Amylolytic Enzymes

2 Amylolytic Enzymes: Glucoamylases S. Negi*, K. Vibha MOTILAL NEHRU NATIONAL INSTITUTE OF TECHNOLOGY , A LLAHABAD , IND IA

2.1 Introduction Starch is one of the most abundant polymers on earth and its industry has a big stake in the market. Starch is composed of unbranched amylose and branched amylopectin, which require three types of amylolytic enzymes for complete hydrolysis into glucose, i.e., a-amylase (4-a-D-glucan glucanohydrolase, EC 3.2.1.1), b-amylase (4-a-D-glucan maltohydrolase, EC 3.2.1.2), and glucoamylase (4-a-D-glucan glucohydrolase, EC 3.2.1.3). a-Amylase cleaves the a-1,4-D-glucosidic linkages between adjacent glucose units in the linear amylose chain; b-amylase cleaves at nonreducing chain ends of amylose, amylopectin, and glycogen molecules; and GA hydrolyzes a-1,4 glycosidic bonds from the nonreducing ends of starch and a-1,6 linkages at the branching points of amylopectin, although at a lower rate than 1,4 linkages, into glucose [1e4]. GA can also catalyze the reverse hydrolysis reaction to produce maltose and isomaltose, which has great significance in industrial processes in which high sugar content is present. GA converts starch and a- and b-limit dextrins into glucose and shows faster reaction on polysaccharides than on oligosaccharides. The rate of hydrolysis depends on the substrate size and the structure, nature, and position of the bond present. GA is ubiquitously present in or produced by all forms of life (plants, animals, bacteria, archaea, and eukaryotes). However it is mainly produced using filamentous fungi, although a host of other microorganisms are also known as good producers of GA. Aspergillus niger, Aspergillus awamori, and Rhizopus oryzae are the commonly used filamentous fungi for industrial production of GA [9,10]. GAs are extensively used in the food and beverage industries. They are used for production of glucose syrup, high-fructose corn syrup, beer, soy sauce, alcoholic beverages, etc. [2,9,11e13]. Most of the GA produced from parent strains catalyzes saccharification efficiently only within a small range of mild temperatures. At high temperatures its catalytic activity reduces sharply because of conformation changes. GA produced from parent fungal sources normally has limited thermostability, catalytic activity, and low pH range, which restrict its application in industrial processes carried out at high temperature and in alkaline medium. At higher temperature the reaction rate is higher; therefore, *

Corresponding Author.

Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products http://dx.doi.org/10.1016/B978-0-444-63662-1.00002-6 Copyright © 2017 Elsevier B.V. All rights reserved.

25

26 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

processing is faster. It also prevents microbial contamination and reduces the viscosity of the reaction mixture. This leads to reduction in process cost. Production of GA that is stable at higher temperatures would be highly beneficial for starch saccharification. Advances in recombinant DNA technology and site-directed and random mutagenesis and other techniques are being used to improve the thermostability and other functional properties of GA [1]. Over the years a lot of research has been carried out to reduce the cost of production of GA and improve its functional properties to suit industrial requirements. Progress in the fields of molecular biology, protein engineering, and bioinformatics has helped to provide it with improved functional properties, such as enhanced thermostability, better selectivity, wider pH range, improved catalytic activity, etc. [14].

2.2 Sources of Glucoamylase GA occurs in a wide range of organisms. It is present in plants [15], animals [16], fungi, bacteria, and yeast. However, microorganisms are the main source explored for GA production.

2.2.1

Microbial Sources

GA is present in a wide range of bacteria, fungi, and yeasts. Some of the microbial sources exploited for the production of GA are shown in Table 2.1.

2.2.1.1 Fungal Sources Fungal species of Aspergillus, Rhizopus, and Endomyces are the most commonly used sources for production of GA. Aspergillus awamori and A. niger are among the most popular microorganisms used by industry for GA production [2,3,17]. Rhizopus oryzae [18], Rhizopus niveus [19], Mucor [6,20], Penicillium [21], and many other fungal species are capable of producing GA. Enzyme production by molds is generally extracellular, which makes its downstream processing cost-effective and less time consuming.

2.2.1.2 Bacterial Sources There are many bacterial strains capable of producing GA. Flavobacterium sp. [22], Bacillus stearothermophilus [23], Sclerotinia sclerotiorum [24], Sclerotium rolfsii [25], and Thermoanaerobacter tengcongensis [26] are some of the bacteria used for production of GA. The amylolytic bacterial strains Clostridium thermosaccharolyticum [27], Clostridium sp. [28], and Lactobacillus amylovorus [29] have also been used for production of GA [2]. Thermostable GA from thermophilic bacteria can be used at higher temperature for saccharification, thereby reducing the production cost of glucose by saving the cost of cooling. For production of glucose from starch, liquefaction of starch is done by a-amylase first, followed by saccharification by GA. Liquefaction can take place rapidly at 95e105
C by a thermostable bacterial a-amylase. But fungal GAs are stable normally up to a temperature range of 55e60
C. Therefore, most of the fungal GAs are used only

Chapter 2  Amylolytic Enzymes: Glucoamylases

27

Table 2.1 Details of Substrates and Microorganisms Used for Production of Glucoamylase Source

Microorganism

Process

Substrate

Yield

References

Fungal

SSF

Corn flour, wheat bran Wheat bran Wheat bran

5582.4 mmol/m g

[37]

