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Microbial Isoamylases: An Overview



Rina Rani Ray
 
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ABSTRACT

An overview presentation is made on current global status of microbial isoamylase, a potent enzyme used in food industries, their production, properties and applications. Although few microbial sources were reported to produce extra cellular isoamylase, the Pseudomonas isoamylases were mostly studied. Microbial isoamylases in general showed affinity mostly towards glycogen and amylopectin but not towards pullulan. The end products were maltotriose and malto oligosaccharides often with maltose and glucose. The purified enzymes showed the molecular weight 60-120 kDa and activity was shown only by the monomers. The isoamylases showed varied temperature and pH optima. Adsorbability onto raw starch by the enzyme made its purification more convenient. Saccharification potential of isoamylases was employed in food industries for preparation of high glucose syrup from starch. The isoamylase gene from Pseudomonas amyloderamosa and Flavobacterium odoratum KU was cloned into Saccharomyces cerevisiae and in E. coli, respectively. Further studies are going on gene sequencing and characterization of the isoamylase produced mainly from bacterial sources.

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  How to cite this article:

Rina Rani Ray , 2011. Microbial Isoamylases: An Overview. American Journal of Food Technology, 6: 1-18.

DOI: 10.3923/ajft.2011.1.18

URL: https://scialert.net/abstract/?doi=ajft.2011.1.18
 
Received: February 24, 2010; Accepted: April 08, 2010; Published: July 05, 2010



INTRODUCTION

Starch is the principal (food) reserve polysaccharide in the plant kingdom and is one of the most abundant energy reserve materials in nature (Ray and Nanda, 1996). It forms an integral part of the multibillion food ingredients market and is indispensable in various food applications where it is used as precursor of sugars, as emulsion stabilizer, water binder, thickener and for production of bread and other bakery goods.

Application of starch in food, bakery, brewery and confectionaries requires starch modification, which is accomplished either by acid hydrolysis or by enzymatic treatment. The use of enzymes is preferred as it offers a number of advantages including improved yields and favourable economics (Satyanarayana et al., 2004) and therefore in modern food processing industries, starch breaking enzymes or amylases are widely used. Among the amylases, α amylase, β amylase, isoamylase, glucoamylase are note worthy, but in comparison to that of other amylases, report of isoamylases is significantly less.

Isoamylase (E.C.3.2.1.68, glycogen-6-glucanohydrolse) is a debranching amylase that hydrolyses 1, 6-α-D-glycosidic linkages (Fig. 1) of glycogen, amylopectin and α and β limit dextrins, producing linear malto oligosaccharides (Fang et al., 1994).

Although, isoamylases are found to be produced by both plant and microbial sources, for commercial purposes microbial strains are used due to their rapid producibility, easy handling and less expensiveness (Burhan et al., 2003).


Fig. 1: Mechanism of starch hydrolysis by Isoamylase (Arrow indicates the cleavage point)

Amongst the microbial sources, maximum studies were carried on Pseudomonas sp. followed by other bacterial and fungal sources. But these isoamylases do differ from one another in their substrate affinity, major end product of hydrolysis and other characteristic features.

Although, a number of reviews are available on production and properties of various amylases (Regulapati et al., 2007; Ray, 2004), no comprehensive study is available on production, properties and other advanced research work done so far on microbial isoamylase,

The present review attempts to highlight production and properties of various microbial isoamylases and biotechnological approaches adopted to increase their production and ameliorate their industrial applications.

Structure of isoamylase: Katsuya and coworkers worked in detail in 1998 on the three dimensional structure of isoamylase from Pseudomonas amyloderamosa by X-ray structure analysis (Fig. 2) which revealed that the enzyme had 750 amino acid residues and a molecular mass of 80 kDa, The structure was elucidated by the multiple isomorphous replacement method and refined at 2.2 A resolution, resulting in a final R-factor of 0.161 for significant reflections with a root-mean-square deviation from ideality in bond lengths of 0.009 A. The analysis revealed that in the N-terminal region, isoamylase had a novel extra domain, the domain N. Katsuya et al. (1998) further detected that the N domain had an incomplete (beta/alpha) 8-barrel-type super secondary structure in the catalytic domain. A long excursed region was present between the third beta-strand and the third alpha-helix of the barrel but it could not be considered to be an independent domain, because this loop formed a globular cluster together with the loop between the fourth beta-strand and the fourth alpha-helix. Isoamylase was found to contain a bound calcium ion, but this was not in the same position as the conserved calcium ion reported in other alpha-amylase family enzymes.

Mode of action: Kjolberg and Manners (1963) described that isoamylase hydrolyses both the anomalous linkages in amylose and the inter chain linkages in amylopectin. It actually triggers at the carbohydrate-amino acid linkage of glycogen, that joins the terminal, reducing-end d-glucose unit of glycogen to the hydroxyl group of tyrosine in glycogenin, the primer protein for glycogen biogenesis (Lomako et al., 1992).


