α-Galactosidases

a-Galactosidases

16 G.S. Anisha

GOVERN MENT COLLEGE FOR WOMEN, TR I V ANDR UM, KE RAL A, INDI A

16.1 Introduction a-Galactosidase, or melibiase (a-D-galactoside galactohydrolase, EC 3.2.1.22), is an exoglycosidase that cleaves the terminal nonreducing a-1,6-linked galactose residues from a-D-galactosides, including galactose oligosaccharides such as melibiose, raffinose, and stachyose and branched polysaccharides such as galactomannans and galacto(gluco)mannans [1]. a-Galactosidases are versatile enzymes with many potentials for biotechnological and medicinal applications. They are widely used for the reduction or removal of antinutritive galactooligosaccharides such as raffinose family sugars that cause flatulence [2], thereby improving the nutritional value of legume-based food. Microbial a-galactosidases are useful enzymes in the sugar industry, in which they eliminate raffinose and/or stachyose, which negatively affect the crystallization of sucrose [3]. Transglycosidase activity was also demonstrated in some of the a-galactosidases [4,5]. The galactooligosaccharides produced by the transferase action of a-galactosidases can be used as a probiotic in functional food [6]. a-Galactosidases have interesting applications in the pulp and paper industry [7]. Furthermore, a-galactosidases are gaining increased interest in human medicine especially in the treatment of Fabry disease, prevention of xenorejection, and blood group transformation. a-Galactosidases are used in enzyme replacement therapy for the treatment of Fabry disease, an X-linked (locus Xq22) lysosomal storage disorder caused by mutations in the a-galactosidase A gene resulting in the defective activity of this enzyme [8,9]. This disease is characterized by progressive accumulation of the enzyme’s substrate, globotriaosylceramide, and related glycosphingolipids [9] in the plasma and particularly in the vascular endothelial lysosomes of hemizygous male victims. In affected males this leads to early death due to occlusive disease of heart, kidney, and brain. a-Galactosidase treatment offers a most attractive alternative to prevent xenorejection, because in vitro treatment of porcine endothelial cells and lymphocytes with green coffee bean a-galactosidase dramatically decreases the binding of human xenoreactive natural antibodies [10]. Additionally, a recombinant taro a-galactosidase is reported not only to hydrolyze a-1,4-linked galactosyl residues, which are accumulated in

Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products http://dx.doi.org/10.1016/B978-0-444-63662-1.00016-6 Copyright © 2017 Elsevier B.V. All rights reserved.

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the tissues from patients with Fabry disease, but also to hydrolyze the a-1,3-linked galactoside of B red blood cells [11]. a-Galactosidases have been classified based on substrate specificity [12] and sequence similarity using hydrophobic cluster analysis [13]. With respect to substrate specificity, group I a-galactosidases hydrolyze oligosaccharides such as melibiose, raffinose, stachyose, and verbascose; group II a-galactosidases are active on polysaccharide substrates, such as galactomannan and galactoglucomannan. By amino acid sequence homology, a-galactosidases are classified into four glycosyl hydrolase (GH) families: GH-4, GH-27, GH-36, and GH-57. a-Galactosidase activity has been demonstrated for only six enzymes of the GH-4 family and two enzymes of GH-57. The majority of the known a-galactosidases belong to the GH-27 and GH-36 families. Most a-galactosidases of eukaryotic origin, including Aspergillus niger AglA [14] and AglB [15], belong to family 27. Family 36 contains primarily bacterial a-galactosidases including a-galactosidase from Bacillus stearothermophilus NUB 3621 [16], Thermus thermophilus [17], and Thermus sp. strain T2 [18]. However, some a-galactosidases of prokaryotic origin are also included in GH-27. Similarly some eukaryotic a-galactosidases are also included in GH-36. For example, a-galactosidase from Streptomyces coelicolor A3(2) (Accession No. CAB54169) is a member of the GH-27 family, whereas that from Trichoderma reesei (Accession No. Z69254) is a member of the GH-36 family. There are also reports documenting the presence of a-galactosidases belonging to both families being produced by the same organism. For example, A. niger ATCC 46890 produces four major a-galactosidase forms (a-Gal IeIV) of which a-Gal I belongs to family 36 and a-Gals II, III, and IV, which appear to be isoforms of the same enzyme, show close similarity to family 27 a-galactosidases [19]. Similarly, Streptomyces griseoloalbus is capable of producing multiple thermostable a-galactosidases from both families of glycosyl hydrolases, GH-27 and GH-36, which differ in their substrate preferences for polymeric galactosides [20,21].

16.2 Substrates for a-Galactosidases The substrates for a-galactosidases are generally called a-galactosides. a-Galactosides are glycosides containing a terminal nonreducing a-D-galactosyl residue whose first carbon atom, or the anomeric carbon atom, is attached to a carbohydrate or noncarbohydrate moiety by an acetal linkage. The a-D-galactosyl groups are ubiquitous in higher plants and are found in a variety of oligosaccharides and polysaccharides and a few nonsugars such as glycerol, inositol, and certain lipids. Such a-galactosides are mainly the substrates for the enzyme a-galactosidase. For preliminary-level studies, nutrient agar plates containing X-a-Gal are used to differentiate a-galactosidase-producing strains from nonproducers [22]. The most commonly used substrate for the routine assay of a-galactosidase is the chromogenic synthetic substrate p-nitrophenyl-a-D-galactopyranoside [21], the hydrolysis of which

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FIGURE 16.1 Hydrolysis of melibiose by a-galactosidase. The solid arrow points toward the a-1,6 linkage, which is the site of action of a-galactosidase.

liberates p-nitrophenol, which is then estimated spectrophotometrically. The natural substrates of a-galactosidase include the oligosaccharides melibiose (a-D-Galp(1 / 6)-DGlu), raffinose (a-D-Galp(1 / 6)-a-D-Glup(1 / 2)-b-D-Fru), stachyose (a-D-Galp(1 / 6) -a-D-Galp(1 / 6)-a-D-Glup(1 / 2)-b-D-Fru), and verbascose (a-D-Galp(1 / 6)-a-DGalp(1 / 6)-a-D-Galp(1 / 6)-a-D-Glup(1 / 2)-b-D-Fru) and polysaccharides like galactomannans and galacto(gluco)mannans [1]. Because of its action on melibiose (Fig. 16.1), a disaccharide of glucose and galactose, a-galactosidase is also known as melibiase. Raffinose, stachyose, and verbascose, commonly called raffinose family oligosaccharides (RFOs), are abundantly found in the seeds, roots, stems, and leaves of the members of the family Leguminoseae, where they serve the purpose of reserve carbohydrate and also protection against frost and drought. The structures of the RFOs raffinose and stachyose and the sites of cleavage by a-galactosidase are shown in Fig. 16.2. The polymeric substrates for a-galactosidase include the galactomannans and galacto(gluco)mannans, which are abundantly present in the members of the family Leguminosae, such as Cyamopsis tetragonoloba and the carob tree Ceretonia siliqua [24]. Cyamopsis tetragonoloba is an annual plant, grown in arid regions of India as a food crop for animals, and its seed endosperm contains the galactomannan called guar gum. Locust bean gum is another galactomannan extracted from the endosperm of the seeds of the carob tree C. siliqua, which grows in Mediterranean countries. These galactomannans contain a b-1,4-linked D-mannopyranose backbone and a-1,6-linked D-galactopyranose side groups. The complete hydrolysis of these polymeric galactoglucomannans requires the concerted action of three different enzymes, a-galactosidase, b-mannosidase, and endo-b-D-mannanase [25e27]. The structures of locust bean gum and guar gum and the sites of enzymatic hydrolysis are shown respectively in Figs. 16.3 and 16.4.

