Chapter - 2

Review of Literature    

26

 

2. Review of Literature _____________________________________________________________________

2.1 Alpha-galactosidases

Alpha-galactosidase is an exoglycosidase that catalyse the

hydrolysis of α-1-6 linked terminal galactose residues from

galactooligosaccharides such as raffinose, melibiose, stachyose and

branched polysaccharides such as galactomannans and

galactoglucomannans (Dey and Pridham, 1972). It is also hydrolyse

glycospingolipids and glycoproteins. Besides hydrolytic activity,

microbial and plant alpha-galactosidases catalyze transgalactosylation

or transferase activity particularly at high substrate concentrations

(Kato et al. 1982) and also hemagglutination or lectin activity by few

plant origin enzymes (Shannon LM and Hankins CN, 1981). Based on

aminoacid sequences, alpha-galactosidases are included in four

different families - GH4, GH27, GH36 and GH57 (Naumoff DG, 2001).

Most of the known bacterial and eukaryotic alpha-galactosidases with

confirmed activity belong to GH27 and GH36, which constitute a

superfamily (clan GH D) (Naumoff DG, 2002) (Naumoff DG, 2004).

Classification of alpha-galactosidases was explained in the Table 2.1.

(http://www.cazy.org/)

27

 

Table 2.1 Classification of alpha-galactosidases

GH 4 Alpha-galactosidase from Escherichia coli and

bifunctional enzymes with alpha-galactosidases and

alpha-glucosidase (EC 3.2.1.20) activities from

Thermotoga maritime and Thermotoga Neapolitan

belong to this family.

GH27 Mainly alpha galactosidases from Eukaryotes

(animals, plants, fungi, etc.) and some Eubacteria.

GH36 Mainly alpha galactosidases from Eubacteria and

Eukaryotes (fungi and plants).

GH57 Alpha-galactosidases from Pyrococcus furiosus and

Thermococcus alcaliphilus.

2.2 Synonyms of alpha-galactosidases

In 1895, Bau and Fischer first classified the enzyme as Melibase

that catalysis the hydrolysis of melibiose, a discaccharide. In

subsequent years, Weidenhagen, 1928 studied the catalytic properties

of melibase enzyme on various galactooligosaccharides and renamed

melibase as alpha-galactosidase. In humans, galactosidase, alpha

(GLA) gene codes for the enzyme and hence labelled as alpha-

galactosidase A (Calhoun et al. 1985). Table 2.2 gives the list of

synonyms of alpha-galactosidase isolated from various organisms.

28

 

Table 2.2 Synonyms of alpha-galactosidase

SYNONYMS ORGANISM

REFERENCE

Aga-Y Yersinia pestis Cao Y et al. 2008

Agalsidase alfa Homo sapiens West M et al. 2009

AglA & AglC Penicillium anescens Sinitsyna OA et al. 2008

alpha-D- galactopyranoside

galactohydrolase

Talaromyces flavus Simerska P et al. 2007

alpha-Gal Lactobacillus fermentum Carrera SEA et al. 2006

alpha-Gal A Homo sapiens Ishii S et al. 2000

MelA Bifidobacterium bifidum Goulas T et al. 2009

Melibiase Streptomyces griseoloalbus Anisha GS and Prema P,

2007

TmGalA Thermotoga maritima Miller ES et al. 2001

2.3 Sources and physiological role of the alpha-

galactosidase:

Alpha-galactosidases are ubiquitious in nature. They are

isolated and purified from various sources comprising of plants,

animals, humans and microorganisms.

2.3.1 a. Plant source:

Alpha-galactosidases are widely distributed in plants and plant

seeds especially legume seeds (Keller F and Pharr DM, 1996). It was

first isolated from sweet almond emulsion (Helferich B and Appel H,

1932). In plants, alpha-galactosidases are classified into- acid and

alkaline alpha galactosidase, which is uncommon among eukaryotes

29

 

(Zamski E and Schaffer AA, 1996). Acidic alpha-galactosidase was

reported in Tachigali multijuga Benth. seeds (Fialho et al. 2008).

Alkaline alpha-galactosidases were isolated from mature leaves of

Cucumber (Cucurbita pepo) (Thomas B and Webb JA, 1977), Ajuga

reptens leaves (Bachmann M and Keller F, 1995), roots of wheat (Zea

mays) (Hadacova V and Benes K, 1977) and melon fruit (Cucumis

melo) (Gao Z and Schaffer AA, 1999). In recent times, novel alpha-

galactosidase enzymes were isolated from grape flesh (Kang HC and

Lee SH, 2001), peanuts (Bryant RJ et al. 2003), tomato fruit (Feurtado

JA, 2001), germinating coffee bean seeds (Haibach F et al. 1991),

sunflower seeds (Kim WD et al. 2003), endosperm of coconut

(Balasubramaniam K and Mathew D, 1986), immature stalks of

sugarcane (Chinen I et al. 1981) and cultured rice (Kim WD et al.

2002).

b. Physiological role of plant alpha-galactosidases:

Acid and alkaline alpha-galactosidases play a significant role

during seed germination. Enzymatic hydrolysis of

galactooligosaccharides like stachyose and cell wall polysaccharides

serves as energy for the plant growth (Dey PM and Pridham JB, 1972).

In coconuts, the enzyme plays a vital role in germination and cell wall

development (Balasubramaniam K and Mathew D, 1986). It was

reported that transgalactosylation activity of alpha-galactosidases

involves in removal of toxic substances and hydrolysis of phenolic

glycosides, thereby providing a mean for monitoring levels for plant

30

 

growth substances (Strobel GA, 1974). It was established that down

regulation of alpha-galactosidases enhances the freeze tolerance in

some transgenic plants (Penny cooke JC et al. 2003).

2.3.2 Animal Source:

Only few alpha-galactosidases were isolated and sequenced

from animals. It was first reported in snails (Helix promatia) (Bierry H,

1913). It was also reported in rat intestine (cytoplasm of Brunner’s

gland) (Suzuki, 1969), sheep pancreas (Bajwa SS and Sastry PS,

1974), mouse embryos (Chapman VM et al. 1978), kidneys of Chinese

hamster (Chang AY, 1978) and Turbo cornutus (Shepard D et al. 1983).

Recently, the gene was isolated, sequenced and characterised in

porcine (similar to pigs) (Yoshimitsu M et al. 2010).

2.3.3 a. Human Source:

Human alpha-galactosidases are the lysosomal enzymes that

catalyse the hydrolysis of glycospingolipids and glycoproteins (Desnick

RJ and Bishop DF, 1989). The gene for alpha galactosidase A (GLA

gene) is located on X chromosome (q22.1) (Calado et al. 2004). It is 12

kb in length and encodes a homodimer glycoprotein (Kornreich R et al.

1989). The enzyme has been isolated from human spleen, liver,

plasma and placenta. It is not present in human intestine and

stomach (Bishop DF and Desnick RJ, 1981) (Dean KJ and Sweeley

CC, 1979).

31

 

b. Physiological role of human alpha-galactosidases:

Deficiency of alpha-galactosidase or mutations in GLA gene

results in Fabry’s Disease (Kint JA, 1970). Fabry’s disease is a rare X-

linked lysosomal storage disease prominently occurring in males

(1:40,000) (Desnick RY, 2006). Deficiency of the enzyme leads to

accumulation of glycospingolipids like globotriaosylceramide (CTH,

Gb3, or GL-3) on various tissues and organs (Karen JK, 2005). The

symptoms of the disease were observed in early childhood. The

disease progressively leads to death.