Fig. 2: Topology diagram of Isoamylase monomer (PDB code: 1bf2)

The splitting of the glycogen-glycogenin bond by isoamylase indicates the α-anomeric configuration of the terminal d-glucose unit. For this specificity, 4-nitrophenyl α-maltotrioside and higher homologs also can act as substrates.

Application of isoamylases: Microbial isoamylase is found to have a number of applications both from research and industrial point of view. Cytophaga isoamylase was used for structural determination of glycogen and starch components (Lee and Whelan, 1972), isoamylase could also be used to examine the structure of the product and to produce branched cyclodextrins via reverse reaction (Kang et al., 2008). Isoamylase is used primarily in the production of food ingredients from starch, like glucose syrup, maltose, maltitol, trehalose, cyclodextrin and resistant starch. Glucose and maltose are widely used in food and pharmaceutical industries as sweetener, whereas maltitol is used as a sugar substitute in the production of non-calorigenic candies, chewing gum and other confectionary (Hirao et al., 1988) and trehalose is used in food as stabilizer, texturizer, humectant and sweetener (Olemposka-Beer, 2007). Cyclodextrins are used as encapsulating agents for food additives, flavours and vitamins, whereas the resistant starches are the non digestible starch used in medical purpose. Isoamylase is useful in the field of saccharification (Yamada et al., 1994). Even an isolated polypeptide with isoamylolytic activity could act on amylaceous substances to produce a high-maltose content product (Amemura and Futai, 1989) and therefore can be advantageously used for industrial purpose. This enzyme in combination with other amylolytic enzymes are used for the industrial production of amylose, maltose and glucose from starch (Sato and Park, 1980b; Norman, 1982; Stominska and Maczynski, 1985). Isoamylase can potentially be used in the elucidation of fine structures of polysaccharides and related α-glucans (Gunja-Smith et al., 1970; Akai et al., 1971; Fujita et al., 2003). It can also be used as effective additives in dishwashing and laundry detergents (Ara et al., 1993; Ito et al., 1998).

Production of microbial isoamylases
Sources of microbial isoamylases:
Isoamylase may be produced intracellularly or extracellularly (Horwath and Rotheim, 1977) depending on the exact conditions of propagation. Intacellular isoamylase was first described in autolysed brewer’s yeast (Maruo and Kobayashi, 1951) and in baker’s yeast (Gunja et al., 1961), later an intracellular glycogen debranching enzyme complex was reported (Lee et al., 1970) from yeast. In 1982, an extracellular isoamylase was reported from yeast, Lipomyces sp. (Spancer Martins, 1982) and the next report came after a decade from Hendersonula toruloidea (Odibo et al., 1992).

First bacterial isoamylase was isolated, purified and characterized from Pseudomonas amyloderamosa (Harada et al., 1968). Since then production and further characterization of isoamylase was continued from a number of wild and mutant strains of Pseudomonas (Yokobayashi et al., 1970; Harada et al., 1972; Fujita et al., 1990; Katsuya et al., 1998) and other bacterial and fungal strains (Table 1).

Carbon source in fermentation media: In most of the cases, starch of variable concentration (0.25 to 3.0% (w/v) was used for the growth and isoamylase production (Spancer Martins, 1982; Takahashi et al., 1996) by various organisms. Dextrin was used as sole carbon source for isoamylase production in the fermentation medium of bacteria like Escherichia intermedia (Ueda and Nanri, 1967) and Pseudomonas amyloderamosa (Olemposka-Beer, 2007). Maltose was also used for the growth of these bacteria (Ueda and Nanri, 1967; Sugimoto et al., 1974; Fang et al., 1994). Production of isoamylase could be induced effectively by the maltose only if the glucose concentration is maintained below the inhibitory level in Pseudomonas amyloderamosa (Lai and Liu, 1996).


Table 1: Various sources of microbial isoamylases

Nitrogen source in fermentation media: Although, nitrogen source in a fermentation medium generally boost up the growth of the microorganism cultivated, it played some role in effecting the production of the enzyme. Hence, various workers optimized the culture media with suitable nitrogen source to get the highest yield of isoamylase from their working strains.

Peptone (Ueda and Nanri, 1967), NaNO3 (Odibo et al., 1992), yeast extract, ammonium sulfate (Ara et al., 1993), peptone and yeast extract (Olemposka-Beer, 2007), Proteimax (Fang et al., 1994), soybean protein (Takahashi et al., 1996; Houng et al., 1989), sodium glutamate and diammonium hydrogen phosphate (Harada et al., 1968), yeast extract (Gomes et al., 2003) were used as major nitrogen source in the culture media of different micro organisms. Yusaku et al. (1970) reported that their isolated strain of Aerobacter aerogenous showed two different isoamylase formation mechanisms representing either way according to the type of the nitrogen source used as the growth and enzyme production kinetics were changed remarkably with the change of nitrogen source from CH3COONH4 to (NH4)2SO4.