45.21 U/mL 2.0 and 1.99 mmol/mL min

[38] [5]

Fungal

Aspergillus oryzae FK-923 A. oryzae Aspergillus niger and Rhizopus A. oryzae

SSF

4.1 IU

[39]

Fungal Fungal Fungal

A. niger A. niger A. oryzae

SSF SSF SSF

31.214 U/mg 152.85 U/mL 330 mg/mL min

[7] [40] [41]

Fungal

Aspergillus niveus

SmF

600 U/mg of protein

[42]

8.3 U/mL 26.3 U/mL

[9] [43]

Fungal Fungal

Fungal Fungal

SSF SSF

Aspergillus awamori Thermomucor indicae-seudaticae Fungal A. awamori Fungal Aspergillus flavus and Thermomyces lanuginosus Fungal A. niger Fungal T. lanuginosus Fungal Colletotrichum gloeosporioides Bacterial Thermoanaerobacter tengcongensis Bacterial Bacillus sp. Bacterial Lactobacillus amylovorus Yeast Candida famata Yeast Pichia subpelliculosa Plant

Liquid

Sugar beet (Beta vulgaris L.)

SSF SSF

Wheat bran and rice bran Potato starch Potato starch Wheat bran and sugarcane bagasse Starch and yeast extract Potato starch Sucroseeyeast extract

SSF SmF

Wheat bran Cassava and corn starch

9420.6 U/gds

[44] [45]

SSF SSF SSF

Rice bran Starch Starch

695 U/g 60 U/mg of protein 0.45 IU/mL

[46] [47] [4]

SSF

Maltose

80 U/mg

[26]

SSF SSF SSF SmF

Starch Dextrin and starch Starch Commercial washed starch MurashigeeSkoog medium

21.6 U/mL 2587.17 mmol/L m 6500 U/L

[30] [29] [31] [32]

102.2 U/mg

[36]

SmF, submerged fermentation; SSF, solid-state fermentation.

after the reaction mixture is cooled down to this temperature. Thermostable GA from thermophilic bacteria can be used for saccharification without much cooling of the liquefied starch. GA produced from aerobic bacteria, such as B. stearothermophilus, Halobacterium sodomense, and Flavobacterium sp., and anaerobic bacteria, such as Clostridium sp. and C. thermosaccharolyticum, have better thermostability compared to fungal GA [26].

28 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

A thermostable GA (TtcGA) from T. tengcongensis was successfully expressed in Escherichia coli by Zheng et al. [26]. Heat treatment and gel-filtration chromatography were used to partially purify the recombinant mature protein, and 30-fold homogeneity was obtained. Maximum activity of the recombinant enzyme was obtained at 75
C and pH 5.0. It was highly thermostable with almost no activity loss at 75
C for 6 h. James and Lee (1995) used L. amylovorus to produce GA in dextrose-free de ManeRogosaeSharpe medium in a 1.5-L fermenter under an optimal dextrin concentration of 1% (w/v), pH 5.5, and 37
C. GA production was maximum at the late logarithmic phase of growth for 16e18 h. Crude enzyme showed maximum activity at pH 6.0 and 60
C. Gill and Kaur (2004) used a bacterial strain of Bacillus for production of TtcGA [30]. Highest production of GA was achieved at 65
C and pH 7.0 after 17e20 h under stationary conditions. Luria broth, supplemented with 0.5% (w/v) soluble starch, yielded highest enzyme activity (21.6 U/mL). GA showed optimum activity at 70
C and pH 5.0. It was highly stable at pH 7.0 with a half-life of 13 h, 8 h, and 3 h 40 min at 60, 65, and 70
C, respectively.

2.2.1.3 Yeast Sources Candida famata [31], Pichia subpelliculosa [32], and Saccharomyces diastaticus [33] are some of the yeast used for production of GA. GA production has also been reported from yeast strains of Saccharomycopsis fibuligera [34] and Lipomyces starkeyi HN-606 [35]. Mohamed et al. isolated C. famata from traditional Moroccan sourdough for production of GA [31]. Starch enhanced GA production, with maximum GA activity at 5 g/L. Yeast extract and (NH4)2HPO4 gave maximum GA and biomass after 72 h of incubation in liquid medium at 30
C, pH 5, at 105 rpm.

2.2.2

Other Sources

There are very few reports of GA production from plant and animal origins. In one such work on GA production from a plant origin, sugar beet (Beta vulgaris L.) was explored for production of GA and a-amylase [36]. They reported the presence of GA and a-amylase in callus and suspension cultures as well as in mature roots of sugar beets (B. vulgaris L.).

2.3 Glucoamylase Production GA is produced by many microorganisms capable of growing in a vast variety of substrates through the fermentation process. Like any other enzyme production, GA also depends on the selection of microbial strain, substrate, medium, and fermentation process and on physicochemical parameters such as incubation time, temperature, pH, relative humidity [in solid-state fermentation (SSF)], inhibitors, oxygen accessibility, etc. Production can be carried out through submerged fermentation (SmF) or SSF depending upon the microbial culture and substrate in use. Until recently, approximately 90% of all industrial enzymes were produced in SmF using a specifically optimized process and genetically manipulated microorganisms. However, scientists have discovered and

Chapter 2  Amylolytic Enzymes: Glucoamylases

29

realized the numerous economical and practical advantages of SSF. Almost all enzymes can be produced in SSF using microorganisms. Some of the sources and GA production details are shown in Table 2.1.