2.3.4 a. Microbial Source:

In recent years, microbes have emerged has a rich source of

industrially important enzymes. The enzyme alpha-galactosidase was

isolated and identified in yeast, fungi, actinomycetes, mesophilic and

thermophilic bacteria. Presence of alpha-galactosidase in higher fungi

has also been reported (Li YT and Shetlar MR, 1964). It was first

isolated from brewers’ yeast (Bau A, 1873) (Fischer E and Lindner P,

1895) and consequently was identified in Saccharomyces

carlsbergensis (Lazo PS et al. 1977), Candida javanaica (Cavazzoni et

al. 1987) etc. In fungi, the enzyme was identified in several Aspergillus

(Somiari RI and Balogh E, 1995) and Pencillium (Luonteri E et al.

1998) species, Trichoderma (Zeilinger S et al. 1993), Mortierella (Galas

E and Miszkiewicz H, 1996), Humicola (Kotwal SM et al 1999) etc. The

enzyme was isolated from numerous bacteria including extreme

32

 

thermophilic bacteria such as Sulfolobus solfataricus isolated from

acidic volcanic pool (Brouns SJ et al. 2006). Microbial alpha-

galactosidases were localized both intracellularly and extracellulary.

Both extracellular as well as intracellular alpha-galactosidases were

reported in Debaryomyces hansenii UFV-1(yeast) (Pollyanna A et al.

2009). Alpha-galactosidases isolated from Aspergillus niger is being

produced commercially as Novozyme SP 230 (Novo Nordisk, Denmark)

(Knap IH et al. 2001). Table 2.3 shows the list of microbial sources of

alpha-galactosidases

b. Physiological role of microbial alpha-galactosidase:

Microbial alpha-galactosidases degrade complex

galactooligosaccharides and polysaccharides into simple sugars,

which serve as carbon source. The microflora present in the human

intestine and stomach degrade galactooligosaccharides present in

different food stuffs into simple sugars, thereby leading to production

of gas or flatulence.

33

 

Table 2.3 Microbial sources of alpha-galactosidases

S.No Source Localization Reference

Bacterial Sources

1. Lactobacillus plantarum Cytoplasm Silvestroni A et al.

2002

2. Bifidobacterium breve Intracellular Xiao M et al. 2000

3. Streptomyces olivaceus Extracellular Crueger A and

Crueger W 1984

4. Saccharopolyspora

erythraea

Culture

supernant

Post DA and

Luebke VE, 2005

5. Sulfolobus

solfataricus(Thermophilic)

Intracellular Brouns SJ et al.

2006

Fungal Sources

6. Thermomyces

lanuginosus(Thermophilic)

Extracellular Puchart V et al.

2000

7. Aspergillus flavipes Extracellular Ozsoy N and

Berkkan H, 2003

8. Aspergillus terreus Extracellular Shankar SK et al.

2009

9. Phlebia radiata Extracellular Prendecka M et al.

2003

Yeast sources

10. Debaryomyces hansenii

UFV-1

Intra&

extracellular

Viana PA et al.

2009

11. Saccharomyces cerevisiae Extracellular Fernandez LR et

al. 2010

12. Saccharomyces cerevisiae Intracelluar Chiba Y et al.

2002

34

 

2.4 Alpha-galactosidase assay:

Alpha-galactosidase can be measured either qualitatively or

quantitatively. A wide range of both artificial and natural substrates

are used to estimate the alpha-galactosidase activity.

2.4.1 Qualitative assays

Recently chromogenic substrates have been developed or

identified for qualitative determination of alpha-galactosidase.

Chromogens like 6-bromo-2-napthyl-α-D-galactopyranoside and X-α-

gal are used for easy screening of microbes producing alpha-

galactosidase enzyme based on the colour zones developed around the

colonies (Andriamainty F, 2000) (Aho S et al. 1997). Another

chromogenic substrate, 5-bromo-4-chloro-3-indolyl-α-D-

galactopyranoside was also used for detection of enzyme activity on

isoelectric focusing gels (Bom I et al. 1998). Resorufinyl α-D-

galactopyranoside, a red flourogenic substrate was employed for high

throughput screening of chaperons required for the treatment of

Fabry’s disease (Zhen DS, 2009).

2.4.2 Quanatiative assays:

Both artificial and natural substrates have been employed for

estimating the hydrolytic activity of alpha-galactosidase. Ο-

nitrophenyl α-D-galactopyranoside and p-nitrophenyl α-D-

galactopyranoside are routinely used as artificial substrates. The

liberated end product p-nitrophenyl was measured

35

 

spectrophotometrically at 400-410nm (Zapater et al. 1990) (Liebl W et

al. 1998). Besides these, phenol glycosides are also used as substrates

(Oishi K and Aida K, 1971) and liberated phenol was measured by

Folin and Denis method (Folin O and Denis W, 1915). Raffinose,

melibiose, stachyose etc., are used as natural substrates (Xiao M et al.

2000) (Sripuan T et al. 2003) (Gheradini F et al. 1985). The liberated

sugars (glucose or galactose) were estimated by Dinitrosalicyclic acid

method (Miller GL, 1959) or Nelson-Somogyi method (Nelson NA,

1944) or coupled enzymatic method (Dahlgyist A, 1961). In some

cases, HPLC analysis was also done to estimate the liberated galactose

upon hydrolysis of raffinose and stachyose (Fridjonsson O et al. 1999).

The most sensitive assay method described in the literature involves

the use of fluorogenic substrate methylumbelliferyl-α-D-galactoside;

the liberated aglycone was measured fluorimetrically (Kilpatrick DC

and Stirling JL, 1977) (Dey PM and Kauss H, 1981). In addition,

polysaccharides like galactomannans and guar gum are also used as

substrates (Li YT et al. 1963) (Suzuki H et al. 1966). The degradation

of guar gum was estimated by measuring viscosity of the reaction

mixture (Yosida S et al. 1997). Table 2.4 lists the details of the assay

conditions using natural and artificial substrates along with the end

products released.

36

 

Table 2.4 List of natural and artificial substrates for quantitative

estimation of alpha-galactosidase

SUBSTRATES REACTION END

PRODUCTS

ORGANISMS Reference

NATURAL SUBSTRATES

Globotriaosylceramide + H2O

D-Galalpha(1,4)D-Glu-ceramide + D-galactose

Homo sapiens Ishii S et al. 2004

melibiose + H2O D-galactose + D-glucose

Oryza sativa, Thermomyces lanuginosus, Bifidobacterium adolescentis, etc

Kobayashi OWD et al. 2002 Puchart V et al. 2000 Holt SM et al. 2008

raffinose + H2O sucrose + alpha-D-galactose

Helianthus annuus Glycine max Streptococcus pneumoniae etc

Kim WD et al. 2003 Guimaraes VM, 2001 Rosenow C et al. 1999

verbascose + H2O alpha-D-galactose + alpha-D-glucopyranosyl-(1,2)-beta-D-fructofuranoside

Oryza sativa Thermotoga maritima etc

Lee R.H. et al. 2009 Miller E.S. et al. 2001

sucrose + melibiose raffinose + D-glucose

Streptococcus equinus Coffea sp.

