amylolytic yeasts: producers of α-amylase and pullulanase

Int. J. Life. Sci. Scienti. Res., 2(4): 339-354 (ISSN: 2455-1716) Impact Factor 2.4 JULY-2016

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Amylolytic Yeasts: Producers of α-amylase and Pullulanase

Djekrif D. S1*, Gillmann L2, Bennamoun L1, Ait-Kaki A1, Labbani K1, Nouadri T1, Meraihi Z1

1Research lab in Microbiology Engineering, Faculty of Biology, Biopole Chaab Erssas, University, Constantine, Algeria 2SONAS-IUT laboratory, University of Angers, Angers, France *Address for Correspondence: Djkrif-Dakhmouche Scheherazed, Lecturer, Department of Natural science and Life

(Biology), University of Frères Mentouri, Constantine, Algeria Received: 01 May 2016/Revised: 31 May 2016/Accepted: 22 June 2016

ABSTRACT- Amylases are among the most important enzymes used in various industries. They represent approximately 30% of the world enzyme production. These are of ubiquitous occurrence and hold the maximum market share of enzyme sales. These comprise hydrolases, which hydrolyze starch to diverse products as dextrins, and progressively smaller polymers composed of glucose units. They are highly demanded in various arrays such as food, pharmaceuticals, textiles, detergents, etc. However, enzymes from mold and bacterial source have dominated applications in industrial sectors while few species of yeast were studied for the amylases production. This review focuses on the amylolytic yeasts and their enzymes and we were interested at α- amylase and pullulanase, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. Key-words- Amylolytic yeast, α-amylase, Pullulanase, Industrial application

-------------------------------------------------IJLSSR----------------------------------------------- INTRODUCTION Different amylases catalyze the enzymatic hydrolysis of starch as α -amylases (EC3.2.1.1), glucoamylases (EC 3.2.1.3), β-amylases (EC 3.2.1.2.) and debranching enzymes such as pullulanases (EC 3.2.1.41). However, with the advances in biotechnology, the amylase application has expanded in many fields such as clinical, medicinal and analytical chemistry, as well as their widespread application in starch saccharification and in the textile, food, detergent, brewing and distilling industries [1-2]. Although they can be derived from several sources, such as plants, animals and microorganisms, fungi and bacteria have been extensively screened for amylases production [1]. Recently, some amylases from yeasts have been found to have the ability to hydrolyze starch: α-amylase and glucoamylase from Schwanniomyces castelli [3], glucoamylases from S. fibuligera [4] and Candida antarctica [5], α-amylase from Cryptococcus sp. S-2 [6],

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Candida lusitaniae, Candida famata [7], amylopullulanase from Clavispora lusitaniae ABS7 [8] and pullulanase from Aureobasidium pullulans [9]. A number of reviews exist on bacterial and fungal amylases and their applications, however, none specifically covers yeast amylases. α-amylases and pullulanase are one of the most popular and important form of industrial amylases and the present review highlights the various aspects of these yeast amylases. Industrial use yeasts or their enzymes Yeasts are used by humans for thousands of years with wide application, both fundamental and industrial, in science, food, medical and agricultural. Yeasts are traditionally involved in many food fermentations and manufacturing products such as beers, ciders, wines, sake, baked goods, cheese, sausages and other fermented foods. Industrial processes involve long since yeast in the production of fuel ethanol from single cell protein (SCP) for animal feed or industrial enzymes, vaccine production and carotenoids [10-11] (Table 1). The yeast extract is an important nutrient (nitrogen source and intake of essential vitamins group B), particularly favorable to the growth of most microorganisms. Yeasts are also involved in the development of agricultural and industrial waste for the production of proteins, enzymes and "SPC" [12]. The enzymes of yeast are increasingly

Review Article (Open access)