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FIGURE 16.2 Structures of (A) raffinose, (B) stachyose, and (C) verbascose, the predominant flatulence-causing oligosaccharides in legumes. The symbol points toward the sites of hydrolytic action of a-galactosidase [23].

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FIGURE 16.3 Structure of locust bean gum and the enzymes that catalyze its hydrolysis.

FIGURE 16.4 Structure of guar gum and the enzymes that catalyze its hydrolysis.

16.3 Sources of a-Galactosidases a-Galactosidases are widely distributed in nature, being found in a variety of plants; in animals, including both vertebrates and invertebrates; and extensively in microorganisms including bacteria, fungi, and yeasts. The majority of a-galactosidases currently used in industry are of microbial origin. Many bacteria, including the filamentous actinomycetes, have been reported to contain a-galactosidase activity. The bacterial sources of a-galactosidase include B. stearothermophilus [28], Lactobacillus acidophilus [29], Lactobacillus fermentum [30], Bifidobacterium adolescentis [31], Bifidobacterium breve [32], etc. The filamentous actinomycetes including Streptomyces erythrus [33] S. coelicolor A3(2) [34], Saccharopolyspora erythraea [35], S. griseoloalbus [20,36,37], and Streptomyces sp. S27 [38] are sources of a-galactosidase. The presence of a-galactosidase is also reported in the extreme thermophilic eubacterium Rhodothermus marinus [39], marine bacterium Pseudoalteromonas sp. [40], and lactic acid bacterium Carnobacterium piscicola [41]. An unusual intracellular a-galactosidase has been isolated from the hyperthermophilic archaeon Sulfolobus solfataricus P2 [42], an aerobic

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microorganism that lives in terrestrial volcanic pools of high acidity. As of this writing, this distinct type of thermostable Sulfolobus a-galactosidase enzyme represents the first member from the Archaea. In fungi it is found in T. reesei [25], Aspergillus oryzae [43], Aspergillus fumigatus [44], Gibberella fujikuroi [45], Mortierella vinacea [46], Penicillium simplicissimum [47], Humicola sp. [48], Thermomyces lanuginosus [49], and Rhizopus oligosporus [50]. A thermophilic a-galactosidase with high specific activity, broad substrate specificity, and significant hydrolysis ability of soy milk is reported to be produced from Neosartorya fischeri P1 [2].

16.4 Production of a-Galactosidases Submerged fermentation for aerobic microorganisms is now the well known and widely used method for the production of a-galactosidase [33,36,51]. Table 16.1 lists the a-galactosidase yield from various microorganisms on various carbon sources in submerged fermentation. Svastits-Du¨csT et al. [51] reported that the a-galactosidase yield was enhanced three to five times in optimized medium with locust bean gum or guar gum as the carbon source. Gurkok et al. [44] reported optimization of culture conditions for Aspergillus sojae expressing the a-galactosidase gene, aglB, of A. fumigatus IMI 385708, which resulted in a fourfold increase in a-galactosidase production. The authors claim the feasibility of industrial large-scale production of a-galactosidase, which is known to be valuable in galactomannan modification. The carbon source used for the induction of a-galactosidases has been found to have a marked effect on the properties of the a-galactosidase produced. Bacteroides ovatus has

Table 16.1

Microbial Production of a-Galactosidase in Submerged Fermentation

Microbial Strain

Carbon Source/Inducer

Thermomyces lanuginosus CBS 395.62/b Aspergillus foetidus ZU-G1 Aspergillus fumigatus Streptomyces griseoloalbus T. lanuginosus CBS 395.62/b Monascus pilosus Trichoderma reesei RUT C-30 Bacillus sp. JF2 strain Bacillus sp. JF strain

Locust bean gum/guar gum Soybean meal þ wheat bran Galactose Locust bean gum Sucrose Galactose Locust bean gum þ galactose Soy effluent stream Wheat flour þ soybean seed flour Soybean meal Galactose Lactose

Bacillus stearothermophilus Streptomyces erythrus Absidia griseola var iguchii ATCC 20431 a

The values given are not necessarily optimal for the relevant enzymes in all cases.

a-Galactosidase Yielda

References

6 U/mL 64.75 U/mL 35.68 U/mL 50 U/mL 90 U/mL 13.9 U/mL 0.195 U/mL 0.6 U/mL 27.4 U/mL

[51] [52] [58] [36] [59] [60] [25] [61] [62]

1.08 U/mL 9.94 U/mL 23 U/mL

[63] [33] [64]