2.3.1

Selection of Fermentation Process

In SmF the substrate is solubilized or suspended as fine particles in a large volume of liquid, whereas in SSF an insoluble substrate is fermented with sufficient moisture but with no free water. SSF is an ideal process when the organism is a filamentous fungus, which is able to withstand the limited water availability. SSF has become popular with scientists as well as industry, particularly with agro-based substrates [2,48,49]. SSF has distinct advantages over SmF in terms of lower production costs, lower energy requirements, higher yield, simple fermentation equipment, and less effluent generation. In SSF, nutrients present in the substrate serve as an anchorage for the microbial cells. In SSF the moisture content varies from 30% to 80% and maximum enzyme production is normally obtained with about 60% moisture content. At lower moisture content microbiological activity ceases.

2.3.2

Composition of Substrate

Composition of the substrate used for production of GA has a big impact on its yield. Selection of substrate is done considering its availability and suitability for the particular strain and process used. Agro-industrial residues like rice husk, wheat bran, rice bran, gram flour, coconut oil cake, sugarcane bagasse, wheat flour, corn flour, tea waste, potato starch, etc., are commonly used as substrates for GA production in SSF [2,17,38,49,50]. Corn starch is a commonly used substrate for production of glucose syrup in the United States and Europe because of its easy availability. The particle size of the substrate has a strong influence on the growth rate and enzyme-producing ability of the organism due to the influence of surface area on growth rates. The optimum particle size is to be decided to ensure maximum mass transfer because bigger particles provide less surface area and smaller particles provide a high degree of solubilization. Pandey reported poor GA activity with a particle size of larger than 1.4 mm and smaller than 180 mm [50]. Wheat bran particles of 425e500 mm were found most suitable for maximizing GA activity. Another vital factor affecting GA production in SSF is water activity, which is an important parameter for mass transfer of the water and solutes across the cell membrane. Pandey et al. reported higher GA yield with higher initial water activity values of substrate [51]. Substrates are normally supplemented with carbon or nitrogen sources to increase the enzyme yield. Supplement can be a simple carbon source like glucose, maltose, or sucrose or a polymeric compound such as starch from some other source, or a nitrogen source such as ammonium or nitrate salts or urea, or a complex source, e.g., corn steep liquor. The requirements for nutrition are normally more complex in SmF compared to SSF. Supplements of fructose, ammonium sulfate, urea, and yeast extract in the substrate,

30 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

such as wheat bran, have been reported to enhance GA production in SSF by Aspergillus sp. and A. niger [2,44,52]. Nyamful et al. (2014) reported wheat bran as the best substrate for production of GA by A. niger and Rhizopus strains. They used wheat bran, rice bran, and groundnut pod as different substrates for investigation. Maximum activities of 2.0 and 1.99 U/mL, respectively, for A. niger and Rhizopus were obtained after 48 h on wheat bran, though GA was produced by both strains on all substrates [5]. Ominyi et al. used Aspergillus sp., Mucor, and Rhizopus sp. for production of GA through SmF using soluble starch as a carbon source [6]. Puri et al. (2013) also used four different substrate compositions, i.e., rice bran, wheat bran, rice bran/wheat bran (1:1), and rice bran/paddy husk (1:1) for GA production from Aspergillus oryzae under SSF. A maximum GA activity of 4.11 IU was obtained with rice bran [39].

2.3.3

Optimization of Physical Parameters

Production of any enzyme in itself is not useful unless it meets the requirements of the industry. For industrial application, the yield of enzyme production, the time of production, and the enzyme’s stability are the basic requirements, so that suitable enzymes can be produced in a cost-effective manner. From the very beginning, various techniques have been used to optimize various parameters and supplements that affect the yield and quality of enzymes. Optimization techniques eliminate wasteful expenditure, experimentation, and calculation. Boas (1962) was the first to develop this technique, wherein optimization of the biological system was based on single-factor search [53,62]. Earlier, this “one-at-a time” method was used by workers for optimization, but was incapable of detecting the true optimum conditions, especially because of probable interaction aspects of enzymes among themselves. Thus, the necessity of an optimization technique that would investigate the interaction factor also was felt. This led to techniques such as the evolutionary operation (EVOP) program. Barnett (1960) reported that EVOP provides a system for exploring the relationships between independent and dependent variables [54,55]. He stated that EVOP consists of the systematic introduction of very small changes in the selected independent variables, which affects the process and statistical selection of the best set of conditions. Response surface methodology (RSM), based on factorial experiments, is another statistical approach to study the effects of test variables on measured response [55,56]. The mathematical model for RSM is derived from orthogonal polynomial fitting techniques. Negi and Banerjee (2006) employed the EVOP factorial design technique to achieve optimal pH, temperature, and humidity for production of GA using A. awamori from wheat bran under SSF. A maximum yield of 9420.6 U/gds GA was achieved at 37
C, pH 4, and 85% humidity [44]. Puri et al. (2013) optimized GA production by varying temperature, moisture content, pH, inoculum, and incubation period of culture. Optimization was carried out by varying temperature from 20 to 40
C, moisture content from 10 to 30 mL/ 5 g, pH from 3 to 7, spore suspension from 1  105 to 1  108 spores/mL, and incubation period from 3 to 6 days. A 30
C incubation temperature, 20 mL/5 g moisture content,

