme required to hydrolyse µmol of fatty acids from biochemical studies were done on L2 isolate. By followingtriglycerides. the bergey’s manual, the isolate was identified likely to be

    Optimization of Media Parameters for ProfoundEnzyme Activity Effect of Incubation Period on Lipase Activity: The L2Effect of Incubation Period on Lipase Activity: L2 was isolate was inoculated in Tributyrin broth and wascultured in Tribuytrin broth containing yeast extract, harvested at 8 hours interval. Maximum enzyme activityNaCl, peptone and 1% (w/v) olive oil at 36°C in an was observed at 48 hours at the early stationary phase.inorbital shaker at agitation speed of 150rpm. The The activity of the enzyme gradually decreased afterculture broth was harvested at 8h intervals by 48 hours (Figure 1).centrifugation at 10,000 g, 30 min, 4°C. The supernatantcollected was used as crude enzyme solution and was Effect of Carbon and Nitrogen Sources on Lipaseassayed for enzyme activity. Activity: For selection of optimum carbon and nitrogen

    Effect of Different Oils as Carbon Source on Lipase for the media. Peak enzyme activity was obtained whenActivity: Olive oil present in the growth media was palm oil and peptone were used as carbon and nitrogenreplaced with different oils like palm oil, ghee, coconut oil, source (Figures 2, 3).ground nut oil, sunflower oil and mustard oil at a finalconcentration of 1% (w/v) by keeping remaining Effect of Temperature, pH and Agitation Speed on Lipaseparameters same. Activity: Lipase activity was observed at broad range of

    Effect of Different Nitrogen Sources on Lipase Activity: was observed at 36°C temperature, pH 7.0 and at agitationDifferent nitrogen sources like yeast extract, soya bean speed of 160rpm (Figures 4,5,6).meal, NaNO , tryptone and peptone were added to the In conclusion, bacterial lipases are one of the3broth at a final concentration of 1% (w/v). Remaining enzymes having huge market demand. The isolatedparameters were unaltered. Staphylococus sps isolate have shown the production

    Effect of Temperature on Lipase Activity: For selection parameters for maximum lipase activity were done onof optimum temperature for the production of lipases, isolated lipolytic bacteria. The isolate has shown a broadthe temperatures varying from 21to 42°C were selected range of pH and temperature. The extracellular lipaseby keeping the remaining parameters same. enzyme can be further purified and used in different

    Effect of pH on Lipase Activity: The optimum pH forenzyme production was selected by varying the pH ofthe tributyrin broth from 5 to 9 whereas the otherparameters were unaltered.

    Effect of Agitation Speed on Lipase Activity: To determinethe optimal agitation speed for peak enzyme activity,the L2 was cultured in an orbital shaking incubator at36°C at varying agitation speed from 120-200 rpm.

    RESULTS AND DISCUSSION

    Screening, Isolation and Identification of LipolyticBacteria: 10 different soil samples collected fromdifferent areas show high bacterial count. The colonylabelled as L2 showed maximum zone of clearance when

    belonging to genus Staphylococcus.

    source, different carbon and nitrogen sources were used

    temperature, pH and agitation speed. Maximum activity

    of extracellular lipases. Optimization studies on media

    industrial applications.

    Table 1: Morphological and biochemical characterisation of L2 isolateSubstrate, Test ResultGram Staining PositiveMorphology CocciCitrate NegativeCatalase PositiveGelatin liquefaction NegativeNitrate reduction Weakly positiveOxidase NegativeFermentation of gas and acid

    Glucose PositiveSucrose PositiveLactose Positive

    Indole NegativeMethyl red NegativeVoges-Proskauer NegativeUrease Negative

  • Advan. Biol. Res., 4 (5): 249-252, 2010

    251

    Fig. 1: Effect of incubation period on lipase activity

    Fig. 2: Effect of different carbon sources on lipase Fig. 5: Effect of pH on lipase activityactivity

    Fig. 3: Effect of different nitrogen sources on lipase Fig. 6: Effect of agitation speed (rpm) on lipase activityactivity

    Fig. 4: Effect of temperature on lipase activity

  • Advan. Biol. Res., 4 (5): 249-252, 2010

    252

    REFERENCES 8. Eugene, W.S., 1974. Industrial application of

    1. Joseph, B., P.W. Ramtekeand and G. Thomas, 2008. 51(2): 12-16.Cold active microbial lipases: Some hot issues and 9. Bajpai, P., 1999. Application of enzymes in the pulprecent developments. Biotech Advances, 26: 457-470. and paper industry. Biotechnol Progr, 15: 147-157.

    2. Snellman, E.A., E.R. Sullivan and R.R. Colwell, 2002. 10. Noureddini, H., X. Gao and R.S. Philkana, 2005.Purification and properties of the extracellular Immobilized Pseudomonas cepacia lipase forlipase, LipA, of Acinetobacter sp. RAG-1. FEBS J., biodiesel fuel production from soybean oil. Bioresour269: 5771-5779. Technol., 96: 769-77.