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 4


used in industries to facilitate the process and reduce the energy cost of the finished product particularly in the food industry. The search for new yeast enzymes having an industrial application potential continues to grow. Yeasts such as Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha are currently used for the industrial production of proteins and enzymes, including pharmaceutical proteins [13]. The yeast Yarwinia lipolytica and Rhodotorula glutinis are used due to their ability to produce lipase in petroleum industries, laundry, detergent industry and the IAA [14]. Table 1: Industrial Enzymes produced by yeasts [13,15-16] Enzyme Yeasts Industry Chymosine Klyveromyces sp. Food processing Saccharomyces

cerevisiae

Galactosidase Saccharomyces sp. Food applications Glutaminase Zygosaccharomyces

rouxii Therapeutic analysis

Inulinases Candida sp. Food applications Klyveromyces

marxianus

Invertase Saccharomyces cerevisiae

Food applications Lactase Candida

pseudotropicalis Food processing

Klyveromyces sp. Lipase Candida rigosa Food processing Pseudozyma

antarctica Aromas

Trichosporon fermentum

Dry cleaning Detergents

Phenylalanine Rhodotorula sp. Pharmaceutical Ammonialyase Rhodosporidium sp. Phenylalanine Candida boidinii Pharmaceutical deshydrogenase Phytase Ogataea polymorpha Nutritional feed In our study, the focus will be on the production of amylolytic enzymes in particular, α-amylase and pullulanase in yeast Clavispora lusitaniae. To date, few studies have been conducted on yeast Clavispora lusitaniae. Amylolytic yeast For their ease of cultivation, amylolytic yeast has attracted the interest of researchers for their application in the bio-industries [17]. Amylolytic yeast can produce different amylolytic enzymes (Table 2). For this reason, their use in enzyme production is increasingly in demand [17]. Furthermore, the excretion amylases depend on the composition of the culture medium [18-19].

Producing yeast extracellular amylase The extracellular amylases of yeast origin are very few listed: Cryptococcus heimaeyensis HA7 [20], Trichosporon pullulans, Saccharomycopsis bispora, Saccharomycopsis capsularis, Saccharomycopsis fibuligera (Endomycopsis fibuligera) [4], Lipomyces starkeyi NCYC 1436 [21], Candida sp. [22] and Candida parapsilosis, Candida glabrata, Rhodotorula mucilaginosa [23] (Table 2). Unlike amylases, few studies are carried out on the production of pullulanase by the yeast. However, since the 80s, the pullulanase activity is demonstrated in Candida, Cryptococcus, Pichia, Schwanniomyces, Torulopsis, Lipomyces, Trichosporon, Endomycopsis, Leucosporidium and Filobasidium [24], Aureobasidium pullulans [9]. Amylolytic system in yeast The amylolytic system of yeast is very diversified: the major enzymes for this system are the α-amylase, glucoamylase, pullulanase and cyclodextrinase [3,7,9,29,54]. Amylolytic enzymes and thermostability The industrial application of amylolytic enzymes requires thermostable enzymes whose optimum temperature is at or above 70°C. Today, the annual market thermostable enzymes represent about $ 250 million and thermostable amylases occupy much [55]. In recent years, much research has been done on the production of amylases by thermophilic microorganisms [56]. Their use in industrial processes offers the advantages of reducing the risk of infection; reduce the reaction time, the cost of external cooling [57-59] and to increase the diffusion rate [60]. The main amylolytic enzymes used in the starch industry are the α-amylase, β-amylase, glucoamylase, pullulanase, maltase and α-1,6 glucosidase. Most commercial amylases are of bacterial origin [61-62] for their interest in the saccharification of starch for the production of glucose, maltose, maltotriose and dextrins [61-62]. Also, it would be judicious to find yeast mixed production of amylase and pullulanase thermostable capable of hydrolyzing the α-1, 4 bonds of starch and amylose and α-1, 6 of pullulan and branched polysaccharides. This pair of endo-amylase is known as the "amylo-pullulanase" [63-65].