Chapter 2  Amylolytic Enzymes: Glucoamylases

31

1  107 spores/mL spore suspension, pH 5.0, and 5-day incubation were found to be optimal conditions for GA production [39]. Keera et al. (2014) used A. oryzae to produce GA by SSF of corn flour. They optimized various parameters, i.e., initial moisture content (50e80% v/w), pH (3e9), and temperature (25e34
C), and used various supplements such as wheat bran (20e50%); carbon supplements (1e3% w/w) of glucose, maltose, sucrose, potato starch, and soluble starch; and inorganic supplements of ammonium sulfate, ammonium oxalate, ammonium, phosphate, diammonium phosphate, triammonium phosphate, ammonium acetate, ammonium nitrate, sodium nitrate, and urea to optimize enzyme yield. Maximum GA production of 5582.4 mmoles of glucose produced per minute per gram of dry fermented substrate was obtained after 72 h at 30
C on corn flour supplemented with 30% (w/w) wheat bran, 1% soluble starch, 0.1% (w/w) urea at pH 5.5 and 60% (v/w) initial moisture [37]. Kumar and Satyanarayana (2001) optimized GA production by the yeast strain P. subpelliculosa and achieved highest yield at 30
C and pH 5.6 in medium containing 0.2% yeast extract, 1% starch, 0.035% NaCl, 0.4% K2HPO4, and 0.1% MgCl2 when agitated at 200 rpm in a shake flask for 11 h. A 15-fold increase in GA secretion was achieved by optimization [32]. Parbat and Singhal optimized GA production under SSF by A. oryzae and maximum yield was obtained at 60
C, pH 5.0, with wheat bran and sugarcane bagasse at a ratio of 1:1 [41]. Gomes et al. (2005) optimized production of GA from starch in SmF by Aspergillus flavus and Thermomyces lanuginosus. Effects of different carbon sources, temperatures, and initial pH of the medium were evaluated using a full factorial design (2  2  3). Cassava starch gave better results than corn starch with A. flavus and exhibited higher activity with starch and maltose mixture. GA produced from A. flavus and T. lanuginosus had high optimum temperature, i.e., 65 and 70
C, respectively, and a wide pH range [45]. Negi and Banerjee (2010) optimized GA production concomitant with protease from A. awamori in a single fermentation and achieved a GA yield of 4528.4  121 U/gds with wheat bran at a Czapek Dox medium ratio of 1:1.5 (w/v), 96 h incubation, 35
C temperature, pH 5.5, and 85% relative humidity through the EVOP factorial design technique. Medium engineered with 1% casein and 1% starch solution further increased the yield by 2.07-fold (9386.5  101 U/gds) [57].

2.4 Purification and Characterization Crude enzyme is needed to be optimally extracted from the fermented mass using a suitable solvent and then purified. The extraction efficiency can be greatly improved by selecting a suitable solvent, concentration of the solvent, soaking time, temperature, and number of washes. The extent of purification depends on the application. Optimization of various extraction conditions such as type of solvent, concentration of the solvent, soaking time, and temperature is done to maximize the extraction of crude extract. Crude enzyme extracted in a suitable solvent normally contains some amount of unfermented mass, microbial cells, cell debris, spores, and other insolubles. Removal of

32 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

such insolubles is normally done by centrifugation and microfiltration, etc. Negi and Banerjee (2009) extracted and purified GA, produced concomitant with a protease, by A. awamori in a single fermenter by SSF, by soaking the fermented mass at room temperature (around 30
C) in 10% glycerol for 2 h and then using acetone in a 1:2 ratio for precipitation. GA was purified up to 4.06-fold with 35.3% recovery. To concentrate the desired extracellular protein, sequential and/or parallel precipitation can be used. Salting out with ammonium sulfate or any other suitable organic solvent can be used to precipitate total protein. Precipitation with cold-water-miscible organic solvents, organic polymers, and isoelectric precipitation are some of the methods adopted by scientists. To separate out the enzyme of interest various centrifugation methods, such as ultracentrifugation, density gradient centrifugation, etc., are also used [8].

2.4.1

Purification Techniques

Various techniques have been developed for the purification of proteins prior to their characterization or use in biotechnological and industrial processes. Purification can be achieved through a series of chromatographic techniques such as ion-exchange and gelfiltration chromatography, or in combination, or affinity chromatography and highperformance liquid chromatography. To check the level of purity and molecular weight determination, sodium dodecyl sulfateepolyacrylamide gel electrophoresis is commonly used. In lab scale, purification is generally preferred through ion-exchange and sizeexclusion chromatography; however, one-step purification affinity chromatography is more economical and preferred. For size exclusion Sephadex-G of different grades and silica gel, and in ion exchange DEAEeSephadex and CM-cellulose, are frequently used materials for GA purification. In affinity chromatographic separation of GA the use of b-cyclodextrinechitosan, amylopectin, alginate, acarbose, etc., is common practice. Other methods such as two-step extraction, ionic liquid extraction, liquideliquid extraction, and biphasic separation are also successfully used to purify GA. Details of the methods and materials used for purification in the past few decades are described in Table 2.2.