    3. Falch, E.A. and E.A. Falch, 1991. Industrial enzymes- 11. Nakamura, K. and T. Nasu, 1990. Enzyme Containingdevelopments in production and application. Bleaching Composition, Japanese Patent, 2: 208-400.Biotechnol Adv., 9: 643-658. 12. Higaki, S. and M. Morohashi, 2003.

    4. Fuji, T., T. Tatara and M. Minagawa, 1986. Studies on Propionibacterium acnes lipase in seborrheicapplication of lipolytic enzyme in detergent dermatitis and other skin diseases and Unsei-in.industries. J. Am. Oil Chem. Soc., 63: 796-799. Drugs Exp. Clin Res., 29: 157-9.

    5. Sharma, R., Y. Christi and U.C. Banerjee, 2001. 13. Sarada Sarkar, B., Sreekanth, Shreya Kant, RintuProduction, purification, characterization and Banerjee and B.C. Bhattacharyya, 1998. Productionapplication of lipases. Biotech Advances, 19: 627-662. and optimization of microbial lipase. Bioprocess

    6. Linko, Y.Y., M. Lamsa, X. Wu, E. Uosukainen, Engineering, 19: 29-32.J. Seppala and P. Linko, 1998. Biodegradable 14. Jensen, R.G., 1983. Detection and determination ofproducts by lipase biocatalysis. J. Biotechnol., lipase (acylglycerol hydrolase) activity from various66(1): 41-50. sources. Lipids, 18: 650-657.

    7. Fariha Hasan, Aamer Ali shah and Abdul Hameed,2006. Industrial applications of microbial lipases.Enzyme and Microbial Tech., 39(2): 235-251.

    microbial lipases: A review. J. Am. Oil Chem. Soc.,

  • 74 

     

    International Conference-2010 UCPS,Kakatiya University OC 2

    STUDIES ON ALPHA-GALACTOSIDASE AND ITS MEDICAL APPLICATIONS

    E. Sirisha1 and *M. Lakshmi Narasu1

    _________________________________________________________________________________

    1Centre for Biotechnology, Institute of Science and Technology,Jawaharlal Nehru Technological University Hyderabad, Andhra Pradesh, India-500085

    ABSTRACT

    Accidental transfusion of ABO incompatible blood leads to fatal blood transfusions. In order to improve safety transfusion and usage of donated blood, B group erythrocytes were enzymatically converted to O group erythrocytes by using enzyme Alpha-galactosidase isolated from Acinetobacter sps. Integrity and stability of ECORBC cell surface was visually examined microscopically.

    Keywords

    B group, erythrocytes, O group, Acinetobacter sps

    1. INTRODUCTION

    Alpha-galactosidases (3.2.1.22) catalysis the hydrolysis of terminal alpha 1-6 linked galactose residues from simple and complex oligosaccharides and polysaccharides. (Kumar  Shivam  &  Sarad Kumar Mishra., 2010) It is widely distributed in plants, animals and micro-organisms.

    α-Galactosidases have variety of applications in medical and food industry. In medical industry, enzyme is applied for the treatment of X-linked lysosomal storage disorder called Fabry’s disease (Fulle et al.,2004), blood group conversion (Yang  et al., 2000) and in xenotransplantation.

    For patients with huge loss of blood and anemia, blood transfusion is a life saving process. Sometimes accidental transfusion of ABO incompatible blood leads to fatal blood transfusions. In order to meet the demand for the safe blood transfusion and to utilize the donated blood efficiently, researchers have developed technology for enzymatic conversion of ‘A’ and ‘B’ blood

    group to ‘O’ group (Gong et al., 2005). The ABO blood groups are determined by cell surface markers present on the surface on the RBC cells. The cell surface markers are characterised by a protein or lipid that has an extension of particular sugar arrangement- N-acetyl galactosamine, N-acetyl glucoseamine, galactose and fucose moiety. ‘B’ group erythrocytes differ from ‘O’ group erythrocytes by alpha linked galactose moiety (Yang et al., 2000). Alpha-galactosidase isolated from coffe bean was successfully applied for the cleavage of the terminal galactose moiety from the surface of ‘B’ group erythrocytes and for enzymatic conversion of ‘B’erythrocytes to ‘O’ group erythrocytes (Zhang et al., 2003).

    Considering the potential applications and economical importance of the alpha-galactosidase enzyme, we have attempted to isolate the enzyme from bacterial source. The searches for the enzyme lead us to isolation of Acinetobacter sps from sugar cane waste dumped soils showing alpha-galactosidase

  • 75 

     

    International Conference-2010 UCPS,Kakatiya University OC 2

    activity. The present study is focused on the application of alpha- galactosidase enzyme purified from Acinetobacter sps

    on conversion of B→O erythrocytes or ECORBC cells. ‘A’ group red blood cells were also used as control for conversion.