Dey PM. and Pridham JB, 1972.

stachyose + H2O alpha-D-galactose + sucrose

Bacteroides ovatus Penicillium canescens Aspergillus niger

Gheradini F et al. 1985 Sinitsyna OA et al. 2008 Ademark P et al. 2001

D-sucrose + H2O D-glucose + D-fructose

Phlebotomus papatasi Umbelopsis vinacea

Jacobson RL et al. 2007 Crueger A. and Crueger, 1984.

galactinol + H2O alpha-D-galactopyranose + myoinositol

Cucurbita pepo Lens culinaris Vicia faba

Gaudreault PR et al. 1983 Dey PM et al. 1983 Dey PM and Pridham JB, 1972

lactose + H2O beta-D-galactose + D-glucose

Bifidobacterium adolescentis

Holt SM et al. 2008

ARTIFICAL SUBSTRATES

p-nitrophenyl-alpha-

D-galactopyranoside

+ H2O

p-nitrophenol +

alpha-D-galactose

Sulfolobus solfataricus

Talaromyces flavus

Brouns SJ et al. 2006 Simerska P et al. 2007

p-nitrophenyl alpha- p-nitrophenol + Penicillium Falkoski DL et al. 2006

37

 

2.5 Strategic Optimization of Fermentation Medium:

The economic feasibility of the industrial enzymes depends on its

production costs. The cost of the enzyme primarily depends on the

D-glucopyranoside +

H2O

alpha-D-glucose griseoroseum

p-aminophenyl alpha-

D-galactoside + H2O

p-aminophenol +

D-galactose

Vicia faba Dey PM and Pridham JB, 1972

p-nitrophenyl alpha-

D-fucoside + H2O

p-nitrophenol + D-

fucose

Vicia faba Dey PM and Pridham

JB, 1972

1-naphthyl alpha-D-

galactoside + H2O

1-naphthol + D-

glucose

Saccharomyces sp. Dey PM and Pridham

JB, 1972

2,4-dinitrophenyl

alpha-D-galactoside +

H2O

2,4-dinitrophenol +

alpha-D-galactose

Thermotoga maritima Comfort DA et al. 2007

2,5-dinitrophenyl

alpha-D-galactoside +

H2O

2,5-dinitrophenol +

alpha-D-galactose

Thermotoga maritima Comfort DA et al. 2007

2-nitrophenyl alpha-

D-galactoside + H2O

2-nitrophenol +

alpha-D-galactose

Aspergillus terreus

Thermotoga maritima

Shankar SK et al. 2009

Comfort DA et al. 2007

4-nitrophenyl alpha-

D-galactopyranoside

+ H2O

4-nitrophenol +

alpha-D-

galactopyranose

Pseudoalteromonas sp.

Penicillium canescens

Balabanova LA et al.

2010

Sinitsyna OA et al. 2008

4-nitrophenyl alpha-

D-galactoside + H2O

4-nitrophenol +

alpha-D-galactose

Aspergillus niger

Aspergillus terreus

Aleksieva P et al. 2010

Shankar SK et al. 2009

m-nitrophenyl alpha-

D-galactoside + H2O

m-nitrophenol + D-

galactose

Cucurbita pepo Gaudreault PR et al.

1983

o-nitrophenyl alpha-

D-galactopyranoside

+ H2O

o-nitrophenol + D-

galactose

Ganoderma lucidum

Thermotoga maritima

Sripuan T et al. 2003

Miller ES et al. 2001

o-nitrophenyl alpha-

D-galactoside + H2O

o-nitrophenol + D-

galactose

Aspergillus tamarii

Cucurbita pepo

Civas A. et al. 1984,

Gaudreault P.R and

Webb J.A,1983

38

 

fermentation process applied to isolate high yielding stable strains

and fermentation media. Hence high yielding and stable strains

should be isolated by using a media containing inducers of the

products and devoid of repressors. In general, no defined medium

was established for the best production of any metabolite because the

genetic diversity present in different microbial sources causes each

organism or strain to have its own special conditions for maximum

product production. Therefore, it is essential to have a detailed

investigation on growth and metabolite production pattern of newly

isolated microbial strain under different environmental conditions to

achieve maximum production benefit (Prakasham RS, 2005).

Microbial alpha-galactosidases are produced by both submerged

and solid state fermentation. Most of industrial alpha-galactosidases

are produced by submerged fermentation compared to other

fermentation methods.

2.5.1 Submerged Fermentation

Notable physical and nutritional factors such as pH,

temperature, agitation, aeration, carbon, nitrogen, metal ions etc.,

influence the metabolism-mediated enzyme production. Therefore,

various physical and nutritional factors were evaluated for

optimization of alpha-galactosidase production using submerged

fermentation technique (Robert Rowlands, 2010). Table 2.5 gives the

list of various fermentation parameters optimized for alpha-

galactosidase production in submerged fermentation.

39

 

2.5.1.1 Physical parameters

Physical parameters are important to promote, stimulate,

enhance and optimize the production of alpha-galactosidases.

a) pH of the medium:

The pH of the medium plays a vital role in microbial growth and

enzyme production. Bacterial alpha-galactosidases are produced in

the optimum pH range of 6.0 to 7.5 (Delente J, 1974). The pH of the

fermentation medium is maintained by addition of buffering agents

like phosphate buffer or phosphate ions (Bridson EY and Brecker A,

1970). In submerged fermentation, Escherichia coli (Nagao Y et al.

1988), Streptomyces erythrus (Elahafei AM et al. 2001) etc., produced

alpha-galactosidase at neutral pH where as extreme thermophilic

bacteria Sulfolobus solfataricus produced the enzyme at pH 4.0 to 5.0

(Brouns SJ, 2006). Most of the fungal alpha-galactosidases

production was carried at acidic pH range of 4.5 to 6.0 (Aleksieya P et

al. 2010; Sumeyra G et al. 2010; Kajji A and Ichimi T, 1969).

b) Temperature of the medium:

Optimum temperature of the medium varies in different

organisms. In mesophilic organisms such as Bifidobacterium breve

etc., an optimum temperature of 30-37°C was maintained for enzyme

production (Xiao M et al. 2000). An optimum temperature of 70°C

was reported for thermophilic bacteria Thermus sp. T2 (Benevides CC

et al. 2007) and 80 to 90°C for extreme thermophilic archaebacteria

Sulfolobus solfataricus (Brouns SJ, 2006).

40

 

c) Aeration and agitation of the medium:

Aeration and agitation increase the dissolved oxygen levels, mass

and heat transfer rates in a medium. Optimum conditions for aeration

and agitation varied with the organism and type of fermentors used.

Optimum aeration and agitation conditions are required for proper

supply of oxygen and to prevent cell damage and lysis. In Bacillus

stearothermophilus, 0.5 vvm of aeration rate and agitation speed of

240 rpm were maintained in 14 L fermentor (Narita S et al. 1975). In

case of alkalophilic bacteria Bacillus megaterium VHM1 agitation

speed was maintained at 180 rpm in a shake flask (Patil AG et al.

2010).

2.5.1.2 Nutritional parameters

Nutritional factors play a vital role in enhancing the growth of

the microorganisms and to attain maximal enzyme activity.

a) Effect of carbon source on alpha-galactosidase production:

Carbon source provide energy and materials for microbial

growth. It also acts as an inducer for alpha-galactosidase production.

Different sugars like galactose (Rio S, 1993), melibiose, stachyose and

raffinose (Rezessy SJM et al. 2002) have been used as carbon source.

In addition agricultural waste materials like soya bean flour or soya

meal extract (Aleksieya P et al. 2010), rice bran (Somiari RI and

Balogh E, 1995), wheat flour (kotwal SM et al. 1995) etc., and complex

carbohydrates like locust bean gum (Duffaud G. D et al. 1997) and

41

 

guar gum (Dey PM et al. 1993) are used as carbon source in the

medium for alpha-galactosidase production. In Lactobacillus

fermentum, stachyose was most effective inducer followed by

melibiose, raffinose, and galactose whereas glucose inhibited the

enzyme production (Marisa SG, 1996). Guar gum as carbon source

was reported to enhance enzyme production in Bacillus mega Bacillus

megaterium VHM1(Patil AG et al. 2010).

b) Effect of nitrogen source on alpha-galactosidase production:

Both organic and inorganic nitrogen sources favoured for

production of alpha-galactosidases. Inorganic nitrogen compound

sodium nitrate was reported to enhance enzyme production in

Streptomyces erythrus (Elshafei AM et al. 2001). However the use of

organic nitrogen sources was found to be favourable for higher levels

of enzyme production as compared to inorganic nitrogen sources.