2.4.2

Characterization

Enzymes are amphoteric molecules containing a large number of acidic and basic groups. Charge on these groups varies with pH according to their acid dissociation constant. Changes in pH affect the activity and structural stability of the enzyme. GA is versatile with respect to pH and has been found active in the pH range 3e11. Negi and Banerjee found GA produced from A. awamori stable at pH 3e9 with optimum pH 4.5 [8]. Amirul et al. reported GA produced by A. niger stable in pH range 3.5e9.0 [90]. A similar pH range for GA from Aspergillus sp. has been reported by others also [91,92]. The thermostability of GA determines its industrial utility. It is directly related to its half-life period (t1/2) at different temperatures. Half-life is defined as the reaction time for the enzyme activity to drop to exactly half the initial activity under optimum conditions. The variation in t1/2 with temperature gives a measure of the temperature

Table 2.2

Details of Extraction and Purification Processes

Extraction and Purification Processes Ultrafiltration, ammonium sulfate precipitation (80%), DEAEeSephacel column chromatography Imarsil, activated charcoal, and Sephadex G-100 chromatography Ammonium sulfate precipitation (80%), Sephadex G-200 gel filtration

DEAEecellulose ion exchange and Sephadex G-100 gel-filtration chromatography Procion Blue H-ERD dye affinity chromatographic separation by elution with a borate solution Ammonium sulfate precipitation (70%), Sephacryl S-200 column gel filtration and S-Sepharose FF cation-exchange and Q Sepharose FF anion-exchange column chromatography Q Sepharose anion-exchange column chromatography, hydrophobic interaction (phenyl Sepharose column) on FPLC Ammonium sulfate precipitation (30%), HiLoad-Q Sepharose column, and monoQ column on FPLC anion-exchange, gel-filtration, hydrophobic interaction (phenyl Superose column) chromatography Sephadex G-75 gel filtration Ammonium sulfate precipitation, Q Sepharose anion-exchange column chromatography, HiLoad 16/60 Sephacryl S-200 gel filtration, and hydrophobic interaction column containing phenyl Sepharose. PM-10 ultrafiltration membrane and an Ultra-Free centrifugal filter device, DEAE Toyopearl 650S column

Optimal Conditions


Yield of GA (%)

References

Aspergillus niger

pH 5, 70 C

54

[58]

Rhizopus oligosporus SK5 mutant A. niger

pH 5, 80
C

57

[59]

pH 8, 40
C pH 6.5, 30
C pH 4.6, 30
C

0.09

[60]

140

[61]

pH 6, 65
C pH 4.5, 75
C

25

[62] [63] [64]

10% glycerol, 40
C pH 4, 60
C

51.9

[8]

9.2 52

[65] [66]

A. niger CCUG 33991 A. niger 2316 Aspergillus flavus HBF34 Rhizopus microsporus var chinensis Aspergillus awamori Paecilomyces variotii A. niger Curvularia lunata

pH 4, 50
C

17.8

[67]

Humicola spp.

pH 4.7, 55
C

33

[68]

Fusarium solani

pH 4.5, 40
C

31.8

[69]

Aureobasidium pullulans N13d Thermomyces lanuginosus

pH 4.5, 60
C

58

[70]

Fomitopsis palustris

pH 5, 70
C

[71]

[72]

33

Continued

Chapter 2  Amylolytic Enzymes: Glucoamylases

Reverse micellar organic phase extraction (by using anionic surfactant, in n-heptane as a solvent) Ni2þ-NTA affinity chromatography Starch affinity chromatography Ammonium sulfate precipitation, aqueous two-phase systems, DEAE-650M chromatography, and Bio-Rad Prep Cell Acetone precipitation, ion exchange, gel filtration

Microbial Source

Details of Extraction and Purification Processesdcont’d

Extraction and Purification Processes Ammonium sulfate precipitation (90%), DEAEeSepharose anion exchange, phenyl Sepharose gel filtration, hydrophobic interaction chromatography ButyleSepharose and Superdex 200 HR gel permeation Macroaffinity ligand-facilitated three-phase partitioning using alginate Lyophilization, acetone precipitation, SP-Sepharose anion-exchange and Sephadex G-50 gel filtration Ultrafiltration and Sephadex G-25, Superdex HiLoad-200 gel filtration Ultrafiltration and Sephadex G-25, Superdex HiLoad-200 gel filtration Ultrafiltration and Sephadex G-25, Superdex HiLoad-200 gel filtration Affinity chromatography Ultrafiltration and Q Sepharose ion exchange Ammonium sulfate precipitation, Sepharose CL-6B, DEAEeSepharose Fast Flow, Q Sepharose Fast Flow, and Superose 12 gel filtration, ultrafiltration Q Sepharose ion exchange, Sephadex G-100 and phenyl Sepharose CL-4B gel filtration Ultrafiltration, DEAEecellulose, CMecellulose, Sepharose-6B column chromatography Affinity precipitation with alginate FPLC using a Bio Sil-SEC-400 filtration column, DEAEecellulose, CMecellulose Ammonium sulfate precipitation (75%), DEAEeSephadex A-50, ion exchange and Sephadex G-25 and G-100 gel filtration Two-step extraction system by using poly(ethylene glycol) and potassium phosphate Mono-Q ion exchanger and Superose-12 gel filtration DEAEecellulose ion-exchange chromatography and concanavalin AeSepharose gel chromatography Protamine sulfate treatment, ammonium sulfate precipitation, gel filtration (Sephadex G-75 sf, Ultrogel AcA 54), DEAEeSephacel chromatography, hydroxyapatite chromatography, and affinity chromatography on acarboseeAHSepharose 4B Acarbose (BAY G-5421) affinity chromatography FPLC, fast protein liquid chromatography; GA, glucoamylase.

