Open access peer-reviewed chapter

α-Amylase Production by Toxigenic Strains of Aspergillus and Penicillium

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Adekunle Odunayo Adejuwon and Victoria Anatolyivna Tsygankova

Submitted: April 12th, 2019Reviewed: May 2nd, 2019Published: June 3rd, 2020

DOI: 10.5772/intechopen.86637

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Abstract

Aflatoxins are produced by a variety of fungal species and these have contributed to devastating health problems globally. However, apart from the capability of the production of aflatoxins, the productions of enzymes by like fungi have been explored. Aflatoxin B1-producing-toxigenic strains of Aspergillus flavus (A1), Aspergillus parasiticus (A2), Penicillium citrinum (P1) and Penicillium rubrum (P2) isolated from rice were grown on a defined medium with varying carbon and nitrogen sources. They were also grown on rice as sole carbon and nitrogen source for fungal growth. In an attempt to purify, the extracellular α-amylases produced were subjected to ammonium sulfate precipitation (40–90% saturation) followed by dialysis. The aflatoxin B1-producing toxigenic strains of Aspergillus flavus (A1), Aspergillus parasiticus (A2), Penicillium citrinum (P1) and Penicillium rubrum (P2) were able to produce α-amylases in both the growth medium with varying C and N sources of fungal and also in the rice medium. The most active α-amylase activity was produced by toxigenic A. flavus (A1) with a value of 3.25 ± 0.15 Units and this was when ammonium sulfate was nitrogen source with starch as carbon source of fungal growth in the defined growth medium. These toxigenic fungal strains can be explored for the industrial production of α-amylases.

Keywords

  • α-amylase
  • toxigenic
  • fungi
  • aflatoxin B1

1. Introduction

1.1 Aspergilliand pathogenicity

Aspergilliare of the taxonomic Division Eumycota, Subdivision Eumycotina, Class Ascomycetes, Order Eurotiales, Family Trichocomaceae [1]. Aspergillusis a filamentous cosmopolitan and ubiquitous fungus commonly isolated from soil, plant debris and indoor air environment [2]. While the teleomorphic state exists for Aspergillus species, some are accepted to be mitosporic without any known sexual spore [3]. The genus Aspergillusincludes over 185 species and about 20 species have so far been reported as causing opportunistic infections in man [4, 5]. Among these species, Aspergillus fumigatusis the most commonly isolated species followed by Aspergillus flavusand Aspergillus niger. Aspergillus clavatus, Aspergillus glaucus, Aspergillus oryzae, Aspergillus terreus, Aspergillus ustusand Aspergillus versicolorare among the other species less commonly isolated as opportunistic pathogens [6, 7]. Food infected by Aspergillus flavusmay be carcinogenic to humans and animals [8]. Aspergillus flavusis a saprophyte of grains. It produces mycotoxins in infected food [2]. Infection of peanuts (Arachis hypogaea) seeds by Aspergillus flavusand Aspergillus parasiticusis a serious problem that can result in aflatoxin contamination in the seed [9]. Aspergillus flavusproduces aflatoxins B, G and cyclopiazonic acid CPA [10].

Beta-1,3-Glucanase activity in peanut seed is induced by infection with Aspergillus flavus[9]. Maize seeds are susceptible to Aspergillus flavusinfection [11]. Aspergillus flavuscauses Ear rot in corn with aflatoxin production. Resistance to aflatoxin production can be controlled by epistasis [12]. Aspergillus flavuscauses kernel infection in maize, the Southwestern corn borer (SWCB) has been reported to substantially increase aflatoxin levels in such infection [13]. Kernels of corn genotype GT-MAS:gk are resistant to Aspergillus flavus[14]. A 14-KDa protein in corn kernel makes it resistant to Aspergillus flavusinfection [15]. Aspergillus flavusfound around corn storage cribs and bins are point sources of inoculum/infection with Aspergillus flavusin the corn agroecosystem [16]. Ear corn rot caused by Aspergillus flavusand Aspergillus parasiticusis severe in areas with high temperatures and drought [2]. Aspergillus flavuscauses the post-harvest disease of Arachis hypogaea[3].

According to Norton [17], carotenoids in endosperm may decrease the amount of aflatoxin produced by Aspergillus flavus. Aspergillus flavuscan be divided into S and L strains on the basis of sclerotial morphology [18]. Atoxigenic Aspergillus flavusL strain reduce formation of both sclerotia and aflatoxin when coinoculated with S strain isolate [18]. Aspergillus flavusL strain reduce formation of both sclerotia and aflatoxin when coinoculated with S strain isolate [18]. Aspergillus flavusproduces aflatoxin in cotton seed with the S strain being highly toxigenic [19]. Aspergillus parasiticusisolated from soil from a corn field produced aflatoxin B(1) B(2) and G(1) G(2) [20]. Aspergillus flavusproduces beta-glucuronidase [21]. Aspergillus flavusand Aspergillus parasiticuscan contaminate agricultural crops with the production of toxic fungal metabolite aflatoxins. An endochitinase which is an inhibitory protein with M(r) of 29,000 is capable of inhibiting growth of Aspergillus flavuson maize [22]. Onion seeds stalk and flowers are susceptible to infection by Aspergillus nigerTiegh [23, 24]. The black-spored Aspergillusisolates that have been found to cause the disease fig smut are Aspergillus nigervar. niger, Aspergillus nigervar. awamori, Aspergillus japonicusand Aspergillus carbonarius[25]. Epiphytic fungi found on table grapes include Epiccocum nigrum, Cladosporium herbarum, Alternaria alternata, Aspergillus niger[26].

1.2 Penicilli and pathogenicity

Penicilli belongs to the taxonomic Division Eumycota, Subdivision Eumycotina, Class Ascomycetes, Order Eurotiales, Family Trichocomaceae [1]. Grape fruit green mold is caused by Penicillium digitatum[27]. Penicilliumis common on citrus, gelly and preservatives. It is abundant in the soil and on decaying materials [3]. Penicilliumspores are present in the air [3]. Studies have shown that Penicilliumis important in the production of antibiotics such as Penicillin and Griseofluvin [5]. Penicillium digitatumcauses the green mold of citrus fruits. Optimum temperature of their mycelia on such fruits is about 25°C [28]. The food borne pathogen Listeria monocytogeneshas been observed to grow on apple infected with Penicillium expansumbut not after 5 days [29]. The blue mold of decayed pear fruit is caused by Penicillium expansum[30]. Penicillium expansumhas been observed to cause gray mold disease in apple and blue mold in pear [31]. Penicilliumspp. have been isolated from pear stem [32] while Penicillium italicumcause citrus blue mold and green mold [33, 34]. Postharvest green mold of oranges is caused by Penicillium digitatum[35, 36]. Penicilliumsp. has been isolated from grapes [26]. Penicillium expansumcauses the blue mold decay of pear [37]. Penicillium digitatumand Penicillium italicumcause postharvest green and blue molds of citrus fruits. Sporulation of both molds can be prevented or reduced by gaseous ozone without noticeable ozone phytotoxicity to the fruits [33, 34]. Apple fruits with stem pulls have been reported to be more susceptible to blue mold decay caused by Penicillium expansumthan fruits with stems [38]. According to Spotts and Holz [39], Penicillium expansumcan infect and cause disease in grape and plum fruits. Aqueous chlorine has been reported to reduce the viable spores of Penicillium digitatum, the causative fungi of the green mold and sour rot of citrus [40]. Sodium bicarbonate has been found to reduce postharvest decay of apple [41]. The level of resistance to decay of apple cultivars, caused by Penicillium expansum, varies from cultivar to cultivar [42]. Ziram but not calcium chloride control gray mold and bull’s-eye rot, the postharvest decay of pear associated with the pathogen Penicillium expansum[43]. Penicillium digitatumhas been associated with the postharvest green mold of oranges. Soda ash was observed to control this post-harvest disease [35, 36]. According to Smilanick et al. [35, 36], the effectiveness of imazalil for the control of citrus green mold caused by Penicillium digitatumimproved significantly when the citrus fruits were treated with heated aqueous solutions of the fungicide as compared with the current commercial practice of spraying wax containing imazalil on the fruits. According to Smilanick et al. [44], fungicide applications with thiabendazole (TBZ) and sodium bicarbonate reduce green mold caused by Penicillium digitatumof citrus fruits and lemon fruits. It was also observed that pre harvest applications of thiophanate methyl to the fruits controlled postharvest green mold. Blue mold caused by Penicilliumis an important postharvest disease of apple. Penicillium expansumand Penicillium solitumhave been identified and isolated from rotten apple and pear fruits [45]. According to Sirois et al. [23, 24], onion seeds are affected by species of Penicillium.