Optimum temperature for enzyme production: Except extremophiles (Fang et al., 2005) and Bacillus stearothermophiles (Prayitno et al., 1996) growing at higher temperatures, the cultivation temperature for isoamylase producer ranged between 28-30°C (Ueda and Nanri, 1967; Ara et al., 1993; Fang et al., 1994; Yamada et al., 1994; Takahashi et al., 1996; Olemposka-Beer, 2007; Ghosh and Ray, 2010). Lipomyces kononenkoae showed a preference towards a lower temperature of 25°C (Spancer Martins, 1982).

Optimum pH for enzyme production: Isoamylase producers showed a broad range of pH preference for synthesizing the enzyme. Pseudomonas amyloderamosa showed optimum growth at pH 6.5-7.5 (Olemposka-Beer, 2007), whereas the mutant WU 2130 (Fang et al., 1994) was cultivated at pH 5. Flavobacterium sp. showed highest isoamylase production at pH 6.8 (Horwath et al., 1977) and 5.3 (Takahashi et al., 1996). The mould Hendersonula toruloidea and mutant strain of bacteria Pseudomonas amyloderamosa MS1 were cultivated at pH 5.5 (Odibo et al., 1992; Sugimoto et al., 1974). Xanthomonas maltophila showed a broad range of pH preference 6-8 (Yamada et al., 1994), whereas Arthrobacter sp and Micrococcus sp. were cultivated at pH 6.8 (Horwath et al., 1977). Higher pH of 8 and 9.2 were preferred for isoamylase synthesis by Rhizopus oryzae (Ghosh and Ray, 2010) and Bacillus sp. (Ara et al., 1993).

Cultivation time for enzyme production: Production of isoamylase took a longer time than other amylases like bacterial and fungal β amylases (Ray et al., 1994; Ray and Chakraverty, 1998), α amylases (Aygan et al., 2008; Ray, 2001). A minimum duration of 48 h were taken by P.amyloderamosa (Chen et al., 1997; Fang et al., 1994), E. intermedia (Ueda and Nanri, 1967). A period of 70-72 h were taken by Bacillus sp. (Ara et al., 1993), Rhizopus oryzae (Ghosh and Ray, 2010), Flavobacterium sp. Micrococcus sp. and Arthrobacter sp. (Yamada et al., 1994). Comparatively longer time of 96 and 120 h was taken by the mould Hendersonula (Odibo et al., 1992) and yeast Lipomyces sp. (Spancer Martins, 1982). respectively.

Properties of microbial isoamylases: Isoamylase is considered as a direct debranching enzyme and is differentiated from other major starch debranching enzyme pullulanse by its ability to cleave all the α-1, 6 linkages of glycogen both inner and outer branching points of soluble amylopectin (Chen et al., 1997) but it is unable to remove 2 and 3 glucose units of the side chain of β and α limit dextrins of oligosaccharides. It has a higher activity than pullulanase and is not inhibited by maltose (Chen et al., 1997). Only the intra cellular isoamylase of E. coli (Jeanningros et al., 1975) was different from the other isoamylases with its inability to hydrolyze glycogen.

Substrate specificity: Kobayashi (1957a) studied the substrate specificity of the purified isoamylase. Being a debranching enzyme, isoamylase was found to cleave glycogen and thereafter glycogen was used for isoamylase assay. In yeasts, isoamylase was found to hydrolyze α-1.6 glucosidic linkage of both starch and glycogen (Kobayashi, 1955, 1957a, b). Glycogen also acted as substrate in the assay system of thermostable isoamylase from Sufolobus sp. (Park et al., 2007), Bacillus sp. (Ara et al., 1993), Pectobacterium (Lim et al., 2001), Rhizopus oryzae (Ghosh and Ray, 2010), Pseudomomas amyloderamosa (Amemura et al., 1980; Kato et al., 1977; Kitagawa et al., 1975; Yokobayashi et al., 1970). Isoamylase from Xanthomonas maltophilia (Yamada et al., 1994) most actively acted on glycogen, followed by amylopectin but hardly on pullulan, with an exception of bacterial isoamylase, described by Ueda and Nanri (1967) that could hydrolyze pullulan. Evans et al. (1979) reported that the isoamylase from a strain of Cytophaga had a very low but significant activity on pullulan and on alpha-dextrins having maltosyl side-chains. Amylopectin was also used as substrate for assaying the isoamylase of Bacillus sp. (Ara et al., 1993), Pectobacterium (Lim et al., 2001), Sufolobus sp. (Park et al., 2007), Pseudomomas amyloderamosa (Kato et al., 1977; Kitagawa et al., 1975; Yokobayashi et al., 1970; Katsuya et al., 1998) and in yeasts like Lipomyces kononenkoae (Spancer Martins, 1982) and in Hendersonula toruloidea (Odibo et al., 1992). Starch was used as substrate for determining the isoamylolytic activities of Pseudomomas amyloderamosa (Olemposka-Beer, 2007 ), E. intermedia (Ueda and Nanri, 1967) and Lipomyces sp. (Spancer Martins, 1982) According to Kainuma et al. (1978) as the Pseudomonas isoamylase could hydrolyze maltotriosyl branches more rapidly than those of the maltosyl branches, it required a minimum of three D-glucose residues in the B- or C-chain. Hence, the favored substrates for Ps. isoamylase were higher-molecular-weight polysaccharides like glycogen, amylopectin and starch.

