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
http://ijlssr.com IJLSSR © 2015 All rights are reserved Page 339
Amylolytic Yeasts: Producers of α-amylase and
Pullulanase
Djekrif D. S1*
, Gillmann L2
, Bennamoun L1
, Ait-Kaki A1
, Labbani K1
, Nouadri T1
,
Meraihi Z1
1
Research lab in Microbiology Engineering, Faculty of Biology, Biopole Chaab Erssas, University, Constantine,
Algeria
2
SONAS-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
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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].

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 b
Figure 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].

Figure 6: Examples of innovation for new biorefineries [169]
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Conflict of interest: Nil

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