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375

been reported to produce two inducible a-galactosidases: a-galactosidase I, which is able to hydrolyze galactomannan, is induced by guar gum but not by other galactosides, whereas synthesis of a-galactosidase II, incapable of acting on guar gum, is induced by galactose, melibiose, raffinose, and stachyose [53]. Similarly Aspergillus tamarii produces two mycelial a-galactosidases when raffinose is used as the carbon source and one secretory a-galactosidase when cultivated in the presence of galactomannan [54]. The production of a-galactosidase I appeared to be regulated coordinately with the mannanase activity, which degrades the backbone of the substrate. Another galactomannan, locust bean gum, has been reported to induce a-galactosidase production by T. reesei Rut C-30 (ATCC 56765) [25] and S. griseoloalbus [36]. As in the case of B. ovatus a-galactosidase I [53], in B. stearothermophilus the maximum a-galactosidase activity did not occur until 5 days after inoculation, suggesting that the mannanase was required to depolymerize the galactomannan to oligosaccharides before a-galactosidase was significantly expressed [55]. In T. reesei a low constitutive amount of a-galactosidase is present and it has been suggested that this enzyme releases galactose from locust bean gum and thereby triggers the production of inducible a-galactosidase [25]. a-Galactosidase has also been reported to be produced constitutively by the thermophilic fungus Humicola sp. [48] and Streptococcus mutans [56], although notable increases in activity have been observed after melibiose, raffinose, or lactose supplementation. Wong-Leung et al. [57] used extracts of sugarcane and soybean wastes as carbon sources for Monascus anka M9 IAM. Both supported the growth of the fungus, but sugarcane waste was superior for the production of a-galactosidase. Cheap agriculture residues, like wheat bran or wheat flour, rice bran, soy flour, soybean cake or soybean meal, sorghum, corn, millet etc., are also used for production of a-galactosidase [41,48,58]. Galactose and several galactose-containing oligosaccharides, such as melibiose, raffinose, and stachyose, have commonly been used for the induction of a-galactosidases, especially when agricultural residues are used as a carbon sources in the medium [58,59]. These low-molecular-weight compounds have been effective inducers for the production of intracellular enzymes by Monascus pilosus [60] and Corynebacterium murisepticum ATCC 21474 [61] and for extracellular a-galactosidase of the actinomycete S. griseoloalbus [37]. In addition to galactose, L-arabinose and corresponding polyols induced a-galactosidase in T. reesei [25]. Foda et al. [62] screened 38 fungal strains for a-galactosidase production using CzapekeDox agar medium supplemented with melibiose or galactose. Only five strains produced appreciable amounts of enzyme, Penicillium janthinellum being superior for the formation of both intra- and extracellular a-galactosidase. In further studies carried out with this fungus, galactose, lupin seed powder, and soybean were shown to be the best carbon sources for a-galactosidase production. In a few cases, waste effluent and waste by-products have been utilized for cultivating the organism and producing a-galactosidase in the fermentation medium [57]. A soy effluent stream, waste liquor generated during the production of dofu, a traditional Chinese soybean bread food, contains 0.69% protein and 0.96% total sugar, of which

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Table 16.2

Microbial Production of a-Galactosidase in Solid-State Fermentation

Microbial Strain

Solid Substrate/Inducer

a-Galactosidase Yielda

References

Aspergillus niger NCIM 839 Aspergillus oryzae A. oryzae A. oryzae Aspergillus foetidus Humicola sp. Penicillium sp. Thermomyces lanuginosus (D2W3) Absidia sp. WL511 Streptomyces griseoloalbus

Wheat bran þ guar flour þ lactose Soy flour (defatted) Pigeon pea plant waste Red gram plant waste Wheat bran þ soybean meal Soy flour Wheat bran þ soy meal þ beet pulp Sorghum straw Soybean meal Soybean flour

87.0 U/gds 10.4 U/gds 5.12 U/gds 3.4 U/gds 2207.19 U/gds 44.6 U/gds 185.2 U/gds 13.4 U/gds 117.8 U/gds 197.2 U/gds

[70] [71] [72] [43] [73] [74] [75] [76] [77] [37]

a

The values given are not necessarily optimal for the relevant enzymes in all cases.

0.3% is raffinose. It has been reported to be effective as an excellent source of carbon and nitrogen for a-galactosidase production by Bacillus sp. JF2 [63]. An unusual a-galactosidase, which was produced exclusively in the presence of a specific inducer, 6-deoxy-Dglucose (quinovose), was reported from the filamentous fungus Talaromyces flavus CCF 2686 [64]. Immobilization of microbial cells is yet another approach widely used in industrial biotechnology for enhancing the production of desired products. Whole-cell immobilization has been reported for a-galactosidase production for the first time from S. griseoloalbus [65]. There have been numerous reports on the production of a-galactosidase by the solidstate fermentation (SSF) process. Table 16.2 lists the various microorganisms reported for producing a-galactosidase by SSF. The first report on a-galactosidase production from M. vinacea by the koji method appeared in 1969 [66]. The organisms reported to be producing a-galactosidase in wheat bran-based SSF are A. niger [67], A. oryzae [43,68,69], Aspergillus foetidus [70], Humicola sp. [71], Penicillium sp. [72], T. lanuginosus [73], Absidia sp. [74], and S. griseoloalbus [37]. Soy flour and soybeans are considered the most ideal substrates for a-galactosidase production in SSF [69]. Sonia et al. [73] reported a sorghum straw-based SSF process for the production of cellulose-free xylanase and associated hemicellulases including a-galactosidase by the indigenous thermophilic T. lanuginosus (D2W3). Shankar and Mulimani [43] reported red gram plant waste mixed with wheat bran as the best solid substrate for a-galactosidase production by A. oryzae.

16.5 Industrial Production Scenario Although several a-galactosidase preparations are produced by SSF, this fermentation technique has not yet been exploited industrially for a-galactosidase production. Both

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377

submerged fermentation and SSF methods have advantages and disadvantages. However, the relative yield and ease of convenience are deciding factors to choose the fermentation method. The microorganisms preferred for commercial production of a-galactosidase are Circinella muscae, Absidia griseola, Absidia hyalospora (Hokkaido Sugar Company, Ltd., Tokyo, Japan) [75], M. vinacea [76], and B. stearothermophilus (Monsanto Company, St. Louis, MO) [77]. It is being produced commercially from A. niger (Novo Nordisk A/S, Bagsvaerd, Denmark) [78]. Many a-galactosidase preparations are available commercially under the trade names Beano (GlaxoSmithKline, USA), Gas-Zyme 3X, TerrainZyme, Jarro-Zymes Plus, EZ-Gest, Bean-Zyme (Mikeska Products LLC, Santa Barbara, CA), Validase AGS (Valley Research, Inc., USA), Nutriteck a-galactosidase (Division of Ultra Bio-Logics, Inc., Rigaud, QC, Canada), Alpha-Gal (Novozymes), etc., and are used as dietary supplements in the human diet. The enzymes from M. vinaceae strain raffinoseutilizer [79] and A. niger (produced by Novo Nordisk A/S) [78] are widely used in in the beet sugar industry and in food and feed processing. a-Galactosidase from A. niger is an enzyme supplement in many digestive enzyme products like U-zyme and Enzalase Group 2 (Therabiotics, Inc.). Highly purified Glyko a-(1e3,4,6)-galactosidase from green coffee bean without any b-galactosidase contamination (Glyko, Inc.) is also available commercially. As of this writing there are two recombinant glycoprotein products, Fabrazyme and Replagal, available for enzyme replacement therapy used in the treatment of Fabry disease [80,81]. Fabrazyme is produced and marketed by Genzyme Corp. (Cambridge, MA) and Replagal by Transkaryotic Therapies (TKT; Cambridge, MA). These two glycoproteins have identical amino acid sequences but are produced in different cell lines, resulting in different glycosylation at the N-linked carbohydrate attachment sites. Fabrazyme is produced in a Chinese hamster ovary cell line, whereas Replagal is a human a-galactosidase A produced by genetic engineering technology in a human cell line. TKT’s gene activation technology is a proprietary approach to the large-scale production of therapeutic proteins, which does not require the cloning of genes and their subsequent insertion into nonhuman cell lines. Replagal contains a greater amount of complex carbohydrate, whereas Fabrazyme contains a higher fraction of sialylated and phosphorylated carbohydrate [82]. Because the polypeptide sequence of the two glycoproteins is identical, these differences in carbohydrate composition are solely responsible for the differences in tissue distribution and dose response of the two enzyme replacement therapies. Genzyme Corp. stands at the center of enzyme replacement therapy of Fabry disease with a-galactosidase. Genzyme Corp. has received US Food and Drug Administration (FDA) approval for marketing of Fabrazyme. Genzyme estimates that only 2000 to 4000 people suffer from the disease worldwide. But even such a small number of patients can mean high revenue if there is no competition and the drug price is high. Genzyme’s leading product, a drug called Cerezyme, which treats another rare inherited disorder called Gaucher disease, has annual sales of about $500 million. Even though only 3000 patients are taking the drug, each one is paying about $170,000 a year. As of October