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Table 2: Production of amylolytic enzymes and other by yeasts

Yeast Amylolytic yeast References Filobasidium capsuligenum α-amylase, glucoamylase [25]

Schwanniomyces castelli α-amylase, glucoamylase [3,26] Candida utilis,

Candida guilliermondi Candida famata

Trichosporon mucoides

α-amylase [7]

C. famata et C. guilliermondii Glucoamylase [27] Candida edax α-amylase, glucoamylase [28]

Candida antartica CBS 6678 α-amylase, glucoamylase [29] C.antartica et C. rugosa Lipases [30]

Wickerhamia sp. α-amylase [31] Saccharomycopsis capsularis α-amylase, glucoamylase [32]

Trichosporon pullulans α-amylase, glucoamylase [33] schwanniomyces alluvius α-amylase [34]

Schwanniomyces occidentalis α-amylase [35] [36] Invertase

Cryptococcus flavus α-amylase [37] Cryptococcus sp. S-2 α-amylase [6]

Saccharomyces diastaticus et Endomycopsis capsularis

Glucoamylase [38] Aureobasidium pullulans Pullulanase [9]

Glucoamylase [39] α-glucosidase, α-amylase [40]

Amylase [41] Cellulase [42]

Lipomyces kononenkoae α-amylase, glucoamylase [12] Pichia burtonii α-amylase [43]

Candida guilliermondii

α-amylase Inulinase

[44] [45]

Esterase [46] Saccharomyces cerevisiae α-amylase [44]

Saccharomycopsis fibuligera α-amylase, glucoamylase [4] Pichia burtonii 15-1 α-amylase [47]

Kluyveromyces fibuligera Inulinase and Pectinase [48-49] Yarrowia lipolytica Lipase [50]

Clavispora lusitaniae α-amylase [7] β-glucosidase

Amylopullulanase [51] [8]

Phytase [52] Naringinase (α-L-rhamnosidase

et β-D-glucosidase) [53]

2.1. Classification of glycoside hydrolases (Amylases) According to [2], [66] and the International Union of Biochemistry and Molecular Biology (UIBMB), the glycoside hydrolases (GH) are classified into three groups according to their mode of action (Figure 1): The endoamylases which hydrolyze α-1,4 linkages of the amylose and amylopectin (the two components of starch) thereby releasing oligosaccharides and dextrins. In this group, we find mainly the α-amylase (EC 3.2.1.1.) The exoamylase, they include the β-amylase (EC 3.2.1.2), the α-glucosidase (EC 3.2.1.20) and glucoamylase or amyloglucosidase (EC 3.2.1.3). Their action releases sugars of low molecular weights such as glucose, maltose and oligosaccharides. The debranching enzymes, they, hydrolyze the α-1,6 bonds of amylopectin. The pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68) belong to this group. The synergistic action found in amylolytic complexes is beneficial for the total hydrolysis of starch, pullulan and branched polysaccharides.



Figure 1: Modes of action of amylolytic enzymes [67] 2.2. Structure of family enzymes amylase Amylases belong to the family 13 glycoside hydrolases of (GH) [68]. The α-amylase and pullulanase have three domains A, B and C in their spatial structure, (Figure 2):

a bFigure 2: Structure of amylases a: Structure of the α-amylase [1]

The area A is shown in red, yellow domain B and the C purple. In the catalytic center, the Ca2+ Ion, shown in blue sphere and chloride ion in the Yellow sphere Green structures are linked to the active site and surface binding sites b: Monomeric structure of the pullulanase of Klebsiella pneumoniae



Domain A is the longest and contains the active site and the substrate binding site. It has the shape of a cylinder called a TIM barrel [58,69] and contains the amino acids Glu and Asp catalysis [70]. These two amino acids also play an important role in heat resistance [71]. Domain B has an irregular structure (rich in β sheets) and varies according to the family of amylase [72]. He is involved in the binding of Ca2+ ions play a structural role and contribute to the stability of the enzyme [58,73-74]. Calcium is also essential to preserve the enzyme from the attack of proteases [1,75]. The C domain, C-terminal side and a so-called sandwich structure of β sheets [76-77] who participates in post-translational folding of the pancreatic amylase rat, guaranteeing and the activity and the secretion of this enzyme [78]. Like all enzymes, α-amylases and pullulanases are glycoproteins [79-80] structure, generally, but some may be monomeric or tetrameric dimeric [81]. The glycosylated portion enzymes protects against enzyme denaturation and proteolysis [82]. The heat resistance of amylase can be partly explained by their high acidic amino acids (Asp and Glu) [71]. The thiol function of the α-amylase is not present in the active site [83] but involved in calcium binding to the enzyme [82]. 2.3. α-amylase This is a glycosidase (EC 3.2.1.1) which breaks osidic α-1, 4 bonds of polysaccharides (starch and glycogen) releasing glucose, maltose and maltodextrins soluble variable size [74]. These maltodextrins contain branch points because the enzyme cannot hydrolyze the branch points α-1,6 [84]. The classic substrate for the α-amylase is starch consisting of amylose and amylopectin (Figure 3):