1.2.1 α-Amylases

Amylases are hydrolytic enzymes that catalyze the degradation of starch molecules and other carbohydrates to yield dextrins and progressively smaller polymers composed of glucose units [46, 47]. Based on their pattern of catalysis and yield of products, amylases can be categorized as: alpha (α) amylase (endoamylase) (α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1); beta (β) amylase (exoamylase) (1,4-α-D-glucan maltohydrolase, EC 3.2.1.2); glucoamylase (exohydrolase) (Glucan-1,4-α-glucosidase, EC 3.2.1.3); pullulanase (α-dextrin endo-1,6-α-glucosidase, EC 3.2.1.41); isoamylase (EC 3.2.1.68). Pullulanases and isoamylases are termed debranching enzymes [46, 48, 49]. They can be of plant and microbial sources [50, 51, 52].

1.2.2 Microbial α-amylases

α-Amylases are produced by bacteria and fungi [53, 54, 55, 56, 57, 58]. The two types of amylases commonly encountered in microbial degradation of starch are α and β amylases.

Degradation of substrate is important in enzymatic hydrolysis [59]. Starch is the substrate used in microbial amylase assay [60]. The starch molecules are hydrolyzed into polymers of glucose units [47]. According to Vihinen and Mantsala [61], starch-degrading enzymes are widely distributed among microbes and several activities are required to hydrolyze the starch into glucose units. Bacillus subtilisisolated from flour mill wastes produced a thermostable α-amylase in a complex medium containing starch [62]. According to Ajayi and Fagade [63], corn starch can be used as substrate for β-amylase production by Bacillus macerans, Bacillus licheniformis, Bacillus circulans, Bacillus coagulans, Bacillus megaterium, Bacillus polymyxa, Bacillus cereusand Bacillus subtilis. According to Reiss et al. [64], approximately 80% of potato starch and 40–50% of grain starch were hydrolyzed by alpha amylase of certain microbes. Lactic acid bacteria have been found to ferment starchy foods to recover RNA though digestion with alpha amylase did not improve extraction [65]. Rhizopus oligosporus, a prolific amylase producer can degrade cassava tuber containing 65% starch into glucose [66]. Thermoactinomyces thalpophilusisolated from flour mill waste has been found to be capable of hydrolyzing 2% soluble starch [62]. A thermostable α-amylase activity from Bacillus subtilisisolated from flour mill waste was found to be more strongly expressed with corn starch than soluble starch [62]. Alpha amylase from Bacillus licheniformis, an hyperthermostable enzyme, is able to hydrolyze starch to medium-size oligosaccharides [67]. Fusarium moniliformewas found to produce alpha amylase in a culture medium containing starch [68].

Certain environmental (physical) factors affect amylase activity [48]. Lactic acid was found to be produced from Lactobacillus delbrueckiisubsp.delbrueckii and defatted rice bran powder containing starch with coupled saccharification with amylase at 37°C and pH 5.0 [69]. An α-amylase produced by Bacillussp. isolated from soil sample was optimally active at 75–80°C [70]. Alpha-amylase from Bacillus licheniformisis able to hydrolyze soluble starch within a temperature range of 60–75°C [71]. A thermophilic, moderately halophilic anaerobic Halothermothrix oreniisynthesized an amylase similar to Bacillus megateriumamylase with optimal activity at 65°C [72]. Thermophilic Thermussp. was reported to produce an extracellular α-amylase able to degrade starch at 70°C [73]. Bacillus stearothermophiluswas found to produce a thermostable α-amylase active at 43°C [74]. According to Saito [75], Bacillus licheniformisproduced a thermophilic extracellular α-amylase stable at 25°C but more active at an optimum temperature of 76°C. Manning and Campbell [76] reported that Bacillus stearothermophilussynthesized a thermostable α-amylase. Rhizopus arrhizusand Rhizopus oryzaewere found to be capable of hydrolyzing starch at 30°C [77]. Bacillus haloduransproduced an alkaline active maltohexaose-forming α-amylase active at 60°C. According to Oh et al. [57], Lactobacillus gasseriis able to synthesize a maltogenic amylase exhibiting optimum activity for β-CD hydrolysis at 55°C. Based on studies carried out by Najafi and Kembhavi [50], a marine Vibriosp. produced an extracellular α-amylase with maximum activity at 55–60°C. According to Ogasahara et al. [78], Bacillus stearothermophiluswas able to produce a thermophilic α-amylase with optimum temperature range of 65–73°C.

α-Amylase from Pyrococcus woeseihas maximal activity at pH 5.6 [79]. A Bacillussp. isolated from piglet cacum produced an extracellular alpha amylase optimally active at pH 7.0 [80]. A microorganism from uncultured soil was observed to produce amylolytic enzyme with optimal pH of 9.0 [81]. A mutant of Bacillus amyloliquefacienshas been reported to synthesize alpha amylase with optimal activity at pH 7.0 [82]. An extracellular alpha amylase isolated from cell free broth of Streptomyces megasporusgrown in glucose, soluble starch and raw starch was stable at a pH range of 5.5–8.5 but with optimum activity at pH 6.0 [83]. Amylases in culture supernatants of an environmentally derived microbial mixed culture selected for its ability to utilize starch-containing plastic films as sole carbon sources produced amylases active at pH 5.5 and 8.0 [84]. Alpha amylase from Thermoactinomyces vulgarishad optimum activity at pH 4.8–6.0 [85]. Starch degradation by Rhizopus oryzaewas favorable at pH 6.0 [77]. Akindahunsi [86] reported that waste water from cassava mash fermented by pure strains of Saccharomyces cerevisiae, Lactobacillus delbrueckiiand Lactobacillus coryniformisproduced amylase after 3 days with maximal activity at pH 6.0.

According to Mijts and Patel [72], the thermophilic, moderately halophilic anaerobic Halothermothrix oreniiis able to synthesize alpha amylase active with specific activity of 2232 U mg−1 requiring CaCl2 for optimum activity and thermostability. The maltooligosaccharide-forming amylase from Bacillus circulansis enhanced by C02+ and Mg2+ [87]. Amylase synthesized by Lipomyces starkeyiwas found to be actively stable in a commercial mouthwash [88]. A salt-tolerant thermostable amylase produced by Bacillus megateriumwas reported to be stable at 5 M NaCl [89]. A thermophilic Thermoascus aurantiacushas been observed to produce amylase with thermostability enhanced by calcium chloride [90]. Amylase production from Bacillus sphaericuswas reported to be maximum with 3 mM divalent cations Mg++ and Ca++ incorporated in a growth medium [91]. Cadmium, Cobalt, Copper, Manganese, Nickel and Lead incorporated into Czapek-Dox liquid medium supported growth and production of amylase by soil yeasts Geotrichum capitatumand Geotrichum candidum[92]. Activity of α-amylase from a marine Vibriosp. was found to be restored by Fe2+, Mn2+, Co2+, Ca2+, Mg2+ and Cu2+ to nearly 25–55% [50]. A Bacillussp. produced an alkaliphilic amylase which was enhanced by Na+ and Co2+ [93]. According to Mishra et al. [94], Bacillus subtilisproduced an α-amylase. Herbizid has been reported to activate amylase production in culture of Fusarium oxysporum, Mucor niemalisand Penicillium chrysogenum[95].

Amylase from Fusarium verticillioideshas been found to be inhibited by a hydrophobic 19.7-KDa inhibitor from corn kernel [96, 97]. Fusarium moniliforme, a mycotoxigenic fungus has been reported to produce an amylase inhibited by a specific amylase inhibitor found in corn [96, 97]. Alpha-amylase inhibitor has been isolated from culture medium of Streptomyces parvullus[98]. Streptomyces aureofaciensproduces a novel polypeptide inhibitor [99]. A strain of Streptomyces nigrifacienshas been reported to produce an amylase inhibitor having inhibitory effects on alpha amylase and glucoamylase [100].