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2002, Fabrazyme was approved for use in 25 countries including all 15 countries of the European Union. Replagal has been approved for commercial use in 27 countries, including the 15 countries of the European Union. In the United States, Replagal is an investigational product. As per the reports of TKT, the sale of Replagal is about $77 million in 2004 and the estimated current market is $190 million in western Europe, which is higher than the US market. Large Scale Biology Corp. (LSBC; Vacaville, CA) has developed a new version of a-galactosidase A trademarked Enzagal using biomanufacturing in plants, which could potentially serve the needs of all segments of the Fabry population worldwide. In January 2003, LSBC received orphan drug designation for Enzagal by the FDA, clearing the way for market protection upon product launch. LSBC’s Enzagal can be produced more efficiently and in higher abundance than competing products, potentially enabling substantial market expansion. Extensive preclinical and manufacturing research and development and regulatory assessments conducted by LSBC and its collaborators have shown that LSBC’s new product could be commercialized rapidly and with significant competitive advantages.

16.6 Purification and Characterization of a-Galactosidases 16.6.1

Molecular Mass and Isoelectric Point

a-Galactosidases have been purified from several strains of fungi, bacteria, and yeast. The characteristics of a-galactosidases purified from various microbial sources are listed in Table 16.3. Most of the a-galactosidases produced by fungi have been isolated from various strains of Aspergillus and Penicillium. Aspergillus a-galactosidases form a heterogeneous group with highly variable molecular properties. The purified a-galactosidase from T. lanuginosus is estimated to be 53 kDa [51]. Most of the fungal a-galactosidases are monomeric proteins, with an average molecular mass of 50 kDa [49,85,88]. Nevertheless, the crystal structure analysis of a-galactosidase from L. acidophilus reveals that it is formed of four identical monomers, which form a tightly packed tetramer in which three monomers contribute to the structural integrity of the active site in each monomer [29]. Despite the heterogeneity in the molecular masses, the isoelectric points determined for Aspergillus a-galactosidases have been rather similar, ranging from 4.2 to 4.8 [19,93]. Penicillium a-galactosidases form a more homogeneous group with molecular masses between 55 and 67 kDa as determined by sodium dodecyl sulfateepolyacrylamide gel electrophoresis (SDSePAGE), but the isoelectric points vary between 4.0 and 7.0 [47,88]. Monascus pilosus [60] produces large a-galactosidase with molecular mass of 150 kDa, as determined by gel filtration. Bacterial a-galactosidases are also a complex heterogeneous group of enzymes with a more complex structure and their molecular masses vary considerably between 45 and 400 kDa [18,94]. a-Galactosidase from C. murisepticum is a homotetramer of 320 kDa [61]. The most complex structure of a-galactosidase has been observed in Thermus sp.

Table 16.3

Characteristics of a-Galactosidases From Various Microorganisms Mr (kDa)

pI

pHopt

Topt ( C)

Active Against

Km for pNPG (mM)

Vmax

References

Rhizopus sp. F78 ACCC30795 Talaromyces flavus

210

n.a.

4.8

50

pNPG, melibiose, raffinose, stachyose

2.9

246.1 mmol/min/mg

[83]

63

n.a.

3.5e4.5

50

0.54

0.21 mM/min

[64]

Thermomyces lanuginosus CBS 395.62/b T. lanuginosus Trichoderma reesei RUT C-30 Mortierella vinacea a-Galactosidase I a-Galactosidase II

93

3.9

5e5.5

65

pNPG, raffinose, stachyose, galactomannans pNPG, raffinose, stachyose

1.13

2498 mmol/min/mg

[84]

57 50

5.2 5.2

4.5e5.0 4

65e70 60

0.5 1.2

52.4 U/mg 30.1 U/mg

[49] [25]

240

5.4

3e4.0

60

pNPG, melibiose, raffinose pNPG, melibiose, raffinose, stachyose, locust bean gum Oligosaccharide chains

n.a.

n.a.

[85]

60

8.5

3e4.0

60

n.a.

n.a.

Aspergillus fumigatus Aspergillus niger ATCC 46890 a-Gal I a-Gal II

54.7 350

4.15

4.5 4.5

55 60

Galactomannooligosaccharides, galactomannans pNPG, melibiose pNPG, melibiose, raffinose, stachyose

0.38 1.4

0.16 mmol/min/mg 18000 nkat/mg

117

4.5

4.5

60

0.22

3600 nkat/mg

a-Gal III a-Gal IV

117 117

4.7 4.8

4.5 4.5

60 60

0.27 0.24

3000 nkat/mg 3200 nkat/mg

M. vinacea

n.a.

n.a.

4e6

n.a.

0.43

143.5 mmol/min/mg

[87]

Penicillium simplicissimum AGLI AGLII

61

5.2

3e4.5

40

n.a.

n.a.

[47]

84

4.4

4e5

60

0.75

26600 nkat/mg

Microorganism

pNPG, melibiose, raffinose, stachyose, galactomannooligosaccharides pNPG, melibiose, raffinose, stachyose pNPG, melibiose, raffinose, stachyose, galactomannooligosaccharides pNPG, oNPG, methyl-a-D-galactoside, melibiose, raffinose, stachyose, 4-o-a-D-galactopyranosyl-D-galactose, 6-o-a-D-galactopyranosyl-o-b-Dgalactopyranosyl-1-glycerol, methylb-L-arabinoside pNPG, raffinose family oligosaccharides, polymeric galacto(gluco)mannans pNPG, raffinose family oligosaccharides

[86] [19]

Continued

Table 16.3

Characteristics of a-Galactosidases From Various Microorganismsdcont’d

Microorganism

Mr (kDa)

pI

pHopt

Topt ( C)

Active Against

Km for pNPG (mM)

Vmax

References

AGLIII

61

7.0

3e4.5

45

pNPG, raffinose family oligosaccharides, polymeric galacto(gluco)mannans pNPG, galactomannooligosaccharides

Penicillium purpurogenum Pycnoporus cinnabarinus Monascus pilosus Ganoderma lucidum Bacillus stearothermophilus B. stearothermophilus NCIM 5146 Lactobacillus plantarum Thermotoga neopolitana 5068 Bifidobacterium adolescentis

67

4.1

4.5

55

n.a.

n.a.