A: Structure of amylose

B: Structure of amylopectine Figure 3: Structure of Starch, A: Amylose, B: Amylopectine

Amylose is a linear polymer consisting of a maximum of 6000 glucose units with α-1, 4 glycosidic linkages. Amylopectin consists of short linear chains of 10 to 60 glucose units linked by α-1, 4 and α-1, 6 bonded to side chains with 15-45 glucose units. 2.3.1. Sources of α-amylase The α-amylases are universally distributed through the animal, vegetable and microbial [2]. Some yeasts produce industrially of α-amylase: Candida tsukubaensis CBS 6389, Filobasidium capsuligenum, Lipomyces kononenkoae, Saccharomycopsis capsularis, Saccharomycopsis fibuligera, Schwanniomyces alluvius, Schwanniomvces casteilli, Trichosporon pullulans and Candida isikubaensis (Table 4). 2.3.2. Characteristics of the α-amylase 2.3.2.1. Molecular weight Despite the large difference in characteristics of microbial α-amylases, their molecular weights are generally in the same range of 40 to 70 kDa [2]. It has been reported that Chloroflexus aurantiacus α-amylase has the molecular weight the higher the α-amylase with 210 kDa [85]. Gupta et al., [2] reported that the α- amylase from Bacillus caldolyticus has a molecular weight of 10 kDa representing the lowest value. This molecular weight can be increased due to glycosylation as in the case of the enzyme of T. vulgaris which reached 140 kDa [86]. In contrast, proteolysis decreases the molecular weight. The α-amylase T. vulganis 94-2A (AmyTV1) is a protein of 53 kDa [87]. 2.3.2.2. pH According to the origin the yeast α-amylases present generally optimum pH between 4 to 6 [88]. The optimum pHs of yeast amylase are summarized in Table 3. 2.3.2.3. Temperature The optimum temperature of the α-amylase also varies according to the origin of the microorganism. It varies from 30 to 70 ° C (Table 3). It is often higher than the growth of the bacteria producing the enzyme. The enzyme is more thermostable than the Bacillus licheniformis CUMC 305 that is stable to heat treatment at 100 ° C for 4 h [89]. 2.3.2.4. Metals Cu2 + ions, Hg2+, Ag2 + and Zn2+ decrease the activity of α-amylase in the yeast Cryptococcus sp. [6], while the Mg2+ ion, Ca2+, Na+ and EDTA have no effect on the enzyme activity of the yeast.



Table 3: Physical and chemical characteristics of some yeast α-amylase

Origin of Amylase Molecular weight (kDa) Optimal temperature

Optimum pH References

Candida antarctica CBS 6678

50 62 4.2 [33]

Saccharomyces kluyveri YKMS - 30 5 [90] Cryptococcius flavus 75 50 5,5 [37]

Saccharomycopsis fibuligera 54 - - [91] Lipomyces kononenkoae 38 50 5,5 [12,88] Lipomyces kononenkoae 76 70 4,5-5 [92]

Wickerhamia sp. 54 50 5-6 [31] Talaromyces pinophilus 1-95 58 55 4-5 [93]