    .

    Figure 1: Carbohydrates present of cell surface of A, B and O red blood cells

    Figure2: A diagramatic presentation of conversion of ‘B’ to ‘O’ group

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    International Conference-2010 UCPS,Kakatiya University OC 2

    2. MATERIALS AND METHODS

    2.1 Purification of the enzyme

    Microorganisms were isolated from sugar cane waste. The soil isolate showing maximum intracellular alpha-galactosidase activity was identified as Acinetobacter sp. The organism was inoculated in 1 litre production media containing 25 g/L raffinose, 10g/L K2HPO4, 10g/L Tryptone, 0.05% (w/v) 1g/L MgSO4.7H2O, 1g/L FeSO4.7H2O (pH 7.0) and was incubated at 360C for 12 hours in shaking incubator at agitation speed of 170rpm. The cells were further disrupted by sonication. Cell debris was removed by centrifugation (10,000 g, 20 minutes, 4°C) and the supernatant was collected. The enzyme was purified by gel filtration chromatography.

    2.2 Conversion of ‘B’ to ‘O’ group

    Alpha galactosidase enzyme was applied for the enzymatic conversion of ‘B’ to ‘O’ group. 4 U of purified enzyme was incubated in 2 ml of ‘B’ group erythrocytes in an isotonic buffer at pH 6.0. The reaction mixture was incubated at 360C for about one hour. The cells

    were washed with 0.5% NaCl to remove enzyme alpha-galactosidase. The same experiment was repeated with other blood groups ‘A’ and ‘O’ and these were taken as controls.

    2.3 Examination of ECORBC

    Enzymatically converted ‘O’ group red blood cells (ECORBC) were examined by adding anti-B antibodies. Integrity of the cell surface of the ECORBC was further examined by microscopic examination.

    3. RESULTS AND DISCUSSION

    3.1 Conversion of B to O group

    Enzyme treated ‘B’ erythrocytes was taken and examined by adding anti-B antibodies. Fig (3) shows the conversion of ‘B’ to ‘O’ erythrocytes. Agglutination was observed at 0 hour and half-hour incubation when anti B antibodies were added. No agglutination was observed after one hour incubation. In case of ‘A’ group agglutination was observed from 0 hour upto 2 hours when anti-A antibodies were added. A group differs from O group by terminal N-acetyl galactosamine and cannot be removed by alpha-galactosidase.

    Fig 3 shows the microscopic examination of B group cells at 0 minutes

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    3.2 Microscopic examination of ECORBC

    Stability and integrity of the surface of ECORBC was visualised microscopically. Fig (4) shows microscopic examination of ‘B’ group erythrocytes with anti-B antibodies at 0 hour and one hour. On microscopic examination at O hour agglutination was observed whereas after one hour incubation, no agglutination was observed and cell surface of ECORBC ie

    enzymatically converted O red blood cells was stable and no cell lysis was observed.

    Fig 4 shows the microscopic examination of ECORBC cells at 60 minutes

    4. CONCLUSION

    Alpha-galactosidase having potential industrial applications was purified from Acinetobacter sps. The purified enzyme was able to convert ‘B’ to ‘O’ RBC

    cells. ECORBC were stable and these can be further used for animal studies and qualitative examinations. These cells can further be used in blood transfusions as universal O group.

    REFERENCES

    Fulle, M., Lovejoy, M., Brooks, D. A., Harkin, M. L., Hopwood, J. J. and Meikle P. J. 2004. Immunoquantificatiion of alpha-galactosidase: evaluation for the diagnosis of Fabry disease. Clin. Chem. 50: 1979-1985. Gong, F., Lü, Q.S., You, Y., Gao, H.W., Bao, G.Q., Gao, X., Li, S.B., Li, L.L., Wang, Y.L., Tian, S.G., Zhang, Z.X., Zhang, P., Zhang, Y.P. 2005. Preparation of transfusable human universal red blood cell with recombinant alpha-galactosidase. 13(2):313-316.

    Kumar Shivam and Sarad Kumar Mishra. 2010. Purification and characterization of a thermostable

    alpha-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796. Process Bio chem 45: 1088-1093.

    Yang, J., Gong, F., Ji, S. P., Ren, H. M., Liu, Z. P. and Gao, X. 2000. Cloning and Expression of Alpha-galactosidase cDNA for seroconversion from Group B to O. Chin J Biochem Mol Biol (Chin).16: 438-442

    Zhang, Y., Yang, J., Gao, X., Jia, Y., Ji, S., Gong, F., Liu, Z., Ren, H., Li, S., Lan, J. and Cao, Q. 2003. B→O blood conversion. Chinese Science Bulletin. 48(3): 269-273

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