[88]

210 150 249 247

3.5 n.a. n.a. n.a.

5 4.5e5 6 7e7.5

75 55 70 60

pNPG pNPG, melibiose, raffinose, stachyose Melibiose, raffinose, stachyose pNPG, melibiose, raffinose, stachyose, galactomannans pNPG, melibiose, raffinose, stachyose, galactomannans (limited activity) pNPG Polymeric galactomannans

0.31 0.8 0.4 0.25

630 mmol/min/mg 39 mmol/min/mg n.a. 195 U/mg

[89] [60] [90] [55]

165.9

4.9

6.5e7

65

0.5

833 U/mg

[28]

194.5 61

n.a. n.a.

5.8 7.5

45 100e105

0.079 n.a.

2838 mmol/min/mg n.a.

[30] [91]

344

n.a.

5.5

55

0.957

n.a.

[31]

37 n.a. 65

pNPG, melibiose, raffinose, stachyose, a-1,3-D-galactobiose, a-1,4-Dgalactobiose, gal-a-1,3-gal-b-1,4-gal, gal-a-1,3-gal-b-1,4-gal-a-1,3-gal Melibiose, raffinose, stachyose pNPG Melibiose, raffinose, stachyose

Bifidobacterium breve Bacteroides fragilis Saccharopolyspora erythraea Streptomyces coelicolor A3(2) Streptomyces griseoloalbus a-Gal I

160 125 45

n.a. 6.2 n.a.

5.5e6.5 5.5 6.1

n.a. n.a. 0.65

n.a. n.a. 31 mmol/min/mg

[32] [92] [35]

58

n.a.

7

40

pNPG, raffinose, stachyose

n.a.

n.a.

[34]

72

4.41

5.0

65

a-Gal II a-Gal III

57 35

5.6 6.13

6.5 5.5

50 55

[20,21] pNPG, melibiose, raffinose, stachyose, polymeric galactomannan pNPG, melibiose, raffinose, stachyose pNPG, melibiose, raffinose, stachyose

n.a., information not available; oNPG, ortho-nitrophenyl-b-D-glucopyranoside; pNPG, para-nitrophenyl-b-D-glucopyranoside.

0.79

693.4 mmol/min/mg

1.0 1.3

297.3 mmol/min/mg 195.3 mmol/min/mg

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strain T2, having a molecular mass of 400 kDa and existing in solution as an octameric form [18]. However, the a-galactosidase from Thermotoga neapolitana is active as a monomer of 61 kDa [95]. The a-galactosidase from B. stearothermophilus NCIM 5146 is a dimeric protein with a molecular mass of 165.9 kDa and pI 4.9 [28]. Streptomyces coelicolor A3(2) family 36 a-galactosidase is a monomeric protein with a molecular mass of 58 kDa [34]. The purified recombinant a-galactosidase from Streptomyces sp. S27 showed a single protein band of w82 kDa on SDSePAGE and three bands of w220, 320, and 480 kDa on nondenaturing gradient PAGE, indicating its native structure of trimer, tetramer, or hexamer [38]. The purified recombinant Rhizomucor miehei a-galactosidase RmgalB in Pichia pastoris is a tetramer and showed a single band corresponding to a molecular mass of 83.1 kDa in SDSePAGE [96]. On the basis of subunit molecular mass, bacterial a-galactosidases have been classified into two groups [18]. The first group consists of a-galactosidases from S. mutans [56], B. stearothermophilus [16], Escherichia coli Raf A [97], B. breve [32], and Pseudomonas fluorescens [94], which have molecular mass of more than 80 kDa. The a-galactosidases from Thermus sp. strain T2, Thermus brockianus [98], Thermotoga maritima [99], and Thermotoga neopolitana [95] belong to the second group, possessing molecular mass ranging from 53 to 65 kDa. The isoelectric points of bacterial a-galactosidases range from 4.5 to 6.9 [28,92].

16.6.2

Multimolecular Forms of a-Galactosidases

The existence of multimolecular forms of a-galactosidases has been reported from a few microorganisms. The underlying biochemical cause of a-galactosidase multiple forms may be posttranslational modifications such as proteolytic cleavage or differential glycosylation of the proteins. Thermomyces lanuginosus a-galactosidase exhibits microheterogeneity due to differential glycosylation of the protein [49]. Sometimes isoenzymes are the products of two distinct genes. Two a-galactosidase isoenzymes, regulated by two different genes, agaA and agaB, were detected in B. stearothermophilus KVE39 [100]. The expression of isoenzymes can vary markedly as a function of the carbon and nitrogen source available in the medium for growth of the microorganism [101]. Streptomyces griseoloalbus produces three forms of a-galactosidases: a-Gal I, a-Gal II, and a-Gal III, of which a-Gal I is active on both galactooligosaccharides and polymeric galactomannans and a-Gal II and a-Gal III are active on only galactooligosaccharides [20]. Two forms of a-galactosidases, one specific for galactooligosaccharides and the other specific for galactomannans, were purified from M. vinacea [87], A. tamarii [54], and B. ovatus [53]. Multiple proteins exhibiting a-galactosidase activity, but differing in molecular mass and pI, have been reported from A. niger [19,93]. In view of the biological economy for an organism, the production of multimolecular forms of an enzyme is wasteful, because they catalyze the same reaction. This is counterbalanced by the specific advantages offered by the multiforms of enzymes to the producing organism. The multiforms of enzymes with different kinetic parameters,

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selectivities, regulatory characteristics, and stabilities provide added flexibility and adaptability to the organism to cope with changing environmental conditions, nutrient availability, and metabolic need.

16.6.3

Glycoprotein Nature

Most of the fungal a-galactosidases are glycosylated proteins [19,48,93]. However, the a-galactosidase purified from Rhizopus sp. is a nonglycosylated protein [83]. Generally bacterial a-galactosidases are nonglycosylated [28]. The carbohydrate content of a-galactosidases has been estimated in only a few cases and very few studies on its composition and structural analysis have been carried out. The a-galactosidase from M. vinacea contains 10.8% neutral sugars and 2.7% D-glucosamine [87]. The a-galactosidases from Cephalosporium acremonium [102] contain about 27% neutral sugars; their carbohydrate composition is N-acetylglucosamine, mannose, galactose, and sialic acid in the molar proportions of 2:7:3:11. The a-galactosidase from T. lanuginosus contains 5.3% carbohydrates, which comprise 56% D-mannose, 8% D-galactose, 36% D-glucosamine, and <1% D-glucose [84]. The carbohydrates are believed to play an important role in stabilizing the enzyme structure, activity, and stability. The carbohydrate moieties also confer resistance to proteolytic attack on the enzymes.