2.4. Pullulanase 2.4.1. Definition and Nomenclature Pullulanase (pullulan α-glucano hydrolase) (EC 3.2.1.41) is a debranching enzyme, capable of hydrolyzing the α-1,6 bonds contained in starch, amylopectin, pullulan and related oligosaccharides. Like most amylolytic enzymes, pullulanase is an extracellular carbohydrase employed in the starch saccharification process [80]. Many microorganisms mesophilic, thermophilic and hyperthermophilic are able to secrete this specific type of glucanase [56]. Pullulan is also known under the name: dextrinase, amylopectin 6-glucanohydrolase, debranching enzyme, alpha-dextrin endo-1,6-alpha-glucosidase, R-enzyme, pullulan alpha-1,6-glucanohydrolase. 2.4.2. Sources pullulanase Pullulanases are produced by plants (the endosperm of the rice seed; [94]) and Sorghum bicolor var. F-2-20 [95] and by microorganisms such as bacteria, fungi and certain yeasts [59]. 2.4.2.1. Bacterial source Many bacteria and archaea (mesophilic, thermophilic and hyperthermophilic) are producing pullulanases [12]. Thermophilic anaerobic bacteria mainly synthesize amylopullulanases. Clostridium thermosulfurogenes [96-97], C. thermohydrosulfuricum Z 21-109 [98], Thermoanaerobacterium thermosaccharolyticum [99] and Thermococcus profundus [100]. Aerobic and thermophilic bacteria, such as species of Bacillus and Geobacillus, are identified as producing amylopullulanase: Bacillus species 3183 [101], Bacillus

circulans F-2 [102], Bacillus sp. TS-23 [60], Bacillus sp. KSM-1378 [80], Bacillus sp. DSM 405 [103], Geobacterium thermoleovorans NP33 [104]. Bacillus sp. US 149 [105] and G. stearothermophilus L14 [64]. Hyperthermophilic archaea Pyrococcus furiosus, P. woesei, Thermococcus litoralis and Thermococcus hydrothermalis are also able to produce highly thermostable amylopullulanases [57,106-109]. 2.4.2.2. Fungal source Few studies are devoted to fungal pullulanases: In yeasts, [24] investigated the production of extracellular enzymes degrading pullulan by several strains of amylolytic yeast. The pullulanase activity, the highest is obtained with species of Endomycopsis, Lipomyces, Filobasidium, Leucosporidium and Trichosporan. [110] have also purified pullulanase type I in yeast Aureobasidium pullulans. In the mold, [137] highlighted in Aspergillus niger ATCC 9642 the presence of a isopullulanase which hydrolyses pullulan at 40 ° C and produces isopanose. 2.4.3. Types of pullulanase and substrate Pullulan is an extracellular glucan synthesized by the yeast Aureobasidium pullulans, when grown on medium containing glucose or sucrose [111]. This polysasaharide consists of polymerized units of maltotriose (α-1 linkage, 6) in linear form (Figure 4) [112] with a 2: 1 ratio of α-1,4 bonds and α-glucano-1 , 6-glucano [111]. Most often it is used as a substrate model for the determination of debranching enzymes [113].



Figure 4: Structure of pullulan Pullulan is only soluble in water to form a transparent, colorless, and viscous [114]. It has potential applications in the food industry, the pharmaceutical and biomedical industries [114-115]. The molecular weight of the pullulan ranges from 1.5 to 810 kDa. Pullulanase attack pullulan by one of two modes of action (shown in Figure 5): An exo-action in which the hydrolysis is limited to the glycosidic bond α-1, 6, the closer to the non-reducing end, with the gradual release of maltotriose. A endo action in which the enzyme hydrolyzes glycosidic bonds α-1, 6 internally and externally, with the mixed output of the hexa, nona and larger oligosaccharides, in addition to maltotriose [116].

Figure 5: Possible Action Mode pullulanase Pullulanase hydrolysis of starch or glycogen, by breaking α -1,6 glycosidic bonds. Pullulanase type II is also capable of hydrolyzing α -1, 4 bonds (Table 4) [117]. Table 4: Specificity of action of pullulanase

Type of pullulanase EC Number hydrolyzed bounds

Substrate Products References

Pullulanase type I

3.2.1.41 α-(1,6) Oligo and Polysaccharides

Maltotriose [67-118]

Pullulanase type II (amylopullulanase)

3.2.1.41 α-(1,6)

α-(1,4) Pullulan, Poly and

Oligosaccharide (Starch)

Malotriose, Mixte of Glucose,

Maltose, and Maltotriose

[64,105,119-120]

Pullulan hydrolase type I (neopullulanase)

3.2.1.135 α-(1,4) Pullulan Panose [80,121]

Pullulan hydrolase type II (isopullulanase)

3.2.1.57 α-(1,4) Pullulan Isopanose [122]

Pullulan hydrolase type III

3.2.1.— α-(1,4) et α-(1,6)