Temperature optima: Four distinct ranges of temperature optima were found among the microbial amylases of which isoamylases extracted from yeasts generally showed low temperature optima and had poor heat stability. The yeast isoamylase showed optimum activity at 25°C (Gunja et al., 1961) 30°C (Kobayashi, 1957a) 30°C (Spancer Martins, 1982; Lee et al., 1970). A slightly higher range of temperature optima of 37-47°C was found in Pectobacterium sp. (Lim et al., 2001), E. intermedia (Ueda and Nanri, 1967), P. amyloderamosa (Olempsoka-Beer, 2007), Hendersonula sp. (Odibo et al.,1992). Most of the isoamylases reported, showed highest activity at a range of 50°-60°C, like 50°C in Xanthomonas maltophilia (Yamada et al., 1994), 52°C in Pseudomonas (Yokobayashi et al., 1970), 55°C in Bacillus (Ara et al., 1993), Rhizopus oryzae (Ghosh and Ray, 2010), 56°C in B. stearothermophilus (Prayitno et al., 1996). The thermophilic isoamylases showed high range of temperature optima of 75-80°C in Sulfolobus sp. (Fang et al., 2005; Park et al., 2007) and 75°C in Rhodothermus marinus (Gomes et al., 2003).

pH optima: In most of the reports, an optimum pH range was mentioned instead of a particular pH. Harada et al. (1968) reported optimum pH range of 5-6 in Pseudomonas sp. Yamada et al. (1994) reported it to be 3-5 for Xanthomonas maltophila, Olemposka-Beer (2007) reported 3-4 for P. amyloderaosa, Krohn et al. (1997) reported 5-8 for Flavobacterium sp. Bacillus isoamylases showed a pH optima of 9 (Ara et al., 1993), or 5 (Castro et al., 1992). Other bacterial isoamylases showed best activities at various pHs like pH 7 for Pectobacterium sp. (Lim et al., 2001), Flavobacterium sp. (Krohn et al., 1997), 6.0 for Flavobacterium sp. (Hizukuri et al., 1996). On the other hand, thermophilic bacteria like Sulfolobus sp. (Fang et al., 2005; Park et al., 2007; Woo et al., 2008) were found to have activities towards acidic pH. Fungal isoamylases like yeast amylase (Kobayashi, 1955; Lee et al., 1970) showed optimum pH at 6.2-6.4, whereas in Lipomyces (Spancer Martins, 1982), Hendersonula (Odibo et al., 1992 ) and Rhizopus oryzae (Ghosh and Ray, 2010 ) the optima were 5.6, 7 and 5, respectively. The optimum pHs of baker's yeast isoamylase isozymes (three fractions) were 6.8, 5.6 and 5.6, respectively (Kawai and Ishibashi, 2009).

Themostability: According to Yamada et al. (1994), isoamylase of yeast and Cytophaga origin has poor heat stability, whereas the same enzyme of Pseudomonas origin does not have the problem of heat stability. Pseudomonas isoamylase reported by Yokobayashi et al. (1970) was stable at 45°C, but 95% activity was lost if exposed at 60°C. Isoamylase from Xanthomonas maltophilia was found to be stable at 45°C for 10 minutes and from Bacillus sp. (Ara et al., 1993) at 30-40°C, but not above 65°C. Complete loss of activity after 10 min at 65°C was found in Pseudomonas isoamylase (Yokobayashi et al., 1970). But the His tagged enzyme of Sufolobus (Fang et al., 2005) was found to be stable at a high temperature of 80°C even after 2 h of exposure but both recombinant wild-type and His-tagged enzyme were more stable at room temperature than at 4°C. Even the isoamylase from Hendersonula (Odibo et al., 1992) was found to be stable at 70°C for 30 min. It was found that Pseudomonas isoamylases were quite stable in maltose-containing buffer (Lin et al., 1994), whereas isoamylase from yeast like Lypomyces sp. was very unstable, even at low temperatures (Spancer Martins, 1982). The thermostability at a pH range of 4-6 was increased after genetic engineering by Bisgard-Frantzen and Svendsen (2008).

pH stability: Isoamylase from Pseudomonas amyloderamosa (Yokobayashi et al., 1973) showed a broad pH stability range of 2.5-7.5, but the same reported by Kato et al. (1977) was active between 3.5-5.5. Isoamylase from Flavobacterium sp. (Krohn et al., 1997) was assayed at 22°C that displayed activity and stability optima of pH 5.0-7.5, whereas, the Bacillus (Ara et al., 1993) isoamylase was active between pH range 5-11.5 and more than 50% of the original activity remained detectable between pH 6.6-10.5. Xanthomonas maltophilia (Yamada et al., 1994) isoamylase was stable at 3.5-6.0. The isoamylase activity of Pseudomonas amyloderamosa WU 5315 was stable over the pH range from 5.5 to 6.25 while only about 30% of the activity remained at pH 6.5 (Wu et al., 1994).