16.6.4

Effects of pH and Temperature

Generally, bacterial a-galactosidases have a pH optimum in the range of 6.0e7.5 [53], whereas the pH optimum of the fungal and yeast a-galactosidases is in the range of 3.5e5.0 [103]. The purified S. coelicolor family 36 a-galactosidase is most active at pH 7.0 and is stable between pH 7.0 and 9.5 over 1 h [34]. Similarly, the purified recombinant a-galactosidase from Streptomyces sp. S27 is optimally active at pH 7.4 [38]. The most acidic pH optimum for a-galactosidase has been observed in fungi, P. simplicissimum [47], M. vinacea [85], and A. niger [19], active at pH 3.0e4.5. The optimal pH range for the action of N. fischeri is pH 4.5 [2]. The T. lanuginosus a-galactosidase is reported to be optimally active at pH 4.6e4.8 and stable in the pH range 4.0e7.0 [51]. The optimum pH of purified recombinant R. miehei a-galactosidase in P. pastoris, with high specific activity, is pH 5.5, and it is stable within pH 5.5e9.5 [96]. A temperature-dependent shift in the pH optima has been observed in the case of a-galactosidase from L. fermentum [104]. Likewise, a-galactosidases from Penicillium duponti [105] show substrate-dependent pH optima. Depending on the source of origin, a-galactosidases differ with respect to their temperature optima and thermal stability. Because of the elevated temperatures used during the sugar manufacturing process, as well as in other industrial applications, stability and activity at high temperatures are important properties of a-galactosidases [17]. It is important that the mode of action and stability of a-galactosidases meet industrial demands, as these enzyme properties reduce expenses for cooling and reheating.

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a-Galactosidase from Penicillium purpurogenum is heat stable only below 40 C [88]. Mortierella vinacea [85] and M. pilosus [60] a-galactosidases are stable below 55 C. The purified recombinant a-galactosidase from Streptomyces sp. S27 is optimally active at 35 C [38]. The optimum temperature of recombinant a-galactosidase from R. miehei has been determined to be 55 C and it is also stable up to 55 C [96]. However, thermostable a-galactosidases have been reported from a wide variety of microbial sources. The T. neapolitana 5068 (TN5068) a-galactosidase is the most thermoactive a-galactosidase hitherto isolated, with a temperature optimum of 100e103 C and a half life of 2 h at 90 C and 3 min at 100 C [91]. An a-galactosidase has been isolated from a hyperthermophilic archaeon, S. solfataricus, with a temperature optimum of 90 C [42]. The a-galactosidase from Thermoanaerobacterium polysaccharolyticum [106] remains stable at 70 C for 36 h. The trimeric and tetrameric forms of a-galactosidase have high thermal stability compared to the monomeric and dimeric forms [28]. The purified a-galactosidase from the thermophilic fungus T. lanuginosus shows maximal catalytic activity in the temperature range 60e66 C and is stable at 70 C [51]. Similarly, the native and recombinant a-galactosidase from N. fischeri are reported to have temperature optima at 60e70 C [2].

16.6.5

Effects of Metal Ions and Sugars

A wide range of chemicals are known to influence the activity of a-galactosidases. The divalent metal cations, like Hg2þ, Ag2þ, and Cu2þ, are found to have considerable inhibitory effects on a-galactosidase [28,98], which usually suggests reaction with thiol groups and/or the carboxyl, amino, and imidazolium group of histidine in the active site [107]. However, there are some reports suggesting a stabilizing/activating effect for Cu2þ [90], Mn2þ, Mg2þ, and Kþ [84,108,109]. Most other metal cations are found to have no or little effect on a-galactosidase activity [28,34,84,90]. The purified a-galactosidase from T. lanuginosus is significantly stimulated by Mg2þ, Mn2þ, and Kþ ions, whereas it is considerably inhibited by the presence of Ca2þ, Agþ, and Hg2þ [51]. a-Galactosidase activity is also influenced by some sugars and sugar derivatives. Generally, galactose, melibiose, raffinose, and stachyose are reported to have an inhibitory effect on a-galactosidase activity when assayed with p-nitrophenyl-a-D-galactopyranoside [28,109]. This may be because the galactosyl residues of these compounds are analogous to the galactosyl residue of para-nitrophenyl-b-D-glucopyranoside (pNPG), with which they compete for the active site of the enzyme. But Suzuki et al. [87] reported a mixed type of inhibition by D-galactose on a-galactosidase from M. vinacea, suggesting its competitive and noncompetitive binding on the enzyme. a-Galactosidases from M. vinacea [87] and Aspergillus ficuum [110] were inhibited noncompetitively by D-glucose, whereas a-galactosidase from A. ficuum showed uncompetitive inhibition by mannose [110]. Luonteri et al. [47] reported three a-galactosidases from P. simplicissimum of which AGLII showed more resistance to product inhibition by galactose than the other two enzymes, AGLI and AGLIII.

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CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

16.6.6

Substrate Specificity

a-Galactosidases are very specific with regard to anomer selectivity of the substrate, though they show some flexibility in glycone and aglycone specificity. The anomeric configuration of the product liberated by the action of a-galactosidase is specifically a-, despite the type of a-galactosidic linkage in the substrate, e.g., a-1,2, a-1,3, a-1,4, a-1,6, etc. [49,102,111]. However, many enzymes do not show absolute specificity for glycone residues and hydrolyze the structural analogues b-L-arabinopyranosides and a-D-fucopyranosides [92]. The aglycone group of the substrate may or may not have a marked effect on hydrolysis; hence many a-galactosidases can hydrolyze for, e.g., methyl, ethyl, n-propyl, or a-naphthyl galactose; p-nitrophenyl-a-D-galactopyranoside; melibiose; and raffinose [94,112]. Most microbial a-galactosidases have in common the fact that they can hydrolyze the synthetic or aryl glycosides like pNPG and oNPG more extensively than the natural a-galactosides like melibiose, raffinose, and stachyose [20], indicating that aryl glycosides are better substrates than the alkyl derivatives. Moreover, a-galactosidase from A. niger hydrolyzed exclusively the synthetic substrate and failed to split off the terminal a-1,6-bound galactose in linear structures like melibiose, raffinose, and stachyose [113]. As mentioned earlier, a-galactosidases are of two types, one group active only on oligomeric substrates and the other group active on both oligomeric and polymeric substrates. Generally GH-27 a-galactosidases are active on both polymeric and oligomeric substrates, whereas GH-36 a-galactosidases are active only on oligomeric substrates. Many of the a-galactosidases in family 36 are large enzymes with a tetrameric structure [16,56,97,98], and hence their ability to release galactose from polymers is probably sterically restricted. The large trimeric or tetrameric a-galactosidases of A. tamarii [54], P. simplicissimum [47], B. ovatus [53], and B. stearothermophilus [55] have little or no activity toward polymeric substrates. The two fungal a-galactosidases in family 36, T. reesei AGLII [114] and A. niger a-Gal I [19], are more specific toward oligosaccharides such as melibiose and raffinose and have little or no activity on polymeric substrates. The a-galactosidases in family 27 are generally smaller and at least some of them are monomers [47,85]. Many of the a-galactosidases in family 27, such as T. reesei AGLI [114], M. vinacea a-galactosidase II [85], P. simplicissimum AGLI [47], and A. niger AglB [15,93], can release galactose from intact galactomannan polymers. An a-galactosidase with negligible hydrolytic activity toward melibiose, but active on locust bean gum and guar gum, was purified from the filamentous fungus T. flavus CCF 2686 [64]. In 2015, Chen et al. [96] reported a recombinant a-galactosidase from R. miehei, which displayed specificity toward raffinose and stachyose and completely hydrolyzed the antinutritive RFOs.