Maximum alpha-galactosidase production was reported in Geobacillus

sp (Sanjay C and Sushma S, 2010) when grown in a medium

containing Soya bean meal. Organic nitrogen sources like peptone

(Lokuge MA and Mathew DC, 2000), tryptone, yeast extract (Delente,

JH, 1974) etc., have been used as nitrogen source in growth medium.

Mostly, a combination of organic and inorganic nitrogen sources are

used in the growth medium. A combination of yeast extract with

inorganic nitrogen ammonium sulphate produced a maximum alpha

galactosidase activity in Bacillus megaterium VHM1 (Patil AG et al.

2010).

42

 

c) Effect of minerals and vitamins on alpha-galactosidase

production:

In addition to carbon and nitrogen source, minerals like CaCl2,

K2HPO4, MgSO4 etc., (liu C et al. 2007) and vitamins like ascorbic acid,

thiamine HCl (Mital BK et al. 1973) are added to the medium to

maintain pH and attain maximum enzyme production. There have

been no reports on the use of alcohols like propanol for alpha-

galactosidase production.

43

 

Table 2.5 Various fermentation parameters optimized for alpha-galactosidase production in submerged fermentation

Microorganism Carbon source

Nitrogen sources

pH Temp

°C

Agitation speed

(rpm)

Fermentation time

Activity

U/ml

Reference

Bacillus stearothermophilus

defatted soybean meal

Yeast extract

6.5–7.0 60 ◦C. - 16hr 1.08 Gote M et al. 2004

Bacillus megaterium VHM1

Guar gum Peptone 7.5 50°C 180 28 h 1.6 Patil et al. 2010

Bacillus sp. JF2 Raffinose Tryptone 7.0 55°C - 24 h 0.6 Li X et al. 1997

Escherichia coli Raffinose Peptone 7.0 30oC 115 6 1.2 mU/ml

Lokuge MA et al. 2000

Klebsiella pneumoniae

Raffinose Peptone 7.0 30oC 115 12 1.2mU/ml

Lokuge MA et al. 2000

Citrobacter freundii Raffinose Peptone 8.0 30oC 115 12 14 mU/ml

Lokuge MA et al. 2000

Lactobacillus curvatus R08

Raffinose Peptone, Yeast extract, Meat extract

7.0 35oC - 12 40 Mi YY et al. 2008

Leuconostoc mesenteriodes JK55

Raffinose Peptone,Yeast extract, Meat extract

7.0 35oC - 12 40 Mi YY et al. 2008

44

 

2.5.2 Solid-state fermentation: Solid-State Fermentation (SSF) holds an immense potential for

production of industrial enzymes. SSF is a fermentation process applied for

the growth of microorganisms on a solid raw material in near to complete

absence of water (Mitchell DA and Lonsane BK, 1992). From ancient times,

SSF has been used for production of several fermentation foods like cheese,

soya sauce, sausages etc. SSF is considered to be more advantages than

submerged process because of its simple and cost-effective process, simple

downstream process and high productivity (Lonsane BK et al. 1992). Natural

raw materials and agricultural wastes like coconut cake, ground nut cake,

wheat bran, rice bran, soya bean flour, red gram flour, soya residue etc.,

have been used as carbon and nitrogen source for growth of various

organisms.

There have been few reports on the alpha-galactosidase production by

SSF. Suzuki H et al in year 1969, first reported alpha-galactosidase

production from Mortierella vinacea by koji method. Mostly fungal species

like Aspergillus oryzae (McKay AM, 1991), Aspergillus niger (Somiari RI

Balogh E, 1992) and Aspergillus awamori (Silman RW, 1980) were grown on

wheat bran based solid substrate for alpha-galactosidase production. Rajoka

M et al. 2009 reported enhanced enzyme production in deoxyglucose-

resistant mutant strains of Aspergillus niger by using corn steep liquor as

nitrogen source. The enzyme produced from Humicola sps (Cruz R and Park

YK, 1982) by using SSF was applied for degradation of oligosaccharides in

soya bean milk. Investigation on alpha-galactosidase production by

45

 

Streptomyces griseoloalbus was carried in a forcefully aerated packed-bed

bioreactor using soya bean flour as a solid substrate (Anisha GS et al.

2010). Table 2.6 gives list of various fermentation parameters optimized for

alpha-galactosidase production in solid-state fermentation.

46

 

Table 2.6 Various fermentation parameters optimized for alpha-galactosidase production in solid-state

fermentation

Microorganism Carbon source Nitrogen

sources

pH Temp

°C

Fermenta

tion

time(hr)

Activity

U/gm

Reference

Aspergillus oryzae red gram plant

waste

- 5.5 30°C 96 3.4 Shankar SK et

al. 2007

Penicillium sp. Wheat bran - 5.5 30°C 75 185.2 Wang CL et al.

2004

Aspergillus foetidus Wheat bran Soya bean

meal

5.5 28°C 96 2207.19 Liu CQ et al.

2007

Humicola sp., soya flour - 45°C 96 44.6 Kotwal M et al.

1998

Streptomyces

griseoloalbus

Soybean flour - - 96 197.2 Anisha GS et al.

2010

Pencillium

chrysogenum LN33

soya meal extract 6.0 30°C 144 1100U/l Aleksieva P et

al. 2010

47

 

2.5.3 Statistical Optimization of fermentation medium: The commonly used medium optimization method is 'one at a time'

method (Prakasham et al. 2006) which ignores interactions among the

different components even after performing of a large number of experiments

(Prakasham et al. 2005a, Sreenivas Rao et al. 2004). Experimental designs

based on statistical tools are known to provide economic and practical

solutions in such cases (Prakasham et al. 2005a; Ravichandra et al. 2007).

Optimization procedures such as response surface methodology (RSM),

orthogonal array, artificial neural network, genetic algorithms, etc., were

developed to optimize the biotechnological processes consisting of an

empirical modeling system developed based on full factorial central

composite parameters that influence the production process (Sreenivas Rao

et al. 2004).

Statistical methods show better performance than one at a time

method even though it has some limitations. For the application of RSM, a

model must be assumed to determine the relative influence of the various

medium components for the objective function and the common model used

was full second order polynomial. The number of experiments is given by LN

(N factor at L levels) therefore in practice only two or three levels could be

applied and plotting was limited to two or three levels. The level of

orthogonal array design or uniform design was also limited by the factor

(Fang et al. 2003). To overcome these problems artificial Feed-Forward

Neural Networks (FFNN) and genetic algorithms (GA) were employed.

48

 

The artificial neural network (ANN) has a similarity like human

decision-making and these are used to solve the nonlinear problems. Many

authors compared the ANN with the statistical design and showed that the

ANN results were much better than the statistical ones (Arulsudar et al.

2005).

Presently, hybrid GA-FNN is becoming popular for the medium

optimization. In many cases GA given data was modeled by the FNN. Hanai

et al. (1999) optimized 21 variables for koji making process while Hongwen

et al. (2005) optimized 1,3 propanediol production with total 29

experiments. Nagata and chu (2003) showed that FNN-GA was the better

optimization design than most of the statistical ones.