Pullulan, Starch, Amylose, and Amylopectin

Mixte of panose, mal-tose and

Maltotriose Maltotriose and

Maltose

[123]



2.4.4. Regulating production of pullulanase Induction / Repression Like most enzymes, regulation of pullulanase is governed by systems induction / repression and knowledge of this mechanism could contribute to the design of an effective medium to fast and economical enzyme induction [124]. The enzyme is inducible by polysaccharides of α- 1, 6 bonds and maltose [125]. Pullulan may improve the synthesis of the pullulanase of B. cereus [124] or inhibit [126], suggesting the suppression of the enzyme by its substrate. 2.4.5. Effect of medium composition on the production of pullulanase 2.4.5.1. Carbon source The carbon source varies with the microorganism. Flour potato at 20% is the best source for the pullulanase production of C. thermosulfurogenes SV2 [127-128]. The starch, dextrin and pullulan stimulate the synthesis of the pullulanase [129]. Pullulan shows an inhibitory effect for the enzyme of T. thalpophilus, B. stearothermophilus KP1064 [130] and A. aerogenes [131] and an activating effect for the A. aerogenes’s enzyme [59]. 2.4.5.2. Nitrogen source The nitrogen source is an important factor in the growth and production of pullulanase. The activity of the pullulanase also appears to be induced by yeast extract [132] and the severe limitation of nitrogen depresses the induction of pullulanase [124]. Tryptone is used for the production of this enzyme from P. furiosus [57], P. woesei [107], T. ethanolicus 39E [133], T. thermosaccharolyticum [99] and C. thermohydrosulfuricum [129]. Peptone is used to the maximum amylopullulanase production from C. thermosulfurogenes SVM17 [63]. 2.4.6. Characteristics of pulluanases 2.4.6.1. Molecular weight Unlike the molecular weight of the α-amylase, the pullulanase is higher and ranges from 55-450 kDa depending on the strains (Table 5): 90-450 kDa for the pullulanase type II. The amylopullulanase of T. thalophilus has a molecular weight of 79-210 kDa [134]. Through these values we can deduce that some pullulanases are monomeric and other oligomeric. 2.4.6.2. pH The pH has a great influence on the metabolism of starch. Most pullulanases have optimum pH acidic or neutral, but some have alkaline pH optima (Table 5). 2.4.6.3. Temperature The temperature is the most important parameter that affects both growth and secretion of extracellular enzymes. The optimum temperature for pullulanase is between 40°C and 50 °C (Table 5). The optimum temperature of the

thermoactive pullulanases hyperthermophilic archaea is 85°C for Pyrococcus woesei [107] and 105°C for Pyrococcus furiosus [57]. The thermostability of the pullulanase is maintained even in the absence of substrate and Ca2+ [67]. 2.4.6.4. Minerals and trace elements some These elements are indispensable to the growth of microorganisms and the production of enzymes. Inorganic salts such as ammonium chloride, ammonium nitrate and ammonium sulphate is a nitrogen source for the cultivation and production of the enzyme of bacteria: Bacillus sp. 3183 [141], Bacillus sp. DSM 405 [103], Bacillus sp. KSM-1378 [80], Bacillus sp. TS-23 [60], B. circulans F-2 [141] and G. thermoleovorans NP33 [104]. The activity of the pullulanase is strongly inhibited by certain cations 0.2 mM: Ni2+, Co2+, Mg 2+, Cu2+, Zn2+. By cons, Ca2+ cations increase the activity of the pullulanase of Bacillus cereus HI5 179%. [142]. 2.5. Economic aspect of enzymes In 2015, the global market for enzymes is approximately $ 7.4 billion and is expected to have an average annual growth rate of 6.5% [143]. Amylases are one of the most important industrial enzymes covering 30.5% of the global enzyme market in 2015 after lipases which constitute 38.5% of the market [144]. From carbohydrases consist of α-amylases, isomerases of, pectic and cellulase is approximately 40%. Food and drinks sectors use 90% of the carbohydrases produced. The annual sale of α-amylase in the market is estimated to be $ 11 million. The world production of α-amylase from B. licheniformis and Aspergillus sp. is about 300 tons pure enzyme per year [145]. 2.6. Industrial application of pullulanase and α-amylase Pullulanase is a debranching enzyme widely used with the α-amylase in the starch industry for the production of various sugar syrups [146]. Biotechnological progress, the implementation of the pullulanase is extended to pharmaceutical chemistry, to the chemical industry (detergents for automatic dishwashers) for bread and production of cyclodextrins molecules to pharmaceutical interest [146].