16.6.7

Transglycosylation Reaction

Many microbial a-galactosidases have been shown to possess transglycosylation activities in addition to hydrolytic activity [4,5,109], especially at high substrate concentrations. In transglycosidase activity the role of acceptor is played by hydroxylic compounds

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other than water, for example, simple alcohols, hydrolysis products [115], saccharides, or a second substrate molecule (substrate transglycosylation) [116]. Acceptor specificity and kinetics of the transglycosidase activity of a-galactosidases have been the focus of research for many investigators [117]. Generally hexoses are better acceptors of galactose molecules. The studies carried out so far showed that water and organic acceptors are bound at the same site, hence hydrolysis and transfer reactions take place on the same site of the enzyme molecule, presumably by identical mechanisms. The a-galactosidases are also known to synthesize (de novo synthesis) oligosaccharides when incubated with high concentrations of monosaccharides [118], and this procedure has been used for the preparation of several glucose and galactose derivatives [119]. An extracellular a-galactosidase with a unique transglycosylation potential was isolated from a filamentous fungus, T. flavus CCF 2686 [64,116,120]. This enzyme showed an unusual regioselectivity in transglycosylation of pNP-a-Gal, with a selective preference for the formation of a-1,3 bonds [116], and tolerates p-nitrophenyl-6-O-acetyl-a-D-galactopyranoside as an acceptor yielding p-nitrophenyl-a-D-galactopyranosyl-(1,3)-6-O-acetyla-D-galactopyranoside [120]. In addition to its peculiar selectivity, this inducible enzyme was found to be active in the transfer of a-galactosyl residues onto sterically hindered acceptors like tert-butyl alcohol [64,120], which is generally quite inert to hydrolases and specifically to glycosidases and consequently used as a cosolvent in transglycosylation reactions to increase substrate solubility [121].

16.6.8

Protease Resistance

The scientific community is always in search of microbial enzymes with novel properties. With this aim several research groups have been searching for organisms endowed with a large repertoire of diverse enzymes with divergent properties. Cao et al. [83] reported the purification of a novel protease-resistant a-galactosidase from Rhizopus sp. F78 ACCC 30795, which showed resistance to both neutral and alkaline proteases, providing a basis for its possible application in the medical, food, animal feed, and sugar industries. Following treatment with a variety of neutral proteases (including subtilisin A, proteinase K, collagenase, trypsin, and a-chymotrypsin), the enzyme retained over 70% of its activity. Enzyme activity was a little activated by alkaline proteases (including proleather and alkaline protease). The purified recombinant a-galactosidase from Streptomyces sp. S27 is reported to be resistant to some neutral proteases (a-chymotrypsin, subtilisin A, and collagenase) [38]. Moreover, it shows hydrolytic ability to natural substrates, including melibiose, stachyose, raffinose, and soybean meal. When combined with intestinal proteases, the enzyme shows higher hydrolytic ability to RFOs in soybean product.

16.6.9

Galactose Tolerance

End-product inhibition is one of the many causes of a decrease in the catalytic efficiency of enzymes. An enzyme with resistance to end-product inhibition would be advantageous

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CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

in this respect. Streptomyces griseoloalbus is reported to produce a novel galactosetolerant a-galactosidase [20]. The a-Gal I produced by this actinomycete can tolerate galactose concentrations as high as 100 mM. In contrast, a-galactosidases from A. niger [19] and B. stearothermophilus [28] are reported to be inhibited by galactose. The a-Gal II and a-Gal III produced by S. griseoloalbus are also inhibited by galactose [20].

16.7 Strain Improvement Research is going on worldwide to find novel sources of a-galactosidase or to improve the potential of microbial sources of a-galactosidase. The a-galactosidase in yeast strains such as Saccharomyces carlsbergensis is encoded by the MEL gene [122]. Nonetheless, strains of Saccharomyces cerevisiae that are used commercially for baking are MEL. Liljestrom et al. [123] has reported the introduction of the MEL gene into S. cerevisiae and as a consequence baker’s yeast started utilizing raffinose present in the beet molasses and produced higher biomass and a-galactosidase commercially. Turakainen et al. [124] reported that in S. cerevisiae strains, the production of a-galactosidase is dose dependent, i.e., the strains containing several MEL genes produce 10- to 100-fold more a-galactosidase than the strains containing only one gene. The enzyme production is further enhanced by galactose induction. Saccharomyces cerevisiae strains are more efficient at a-galactosidase production than other strains of Saccharomyces. Glucose repression is very tight in strains carrying one MEL locus but less tight in strains with several MEL loci, so that even the presence of glucose did not fully repress MEL gene expression. Such a dose-dependent enhancement in enzyme production suggests the possibility of improving the industrial production of a-galactosidase by cloning and overexpression of the gene encoding a-galactosidase. Several researchers have reported the cloning and expression of genes from various microorganisms in heterogeneous host. Escherichia coli and Saccharomyces are most commonly used as expression hosts for the enhanced production of a-galactosidase [16,94,125]. The cloning and overexpression of the a-galactosidase gene is found to increase the enzyme yield remarkably. In 2015, Chen et al. [96] cloned the a-galactosidase gene, designated as RmgalB, from the thermophilic fungus R. miehei and expressed it in P. pastoris. It was reported that the recombinant a-galactosidase (RmgalB) was secreted at high levels of 1953.9 U/mL in a high-cell-density fermenter. The recombinant enzyme also exhibited a very high specific activity of 505.5 U/mg. In another study, an extracellular a-galactosidase (Gal27A) belonging to the GH-27 family and with high specific activity of 423 U/mg was identified in the thermophilic N. fischeri P1, and its coding gene (1680 bp) was cloned and functionally expressed in P. pastoris [2]. The authors reported that considering the high yield (3.1 g/L) in P. pastoris, recombinant rGal27A is more favorable for industrial applications. There are studies reporting the cloning and expression of thermostable a-galactosidase-encoding genes from hyperthermophilic microorganisms such as Thermus sp.