Bacillus subtilisisolated from soil produced a starch degrading amylase with molecular weight of 50 KDa and an isoelectric point of 4.9 [101]. Streptomyces lividanshas been reported to have a molecular weight of 107,054 KDa [102]. Alpha amylases from some Bacillusspp. were detected to possess molecular weight of approximately 65,5854 and 49 KDa [103]. Alpha-amylase of Clostridium thermosulfurogeneshas been reported to have a molecular mass of 75,112 Da [104]. According to Kang et al. [105], Bacillus stearothermophilusproduces an alpha-amylase which was glycosylated and with molecular weights of approximately 61–75 KDa.

According to Lorentz [106], Protected 4-Nitrophenyl-1,4–1-D-maltoheptaoside can be used in routine amylase assay. A simple and rapid method using Remazol Brilliant Blue-starch as substrate which is non-destructive allows direct visualization and isolation of amylolytic microorganisms from the environment [107].

Alpha amylase can be used in improving anaerobic solid waste treatment [108]. Carbohydrate-hydrolyzing enzymes have long been used by industrial product markers as major catalysts to transform raw materials into end products in such areas as food processing, beverage production, animal nutrition, leather and textiles [109].

With the advent of new frontiers in biotechnology, the spectrum of amylase application has widened in many fields such as clinical, medicinal and fine-chemical industries, as well as a widespread application of starch saccharification in the textile, food, brewing and distilling industries [110].

1.2.3 Aflatoxin B1 and α-amylase production

According to Mellon et al. [111], an aflatoxin B1 producer strain of Aspergillus flavusseem to possess the ability to produce numerous extracellular hydrolases (α-amylase inclusive). Aflatoxin B1 have been detected in groundnut and maize contaminated with Aspergillus flavus[112]. The removal of lipids from ground substrates significantly reduced the substrate’s potential for aflatoxin B1 (AFB1) production by Aspergillus flavus. However, maltose, glucose, arginine, glutamic acid, aspartic acid and zinc significantly induced the AFB1 production up to 1.7–26.6 fold [113]. Aflatoxins B1, B2, G1 and G2 and α-amylase were detected in Aspergillus oryzae, Aspergillus flavus, Talaromyces spectabilis, Pacilomyces variotiiand Lichtheimiasp. isolated from nuruks in several regions of Korea [114]. According to Fakhoury and Woloshuk [115], a mutant strain of Aspergillus flavusfailed to produce extracellular α-amylase when the Amy1 gene necessary for the production of α-amylase was disrupted in an aflatoxigenic strain (an aflatoxin B1 producing strain) of the fungus. Mycotoxigenic strains (aflatoxin B1 producing strains) of Fusarium moniliformeand Aspergillus flavuswere capable of α-amylase production in a medium composed of 2% ground corn in milky stage corn [116]. In their attempt to increase aflatoxin B1 resistance in maize, Rajasekaran et al. [117] discovered that the α-amylase inhibitor-like protein (AILP) seem to play a role in the inhibition of Aspergillus flavusα-amylase and fungal growth. Fountain et al. [118] reviewed the nature of the interaction occurring between aflatoxin production by Aspergillus flavus, the environment in which the fungus thrives and its susceptibility to crop host before harvest. They proposed future directions for elucidating future relationship between resistance and susceptibility to the fungus’ colonization, abiotic stress and its relationship to oxidative stress in which its aflatoxin B1 production may function as a form of antioxidant protection to the producing fungus. In a known positive transcriptomic database, E-probe Diagnostic for Nucleic acid Analysis (EDNA), a bioinformatic tool, originally developed to detect plant pathogens in mutagenomic databases, is capable of discriminating between production and non-production of aflatoxin B1 by Aspergillus flavus[119]. Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block “acetate” for aflatoxin B1 synthesis in aflatoxin-producing Aspergillus flavusand Aspergillus parasiticus[120]. According to Smith et al. [121], silencing of the aflatoxin gene cluster in a certain strain of aflatoxin B1 producing Aspergillus flavusis suppressed by ectopic aflR gene (the transcriptional regulator of the aflatoxin biosynthetic gene cluster) expression.

1.2.4 Rice (Oryza sativa)

Rice (Oryza sativa) is a monocotyledonous cereal which belongs to the Grass family Gramineae or Poaceae [122]. With over 7000 varieties of rice, its pericarp and embryo contain 70–80% starch, 7% proteins, 1.5% oils, some vitamins (mostly A, B and C) and some essential minerals [3]. According to Sizer and Whitney [123], rice contains fiber and the vitamin folate and provides 80% of the calories consumed by humans worldwide [122]. It contains 12 chromosomes in a haploid set [124]. The domestication of rice formed part of the basis for civilization in the near East, far East and the New World [125]. Feeding more people worldwide than any other crop, rice is the only crop grown exclusively for human consumption [125]. Sedentary irrigated rice production in tropical lowlands can support hundreds of people per square kilometer, explaining the wide spread importance of rice crops in the tropics [126]. The discovery of Gibberellins arose from infected rice [122]. Oryza sativais the main cultivated rice species but over 20 species in the genus are known [125].

This research was designed to examine the production and activity of α-amylases by some toxigenic aflatoxin B1-producing strains of Aspergillusand Penicilliumisolated from deterioration rice. Attempts were made to purify the α-amylases.

1.2.5 Contribution to knowledge

The present research will establish the presence of α-amylases in rice during mycotic spoilage by toxigenic strains of Aspergillus flavus(A1), Aspergillus parasiticus(A2), Penicillium citrinum(P1) and Penicillium rubrum(P2). These fungi, being capable of producing these enzymes can be used in the production of amylases. Rice as substrate can be explored in such production.

Amylases are used in clinical chemistry most especially in diagnosis. Their combination with proteases and lipases are also employed industrially in the bioremediation of recalcitrants/organic pollutants and the hydrolytic digestion of the peptidoglycan layers of both gram positive and gram negative bacteria in wastewaters before chlorination [109].

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2. Materials and methods

2.1 Sources and identification of isolates

The isolates of aflatoxin B1-producing-toxigenic strains of Aspergillus flavus(A1), Aspergillus parasiticus(A2), Penicillium citrinum(P1) and Penicillium rubrum(P2) for this research were from deteriorated rice and identified at the Seed Health Unit of the International Institute for Tropical Agriculture, Ibadan, Nigeria using techniques contained in the illustrated Handbook of fungi [127, 128].The identification was done by observing cultural and morphological characteristics. Each isolate was cultured on Potato Dextrose agar. The nature of growth, rate of growth, colony color and sporulation patterns were carefully observed. Sporulating mature cultures was used in microscopic examination. Fungal samples were taken from advancing margins and centers of the growth regions with the aid of sterile inoculating needle. The samples were smeared on glass slides and stained with lactophenol cotton blue. After placing the cover slips, macroscopic and microscopic morphological characteristics like arrangement and shape of spores, type of sporangia, type of hyphae, presence or absence of septa on hyphae was examined under the high power objective of a compound binocular microscope.

2.2 Culture conditions and preparation of inocula

The isolates were subcultured and maintained on Potato Dextrose agar plates and slants. Each fungus was further subcultured into test tubes of the same medium and incubated at 25°C. A 96-hr-old culture of toxigenic strains of Aspergillus flavus(A1), Aspergillus parasiticus(A2) and Penicillium rubrum(P2) and 120-h-old culture of Penicillium citrinum(P1) was used as inocula. According to the modified method of Olutiola and Ayres [129], cultures was grown in a defined medium of the underlisted composition: MgSO4.7H20, K2HPO4, KH2PO4, L-cysteine, biotin, thiamine and FeSO4.7H20 with added carbon and nitrogen sources (Sigma). Conical flasks (250 ml) containing 100 ml growth medium will be inoculated with 1 ml of an aqueous spore suspension containing approximately 6 × 104 spores per ml of each isolate. Experimental and control flasks was incubated without shaking at 25°C [130].

2.3 Rice as a source of carbon

Rice (Caprice) from Spain was bought at the main market, Bodija, Ibadan, Nigeria. The rice was added to distilled water (1% w/v) and autoclaved at 15Ib/in2 at 121°C. Experimental Conical flasks (250 ml) containing 100 ml of the rice medium was inoculated with 1 ml of an aqueous spore suspension containing approximately 6 × 104 spores per ml of each isolate. Control flasks contained sterilized rice medium not inoculated with aqueous spore suspension of the isolate. Experimental and control flasks was incubated without shaking at 25°C.