Incubation time: A reaction time of 10 to 15 min was required for assaying the activities of isoamylase from Hendersonula toruloidea, (Odibo et al., 1992), Xanthomonas maltophila (Yamada et al., 1994), Rhizopus oryzae (Ghosh and Ray, 2010) and Bacillus sp. (Ara et al., 1993), but a longer duration of 30 and 60 min was needed for determining the activity of isoamylase from Pseudomonas amyloderamosa (Olemposka-Beer, 2007) and E.intermedia (Ueda and Nanri, 1967), respectively.

Inhibition of enzyme: The enzyme could be inhibited by a number of inhibitors that included metal ions, thiol inhibitors, saccharides etc. Treatment of isoamylase of Pectobacterium crysanthemi with metal ions Zn2+, Mg2+, Mn2+ at a concentration of 5 mM resulted in 20, 80, 70% residual activities respectively (Lim et al., 2001). Hg2+ caused a drastic reduction in the isoamylase activities of Bacillus sp. (Ara et al., 1993) and Pseudomonas amyloderamosa (Kitagawa et al., 1975). Other metal ions acting as potent inhibitors of isoamylase activities were Ag2+ and Cu2+ presumably due to their interference with the active site of the enzyme. Activities were completely lost after treatment with detergents like SDS in Pectobactrium sp. (Lim et al., 2001), Pseudomonas sp. (Kitagawa et al., 1975) Thiol inhibitors like pCMB although inhibited the isoamylase activity in Pseudomonas (Yokobayashi et al., 1970) and Xanthomonas maltophilia (Yamada et al., 1994) but did not affect the samein Hendersonula (Odibo et al., 1992). EDTA, NaF, N-Bromosuccinimide, 2,4-dinitrofluorobenzene, 2-hydroxy-5-nitrobenzyl bromide, guanidine hydrochloride, β-mercaptoethanol, were found to act as potent inhibitors of Pseudomonas isoamylase (Yokobayashi et al., 1970). Some poly and oligosaccharides like cyclomaltoheptaose, glucose, xylose, maltose, isomaltose, maltotriose also reduced the enzyme activity of isoamylase from Pseudomomas sp. (Kitagawa et al., 1975).

Assay method: A method for determining the isoamylase activity was devised by Kobayashi (1955). A properly diluted enzyme solution was allowed to act at 20°C on a 1% solution of glutinous-rice starch buffered to pH 6.2. After 24 h, 0.2 mL of then reaction mixture was mixed with 2 mL of 0.01 N I2 solutions and diluted to 25 mL. Optical density (E) of the solution at 620 μm was read in a Pulfrich photometer, using a 1 cm cell. The increment of E at 620 μm was proportional to enzyme activity within certain ranges. The amount of enzyme was expressed in the isoamylase unit, i.e., an increase of E 0.200 being taken as 10 units. Isoamylase activity was also assayed with amylopectin as substrate, by determining the increase in iodine staining power (Maruo and Kobayashi, 1951). Ghosh and Ray (2010) measured isoamylase activity by incubating the assay mixture (1 mL) containing an equal volume of enzyme and 1% (w/v) Oyster glycogen in 0.1 M phosphate buffer (pH-5) at 55°C for 5 min. The reducing sugar released was measured by the dinitrosalicylic acid method (Bernfeld, 1955) taking glucose as standard. Blanks were prepared with inactivated enzymes. One unit of isoamylase was defined as that amount of enzyme that liberated 1 μmole of glucose/mL/min of reaction (Ara et al., 1993). A method for detection of isoamylase in polyacrylamide gel by using a two step replica gel revealing assay was devised by Gonzalez (1994).

End product analysis: Maltose, maltotriose and maltotetraose were the main hydrolysis product of Hendersonula isoamylase (Odibo et al., 1992), maltose along with maltooligosaccharides were detected as the major end product of isoamylase extracted from Bacillus sp. (Ara et al., 1993) and from Lipomyces (Spancer Martin, 1982). Degradation product of glycogen by isoamylase of Bacillus stearothermophilus was detected as a single spot of glucose on thin layer chromatogram (Prayitno et al., 1996). Only maltotriose, but no maltose or glucose could be detected as the end product of isoamylolysis in Flavobacterium sp. (Sato and Park, 1980a). In Sulfolobus solfataricus linear maltooligosaccharides (Park et al., 2007) and maltose and beta-cyclodextrinin (Kang et al., 2008) were found to be the end products of isoamylase action. Isoamylase from various strains of Pseudomonas amyloderamosa were reported to cleave the branching points completely in glycogen or amylopectin to produce maltose + maltooligosaccharides (Yokobayashi et al., 1970; Kitagawa et al., 1975; Kato et al., 1977; Amemura et al., 1980, 1988; Katsuya et al., 1998; Fujita et al., 1990). Yeast isoamylase acted on α, β-limit dextrin (DP 9.6) to liberate glucose as well as maltose and higher oligosaccharides (Sakano et al., 1969).