Statistical optimization for alpha-galactosidase production was carried

out both in submerged and solid state fermentations. Alpha-galactosidase

produced by submerged fermentation from Streptomyces griseoloalbus was

optimized using response surface methodology (Box-Behnken design) where

as screening of the variables was done by Plackett-Burman design resulted

an increase of 194% in enzyme production (Anisha GS et al. 2008). Alpha-

galactosidase produced from Streptomyces griseoloalbus (Anisha GS et al.

2008) and Aspergillus foetidus ZU-G1 (Liu CQ et al. 2007) using solid-state

fermentation were optimized by using response surface methodology. In case

of Aspergillus niger MRSS 234, screening for various nitrogen sources,

minerals, growth and enzyme inducers was done by placket-burman design

(Srinivas MRS et al. 1994). To the best of our knowledge no reports were

49

 

available for optimization of intracellular alpha-galactosidase. Table 2.7

gives list of statistical method used to enhance alpha-galactosidase

production in various microorganisms.

50

 

Table 2.7 Statistical methods employed to enhance alpha-galactosidase production in various microorganisms.

S.No Organism

name

I/E

Type of

fermentation

Design Design Variables Activity

U/ml

Reference

1 Streptomyces

griseoloalbus

E Submerged RSM

(Box-

Behnken

Design)

pH, temperature, inoculum size,

inoculum age,

incubation period, agitation speed,

carbon source,

yeast extract, MgSO4, FeSO4 and

salinity

50 U/ml Anisha GS et al, 2008b

2 Streptomyces

griseoloalbus

E Solid-state RSM Inoculum size,

Moisture,Galactose

(117 U /g of

dry

fermented

substrate

Anisha GS et al, 2008a

3 Aspergillus foetidus

ZU-G1

E Solid-state RSM wheat bran, soybean meal, KH2PO4,

MnSO4. H2O, CuSO4.5H2O

2207.19 U

g(-1) dry

matter

Liu CQ et al, 2007

4 Aspergillus foetidus

ZU-G1

E Submerged RSM soybean meal, wheat bran, KH2PO4,

FeSO4.7H2O and the medium initial

pH

64.75 U/mL Liu C et al, 2007

51

 

2.6 Purification studies of alpha-galactosidase:

Enzyme purification is vital to study the structure, function and

interactions of the enzyme of interest. Each enzyme requires a specific

strategy of purification. The degree of purity intends on the end use of the

enzyme. Isolation of enzyme depends on the source of the enzyme and

localization. Intracellular enzymes were isolated by disrupting the cells

using different physical, mechanical and enzymatic methods whereas

extracellular enzymes were isolated by removing cells, solids etc using

centrifugation or filtration procedure.

Alpha-galactosidases from various sources have been purified to

homogeneity. Alpha-galactosidases were isolated intracellularly from

Streptococcus bovis (Bailey RW, 1963), Debaryomyces hansenii UFV-1 (Viana

PA et al. 2009) etc., and extracellularly from Citrobacter freundii (Lokuge MA

and Mathew DC, 2000), Bacillus megatrium VHM1 (Patil AG et al. 2010) etc.,

Intracellular alpha-galactosidases were isolated by lysing the cells

either by cell homogenizer (Rios S et al. 1993) or ultrasonicator (Kocabas EE

and Dizbay M, 1999) or by grinding the cells with abrasives (Galas E and

Miszjiewicz H, 1996). In some cases the enzyme was extracted by using

organic solvents like ethanol (Kotwal SM et al. 1999). The crude enzyme

was further concentrated by using any one of the methods or combination of

methods like ammonium sulphate precipitation (Garro MS et al. 1996) or

acetone/ethanol precipitation (Shibuya H et al. 1995) or by ultrafiltration

(Kotwal SM et al. 1999). In thermophilic organisms, an additional heat

52

 

treatment step was introduced to denature the unwanted thermolabile

soluble proteins (Fridjonsson O et al. 1999). Further purification of the

enzyme is usually carried out by combination of either one or more of the

chromatographic techniques viz. ion exchange chromatography, gel filtration

chromatography, hydrophobic interaction chromatography and affinity

chromatography (Zapater IG et al. 1990; King MR et al. 2002). Other

methods used to purify multi-molecular forms of alpha-galactosidase

include isoelectric focussing (Berg JO et al. 1980), preparative gel

electrophoresis (Pederson DM and Goodman RE, 1980) and

chromatofocussing (Talbot G and Sygusch J, 1990)

Affinity chromatography has been used effectively to purify

recombinant and multi-molecular forms of alpha-galactosidase. Hybrid

affinity chromatography using an immobilized metal affinity matrix and

substrate analogs as ligands was used to purify alpha-galactosidase isolated

from roots of Verbascum thapsus L (Bom I et al. 1998). Synthetic alpha D-

galactosylamine linked to 6-aminohexanoic and coupled to carboxyl-

Sepharose was successfully used as affinity support to purify alpha-

galactosidase A from spleen, placenta and plasma of humans (Bishop DF

and Desnick RJ, 1981). Recently recombinant alpha-galactosidases from

Pseudomonas fluorescens subsp. cellulosa and Thermus sp. strain T2 have

been produced as fusion proteins with affinity tags, which enabled simpler

one step purification by its respective affinity ligands like histidine

molecules (Halstead JR et al. 2000; Ishiguro M et al. 2001). Summary of

purification strategies of alpha-galactosidase presented in table 2.8.

53

 

Table 2.8 Summary of purification strategies of alpha-galactosidase from various microorganisms

S. No Microorganism Conc. technique

Colum Chromatography Reference

Ion exchange

Gel filteration

1. Thermoanaerobacterium

polysaccharolyticum

- DEAE Fractogel SO3- King M.R et al. 2002

2. Bifidobacterium longum

JCM 7052

Ammonium

sulphate ppt.

Q-Sepha-

rose

Sepharose 4B Naoko S et al. 2010

3. Bacillus stearothermophilus Ultrafiltration

followed by ethanol

precipitation

- Phenyl Sepharose CL-4B (hydrophobic column

chromatography)

Gote MM et al. 2006

4. Bacteroides ovatus - - Sephacryl S-200 Frank G et al. 1985

5. Lactobacillus fermentum

CRL722

Ammonium sulfate

precipitation

Q Sepharose - Carrera SEA et al. 2006

6. Sulfolobus solfataricus - Q-Sepharose Superdex 200 HR 10/30 gel

filtration

Brouns SJ et al. 2006

7. Thermus sp. strain T2 Ammonium sulfate

fractionation

Sepharose

CL-6B

Superose 12 Ishiguro M et al. 2001

54

 

2.7 Characterization studies of alpha-galactosidase:

Alpha-galactosidase isolated in pure form from plant, animal and

microbial sources have been studied extensively for its industrial

applications. Biochemical and molecular properties of alpha-galactosidases

were studied in detail. Table 2.9 presents characterization properties of

alpha-galactosidase enzyme isolated from various sources.

2.7.1 Biochemical properties of enzyme alpha-galactosidase:

a) Effect of pH on enzyme activity:

The pH of the environment has a profound effect on enzyme activity

and stability. Alpha-galactosidases from yeast, fungi and plant seeds were

reported to have a broad optimum pH range of 3.0 to 6.0 (Dey PM and

Pridham JB, 1972; Church FC et al. 1980; Ulezlo IV and Zaprometova,

1982). Bacterial alpha-galactosidases have a rather narrow and neutral pH

optimum range of 6.0 to 7.5 (Akiba T and Horikoshi K, 1976; King MR et al.

1998; Fridjonsson O et al. 1999). Substrate dependent pH optima was

reported in alpha-galactosidases from Pencillium duponti (Arnaud N et al.