On a daily basis, the contents of each flask was filtered through glass fiber filter paper (Whatman GF/A). The protein content of the filtrates was determined using the method of Lowry et al. [131]. The filtrates were analyzed for amylase activity using the modified methods of Pfueller and Elliott [132] and Xiao et al. [133]. The filtrates were used as crude preparation.

2.4 Ammonium sulfate fractionation

The crude enzymes were treated with ammonium sulfate (analytical grade) within the limits of 40–90% saturation. Precipitation was allowed to continue at 4°C for 24 h. The mixtures were then centrifuged 10,000 g for 30 min at 4°C using a high speed cold centrifuge (Optima LE-80 K Ultracentrifuge, Beckman, USA). The supernatant was discarded. The precipitate was re-dissolved in 0.2 M citrate phosphate buffer, pH 6.0. The protein contents were determined using the Lowry et al. [131] method while amylase activity was determined using the modified methods of Pfueller and Elliott [132] and Xiao et al. [133].

2.5 Dialysis

Using acetylated dialysis tubings (Visking dialysis tubings, Sigma) [134] and a multiple dialyser (Pope Scientific Inc. Model 220, USA), the enzyme preparations were dialysed under several changes of 0.2 M citrate phosphate buffer pH 6.0 at 4°C for 24 h. The protein contents of the dialysed enzymes were determined using the Lowry et al. [131] method while amylase activity was determined using the modified methods of Pfueller and Elliott [132] and Xiao et al. [133].

2.6 Enzyme assay

Both experimental (fungal isolate inoculated) and control (un-inoculated) flasks were assayed for amylase activity.

2.6.1 α-Amylase

α-Amylase activity was determined using the modified methods of Pfueller and Elliott [132] and Xiao et al. [133]. The reaction mixtures consisted 2 ml of 0.1% (w/v) starch (Sigma) in 0.2 M citrate phosphate buffer, pH 6.0 as substrate and 0.5 ml of enzyme. These were the experimentals in the assay procedure. The controls in the assay procedure consisted only 2 ml of the prepared substrate. The contents of both experimental and control tubes were incubated at 35°C for 30 min. The reactions were terminated with 3 ml of 1 N HCl. Enzyme (0.5 ml) was added to the contents of each control. About 2 ml of the mixture from each of the sets of experimentals and controls was transferred into new sets of clean test tubes. About 3 ml of 0.1 N HCl was added into the contents of each test tube after which 0.1 ml of iodine solution was added. Optical density readings were taken spectrophotometrically at 620 nm. Enzyme activity was defined in units and specific activity as enzyme units per mg protein.

One unit of α-amylase activity was defined as the amount of enzyme which produced 0.1% reduction in the intensity of the blue color of starch-iodine complex under conditions of the assay.

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3. Results

3.1 Amylase activities of isolates on growth media

Toxigenic strains of Aspergillus flavus(A1), Aspergillus parasiticus(A2), Penicillium citrinum(P1) and Penicillium rubrum(P2) grew and exhibited amylase activities, varyingly, in modified growth medium used for this research.

Using different carbon sources (rice, starch, maltose, sucrose, lactose, glucose and galactose) in the growth medium, amylase activity expressed by each isolate on the tenth day of incubation is shown in Table 1 .

Carbon sourceIsolateAmylase activity (Units)
RiceAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.54 ± 0.01
0.72 ± 0.04
0.43 ± 0.23
0.62 ± 0.06
GalactoseAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.06 ± 0.01
0.53 ± 0.13
0.36 ± 0.05
0.09 ± 0.04
GlucoseAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.50 ± 0.04
0.63 ± 0.08
0.44 ± 0.08
0.32 ± 0.11
LactoseAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.10 ± 0.00
0.66 ± 0.10
0.40 ± 0.17
0.37 ± 0.08
MaltoseAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.52 ± 0.03
0.68 ± 0.04
0.75 ± 0.01
0.58 ± 0.12
StarchAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.45 ± 0.04
0.57 ± 0.12
0.68 ± 0.03
0.60 ± 0.14
SucroseAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.46 ± 0.05
0.69 ± 0.03
0.59 ± 0.13
0.39 ± 0.06

Table 1.

Effect of carbon sources on activity of amylase produced by isolates.

Each value represents the mean of three replicates with standard error.

With different sources of nitrogen (NH4Cl, urea, KNO3, ammonium sulfate, glycine, sodium nitrate, tryptone and peptone) in the growth medium, amylase activity expressed varyingly by each isolate on the tenth day of incubation is shown in Table 2 .

Nitrogen sourceIsolateAmylase activity (Units)
Ammonium sulfateAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
3.25 ± 0.15
0.05 ± 0.00
0.38 ± 0.13
0.13 ± 0.03
GlycineAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.38 ± 0.13
2.48 ± 0.03
0.00 ± 0.00
1.53 ± 0.48
Potassium nitrateAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.50 ± 0.00
1.30 ± 0.10
0.25 ± 0.25
0.68 ± 0.03
Ammonium chlorideAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
3.02 ± 0.18
0.05 ± 0.05
0.13 ± 0.13
0.13 ± 0.03
PeptoneAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.25 ± 0.00
1.50 ± 0.15
0.13 ± 0.13
2.48 ± 0.03
Sodium nitrateAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.38 ± 0.13
1.48 ± 0.13
0.25 ± 0.00
0.93 ± 0.73
TryptoneAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.25 ± 0.00
0.20 ± 0.05
0.38 ± 0.13
2.40 ± 0.10
UreaAspergillus flavus(A1)
Aspergillus parasiticus(A2)
Penicillium citrinum(P1)
Penicillium rubrum(P2)
0.15 ± 0.00
2.32 ± 0.03
0.00 ± 0.00
2.33 ± 0.08

Table 2.

Effect of nitrogen sources on activity of amylase produced by isolates.

Each value represents the mean of three replicates with standard error.

Toxigenic P. citrinum(P1) produced active α-amylase (0.75 ± 0.01 Units) and this was when potassium nitrate was nitrogen source with maltose as carbon source of the defined growth medium. Toxigenic A. parasiticus(A2) also expressed an α-amylase activity value of 0.72 ± 0.04 Units when rice was both carbon and nitrogen source of medium for fungal growth ( Table 1 ).

Toxigenic A. flavus(A1) produced the most active α-amylase (3.25 ± 0.15 Units) and this was when ammonium sulfate was nitrogen source with starch as carbon source of the defined growth medium. Toxigenic A. flavus(A1) also expressed an α-amylase activity value of 3.02 ± 0.18 Units when starch was carbon source and ammonium chloride was nitrogen source of the defined fungal growth medium ( Table 2 ).

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4. Discussion

The results of this investigation show that the toxigenic strains of A. flavus(A1), A. parasiticus(A2), P. citrinum(P1) and P. rubrum(P2) grew in a synthetic medium with varying carbon and nitrogen sources exhibiting α-amylase activities. α-Amylase activities were detected in the extracts of growth medium with rice as carbon source, infected with the toxigenic strains of A. flavus(A1), A. parasiticus(A2), P. citrinum(P1) and P. rubrum(P2). When the carbon source was varied, potassium nitrate was the nitrogen source. When the nitrogen source was varied, starch was the carbon source for fungal growth. According to Olutiola [135], Aspergillus chevalierifrom moldy maize produced extracellular amylase when grown in a liquid medium containing starch as carbon source. According to Barnett and Fergus [136], increasing the amount of starch-yeast extract medium increased the extracellular amylase produced by Humicola lanuginosa. Studies carried out by Okafor et al. [137] revealed that Lactobacillus delbrueckii, Lactobacillus coryniformisand Saccharomycessp., isolated from cassava processing environments were high amylase producers. Among a series of starch sources of carbon, wheat and soluble starch were inducers of a thermostable amylase by a yeast strain isolated from starchy soil [138]. According to Bluhm and Woloshuk [139], amylopectin, an important constituent of starch, induces fumonisin B(1) production in Fusarium verticillioidesduring colonization of maize. According to Coleman [140], extracellular α-amylase was secreted by Bacillus subtilisin a complex medium containing maltose, starch, glycerol or glucose as carbon source; the general characteristics of secretion indicated a low but definite production of exoenzyme from the moment the cells of the organism started to grow until the end of the logarithmic phase after which, the rate of increase in cell mass decreased, the rate of enzyme secretion increased to a high linear value which was maintained even in the stationary phase.