Enzyme activity: Isoamylase from Bacillus (Ara et al., 1993) showed a specific activity of 470 U mg-1 after reacting with oyster glycogen, which was increased respectively to 522 and 667 U mg-1 when bovine muscle glycogen and corn amylopectin were used as respective substrates. From shake culture of a strain of Aerobacter aerogenous Yusaku et al., 1970 obtained isoamylase with activity of 500 U mL-1. Kato et al. (1977) reported the isoamylase activity in Pseudomonas sp was 190 U mg-1. Krohn et al. (1997) detected the variations of activity of isoamylase from Flavobacterium with the change in substrates, as in presence of oyster glycogen, recombinant isoamylase showed a specific activity of 182 U mg-1, which was changed to 120, 154 and to 174 U mg-1 in presence of starch, amylopectin and rabbit muscle glycogen respectively. A specific activity of about 59 U mg-1 was reported by Yokobayashi et al. (1970) and Fujita et al. (1990) from Pseudomonas amyloderamosa. Horwath and Rotheim (1977) reported the isoamylase activities of the strains of Flavobacterium sp. ATCC 21918, Micrococcus so ATCC 21919 and Arthrobacter sp. ATCC 21920 showed activities of 28.2, 12.9 and 6.3 U mL-1, respectively. Yamada et al. (1994) opined that isoamylases of Pseudomonas had a low productivity and researchers were trying to remove this disadvantage by adopting various biotechnological approaches and finding new strains with high isoamylase activities. Wu et al. (1993) was able to increase the yield of his working strain of P. amyloderamosa by classical mutagenesis upto a titer of 5100 U mL-1. The extracellular isoamylase activity of transformed Saccharomyces cerevisiae could reach 86 U mL-1 after 4 days cultivation (Chen et al., 1998).

Molecular weight of purified enzyme: Purified isoamylases from various microbes showed the molecular weight of 60-120 kDa and in most of the cases these enzymes were a monomer or a dimer. It may be mention worthy that the rice isoamylase is a homo tetramer to homo hexamer (Fujita et al., 1999). Isoamylase from Pseudomonas amyloderamosa purified by raw starch adsorption-desorption (Fang et al., 1994) showed four protein bands on SDS PAGE corresponding to monomer, dimer, trimer and tetramer of the enzyme, where enzyme activity was shown by the monomer (M.W 78,000) only. The molecular weight of Pseudomonas amyloderamosa was determined by Yokobayashi et al. (1970) by gel electrophoresis and low speed sedimentation was 95,000. Amemura et al. (1980) detected it as monomer, having a molecular weight of 94,000. Kitagawa et al. (1975) found it to be a composition of two subunits with mol wt 105,000 and 50,000, not linked covalently with each other. The same enzyme studied by Katsuya et al. (1998), was found to have the mol wt of 80, 000. Sato and Park (1980a) purified isoamylase from Flavobacterium sp. by fractionation with ammonium sulfate, chromatography with DEAE cellulose, DEAE sephadex and CM-cellulose and obtained a single band in SDS PAGE. With the help of SDS PAGE, Cheng and Chang (1986) demonstrated that the isoamylase was a polymorph of protein aggregates of 82, 160, 250, 340, 420 and 537 kDa size species on basic PAGE. Krohn et al. (1997) detected it’s molecular weight 83,000. The same molecular weight was found by Park et al. (2007) in Sulfolobus isoamylase, which was a dimmer or a tetramer. According to Woo et al. (2008) in Sulfolobus solfataricus, the enzyme existed in two oligomeric states in solution, as a dimer and tetramer. Bacillus isoamylase (Ara et al., 1993) purified by gel filtration technique indicated that it was a monomer of mol wt 65,000. In Xanthomonas maltophila (Yamada et al., 1994) the mol wt of the isoamylase was 105,000. The yeast isoamylase was purified by Sakano et al. (1969) by DEAE cellulose and gel filtration on sephadex G 100 and CM-cellulose chromatography. Lee et al. (1970) detected that the purified yeast isoamylase was made up of at least two subunits. Isoamylase from another yeast strain Lipomyces sp. had a molecular weight of around 65,000. Odibo et al. (1992) found the purified isoamylase of Hendrsonula toruloidea had a molecular weight of 83,000. Cytophaga sp. Lee et al. (1972) was found to secrete the isoamylase with molecular weight of 120,000. The molecular weight of the enzyme from Pectobacterium carotovorum subsp. carotovorum (Cho et al., 2007) was also estimated to be 74 kDa by activity staining of a SDS-PA gel.