1976) and coffee beans seeds (Dey PM and Pridham JB, 1972).

Thermoanerobacterium polysaccharolyticum was reported to have a broad pH

range of 5.5 to 10.0 showing 60% of maximal activity at pH 5.5 and 45% of

maximal activity at pH 10 (King MR et al. 2002).

In general alpha galactosidases are stable over a broad pH range.

Alpha-galactosidase from Bifidobacterium breve was reported to be stable for

overnight at pH 12 when maintained at 4°C (Xiao M et al. 2000). In another

55

 

case, alpha-galactosidase from Thermus sp.T2 was stable at pH 13 and

temperature 40°C for one hour (Ishiquro M et al. 2001). Plant alpha-

galactosidases were reported to be most stable at acidic pH range (Dey PM

and Wallenfels K, 1974; Balasubramaniam, KM, 1986).

b) Effect of temperature on enzyme activity:

Temperature stability of the enzyme has a huge impact on enzyme

activity and stability. Optimum temperature and thermal stability of enzyme

generally depends on source. Alpha-galactosidases have optimal

temperature range of 35°C to 70°C. Enzymes isolated from thermophilic

fungi and bacteria have an optimal temperature range of 65°C to 95°C.

Thermal stability of an enzyme plays a crucial role in commericializing

the enzyme. Alpha-galactosidase from Geobacillus stearothermophilus

(Fridjonsson O et al. 1999), Sulfolobus solfataricus (Brouns SJ et al. 2006)

etc., are thermostabile above 70°C. Most thermostable alpha-galactosidase

was reported to be stable at 70°C for 36 hours in Thermoanaerobacterium

polysaccharolyticum (King MR et al. 2002). The enzymes isolated from

Escherichia coli (Schmitt R and Rotman B, 1966) and Human (isozyme-A)

(Kusiak JW et al. 1978) were reported to be heat labile.

c) Effect of activators and inhibitors on enzyme activity:

Metal ions play a vital role in biological function of the enzyme. They

act as electron acceptors or donors. They can either activate or inhibit the

activity of enzyme. Metal ions like Ca2+, Mg2+, Na+, K+ were reported to

strongly activate or increase the activity of enzyme alpha-galactosidase from

56

 

Aspergillus terreus at final concentration of 10mM (Falkoski DL et al. 2006).

Mn2+ ions have found to be stabilizing as well as activating the enzyme in

Pencillium janthinellum (Elshafei AM et al. 1993) and Escherichia coli

(Burstein C andKepes A, 1971). In addition, organic compounds like EDTA,

glucose were reported to increase the enzyme activity in Aspergillus terrus

(Falkoski DL et al. 2006) and NAD+, NADP+ in Bacillus halodurans

(Anggraeni AA et al. 2008).

A substance that causes partial or complete loss of enzyme activity are

known as inhibitors. In general, metal ions and organic compounds act as

enzyme inhibitors. Divalent ions like Ag2+, Cu2+ and Hg2+ are potent

inhibitors of alpha-galactosidase. Hg2+ ions react with amino, thiol, carboxyl

and imidazol groups whereas Ag2+ ions react with carboxyl or histidine

groups at the active site of alpha-galactosidase, thereby strongly inhibiting

the enzyme activity (Dey PM and Pridham JB; 1972; Dey PM, 1969). Various

sugars were also reported to inhibit the alpha-galactosidase activity. Alpha-

galactosidase from Aspergillus nidulans was competitively inhibited by

melibiose, raffinose, D-galactose and D-glucose (Rios S et al. 1993). Three

different types of inhibition patterns-competitive, noncompetitive and mixed,

was observed in alpha galactosidase from Bacillus stearothermophilus, when

incubated with galactose, lactose and cellobiose (Pederson DM and

Goodman RE, 1980). Detergents like Sodium dodecyl sulphate were reported

to strongly inhibit the enzyme activity in Glycine max and Pencillium

griseoroseum (Falkoski DL et al. 2006).

57

 

2.7.2 Molecular properties of enzyme alpha-galactosidase:

a) Multiple forms of alpha galactosidase:

In 1964, IUPAC recommended the use of term ‘Multiple forms of

enzymes’ for isoenzymes existing in a same species. The multiforms of

alpha-galactosidase were reported to be predominantly present in plants

and few microorganisms. Multiple forms of alpha-galactosidase are mainly

due to differential glycosylation during post translational modifications of an

enzyme or due to proteolytic cleavage (Dey PM and Del CE, 1984). Alpha-

galactosidase I and II were detected in Bacteroides ovatus. Alpha-

galactosidase I was produced when grown on guar gum whereas alpha-

galactosidase II was produced when grown on galactose, raffinose, melibiose

or stachyose (Frank G et al. 1985). Similarly two isoenzymes, regulated by

two different genes, agaA and agaB were detected in Bacillus

stearothermophilus KVE39 (Ganter C et al. 1988). In humans, two multiple

forms of alpha-galactosidase A and B were detected in normal tissues where

as alpha-galactosidase A is absent in Fabry’s disease (Kusiak JW et al.

1978).

b) Molecular weight of alpha-galactosidase:

Microbial alpha-galactosidases are complex in structure with

molecular masses varying between 30KDa to 400KDa (Halstead JR et al.

2000; Ishiguro M et al. 2001). Fungal alpha-galactosidases are monomeric

with a molecular weight of 50KDa (Shibuya H et al. 1995; Shibuya H et al.

1997). Most complex structure of alpha-galactosidase has been detected in

Thermus sp. Strain T2 having a molecular mass of approximately 400KDa

58

 

and existing in solution as an octomeric form (molecular weight of one

subunit is 53,514Da) (Ishiguro M et al. 2001).

c) Isoelectric point of alpha-galactosidase:

The pI value of alpha-galactosidase purified from various sources

varies from 3.5 to 9. Multi-molecular forms of alpha-galactosidase were

reported to have different isoelectric point. Alpha-galactosidase I and II from

Bacteroides ovatus were reported to have pI values of 5.6 and 6.9 (Frank G

et al. 1985).

59

 

Table 2.9. Properties of alpha-galactosidases from various microorganisms

S. No

Microorganism Multiforms Mol.wt (KDa)

pH Temp (°C)

Isoelectric point

Reference

1. Streptomyces griseoloalbus

I II III

72 57 35

5 6.5 5.5

65 50 55

4.41 5.6 6.13

Anisha GS et al. 2009

2 Bacteroides ovatus I II

85 80

5.9 6.3

50 50

5.6 6.9

Frank G et al. 1985

3. Bacillus stearothermophilus

I II

81 84

6 7

65 65

- -

Pederson DM and Goodman, 1980

4. E. coli K12 I II

329 200

- -

- -

5.1 --

Schmid K and Schmitt R 1976

5. Penicillium simplicissimum

I II III

61 84 61

3.0 4.0-5.0

4.5

- - -

5.2 4.4 7.0

Luonteri E et al. 1998

6 Aspergillus niger I II III IV

350 117 117 117

5.0-6.0 2.5-5.0 2.5-5.0 2.5-5.0

50-60 50-60 50-60 50-60

4.15 4.5 4.7 4.8

Ademark P et al .2001

7. Candida guilliermondii

I II

270 270

- -

- -

6.16 6.21

Hashimoto H et al. 1993

8 Aspergillus tamarii I II III

26.5 25.4 56

3.5-6.5 3.5-6.5

4.8

45-50 37 50

- - -

Civas A et al. 1984

60

 

2.8 Kinetic Studies of alpha-galactosidase:

To develop any enzyme-based process, knowledge of the kinetic

parameters of the enzyme under study is of utmost importance. To be

precise, kinetic properties like Vmax, Km, Kcat, and Ea knowledge are essential

for designing enzyme reactors or quantifying the applications of the enzyme

under different conditions. This information also helps in understanding the

catalytic behaviour related to enzyme-, substrate- and environment

specificity. The synthetic substrates are much more popular than complex

substrates for defining Km and Vmax as they are convenient (Kumar CG,

2002; Larcher et al. 1996). Various natural substrates like raffinose,

melibiose and stachyose etc., and synthetic substrates such as 4-

nitrophenyl galactopyranoside etc., are used for determining kinetic

parameters for alpha-galactosidases. For an alpha-galactosidase from

Escherichia coli the Km value was 0.12 for ρ-nitrophenyl alpha-D-galactoside,

2.33 and 3.65 for melibiose and raffinose (Kawamura S et al. 1976).