Table 5: Some properties of pullulanase Micro-organisms MW (kDa) Isoelectric

point (pI) Optimal

pH Optimale

temperature (°C)

Reference

Type I Bacillus flavocaldarius KP 1228 55 - 7 75-80 [135]

Fervidobacterium pennavorans Ven 5 240 - 6 85 [136] Aureobasidium pullulans 73 - - - [110]

Type II Thermoanaerobacter sp. B6A 450 4.5 5 75 [98]

Pyrococcus furiosus 110 - 5.5 98 [57,140] Pyrococcus woesei 90 - 6 100 [107]

Desulfurococcus mucosus - - 5 100 [137] Isopullulanase

Aspergillus niger ATCC 9642 69–71 - 3.5 40 [138] Bacillus sp. US 149 200 - 5 60 [105]

Alkaline pullulanase Bacillus sp. KSM-1876 120 5.2 10-10.5 50 [139] Bacillus sp. KSM 1378 210 4.8 9.5 50 [80]

Pullulane hydrolase type III Thermococcus aggregans expressed

in E. coli - - 6.5 95 [123]

2.6.1 Glucose production In sugar syrup industries, α-amylase leads to the preparation of linear and branched oligosaccharides mixtures known commercially as maltodextrins or glucose syrup. As for the pullulanase, it completes the starch hydrolysis initiated by the α-amylase which increases the quality of sugar syrups. Treatment of starch simultaneously with the α-amylase and pullulanase stimulates the reaction efficiency of saccharification and generates higher yields of the final product [132,147,148]. Amylases (α-amylase and pullulanase) having pH values above 8.0, have potential applications for the saccharification of starch in the starch industries [149]. Pullulanase debranching allows the corn starch in the production of certain corn sweeteners [67,150]. 2.6.2. In processing industry starch The pullulanase is used for preparing starches, high amylose. They are subject to a huge demand on the market [151]. The high amylose starches are of great interest and can be converted into "resistant starch" which presents nutritional benefits [152]. Unlike normal starch, resistant starch is undigested in the small intestine but is fermented in the large intestine by bacteria of the intestine to form short-chain fatty acids (butyrate), health benefits colon [153]. 2.6.3. In Bread making The starch and enzymes responsible for modification is used by the baking industry in large quantities in the world. One of the major problems confronted with the baking

industry is the staling effect [154]. This leads to an increase in crumb firmness, loss of sharpness of the crust, a reduction of the moisture content of the crumb and taste of bread. This greatly reduces the quality of bread and other products [155]. This problem is essentially solved by chem-ical treatments. But these days, consumers prefer the en-zyme treatment, so biological, with less adverse effects on health. 2.6.4. In brewery During the brewing of beer, starch is decomposed by α-amylase into dextrins and fermentable sugars to form sweet wort. Hops are boiled with sweet wort to produce the wort. Yeasts then act on the wort to ultimately produce beer [146]. The pullulanase is added during the mashing step of the beer production to break the points of connection of the α-1,6 starch, which leads to the maximum fermentability of the wort [156]. Accordingly, there has been an increase in the percentage yield of the production of beer low calorie [157]. 2.6.5. In detergent industry Various alkaline enzymes (pH ≥ 8.0) (proteases, α-amylases, pullulanases, lipases, cellulases) are used as additives in detergents [149,158-159]. The use of enzymes in detergents has increased in recent years. Today, 90% of all these detergents contain enzymes [2,160]. Amylase (α-amylase and pullulanase at pH ≥ 8.0) have potential applications for the saccharification of starch and textiles and are also used in the detergent industry for automatic