4.1 Significance of study

Aflatoxin B1-producing-toxigenic strains of Aspergillus flavus, Aspergillus parasiticus, Penicillium citrinumand Penicillium rubrumcan be explored industrially for α-amylase production using the specific growth medium and the rice medium used in this investigation. Varying the specific C and N source of this growth medium is of upmost significance in such an exploration.

4.2 Limitations

The genetic make-ups of these aflatoxin B1-producing-α-amylase-producing fungal strains are important in their ability to produce the enzyme α-amylase. Specific genes are necessary and important in the production of this enzyme. Mutant strains lacking the specific genes for α-amylase production will not be ideal in the exploration for production of the enzyme. More so, there seems to be a significant relationship between the ability to produce α-amylase and aflatoxin B1 production in mycotoxigenic fungi from literature.

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5. Conclusion

The toxigenic strains of A. flavus(A1), A. parasiticus(A2), P. citrinum(P1) and P. rubrum(P2) can be explored in the industrial production of α-amylases.

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Acknowledgments

Authors are thankful to the British Mycology Society (BMS), United Kingdom for Grant supports.

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A. Appendix

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A.1 Acetylation of cellophane tubings [134]

Material

Visking dialysis tubings (Sigma- Aldrich).

Reagents

  1. Aqueous ethanol (50% V/V)

  2. Absolute ethanol

  3. Diethyl ether

  4. A mixture of benzene, acetic anhydride and pyridine in the ratio 5:4:2 (V/V)

  5. 10% KCl (10 g of KCl in 100 ml distilled water)

Procedure

The cellophane tubings were filled with distilled water and soaked in distilled water for 24 hours. The tubings were then soaked in turn, for 30 min each time in 50% ethanol, absolute ethanol and diethyl ether successively. The tubings were thereafter soaked in the mixture of benzene, acetic anhydride, and pyridine, prepared as described above, for 18 hours. Each tubing was then properly rinsed in distilled water and stored in 10% KCl solution at 4°C until required.

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A.2 Protein content determination [131]

Reagents

  1. Reagent A—2% Na2CO3 in 0.1 N NaOH

  2. Reagent B—0.5% CuSO4.5H2O in 1% Sodium Potassium tartrate

  3. Reagent C—50 ml of reagent A mixed with 1 ml of reagent B

  4. Folin-Ciocalteu’s phenol reagent (Sigma-Aldrich Chemie GmbH, Fluka Biochemika) diluted with distilled water in the ratio 1:1(V/V). This is labeled reagent D.

Procedure

5 ml of reagent C was added to 1 ml of the test sample. This was thoroughly mixed and left at room temperature for 10 min. Thereafter, 0.5 ml of reagent D was added and allowed to remain at room temperature for 30 min. Absorbance was determined at 620 nm.

Serial dilutions of Bovine serum albumin (Sigma) were treated likewise and used to plot standard graph. The unknown protein value in each test sample is meant to be extrapolated from the standard graph.

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A.3 Iodine solution

(0.3% Iodine in 3% KI)

Reagents

  1. Iodine

  2. Potassium iodide (KI)

Procedure

3 g of KI was dissolved in 100 ml of warm distilled water. 0.3 g of Iodine was thereafter added and allowed to dissolve in the solution by mixing and warming.