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strain T2 [18], T. brockianus ITI 360 (agaT) [98], and T. neapolitana (agaA) [95]. Halstead et al. [94] demonstrated the cloning and high level expression of a-galactosidase A from P. fluorescens subsp. cellulose and its role in galactomannan hydrolysis. Cao et al. [38] cloned a full length a-galactosidase gene (2226 bp) from Streptomyces sp. S27 ACCC 41168 and overexpressed it in E. coli. The recombinant enzyme showed higher similarity with a-galactosidase belonging to GH family 36 and showed optimal activity under conditions similar to the intestinal conditions of mammals and poultry. It also showed resistance to neutral proteases (a-chymotrypsin, subtilisin A, and collagenase) and showed hydrolytic ability to natural substrates, including melibiose, stachyose, raffinose, and soybean meal. When combined with intestinal proteases, the enzyme showed higher hydrolytic ability to RFOs in soybean product. The authors affirm that these favorable properties make the Streptomyces sp. S27 a-galactosidase a good prospective candidate for soybean processing in the food and feed industries.

16.8 Conclusion and Perspectives Enzymes hydrolyzing a-linked galactosidic bonds are widespread in microorganisms. Owing to its importance in many diverse applications, a-galactosidase has been extensively studied to understand the precise mechanisms by which it hydrolyzes a-glycosidic bonds and to possibly improve its catalytic properties. There are numerous microorganisms in nature that are yet to be isolated and identified. They remain untapped resources and their potential should not be overlooked. Any attempt to identify a novel source of an enzyme like a-galactosidase with improved catalytic properties for diverse applications would be worthwhile because it can have a significant impact on the industrial processes that take advantage of the action of a-galactosidases. Moreover, strain improvement and optimization of production technologies can offer significant advancement in the industrial-scale production of this versatile enzyme.

References [1] Naumoff DG. Phylogenetic analysis of a-galactosidases of the GH27 family. Molecular Biology 2004;38:388e99. [2] Wang H, Shi P, Luo H, Huang H, Yang P, Yao B. A thermophilic a-galactosidase from Neosartorya fischeri P1 with high specific activity, broad substrate specificity and significant hydrolysis ability of soymilk. Bioresource Technology 2014;153:361e4. [3] Ohtakara A, Mitsutomi M. Immobilization of thermostable a-galactosidase from Pycnoporus cinnabarinus on chitosan beads and its application to the hydrolysis of raffinose in beet sugar molasses. Journal of Fermentation Technology 1987;65:493e8. [4] Anisha GS. a-Galactosidase from Streptomyces griseoloalbus: an enzyme with versatile applications. In: Proceedings of the first Kerala women science congress; 2011. p. 19e25. [5] Kurakake M, Okumura T, Morimoto Y. Synthesis of galactosyl glycerol from guar gum by transglycosylation of a-galactosidase from Aspergillus sp. MK14. Food Chemistry 2015;172:150e4.

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[6] Rivero-Urgell M, Santamaria-Orleans A. Oligosaccharides: application in infant food. Early Human Development 2001;65(Suppl.2):43e52. [7] Clarke JH, Davidson K, Rixon JE, Halstead JR, Fransen MP, Gilbert HJ, et al. A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanse and a-galactosidase. Applied Microbiology and Biotechnology 2000;53:661e7. [8] Daitx VV, Mezzalira J, Moraes VC, Breier AC, Ce´ J, Coelho JC. Comparing the alpha-galactosidase A biochemical properties from healthy individuals and Fabry disease patients. Clinica Chimica Acta 2015;445:60e4. [9] Desnick RJ. Fabry disease: a-galactosidase A deficiency. In: Rosenberg RN, Pascual JM, editors. Rosenberg’s molecular and genetic basis of neurological and psychiatric disease. Academic Press; 2015. p. 419e30. [10] Watier H, Guillaumin JM, Piller F, Lacord M, Thibault G, Lebranchu Y. Removal of terminal alpha galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity by persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity. Transplantation 1996;62:105e13. [11] Chern MK, Li HY, Chen PF, Chien SF. Taro a-galactosidase: a new gene product for blood conversion. Biocatalysis and Agricultural Biotechnology 2012;1:135e9. [12] Dey PM, Patel S, Brownleader MD. Induction of alpha-galactosidase in Penicillium ochrochloron by guar (Cyamopsis tetragonoloba) gum. Biotechnology and Applied Biochemistry 1993;17:361e71. [13] Henrissat B, Bairoch A. Updating the sequence-based classification of glycosyl hydrolases. Biochemical Journal 1996;316:695e6. [14] den Herder IF, Rosell AMM, van Zuilen CM, Punt PJ, van den Hondel CAMJJ. Cloning and expression of a member of the Aspergillus niger gene family encoding a-galactosidase. Molecular and General Genetics 1992;233:404e10. [15] de Vries RP, van den Broeck HC, Dekkers E, Manzanares P, de Graaff LH, Visser J. Differential expression of three a-galactosidase genes and a single b-galactosidase gene from Aspergillus niger. Applied and Environmental Microbiology 1999;65:2453e60. [16] Fridjonsson O, Watzlawick H, Gehweiler A, Mattes R. Thermostable a-galactosidase from Bacillus stearothermophilus NUB3621: cloning, sequencing and characterization. FEMS Microbiology Letters 1999;176:147e53. [17] Fridjonsson O, Mattes R. Production of recombinant a-galactosidases in Thermus thermophilus. Applied Environmental Microbiology 2001;67:4192e8. [18] Ishiguro M, Kaneko S, Kuno A, Koyama Y, Yoshida S, Park GG, et al. Purification and characterization of the recombinant Thermus sp. strain T2 a-galactosidase expressed in Escherichia coli. Applied Environmental Microbiology 2001;67:1601e6. [19] Ademark P, Larsson M, Tjerneld F, Stalbrand H. Multiple a-galactosidases from Aspergillus niger: purification, characterization and substrate specificity. Enzyme and Microbial Technology 2001; 29:441e8. [20] Anisha GS, John RP, Prema P. Biochemical and hydrolytic properties of multiple thermostable a-galactosidases from Streptomyces griseoloalbus: obvious existence of a novel galactose-tolerant enzyme. Process Biochemistry 2009;44:327e33. [21] Anisha GS, John RP, Prema P. Substrate specificities and mechanism of action of multiple a-galactosidases from Streptomyces griseoloalbus. Food Chemistry 2011;124:349e53. [22] Tubb RS, Liljestrom PL. A colony-colour method which differentiates a-galactosidase-positive strains of yeast. Journal of the Institute of Brewing 1986;92:588e90. [23] Anisha GS, John RP, Pandey A. Alpha-galactosidase: a food and feed enzyme. In: Haugen S, Meijer S, editors. Handbook of nutritional biochemistry: genomics, metabolomics and food supply, Nutrition and diet research progress series. New York: Nova Science Publishers Inc.; 2010. p. 365e84.

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