Microbial Source Chaetomium thermophilum Sulfolobus solfataricus A. niger Thermomucor indicaeseudaticae Thermoplasma acidophilum Picrophilus torridus Picrophilus oshimae Thermobacterium thermosaccharolyticum A. niger Bo-1 T. lanuginosus

Optimal Conditions

Yield of GA (%)

References

pH 4, 65 C

3.57

[73]

pH 5.5e6, 90
C

4.6 83 1.03

[74] [75] [76]




pH 7, 60
C pH 2, 90
C

[77]

pH 2, 90
C pH 2, 90
C pH 5, 55
C

[77] [77] [78]

pH 4.4e5.6, 70
C 23

[79] [47]

T. lanuginosus F1

pH 6, 70
C

[81]

Scytalidium thermophilum

pH 5.5, 70
C

51

[82] [80] [83] [84]

A. niger, Bacillus amyloliquefaciens S. thermophilum Arthrobotrys amerospora ATCC 34468 A. awamori NRRL 3112

pH 6.5, 60
C pH 5.6, 55
C

81 78 41.6 3.2

pH 4.2, 60
C

100

[85]

Lactobacillus amylovorus Myrothecium strain M1

pH 6, 45
C pH 4, 70
C

21

[86] [87]

Candida antarctica CBS 6678

pH 4.2, 57
C

A. niger, Rhizopus sp.

[88]

80

[89]

34 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

Table 2.2

Chapter 2  Amylolytic Enzymes: Glucoamylases

35

stability of the enzyme under actual reaction conditions and allows easy and quantitative comparison of enzyme stability [93]. This provides a way of selecting the most efficient enzyme from a group of enzymes and, hence, gives valuable input for suggesting a suitable enzyme for an industrial process. Negi and Banerjee (2009) observed that GA produced from A. awamori was stable in the temperature range 25e70
C. The highest amylolytic activity was obtained around 70
C; beyond that there was a fall in activity. This happens because, like any other chemical reaction, the rate of catalytic reaction of an enzyme increases with rising temperature, but at higher temperatures denaturation of the enzyme also takes place [8]. Negi et al. reported the half-life period for GA as 210, 120, 60, and 35 min at 50, 60, 70, and 80
C, respectively [17]. Nguyen et al. reported a thermophilic amylase of T. lanuginosus with half-life times longer than 1 day at 60
C [47]. The MichaeliseMenten constant (Km) is another important kinetic parameter and is defined as the concentration of substrate that gives half-maximal velocity (Vmax). Km and Vmax are significant in the biochemical characterization of an enzyme. The more firmly the enzyme binds to its substrate the smaller will be the value of Km. Moreover, Km is independent of enzyme concentration and is a true characteristic of the enzyme under defined conditions of temperature, pH, etc.; and, thus, it can be used as a genetic marker to identify a particular enzyme protein. Normally, higher Vmax and lower Km are the two desirable conditions for efficient enzyme hydrolysis. Negi and Banerjee reported Km and Vmax for GA produced from A. awamori as 9.8 mg/mL and 56.2 mg/mL min, respectively [94]. Nguyen et al. reported Km and Vmax of a-amylase from T. lanuginosus on soluble starch as 0.68 mg/mL and 45.19 U/mg, respectively [47]. For industrial use, the stability of an enzyme in the presence of metal salts is very important. In fact more than 75% of enzymes require metal ions to express their full catalytic activities. The optimum concentration of metal ions can be used to enhance the catalytic activity of an enzyme in that metal ions act as cofactors of many enzymes, but a high concentration of metal ions generally causes denaturation of the enzyme [95]. Negi and Banerjee (2009) reported enhanced activity of GA produced by A. awamori in low amounts of Ca2þ, Co2þ, Cu2þ, Fe3þ, Mg2þ, Zn2þ, and Hg2þ, excepting MnCl2 [94]. Chen et al. [73] also observed that Ca2þ, Mg2þ, Naþ, and Kþ enhanced the GA activity of Chaetomium thermophilum, whereas Fe2þ, Agþ, and Hg2þ inhibited it, and similar results were also observed by Tunga et al. [96], Quang et al. [47], and Spinelli (1996) [97]. Surfactant also influences enzyme activity in several ways. Negi and Banerjee reported an increase in GA activity in the presence of low concentrations (0.03% w/v) of Sodium Lauryl Sulfate and Triton X-100, whereas Tween 40, Tween 60, and Tween 80 inhibited the activity [94]. Negi and Banerjee (2010) [98] studied changes in the patterns or motifs of secondary structures of GA from A. awamori in the presence of denaturants such as urea and guanidine-HCl (Gdn-HCl) and at different pH through circular dichroism (CD) spectroscopy. They reported that 0.5 M concentration of urea increased GA activity and at 6 M concentration of urea GA loses its activity. CD spectra of GA in the presence of GdnHCl also followed a pattern similar to urea. At lower concentration (0.1 M) of Gdn-HCl the negative peak shifted from 208 to 219 nm to a very sharp peak at 198 nm with lower

36 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

intensity than the control, and in 3 M Gdn-HCl the spectrum was totally disrupted. Selvakumar et al. reported four different forms of GA produced by A. niger, i.e., GA I, GA I0 , GA II, and GA III, having apparent molecular masses of 112.0, 104.0, 74.0, and 61.0 kDa, respectively [91].