References

  1. 1.Alexopoulos CJ. Introductory Mycology. New York/London: John Wiley and Sons Inc.; 1962. pp. 263-273
  2. 2.Streets RB. Diseases of the Cultivated Plants of the Southwest. Tucson, Arizona: The University of Arizona Press; 1969
  3. 3.Dutta AC. Botany for Degree Students. New Delhi: Oxford University Press; 2007. 708pp
  4. 4.Prescott LM, Harley JR, Klein DA. Microbiology. New York: McGraw Hill; 2005. 992pp
  5. 5.Tortora GJ, Funke BR, Case CL. Microbiology: An Introduction. San Francisco, California: Pearson Education Inc.; 2004. 898pp
  6. 6.Brock DT, Madigan MT. Biology of Microorganisms. Prentice-Hall International Inc.; 1991. 900pp
  7. 7.Brock DT, Madigan MT, Martinko JM, Parker J. Biology of Microorganisms. Eaglewood Cliffs, New Jersey: Prentice-Hall, Inc.; 1994. pp. 528-530
  8. 8.Willey JM, Linda MS, Woolverton CJ. Prescott, Harley and Klein’s Microbiology. New York: McGraw Hill Companies Inc.; 2008. 1088pp
  9. 9.Liang XQ , Holbrook CC, Lynch RE, Guo BZ. Beta-1,3-glucanase activity in peanut seed (Arachis hypogaea) is induced by inoculation withAspergillus flavusand copurifies with a conglutin-like protein. Phytopathology. 2005;95:506-511
  10. 10.Novas MV, Cabral D. Association of mycotoxin and sclerotia production with compatibility groups inAspergillus flavusfrom peanut in Argentina. Plant Disease. 2002;86:215-219
  11. 11.Windham GL, Williams WP.Aspergillus flavusinfection and aflatoxin accumulation in resistant and susceptible maize hybrids. Plant Disease. 1998;82:281-284
  12. 12.Walker RD, White DG. Inheritance of resistance to Aspergillus ear rot and aflatoxin producton of corn from C12. Plant Disease. 2001;85:322-327
  13. 13.Windham GL, Williams WP, Davis FM. Effects of the southwestern corn borer onAspergillus flavuskernel infection and aflatoxin accumulation in maize hybrids. Plant Disease. 1999;83:535-540
  14. 14.Russin JS, Guo BZ, Tubajika KM, Brown RL, Cleveland TE, Widstrom NW. Comparison of kernel wax from corn genotypes resistant or susceptible toAspergillus flavus. Phytopathology. 1997;87:529-533
  15. 15.Chen ZY, Brown RL, Lax AR, Guo BZ, Cleveland TE, Russin JS. Resistance toAspergillus flavusin corn kernels is associated with a 14-KDa protein. Phytopathology. 1998;88:276-281
  16. 16.Olanya OM, Hoyos GM, Tiffany LH. Waste corn as a point source of inoculum forAspergillus flavusin the corn agroecosystem. Plant Disease. 1997;81:576-581
  17. 17.Norton RA. Effect of carotenoids on aflatoxin B(1) synthesis byAspergillus flavus. Phytopathology. 1997;87:814-821
  18. 18.Garber RK, Cotty PJ. Formation of sclerotia and aflatoxins in developing cotton balls infected by the S strain ofAspergillus flavusand potential for biocontrol with an atoxigenic strain. Phytopathology. 1997;87:940-945
  19. 19.Orum TV, Bigelow DM, Cotty PJ, Nelson MR. Using predictions based on geostatics to monitor trends inAspergillus flavusstrain composition. Phytopathology. 1999;89:761-769
  20. 20.McAlpin CE, Wicklow DT, Platis CE. Genotypic diversity ofAspergillus parasiticusin an Illinois corn field. Plant Disease. 1998;82:1132-1136
  21. 21.Brown RL, Chen Y, Cleveland TE, Russin JS. Advances in the development of host resistance in corn to aflatoxin contamination byAspergillus flavus. Phytopathology. 1999;89:113-117
  22. 22.Moore KG, Price MS, Boston RS, Weissinger AK, Payne GA. A chitinase from Tex 6 maize kernels inhibits growth ofAspergillus flavus. Phytopathology. 2004;94:82-87
  23. 23.Sirois KL, Loparco DP, Lobeer JW. Systemic infection of onion seedlings byAspergillus nigerandFusariumsp. Phytopathology. 1999;89:S73. Publication No: P-1999-0520-AMA
  24. 24.Sirois KL, Lorbeer JW, Holcomb MA. Onion seed infection levels subsequent to sequential exposure of onion seed stalk and flower parts toAspergillus niger. Phytopathology. 1999;89:S73. Publication No.: P-1999-0521-AMA
  25. 25.Doster MA, Michailides TJ, Morgan DP.Aspergillus speciesand mycotoxins in Figs from California orchards. Plant Disease. 1996;80:484-489
  26. 26.Thompson JR, Latorre BA. Characterization ofBotrytis cinereafrom table grapes in Chile using RAPD-PCR. Plant Disease. 1999;83:1090-1094
  27. 27.Shellie KC, Skaria M. Reduction of green mold on grape fruit after hot force-air quarantine treatment. Plant Disease. 1998;82:380-382
  28. 28.Zhang J, Swingle PP. Effects of curing on green mold and stem-end rot of citrus fruit and its potential application under Florida packing system. Plant Disease. 2005;89:834-840
  29. 29.Conway WS, Leverentz B, Saftner RA. Survival and growth ofListeria monocytogeneson fresh-cut apple slices andPencillium expansum. Plant Disease. 2000;84:177-181
  30. 30.Spotts RA, Cervantes LA. Disease incidence-inoculum close relationships forBotrytis cinereaandPenicillium expansumand decay of pear fruit using dry, air borne conidia. Plant Diesease. 2001;85:755-759
  31. 31.Tian S, Fan Q , Xu Y, Liu H. Biocontrol efficacy of antagonist yeasts to gray mold and blue mold on apples and pears in controlled atmospheres. Plant Disease. 2002;86:848-853
  32. 32.Meyer UM, Spotts RA. Detection and quantification ofBotrytis cinereaby ELISA in pear stems during cold storage. Plant Disease. 2000;84:1099-1103
  33. 33.Palou L, Smilanick JL, Crisostco CH, Mansour M. Effects of gaseous ozone exposure on the development of green and blue molds on cold stored citrus fruit. Plant Disease. 2001;85:632-638
  34. 34.Palou L, Smilanick JL, Usall J, Vinas I. Control of post harvest blue and green molds of oranges by hot water, sodium carbonate and sodium bicarbonate. Plant Disease. 2001;85:371-376
  35. 35.Smilanick JL, Mackey BE, Reese R, Usall J, Margosan DA. Influence of concentration of soda ash, temperature and immersion period on the control of post harvest green mold of oranges. Plant Disease. 1997;81:379-382
  36. 36.Smilanick JL, Michael IF, Mansour MF, Mackey BE, Margosan DA, Flores D, et al. Improved control of green mold of citrus with imazalil in warm water compared with its use in wax. Plant Disease. 1997;81:1299-1304
  37. 37.Sugar D, Spotts RA. Control of postharvest decay in pear by four laboratory-grown yeasts and two registered biocontrol products. Plant Disease. 1999;83:155-158
  38. 38.Janisiewicz WJ, Peterson DL. Susceptibility of the stem pull area of mechanically harvested apples to blue mold decay and its control with a biocontrol agent. Plant Disease. 2004;88:662-664
  39. 39.Spotts RA, Holz G. Adhesion and removal of conidia ofBotrytis cinereaandPenicillium expansumfrom grape and plum fruit surfaces. Plant Disease. 1996;80:688-691
  40. 40.Smilanick JL, Aiyabei J, Gabler FM, Doctor J, Sorenson D, Mackey B. Quantification of the toxicity of the aqueous chlorine to spores ofPenicillium digitatumandGeotrichum citri-aurantii. Plant Disease. 2002;86:509-514
  41. 41.Janisiewicz WJ, Peterson DL, Yoder KS, Miller SS. Experimental bin drenching system for testing biocontrol agents to control post harvest decay of apple. Plant Disease. 2005;89:487-490
  42. 42.Spotts RA, Cervantes LA, Mielke EA. Variability in post harvest decay among apple cultivars. Plant Disease. 1999;83:1051-1054
  43. 43.Sugar D, Benbow JM, Powers KA, Basile SR. Orchard sprays on post harvest decay of pear. Plant Disease. 2003;87:1260-1262
  44. 44.Smilanick JL, Mansour MF, Sorenson D. Pre- and post harvest treatments to control green mold of citrus fruit during ethylene degreening. Plant Disease. 2006;90:89-96
  45. 45.Pianzzola MJ, Moscatelli M, Vero S. Characterization of Penicillium isolates associated with blue mold on apple in Uruguay. Plant Disease. 2004;88:23-28
  46. 46.Bohinski RC. Modern Concepts in Biochemistry. 4th ed. Boston/London/Sydney/Toronto: Allyn and Bacon Inc.; 1983. 531pp
  47. 47.Reddy NS, Nimmagadda A, Sambasiva Rao KRS. An overview of the microbial α-amylase family. African Journal of Biotechnology. 2003;2(12):645-648
  48. 48.Dixon M, Webb EC. Enzymes. London: Longmans; 1971. 950pp
  49. 49.Robyt JF. Enzymes in hydrolysis and synthesis of starch. In: Starch Chemistry and Technology. New York: Academic Press; 1984. pp. 88-90
  50. 50.Najafi MF, Kembhavi A. One step purification and characterization of an extracellular α-amylase from marineVibriosp. Enzyme and Microbial Technology. 2005;36(4):535-539
  51. 51.Rahardjo YSP, Jolink F, Haemers S, Tramper J, Rinzema A. Signficance of bed porosity, bran and specific surface area in solid-state cultivation ofAspergillus oryzae. Biomolecular Engineering. 2005;22(4):133-139