dishwashers and laundries [149,158-159,161]. The effectiveness of alkaline debranching enzymes can be improved in the wash water when used in combination with alkaline α-amylase [80], as chlorine is an activator of the enzyme [162]. 2.6.6. In textile industry It forms a protective layer surrounding the son to avoid their disintegration during weaving. It is used for finishing of clothes to make them firmest, stiffer and heavier. It allows printing of fabric or creation of certain colors on the fabric surface. Alkaline amylase (α-amylase and pullulanase at pH ≥ 8.0) and thermostable ideal for the textile industry for starch saccharification, in laundry to remove starchy stains and primer [1]. 2.6.7. In industries cyclodextrins (CDs) CD production is very simple and includes a processing ordinary starch with a set of enzymes modified starch. Commonly, the cyclodextrin glycosyltransferase (CGTase) is used with α-amylases but saccharide α-1 links, 6 amylopectin block the action of CGTase [146,163]. These bonds are broken with pullulanase and, therefore, the production yield of cyclodextrins increases [10]. These products are used in the medical and pharmaceutical field as stabilizers for masking odors [146]. 2.6.8. In the diagnostic and pharmaceutical industries In the medical diagnostic Domain the rate of α-amylase in biological fluids is a marker to detect certain diseases: kidney failure, heart failure, mumps, pancreatic cancer, etc. [164]. In the pharmaceutical field the α-amylase is an anti-inflammatory [164]. Fungal α-amylase (resistant to acidity) in combination with celluloses is digestive aids drugs to prevent dyspepsia and intestinal fermentation [165]. Cyclodextrins (CD), produced after action pullulanases possible to increase the solubility and absorption of drugs. The required quantity of product is thus very small; it results in decreased side effects (stomach irritation) and financial costs [146]. 2.6.8. In the production of food gums Food gums are polysaccharides obtained from natural sources. These substances added even at low concentrations to solutions, cause an increase in viscosity. They are used as thickening agents, gelling agents, emulsifiers and stabilizers in the food industry [146]. In addition, they are used in other industries as binders adhesives, clarifying agents, encapsulating agents, flocculent agents, foam stabilizers and blowing agents [146]. A natural gum is locust bean gum (LBG: locust bean gum). This is a galactomannan [166] extracted from the seeds of the carob tree whose extraction is difficult hence its high price.

This problem is solved by the use of the pullulanase on the guar galactomannan. Pullulanase removes galactose residues from guar galactomannan to produce modified guar galactomannans [167]. They have the same functional properties as the locust bean gum with improved rheological properties such as viscosity and elasticity [168]. These modified galactomannans may thus be used for various food and non-food gum carob. 2.6.9. In biorefinery The demand amylolytic enzymes increased following the oil energy crises. To cope with the shortage of oil, the biorefinery has emerged since 2006 [169]. The biorefinery concept or refinery plant is based on the total enzymatic hydrolysis of polysaccharides, cellulose and starch, glucose. The glucose is then converted to succinic acid in the manufacture of agricultural films and coating or sorbitol and then isosorbide for the manufacture of plasticizers and performance materials (Figure 6). To enable the development of biorefineries, research efforts are needed to increase the productivity of commercial enzymes, including amylolytic thermostable enzymes (α-amylase and pullulanase) and reduce the cost of production of enzymes, using raw materials abundant and cheap [170]. 2.6.10. In paper industry Amylases solubilize starch for glueing and paper coating [71,122]. Starches, high amylose, obtained through the action of the pullulanase are used in adhesives and in the production of corrugated cardboard and paper [84]. 2.6.11. In the production of bioethanol The total content of the starch in duckweed may vary from 3 to 75% of dry weight depending on the species of duckweed [171]. Some species such as Spirodela polyrrhiza grown on sewage may contain a starch content of about 59% [172]. The duckweed biomass is hydrolyzed by the action of the enzyme pullulanase and amyloglucosidase to produce reducing sugars [173]. These sugars are then converted into bioethanol. The overall rate of conversion of starch by the action of the pullulanase is very high [146]. 2.6.12. In the beverage industry In the beverage industry, α-amylase and pullulanase hydrolyze starch into fermentable sugars in the manufacture of ethyl alcohol, soft drinks and sweetened fruit juices [174-175].



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amylolytic yeasts: producers of α-amylase and pullulanase

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