2.5 Enzyme Assay Enzyme assay is carried out to confirm the presence of the targeted enzyme in the source organism and to evaluate the enzyme activity in the reaction mixture. Most of the substances show absorption in a UV range that can be detected by a spectrometer. Spectrophotometric assay is the commonly used technique for observing the enzyme reaction with the mixture. Photometric assays are preferred because they are easy to perform and less susceptible to disturbances [99]. Enzyme assay is carried out at a temperature in the stable zone of the enzyme activityetemperature characteristic curve. Assay of thermophilic enzymes is carried out at higher temperatures, at which their activity is stable [99]. The isolates are screened for starch-hydrolyzing ability. Fungal isolates are normally inoculated on starch potato dextrose agar (PDA) plates. Ominyi et al. [6] inoculated fungal isolates on a 1% starch PDA plate and after 3e4 days of fungal growth the plates were flooded with iodine solution. A dark blue starcheiodine complex covered the entire agar because of the reaction of starch with iodine. Clear zones surrounding streaked lines were seen when the starch was broken down into sugars, indicating starch hydrolysis [100]. Units of enzyme activity: Enzyme activity is the measurement of the amount of desired product converted from substrate per unit time per unit of enzyme. The SI unit of enzyme activity is the “katal,” which is defined as moles (product) per second. One katal is a very large unit for practical purpose, hence, micromoles per minute (mmol/min) is used to express enzyme activity. In fact, the more commonly used unit of enzyme activity is IU (International Unit), given by the Nomenclature Committee of the International Union of Biochemistry (1982). IU is defined as the amount of enzyme required to catalyze conversion of 1 mmol/min of substrate. The parameter observed to determine the rate of substrate conversion is the change in concentration of the assay; hence, assay volume is also measured to determine the enzyme unit from the rate of change in concentration. Lakshmi and Jyothi (2014) used A. oryzae to produce GA using wheat bran as substrate. GA activity was determined by incubating the reaction mixture consisting of enzyme with 1% soluble starch solution in 50 mM citrate buffer (pH 5.5) at 50
C for 20 min [38]. The liberated glucose was measured with 3,5-dinitrosalicyclic acid reagent using glucose as a standard [101]. The GA activity unit (U) was expressed as the amount of enzyme releasing 1 mol of glucose equivalent per minute per milliliter. Keera et al. used A. oryzae to produce GA by SSF on corn flour [37]. The dinitrosalicylic acid method was used to determine the GA activity by measuring released reducing sugars using glucose as a standard. Enzyme assay was performed at 50
C in 1.0% (w/v) soluble starch

Chapter 2  Amylolytic Enzymes: Glucoamylases

37

in 50 mM phosphate buffer at pH 5.0. An enzyme unit (U) was defined as the amount of enzyme that released 1 mmol of reducing sugars per minute, and enzyme activity was given in terms of units per gram dry original substrate (U/g). Nguyen et al. (2002) used T. lanuginosus for production of GA on starch-based medium. For GA assay, 1 mL reaction mixture containing 0.25 mL 0.1 M sodium acetate buffer, pH 4.6, and 0.25 mL 1% (w/v) soluble starch solution were preincubated at 50
C for 10 min. Incubation was continued for a further 15 min after addition of the appropriately diluted culture filtrate (0.5 mL) [47]. The reaction was terminated by placing the tubes in a boiling bath for 30 min and then allowed to cool. Glucose concentration was estimated by the glucose oxidase/peroxidase method using a standard glucose curve prepared under the same conditions [102]. A TtcGA from T. tengcongensis MB4 was successfully expressed in E. coli by Zheng et al. [26]. They determined GA activity in 50 mM HOAceNaOAc buffer (pH 5.0) containing 2% (w/v) maltose. The reaction mixture was incubated at 75
C and samples were removed at various time durations and the reaction was stopped by boiling at 100
C for 3 min. Removal of denatured proteins was done by centrifugation at 12,000 g for 5 min and measurement of liberated glucose was done using a D-glucose kit. One unit of enzyme activity was defined as the amount of enzyme that liberates 1 mmol of glucose per minute under assay conditions. Maximum enzyme activity was found at 75
C and pH 5.0.

2.6 Strain Improvement Selection of a particular strain out of many microbial cultures is a very vital but tedious task, especially when a commercially competent enzyme yield is to be achieved. For example, a strain of A. niger is capable of producing a large number of different enzymes. Selection of a suitable strain for the required purpose depends upon a number of factors, such as type of fermentation process, nature of the substrate, nutrients, functional characteristics of the desired enzyme, environmental conditions, etc. Microbial strains isolated from the living organism are required to be screened, grown, and maintained in a suitable environment before they are utilized for fermentation. Selective media prepared by supplementing the base medium with other compounds help in the growth of the targeted microorganism while inhibiting growth of other organisms. GA produced from conventional methods of using the parent strain of the microorganism normally exhibits normal catalytic activity in a temperature range of 50e60
C and pH range of 4e7. Many industrial process conditions are much more stringent, and in them the GA produced from parent strains is ineffective. Moreover, the cost of the process increases when bringing the temperature, the pH, etc., of the process into the range of the GA. With new techniques of protein engineering, microbiology, and molecular biology, novel strains capable of producing GA with better thermostability, better pH range, and better catalytic activity can be synthesized from the parent strain. In