  52. 52.Wu YB, Ravindran V, Pierce J, Hendriks WH. Influence of three phytase preparations in broiler diets based on wheat or corn:In vitromeasurement of nutrient release. International Journal of Poultry Science. 2004;3(7):450-455
  53. 53.Ajayi AA, Adejuwon AO, Olutiola PO. Extracellular α-amylase production by the yam (Discoreaspp.) rot organism,Penicillium sclerotigenumYamamoto Science. Focus. 2005;10(3):197-203
  54. 54.Douzdijian V, Gugliuzza KK. The impact of midline versus transverse incisions on wound complications and outcome in simultaneous pancreas-kidney transplant: A retrospective analysis. Transplant International. 1996;9(1):62-67
  55. 55.Hashim SO, Delgado OD, Martinez MA, Kaul RH, Mulaa FJ, Mattiasson B. Alkaline active maltohexaose-forming α-amylase fromBacillus haloduransLBK 34. Enzyme and Microbial Technology. 2005;36(1):139-146
  56. 56.Hemker M, Stratmann A, Goeke K, Schroder W, Lentz J, Piepersberg W, et al. Identification, cloning, expression and characterization of the extracellular acarbose-modifying glycosyltransferase, AcbD fromActinoplanessp. strain SE 50. Journal of Bacteriology. 2001;183(15):4484-4492
  57. 57.Oh KW, Kim MJ, Kim HY, Kim BY, Baik MY, Auh JH, et al. Enzymatic characterization of a maltogenic amylase fromLactobacillus gasseriATCC 33323 expressed inEscherichia coli. FEMS Microbiology Letters. 2005;252(1):175-181
  58. 58.Papi RM, Chaitidou SA, Trikka FA, Kyriakids DA. EncapsulatedEscherichia coliin alginate beads capable of secreting a heterologous pectin lyase. Microbial Cell Factories. 2005;4:35
  59. 59.Lehninger AL. Principles of Biochemistry. New York: Worth Publishers, Inc.; 1982. 1011pp
  60. 60.Howling D. Mechanisms of starch enzymolysis. International Biodeterioration. 1989;25:15-19
  61. 61.Vihinen M, Mantsala P. Microbial amylolytic enzymes. Critical Reviews in Biochemistry and Molecular Biology. 1989;24(4):329-418
  62. 62.Uguru GC, Akinyanju JA, Sani A. The use of sorghum for thermostable amylase production fromThermoactinomyces thalpophilus. Letters in Applied Microbiology. 1997;25:13-16
  63. 63.Ajayi AO, Fagade OE. Utilization of corn starch as substrate for β-amylase byBacillusspp. African Journal of Biomedical Research. 2003;6(1):37-42
  64. 64.Reiss M, Heibges A, Metzger I, Hartmeier W. Determination of BOD-values of starch containing waste water by a BOD-biosensor. Biosensors and Bioelectronics. 1998;13(10):1083-1090
  65. 65.Ampe F, Ben Omar N, Guyot JP. Recovery of total microbial RNA from lactic acid fermented foods with a high starch content. Letters in Applied Microbiology. 1998;27(5):270-274
  66. 66.Doelle HW. Socio-economic microbial process strategies for a sustainable development using environmentally clean technologies: Sagopalm a renewable resource. Livestock Research for Rural Development. 1998;10(1):23-32
  67. 67.Rivera MH, Lopez-Munguia A, Soberon X, Saab-Rincon G. Alpha-amylase fromBacillus licheniformismutants near to the catalytic site: Effects on hydrolytic and transglycosylation activity. Protein Engineering. 2003;16(7):505-514
  68. 68.Zangrando FEL, Yoko HE. Culture media for amylase production by toxigenic fungi. Brazilian Archives of Biology and Technology. 2000;43(5):461-467
  69. 69.Tanaka T, Hoshina M, Tanabe S, Sakai K, Ohtsubo S, Taniguchi M. Production of d-lactic acid from defatted rice bran by simultaneous saccharification and fermentation. Bioresource Technology. 2005;97(2):211-217
  70. 70.Sejedi RH, Naderi-Manesh H, Khajeh K, Ahmadvand R, Ranjbar B, Asoodeh A, et al. A Ca-independent α-amylase that is active and stable at low pH from theBacillussp. KR-8104. Enzyme and Microbial Technology. 2005;36(5-6):666-671
  71. 71.Rodriguez VB, Alamenda EJ, Gellegos JFM, Lopez AIG. Thermal deactivation of a commercial α-amylase fromBacillus licheniformisused in detergents. Biochemical Engineering Journal. 2006;27(3):299-304
  72. 72.Mijts B, Patel BKC. Cloning, sequencing and expression of an alpha-amylase gene, amy A, from the thermophilic halophileHalothermothrix oreniiand purification and biochemical characterization of the recombinant enzyme. Microbiology. 2002;148:2343-2349
  73. 73.Shaw JF, Lin FP, Chen SC, Chen HC. Purification and properties of an extracellular α-amylase fromThermussp. Botanical Bulletin of Academia Sinica. 1995;36:195-200
  74. 74.Yutani K. Molecular weight of thermostable α-amylase fromBacillus stearothermophilus. Journal of Biochemistry. 1973;74:581-586
  75. 75.Saito N. A thermophilic extracellular α-amylase fromBacillus licheniformis. Archives of Biochemistry and Biophysics. 1973;155:290-298
  76. 76.Manning GB, Campbell LL. Thermostable α-amylase ofBacillus stearothermophilus. I. Crystallization and some general properties. The Journal of Biological Chemistry. 1961;236(11):2952-2957
  77. 77.Huang LP, Jin B, Lant P, Zhou J. Simultaneous saccharification and fermentation of potato starch waste water to lactic acid byRhizopus oryzaeandRhizopus arrhizus. Biochemical Engineering Journal. 2005;23:265-276
  78. 78.Ogashara K, Imanishi A, Isemura T. Studies on thermophilic α-amylase fromBacillus stearothermophilus. I. Some general and physico-chemical properties of thermophilic α-amylase. The Journal of Biochemistry. 1970;74:581-596
  79. 79.Synowiecki J, Grzybowska B, Zdzieblo A. Sources, properties and suitability of new thermostable enzyme in food processing. Critical Reviews in Food Science and Nutrition. 2006;46(3):197-205
  80. 80.Peng P, Wu J, Cheng AC, Gao QY, Zhang SZ. Cloning and expression of the alpha-amylase gene from aBacillussp. WS06 and characterization of the enzyme. Wei Sheng Wu Xue Bao. 2005;45(6):876-880
  81. 81.Yun J, Kang S, Park S, Yoon H, Kim MJ, Heu S, et al. Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library. Applied and Environmental Microbiology. 2004;70(12):7229-7235
  82. 82.Bessler C, Schmitt J, Maurer KH, Schmid RD. Directed evolution of a bacterial alpha-amylase: Towards enhanced pH-performance and higher specific activity. Protein Science. 2003;12(10):2141-2149
  83. 83.Dey S, Agarwal SO. Characterization of a thermostable alpha-amylase from a thermophilicStreptomyces megasporusstrain SD12. Indian Journal of Biochemistry and Biophysics. 1999;36(3):150-157
  84. 84.Burgess-Cassler A, Imam SH, Gould JM. High-molecular weight amylase activities from bacteria degrading starch-plastic films. Applied and Environmental Microbiology. 1991;57(2):612-614
  85. 85.Heese O, Hansen G, Hohne WE, Korner D. A thermostable alpha amylase fromThermoactinomyces vulgaris.Purification and characterization. Biomedica Biochimica Acta. 1991;50(3):225-232
  86. 86.Akindahunsi AA. Physico-chemical studies on amylases from fermented cassava waste water. In: Scientific Preprints Automized List. Trieste, Italy: The Abdus Salam International Centre for Theoretical Physics; 2001
  87. 87.Dey G, Palit S, Banerjee R, Maiti BR. Purification and characterization of malto oligosaccharide-forming amylase fromBacillus circulansGRS 313. Journal of Indian Microbiology and Biotechnology. 2002;28(4):193-200
  88. 88.Doman K, Ryu SJ, Heo SJ, Kim DW, Kim HS. Characterization of a novel carbohydrase fromLipomyces starkeyiKSM 22 for dental application. Journal of Microbiology and Biotechnology. 1999;9(3):260-264
  89. 89.Jana M, Chattopadhyay DJ, Patti BR. Thermostable, high salt-tolerant amylase fromBacillus megateriumVUMB-109. Acta Microbiologica et Immunologica Hungarica. 1997;44(3):281-289
  90. 90.Ohno N, Fukuda H, Wang H, Kasamura M, Shinoyama H, Fuji T. Amylases produced by a thermophilic fungus.Thermoascus aurantiacusand some of their properties. Sebutsu-Kogaku Kaishi. 1998;76(3):111-117
  91. 91.Shekhar HU, Ali MM, Hossain MA. Extracellular amylase production by locally isolated mosquito-pathogenicBacillus sphaericusN11. Bangladesh Journal of Microbiology. 1997;14(1-2):17-23
  92. 92.Falih AM. Effect of heavy-metals on amylolytic activity of the soil yeastsGeotrichum capitatumandGeotrichum candidum. Bioresource Technology. 1998;66(3):213-217
  93. 93.Bernhardsdotter ECMJ, Ng JD, Garriott DK, Pusey ML. Enzymatic properties of an alkaline, chelator-resistant alpha-amylase from an alkaliphilicBacillussp. isolate L1711. Process Biochemistry. 2005;40:2401-2408
  94. 94.Mishra S, Noronha SB, Suraishkumar GK. Increase in enzyme productivity by induced oxidative stress inBacillus subtiliscultures and analysis of its mechanism using microarray data. Process Biochemistry. 2005;40(5):1863-1870
  95. 95.El-Said AH, Abdel-Hafez SI, Saleem A. Effect of herbizid and touchdown of some extracellular enzymes. Acta Microbiologica et Immunologica Hungarica. 2005;52(1):105-130