2.9 Immobilization of enzyme alpha-galactosidase:

Enzyme immobilization is found to be one of the strategic methods to

increase the stability and reusability of the enzyme. Till date various

methods have been reported for immobilization of alpha-galactosidase

enzyme such as covalent binding onto modified silica (crosslinked with

glutaraldehyde) natural chitin, anion exchange resins etc and entrapment in

gels/beads/fibres like polyacrylamide (Thippeswamy S and Mulimani VH

2002), k- Carrageenan (Girigowda K et al. 2007), calcium alginate, gelatin

61

 

(Naganagouda K and Mulimani VH, 2006) and agarose. A hexameric alpha-

galactosidase from Thermus sp. T2 strain was reversibly immobilized on

polymeric ionic exchangers (Miguel F et al. 2008).

In addition, whole cell immobilization of organisms producing alpha-

galactosidase was reported. Papaver somniferum L and Humicola lutea 120-

5 strain cells producing extracellular alpha-galactosidase were reportedly

to be immobilized on glutaraldehyde (Stano IJ et al. 1997) and sol-gel

nanomatrices (Djambaski et al. 2010) for enhanced production of the

enzyme.

Immobilized alpha-galactosidases were reported to be applied for

degradation of galactooligosaccharides in soya bean milk in batch, repeated

batch and continous fluidized bed reactors. They were also used for

degradation of raffinose in sugar beet industry.

2.10 Applications of Alpha-galactosidase:

Alpha-galactosidases have immense potential applications in different

industries. The enzyme, though originally identified as potent drug with

possible applications in enzyme therapy for treatment of Fabry’s disease,

has been used in food industry for degradation of galactooligosaccharides in

legumes, galactomannans processing and animal feed processing. Recent

applications of enzyme include in xenotransplantation, blood group

transformation and as digestive enzymes. The applications of alpha-

galactosidase are broadly cateogorized into two groups- medical applications

and industrial applications. Alpha-galactosidases isolated from Mortierella

62

 

vinaceace and Aspergillus niger were commercially used in different food

industries.

2.10.1 Medicinal Applications of Alpha-galactosidase

2.10.1.1 Enzymatic treatment for Fabry’s Disease:

Fabry’s disease also known as Anderson-Fabry disease or alpha-

galactosidase A deficiency is a rare X-linked recessive lysosomal storage

disorder (James WD et al. 2006). It is caused due to deficiency of lyosomal

enzyme, alpha-galactosidase A (alpha-Gal A) (Peters FPJ et al. 2001). Alpha-

Gal A is responsible for degradation of glycosphingolipids and

globotriaosylceramide (GL-3) present in cell membranes. Deficiency of the

enzyme leads to accumulation of mainly GL-3 in lyososmes and plasma of

most cells in the body (Karen JK et al. 2005). Fabry’s disease primarily

affects hemizyous males (1:40,000 to 60,000) and heterozygous females

(Kotnik J et al. 2005). Symptoms like hypohidrosis, angiokeratomas, severe

pain, corneal opacities etc were observed in childhood in males. As age

advances it leads to renal failure, brain and heart diseases (Desnick RJ and

Brady RO, 2004). Females may have the same symptoms as males but to a

more variable degree (Beck M, 2002).

Enzyme replacement therapy is suggested as a specific treatment for

fabry’s disease. Two commercially produced recombinant enzymes were

currently available in the market for treatment of fabry’s disease. One

enzyme is agalsidase beta available in trade name of Fabrazyme (Genzyme

Corporation) is based on expression of the human GLA gene in Chinese

63

 

hamster ovary cells recombinantly is available in USA. Other enzyme,

agalsidase alfa available in trade name Replagal (Transkaryotic Therapies,

Inc) is produced by cultured human skin fibroblasts with activated human

alpha-galactosidase A gene promoter is available in Europe (Bengt AB et al.

2003). Within few weeks of treatment with these enzymes, patients showed

complete disappearance of accumulated GL3 deposits from the kidney blood

vessel walls and skin (Eng CM et al. 2001; Thurberg BL et al. 2002). Even

after 30-36 months the long-term effects are minimal, GL3 levels remained

low, and kidney function remained stable. After 4 to 4.5 years of study,

patients continue to do well (Wilcox WR et al. 2004).

2.10.1.2 Digestive Enzyme:

Most of legumes and cruciferous vegetables are made up of complex

galacto-oligosaccharides. In humans, due to the absence of intestinal alpha-

galactosidase the complex galactose containing sugars were not metabolized

into simple sugars in small intestine. The undigested sugar molecules on

entering the large intestine are metabolized by fermenting microbial flora

releasing gas or flatulence (Levine B and Weisman S, 2004; Naczk M et al.

1997). Alpha-galactosidases isolated from Aspergillus niger is commercially

used as digestive enzyme for treatment of gas, bloating and flatulence

(Solomons NW et al. 1991; Ganiats TG et al. 1994). The enzyme is

commercially available single or in combination with other digestives

enzymes under different trade names like Beano (Alan EK, 1999) (Glaxo-

smith), EZ-Gest (Shaklee, plant based product), Bean-zyme etc.

64

 

2.10.1.3 Conversion of ‘B’ to ‘O’ blood group:

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 F et al., 2005; Martin LO et al. 2004). 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 J et al., 2000). Cell surface markers of red blood cells

are given in Figure 2.1). Alpha-galactosidase isolated from coffee 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 Y et al., 2003). Figure 2.2

represents diagrammatic presentation of enzymatic action of alpha-

galactosidases on ‘B’ group cells.

65

 

Figure 2.1 Cell surface markers of RBC cells

Figure 2.2 Enzymatic blood group conversion

66

 

2.10.1.4 Xenotransplantation:

Organ transplantation has emerged as one of the life saving medical

process. Due to acute shortage of organs, xenotransplantation of organs

from pigs to humans is considered as potential solution. However,

consequent xenorejection prevents xenotransplantation from the clinical

application (Bach FH et al. 1995). One of the causes for xenorejection is Gal

alpha 1,3 Gal (G antigen or Gal epitope), a disaccharide expressed

abundantly in mouse, pigs and monkeys (Good AH et al. 1992). The G

antigens binds to human xenoreactive natural antibodies (XNA) thereby

leading to xenorejection (Azimzadeh A et al. 1997). One of the most

attracting attempts to prevent xenorejection is to inhibit the synthesis of Gal

alpha 1,3Gal. In vitro treatment of porcine endothelial cells and lymphocytes

with green coffee bean alpha galactosidase dramatically decreased the

binding of human xenoreactive natural antibodies without any nonspecific

immunoglobulin binding sites induced (Yan JL et al. 2003). Application of

enzyme alpha-galactosidase to prevent xenorejection can be one of the

potential solutions for xenotransplantation.