Genetic analysis: Since, cloning of isoamylase gene and its expression in vector is a prior requirement to detect the sequence of the enzyme, the P.amyloderamosa gene pmi encoding isoamylase was cloned and sequenced (Amemura et al., 1988). The Isoamylase Gene (ISO) of Pseudomonas amyloderamosa JD210, an isoamylase-hyperproducing mutant, was cloned in an isoamylase-deficient and transformable mutant strain K31 (Chen et al., 1990). Its nucleotide sequence contained an open reading frame of 2328 nucleotides (776 amino acids) encoding a secreted isoamylase precursor. Isoamylase gene (iso) of Pseudomonas amyloderamosa was amplified by polymerase chain reaction and cloned into Saccharomyces cerevisiae vectors under the control of alcohol dehydrogenase gene and glyceraldehyde-3-phosphate dehydrogenase gene promoters (Chen et al., 1998). On the other hand, two plasmids, designated pRTI and pTI, were constructed to allow the integration of a bacterial isoamylase gene (iso) into Saccharomyces cerevisiae G23-8 chromosome by Ma et al. (2000). Transcription of iam gene from P. amyloderamosa was studied by Fujita et al. (1989). Cloning and nucleotide sequence of the isoamylase gene from a strain of Pseudomonas sp. was done by Tognoni et al. (1989). Isoamylase gene of Flavobacterium odoratum KU was expressed in E. coli (Abe et al., 1999). Isoamylase gene from Flavobacterium sp. was extensively studied by Barry et al. (1996). In further study, the sequence analysis of iam from Flavobacterium sp. suggested that transcriptional control of this gene was mediated through the product of another gene, malt regulatory gene (Krohn et al., 1997). In P. amyloderamosa, Fujita et al. (1990) found that after introducing the plasmid pIAM275, pUC9 carrying iam gene in Escherichia coli, recombinant plasmid did not direct the synthesis of isoamylase in it. An intracellular isoamylase gene from Pectobacterium chrysanthemi P35 was cloned and characterized by Lim et al. (2001). The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli DH5α by Cho et al. (2007). The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues. According to them isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and the sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis. A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 was reported to produce TreX, which hydrolyzed the alpha-1, 6-branch portion of amylopectin and glycogen (Park et al., 2007). In the subsequent experiment, Park et al. (2008) found the TreX existed as a tetramer in the presence of DMSO at pH 5.5-6.5 which showed a 4-fold higher catalytic efficiency than the dimmer and they presumed that TreX might remain associated with glycogen metabolism by selective cleavage of the outer side chain. The isoamylase gene was cloned to an expression vector with a T7lac promoter. Both wild-type and His-tagged isoamylases were expressed in Escherichia coli (Fang et al., 2005). Although generally similar to the monomeric structure of isoamylase, TreX exhibited two different active-site configurations depending on its oligomeric state. The N terminus of one subunit was located at the active site of the other molecule, resulting in a reshaping of the active site in the tetramer. This was accompanied by a large shift in the flexible loop (amino acids 399-416), creating connected holes inside the tetramer. Mutations in the N-terminal region resulted in a sharp increase in alpha-1,4-transferase activity and a reduced level of alpha-1,6-glucosidase activity. On the basis of geometrical analysis of the active site and mutational study, Woo et al. (2008) suggested that the structural lid (acids 99-97) at the active site generated by the tetramerization was closely associated with the bifunctionality and in particular with the alpha-1,4-transferase activity. Few information regarding isoamylase gene isolated from different microbes are given herewith: form Sulfolobus solfatricus P2 ( Uniport AAK42273.1 CAA69504.1, CAC23738.1, NP_343483.1 andPBD/3D P95868) from Escherichia coli K-12 MG1655 (Uniport:AAC76456.1, CAP11380.1, NP_417889.1 and PBD/3D P15067.3) and Pseudomonas amyloderamosa SB-15 (Uniport AAA25854.1, CAA31754.1 and PBD/3DP10342.3).

Biotechnological approaches
Mutagenesis: In order to increase the enzyme production hyperproducing strains of Pseudomonas amyloderamosa were bred by stepwise classical mutagenesis using UV and MNNG as mutagens whereby a 22 fold increase in enzyme production could be achieved (Wu et al., 1993). To identify the essential residues of isoamylase in Flavobacterium odoratumx KU, site directed mutagenesis was done by Abe et al. (1999).

Raw starch adsorption: One of the characteristic features of isoamylase is its adsorbability on raw starch, which was effectively utilized for various purposes including purification. Kato et al. (1977) showed that isoamylase can be effectively adsorbed onto cross linked amylase gel and eluted by maltose containing buffer. Fang et al. (1994) described the recovery of isoamylase by adsorption on raw starch and desorption thereof. Later the method of purification by affinity separation followed by raw starch adsorption and desorption was patented by Fang et al. (1988). Lai et al. (1998) found that Pseudomonas isoamylase immobilized physically by raw starch adsorption resulted in an increase in pH and temperature stability and a retention of about 75% of activity even after 75 days. Chou et al. (1999) showed that P. amyloderamosa isoamylase could be preserved in adsorbed form at room temperature for eight months with high stability. According to Lin et al. (1994), the raw starch-enzyme suspension of Pseudomonas amyloderamosa when packed into a funnel-type glass filter instead of conventional flask to elute the raw starch adsorbed isoamylase, the recovery was increased from 53.6% to about 81% and the concentration of isoamylase was also increased 42-fold. Further, the addition of 1% potassium sorbate into it increased the self life of the enzyme.