  96. 96.Figueira ELZ, Hirooka EY, Mendiola-Olanya E, Blanco-Labra A. Characterization of a hydrophobic amylase inhibitor from corn (Zea mays) seeds with activity against amylase fromFusarium verticilliodies. Phytopathology. 2002;93:917-922
  97. 97.Figueira ELZ, Ono EYZ, Mendiola-Olanya E. New amylase inhibitor present in corn seeds activein vitroamylase fromFusarium verticilliodes. Plant Disease. 2002;87:233-240
  98. 98.Hofmann O, Vertesy L, Braunitzer G. The primary structure of alpha-amylase inhibitor Z-2685 fromSteptomyces parvullusFH-1641. Sequence homology between inhibitor and alpha-amylase. Biological Chemistry Hoppe-Seyler. 1985;366(12):1161-1168
  99. 99.Vertesy L, Tripier D. Isolation and structure elucidation of an alpha-amylase inhibitor, A1-3688, from Streptomyces aureofaciens. FEBS Letters. 1985;185(1):187-190
  100. 100.Su YC, Chiu RJ, Yu N, Chang WR. The microbial production of amylase inhibitor and its application. I. Isolation and cultivation ofStreptomyces nigrifaciensNTU-3314. Proceedings of the National Science Council, Republic of China. 1984;B8(4):292-301
  101. 101.Lin J, Lu C, Yu IT, Wu JF. Effects of starch sources on the production and activity of amylase fromBacillus subtilis. Journal of the Agricultural Association of China. 1999;185:22-31
  102. 102.Yin XH, Gerbaud C, Francou FX, Guerineau M, Virolle MJ. Ami C, another amylolytic gene maps close to the ami B locus inStreptomyces lividansTK 24. Gene (Amsterdam). 1998;215(1):171-180
  103. 103.Lo HF, Lin LL, Chiang WY, Chie MC, Hsu WH, Chang CT. Deletion analysis of the C-terminal region of the alpha-amylase ofBacillussp. strain TS-23. Archives of Microbiology. 2002;178(2):115-123
  104. 104.Bahl H, Burchhardt G, Spreinat A, Haeckel K, Wienecke A, Schmidt B. Alpha-amylase ofClostridium therosulfurgenesEMI: Nucleotide sequence of the gene, processing of the enzyme and comparison of alpha amylases. Applied and Environmental Microbiology. 1991;57(5):1554-1559
  105. 105.Kang DO, Hwang IK, Kim BY, Ahn SC, Mheen TI, Ahn JS, et al. Secretion of Bacillus alpha-amylase from yeast directed by glucoamylase I signal sequence ofSaccharomyces diastaticus. Biochemistry and Molecular Biology International. 1996;39(1):181-190
  106. 106.Lorentz K. Routine alpha-amylase assay using protected 4-nitrophenyl-1,4-alpha-D-maltoheptaoside and a novel alpha-glucoside. Clinical Chemistry. 2000;46:644-649
  107. 107.Akpan I, Bankole M, Adesemowo AM. A rapid plate culture method for screening of amylase producing microorganisms. Biotechnology Techniques. 1999;13(6):411-413
  108. 108.Higuchi Y, Ohashi A, Imachi H, Harada H. Hydrolytic activity of alpha-amylase in anaerobic digested sludge. Water Science and Technology. 2005;52(1-2):259-266
  109. 109.Uhlig H. Industrial Enzymes and their Applications. New York: Wiley and Sons; 1998. 472pp
  110. 110.Pandey A, Nigman P, Soccol CR, Soccol VT, Singh D, Mohan R. Advances in microbial amylase. Biotechnology and Applied Biochemistry. 2000;31(2):135-152
  111. 111.Mellon JE, Cotty PJ, Dowd MK.Aspergillus flavushydrolases: Their roles in pathogenesis and substrate utilization. Applied Environmental Microbiology and Biotechnology. 2007;77:497-504
  112. 112.Jallow EAA, Twumasi P, Mills-Robertson FC, Dumevi R. Assessment of aflatoxin-producing fungi strains and contamination levels of aflatoxin B1 in groundnut, maize, beans and rice. Journal of Agricultural Science and Food Technology. 2018;4(4):71-79
  113. 113.Liu J, Sun L, Zhang N, Zhang J, Guo J, Li C, et al. Effects of nutrients in substrate of different grains on aflatoxin B1 production byAspergillus flavus. Biomedical Research International. 2016;2016:10. Article ID 7232858.http://dx.doi.org/10.1155/2016/7232858
  114. 114.Kim HR, Kim JH, Bai DH, Ahn BH. Identification and characterization of useful fungi with α-amylase activity from Korean traditional nuruk. Microbiology. 2011;39(4):278-282
  115. 115.Fakhoury A, Woloshuk CR. Amy1, the α-amylase gene ofAspergillus flavus: Involvement in aflatoxin biosynthesis in maize kernels. Phytopathology. 1999;89(1):908-914
  116. 116.Figueira ELZ, Hirooka EY. Culture medium for amylase production by toxigenic fungi. Brazilian Archives of Biology and Technology. 2000;43(5):461-467
  117. 117.Rajasekaran K, Sayler RJ, Majumdar R, Sickler CM, Cary JW. Inhibition ofAspergillus flavusgrowth and aflatoxin production in transgenic maize expressing the α-amylase inhibitor fromLablab purpureusL. Journal of Visualized Experiments. 2019;144:e59169. DOI: 10.3791/59169
  118. 118.Fountain JC, Scully BT, Ni X, Kemerait RC, Lee RD, Chen ZY, et al. Environmental influences on maize-Aspergillus flavusinteractions and aflatoxin production. Frontiers in Microbiology. 2014;5:40. DOI: 10.3389/fmicb.2014.00040
  119. 119.Espindola AS, Schneider W, Cardwell KF, Carrillo Y, Hoyt PR, Marek SM, et al. Inferring the presence of aflatoxin-producingAspergillus flavusstrains using RNA sequencing and electronic probes as a transcriptomic screening tool. PLoS One. 16 Oct 2018;13(10):e0198575. DOI: 10.1371/journal.pone.0198575
  120. 120.Yu J, Mohawed SM, Bhatnagar D, Cleveland TE. Substrate-induced lipase gene expression and aflatoxin production inAspergillus flavus. Journal of Applied Microbiology. 2003;95:1334-1342
  121. 121.Smith CA, Woloshuk CP, Robertson D, Payne GA. Silencing of the aflatoxin gene cluster in a diploid strain ofAspergillus flavusis suppressed by ectopic aflR expression. Genetics. 2007;176(4):2077-2086
  122. 122.Stern KR, Jansky S, Bidlack JE. Introductory Plant Biology. New York: McGraw Hill Higher Education; 2003. 624pp
  123. 123.Sizer FS, Whitney EN. Nutrition: Concepts and Controversies. London: Wadsworth, Thomson Learning; 2000. 567pp
  124. 124.Mauseth JD. Botany: An Introduction to Plant Biology. London: Saunders College Publishing; 1995. 794pp
  125. 125.Leventin E, McMahon K. Plants and Society. New York: WCB McGraw Hill; 1999. 477pp
  126. 126.Graham LE, Graham JM, Wilcox LW. Plant Biology. Upper Saddle River: Pearson Education, Inc.; 2006. 670pp
  127. 127.Cannon PF, Kirk PM. Fungal Families of the World. Wallingford, Oxfordshire: CAB International Publishing; 2007. 4556pp
  128. 128.Hanlin RT. Illustrated Genera of Ascomycetes. St. Paul, Minnesota: American Phytopathological Society Press; 1990. 263pp
  129. 129.Olutiola PO, Ayres PG. Utilization of carbohydrates byRhynchosporium secalis.I. Growth and sporulation on glucose, galactose and galacturonic acid. Physiologia Plantarum. 1973;29:92-96
  130. 130.Olutiola PO, Nwaogwugwu RI. Growth, sporulation and production of maltase and proteolytic enzymes inAspergillus aculeatus. Transactions of the British Mycological Society. 1982;78(1):105-113
  131. 131.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry. 1951;193:265-275
  132. 132.Pfueller SL, Elliott WH. The extracellular α-amylase ofBacillus stearothemophilus. Journal of Biological Chemistry. 1969;244:48-54
  133. 133.Xiao Z, Storms R, Tsang A. A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Analytical Biochemistry. 2006;351(1):146-148
  134. 134.Whitaker DR, Hanson KR, Datta PK. Improved procedure for purification and characterization of Myrothecium cellulase. Canadian Journal of Microbiology and Physiology. 1963;41:671-696
  135. 135.Olutiola PO. α-Amylase activity ofAspergillus chevalierifrom mouldy maize. Indian Phytopathology. 1982;35(3):428-433
  136. 136.Barnett EA, Fergus CL. The regulation of extracellular amylase mycelium and time, in some thermophilic and mesophilicHumicolaspecies. Mycopathologia et Mycologia Applicata. 1971;44(2):131-141
  137. 137.Okafor N, Umeh C, Ibenugbu C. Amelioration of garri, a cassava based fermented food by the inoculation of microorganisms secreting amylase, lysine and linamarase into cassava mash. World Jounal of Microbiology and Biotechnology. 1998;14(6):835-838
  138. 138.Fossi BT, Tavea F, Ndjouenkeu R. Production and partial characterization of a thermostable amylase from ascomycetes yeast strain isolated from starchy soils. African Journal of Biotechnology. 2005;4(1):14-18
  139. 139.Bluhm BH, Woloshuk CP. Amylopectin induces fumonisin B(1) production byFusarium verticilliodesduring colonization of maize kernels. Molecular Plant-Microbe Interactions. 2005;18:1333-1339
  140. 140.Coleman G. Studies on the regulation of extracellular enzyme formation byBacillus subtilis. Journal of General Microbiology. 1967;49:421-431

Written By

Adekunle Odunayo Adejuwon and Victoria Anatolyivna Tsygankova

Submitted: April 12th, 2019Reviewed: May 2nd, 2019Published: June 3rd, 2020