2.10.2 Industrial applications of alpha-galactosidase

2.10.2.1 Increasing the nutritive value of legumes

Legumes such as beans, lentils, peanuts, soya beans, cow peas etc.,

are used in different cuisines and is a main staple food in different countries

like Africa etc. Some of the legumes are used in animal feeds of poultry and

cattle. These are good source of fibre, dietary protein, iron, folic acid,

potassium and magnesium for humans and animals. These foods are

67

 

available as fresh, canned, frozen or in dried versions. One of the problems

with legumes is increased intestinal flatulence (gas). In addition to the

above mentioned nutrients, legumes are rich source of raffinose family of

oligosaccharides like raffinose, stachyose, mellibose etc. Degradation of

these raffinose oligosaccharides by small intestinal microflora leads to

production of gas (Delumen BO, 1992; Naczk M et al. 1997). Traditional

methods like soaking and sprouting degrades the oligosaccharide but not to

an extent of 100% (Mulimani VH and Devender S, 1998). Processing of

legumes by using enzyme alpha galactosidase is one of the solutions to

increase the nutritive value of the legumes (Figure 2.3).

Industrially, both soluble and immobilized enzymes were used in

processing of legumes. Soya milk was processed by soluble enzyme from

Lactobacillus fermentum CRL 722 (LeBlanc et al. 2004) and Mortierella

vinacea (Thananunkal et al. 1976). Soya milk was also processed by soluble

and immobilized enzymes entrapped in calcium alginate, gelatin alginate, k-

carrageenan and poyacrylamide matrix from Aspergillus oryzea (Girigowda K

et al. 2007; Naganagouda K and Mulimani VH, 2006). Galacto-

oligosaccharides in chick peas were degraded by alpha-galactosidase

enzyme isolated from Gibberella fujikuroi (Mulimani VH and Ramalingam,

1997). Somiari and Balogh have reported the hydrolysis of stachyose in

cowpea flours using soluble alpha-galactosidase from Aspergillus niger

(Richard IS and Esther B, 1993).

68

 

Figure 2.3 Enzymatic degradation of galactooligosaccharides

2.10.2.2 Sugar beet industry:

Sugar beet is used worldwide for commercial production of sugar.

Sugar beet molasses contains small amounts of Raffinose. In sugar beet

processing, raffinose concentration gradually increases and accumulates at

65°C thereby hindering the crystallization of sugar (Suzuki H et al. 1969).

Elimination of raffinose by alpha-galactosidases improves crystallization

process and also increase the yield of sucrose (Yamane T, 1971). Alpha

galactosidases extracted from Penicillium duponti (Arnaud N and Bush D,

1976) etc., were reported to hydrolysis raffinose present beet sugar and are

finding utility in the beet sugar manufacturing industry. James CL, (1982)

reported the use of immobilized alpha-galactosidase from mycelia in beet

sugar industry.

2.10.2.3 Galactomannan processing:

Galactomannans are polysaccharides made up of backbone mannose

residues linked by β 1-4 linkage to which galactose side chains are attached

69

 

at O-6 position. The ratio of mannose/galactose vary from plant to plant

(Dea ICM, 1975; Reid JSG and Edwards ME, 1995).

a) Guar gum processing:

In food, cosmetic and pharmaceutical industries, guar gum is used a

thickener, emulsifier and stabilizer. Guar gum is a galactomannan extracted

from guar beans grown in India and Pakistan. The mannose/galactose ratio

is 2 for guar gum and 4 for locust bean gum (extracted from locust bean).

The increase in number of galactose substitutes causes guar gum to be

more soluble and viscous than locus bean gum. But it lacks self-gelling

capacity similar to locust bean gum (Frederick NE et al. 2008). Partial

enzymatic hydrolysis of guar gum with alpha-galactosidase enzyme removes

the addition galactose side chains without disturbing the mannose

backbone (Pai VB and Khan SA, 2002) (Figure 2.4). Plant and microbial

alpha galactosidases were reported to modify galactomannan polymers.

(Bulpin PV et al. 1990).

70

 

Figure 2.4 Enzymatic hydrolysis of Guar gum

b) Animal feed processing:

Legumes are added in animal feed of cattle, pigs and poultry. Presence

of galactomannans in feed is considered disadvantages because of its

viscosity inducing capacity. A combination of alpha-galactosidase and Beta-

mannase is effective in hydrolysing galactomannas. Alpha-galactosidase

cleaves the galactose side chains from mannose backbone, thereby

liberating space for beta-mannase to hydrolyse the mannose units. This

effectively reduces the viscosity of the digesta in the intestine (Henk G,

2004). An alpha-galactosidase supplement was added to corn and soya

based diets to increase digestive efficiency of broilers and pigs (Kidd MT et al

2010; Pan B et al. 2002).

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c) Paper and Pulp industry:

Mannases are enzymes that degrade mannans, a complex

polysaccharide present in hardwood, soft wood and leguminous seeds.

Mannases are one of the enzymes used for enzymatic bleaching and for

pretreatment of pulp. In softwood and leguminous seeds, galactose is linked

at O-6 position of mannose moieties (Galactomannas). Alpha-galactosidases

catalyse the release of galactose molecules from soft wood and leguminous

seeds. Alpha galactosidase isolated from Pseudomonas flourescens was

reported to enhance bleaching effect when used along with mannoses

(Clarke JH et al. 2000).

2.10.2.4 Alpha-galactosidases as detergents:

Recombinant alpha-galactosidase from Trichoderma ressi was reported

to be added to laundry, surface and dish cleaning compositions along with

other ingredients. The enzyme was effectively able to remove chocolate

cream, salad dressing, guar gum pigment and ice cream stains (Mcdonald

HC and Poulose AJ, 2010).

2.11 The Genus Acinetobacter:

The term Acinetobacter is derived from scientific greek word ‘non-

motile rods’. In general Acinetobacter species are aerobic, non-fermentative,

gram negative bacilli. Acinetobacter sps are predominately present in soil

and water. Till date only 17 species have been identified where as remaining

14 species were unidentified. This is because identification of Acinetobacter

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at species level is difficult by using biochemical studies. Different molecular

methods like AFLP, PCR-RFLP and other sequences are required for

identification at species. They are oxidase and nitrate negative, non-motile

and grow well on MacConkey medium without salt (Dijkshoorn L, 2008).

Scientific classification of the organisms is as follows.

Recent studies on Acinetobacter species has gained a lot of interest in

its biotechnological and environmental applications. Some strains of this

genus are known to be involved in biodegradation of a number of different

pollutants such as biphenyl and chlorinated biphenyl, amino acids

(analine), phenol, benzoate, crude oil, acetonitrile, and in the removal of

phosphate or heavy metals. Futhermore, several economic products like

proteases, lipases, bioemulsifiers, cyanophycine and biopolymers were

produced by different strains of Acinetobacter. These organisms were also

used as model system for modern genetic manipulations and protein

engineering (Desouky AH, 2003).

Kingdom- Bacteria

Phylum- Proteobacteria

Class- Gammproteobacteria

Order- Pseudomonadales

Family- Moraxellaceae

Genus- Acinetobacter

(Brisou and Prevot, 1954)

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Some of the strains of Acinetobacter are known human pathogens

although they did not cause any infection to healthy individual.

Acinetobacter baumannii is an opportunistic pathogen present mainly in

hospital environment causing serious infections to the patients (Gerischer U,

2009). Some isolated strains of Acinetobacter baumannii are multidrug

resistant and often fatal.

Till date no alpha-galactosidase producers among Acinetobacter sps

have been reported. In present study, screening and isolation followed by

identification has resulted in a new strain of Acinetobacter sps that produces

two alpha-galactosidases which are discussed in the later part of this thesis.