Immobilization: For judicious exploitation of enzymes and further characterization, it may be immobilized onto various matrices. Isoamylase recovered from the fermentation broth of Pseudomonas amyloderamosa was immobilized by Chen et al. (1997) onto water-insoluble carriers like chitin, CM-cellulose and a temperature-sensitive reversibly soluble copolymer (N-isopropylacrylamide-co-N-acryloxysuccinimide). The debranching actions of immobilized and free isoamylase, was checked by Sunarti et al. (2001). Waxy maize amylopectin was treated with Pseudomonas isoamylase immobilized on magnetic supports in order to prepare various kinds of partial hydrolysates to check the arrangement and size of the amylose chains (Hisamatsu et al., 1995).

Saccharification: As isoamylase ameliorates sugar production, to find out new microbial isoamylases with high saccharifying potential and ability to convert native, non expensive starch residues to sugar become the need of the hour. Extra cellular isoamylase from Rhizopus oryzae PR7 MTCC 9642 was found to saccharify soluble potato starch and various native raw starches collected from domestic effluents, of which arrow root, tamarind kernel, tapioca and oat were noteworthy (Ghosh and Ray, 2010). Bioconversion of starch hydrolysate to a high DX glucose syrup at pH 3-5 by the enzyme mixture of a glucoamylase and an acidophilic Pseudomonas isoamylase (Hayashibara, Japan) was successfully done by Norman (1982). According to Castro et al. (1992) the properties of extracellular isoamylase of Bacillus circulans would allow its use in normal saccharification processes in the starch industries. Thermophilic enzyme of Sulfolobus sp. showed a potential to be used in industry to degrade the debranching points of starch at a high temperature (Fang et al., 2005) and could be employed for the production of high-maltose syrups and highly purified maltose by a combination of debranching enzymes (Spancer Martins, 1982). According to Krohn et al. (1997), isoamylase could be used for industrial production of various syrups from starch. Amemura et al. (1980) and Kato et al. (1977) opined this enzyme was applicable for industrial production of amylose, maltose and D-glucose from starch, alone or in combination with beta-amylase and for glucoamylase production.

Analysis of toxicity: Checking of toxicity is a must prior to be used in food industry and the isoamylase enzyme preparation was tested to confirm compliance with the General Specifications and considerations for Enzyme Preparations used in Food Processing by Hayashibara, Japan (Olemposka-Beer, 2007). The isoamylase preparation from P. amyloderamosa has been used in food industry in countries such as Japan for more than two decades. No acute toxicity, no toxicity after short term (13 weeks) toxicity study, no genotoxicity and pathogenecity were reported. The recommended use levels range from 50-500I AU g-1 starch for the production of food ingredients.

Other sources of isoamylases: Besides microbial sources other isoamylase producing sources are plant like Arabidopsis (Zeeman et al., 1998), rice endosperm (Fujita et al., 2003), Chlamydomonas reinhardtii (Dauvillee et al., 2001), Phaseolus vulgaris (Takashima et al., 2007), rye ( Xu et al., 2009), potato tuber (Bustos et al., 2004); maize (Rahman et al., 1998) Homo sapiens (Clave et al., 1995) and Rhizopthera (Cinco-Moroyoqui et al., 2008.) In plant, the enzyme is required for normal synthesis of amylopectin (Hussain et al., 2003), whereas it is a normal secretion from human pancreas.

Concluding remark: Enzymes have become an integral part of human need in day to day life, particularly in the modern food, pharmaceutical, detergent and textile industries. In food processing industry, amylase predominantly are applied during processing of raw and gelatinized starch. In order to accelerate the splitting of starch, various industrially prepared microbial amylase are being added. This requires extensive researches on microbial amylases. Despite so many commercial values, research on microbial isoamylase is found to remain restricted within few papers and patents and in comparison to that of α-, β- and glucoamylases and also plant isoamylases, research on microbial isoamylase remain somewhat neglected. Hence a comparative account of isoamylases reported from various microbial sources, difference in their characteristic features and recent biotechnological advancement for the enhancement of productivity and industrial applicability of microbial isoamylases becomes extremely necessary. Although isoamylase related sequences of only few micro organisms are available and x-ray crystallographic structure of the enzyme is revealed (Katsuya et al., 1998), still a number of behavioral contradictions exist among these isoamylases, like affinity towards pullulan (Ueda and Nanri, 1967), pH and temperature optima, chemostability etc, which needs further clarification. Future work should focus on deduction of the amino acid sequences of these isoamylases that may be helpful in identifying the protein motifs (Ara et al., 1993) which will further determine the characteristic features of these enzymes. Moreover, from theoretical point of view various types of BLAST searching may be employed to identify the relationships of homology, or through searching the dictionary of protein motifs of the active sites, the characteristic feature of a newly isolated isoamylase may be predicted. But above all, isoamylase an enzyme of immense practical importance must be made industrially applicable for production of various sugars and allied products.

ACKNOWLEDGMENT

The author is thankful to University Grants Commission, New Delhi, India for financial support.

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