38 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

industrial microbiology, induced mutagenesis using physical or chemical mutagens followed by selection is one of the effective strain improvement techniques. Pavezzi et al. produced A. awamori GA expressed in Saccharomyces cerevisiae by SmF in starches from various sources [1]. A mutant GA with improved thermostability was produced by a mutagenic polymerase chain reaction. Gene mutation fashioned three amino acid alterations in the protein structure (Ser54 / Pro, Thr314 / Ala, and His415 / Tyr) leading to an increase in the thermostability of mutant GA. A 7
C increase in the optimum temperature and 3.6 kJ/mol increase in the free energy of thermoinactivation (DG) at 65
C, and 1.8 kJ/mol at 80
C, was exhibited by the mutant GA compared to parent GA. The mutant GA showed better activity on potato starch, better yield, and a half-life twice that of the parent GA at 65
C. Kumar and Satyanarayana used nitrous acid and g-irradiation (60Co) to obtain a mutant Thermomucor indicae-seudaticae for production of GA [14]. Nitrous acid treatment was carried out by mixing the spore suspension of T. indicae-seudaticae in a 0.07 M solution of NaNO2 prepared in acetate buffer (0.2 M, pH 4.5). The samples were diluted with phosphate buffer (0.1 M, pH 7.0) to stop the reaction. In the next phase of mutagenesis, spore suspensions were exposed to 60Co g-irradiation in a dose range of 0e150 KR. A mutant strain of T. indicae-seudaticae produced 1.8-fold higher GA, retaining all the functional properties of the parent strain. Riaz et al. investigated g-ray-mediated mutagenesis of A. niger for enhancing the production of GA [7]. Mutant GA was more efficient and more stable at temperatures higher than 60
C than the parent GA. A comparison of the properties of mutant and wild-type GAs after strain improvement is listed in Table 2.3.

2.7 Commercially Available Glucoamylases GA has a wide variety of applications in various food industries. It is used in saccharification processes of starch or dextrin to produce glucose, which is further utilized by many industries like the beverage and baking industries as a substrate for various products. Because of its high demand and applications in various fields, many industries are also taking interest in industrial production of GA. For commercial purposes GA has been traditionally produced by using filamentous fungi such as Aspergillus and Rhizopus spp. Both SSF and SmF techniques are involved in industrial production of GA. One of the commercial forms of GA is available on the market by the name of Enzeco Glucoamylase Powder RO. The source of this commercial enzyme is R. oryzae. It shows GA activity and also possesses a considerable amount of protease and amylase activity. Another example of a commercial GA is Enzeco Glucoamylase-L, a liquid GA derived from A. niger. The manufacturer of these enzymes is Enzyme Development Corporation, which is a New York-based company. Dextrozyme is among one of the commercially available GAs used in various fermentation processes and is produced by A. niger. This enzyme is produced by

Chapter 2  Amylolytic Enzymes: Glucoamylases

Table 2.3

39

Comparison of Activities of Mutant and Wild-Type Glucoamylase

Source

Mutation

Effect on Mutant Type

Reference

Aspergillus niger

g-Ray-mediated mutagen

[7]

Aspergillus awamori

Mutagenic polymerase chain reaction

Thermomucor indicae-seudaticae

Using nitrous acid and g-irradiation

Sulfolobus solfataricus A. awamori

Gene cloning and expression in Escherichia coli Cys Ala246

A. awamori

Cys, chemical Gln400 modification to cysteine sulfonic acid Ala Ser411

Improved kinetic properties, Kcat ¼ 343 and 727/s, Km ¼ 0.25 and 0.16 mg/mL, Kcat/Km ¼ 1374 and 4510 mg/mL s, for mutant and parent enzyme, respectively Better yield and thermal stability at 65
C with increased enzyme activity Improved productivity, i.e., >23 U/mL, versus parent strain (18 U/mL) and higher specific growth rate than parent strain Extremely thermostable GA having optimum temperature of 90
C Thermal stability increased (stable at 66
C) Activity increased to 160% of wild type Specific activity increased by 32% compared to wild type Mutant GA showed increase in specific activity from 45 to 67.5
C. Yield increased (by 100-fold)

A. awamori A. awamori Pichia pastoris

P. pastoris A. awamori

60

Co

Disulfide bonds introduced through protein engineering Cloning and expression of catalytic domain of GA from A. awamori Heterologous expression, vector used was pHIL-D2 Gly137 Ala

Aspergillus candidus Link var aureus

Chemical mutagenesis using MNNG

A. awamori var kawachi

Protease-less mutant

Higher thermal stability and yield increases 10% higher activity than wild type, i.e., 24.5 IU/mg Mutant produces 111 U/mL GA compared to 50 U/mL GA produced from parent strain Higher GA activity

[1]

[14]

[74] [103] [103] [104] [105] [106]

[107] [108] [109]

[110]

GA, glucoamylase; MNNG, N-methyl-N0 -nitro-N-nitrosoguanidine.

Novozymes, located in Denmark. There are many industries present in China that are extensively involved in the production of the GAs, for example, DuPont Genencor Science, producing an enzyme by the name of Glucoamylase GA-L New from A. niger; Wuxi Syder Bioproducts Co., Ltd., which provides GA by the name of Syder Brand

40 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

Table 2.4

Some Commercially Available Glucoamylases [14]

Commercial Name

Source of Enzyme

Manufacturer

Glucoamylase GA-L New Amyloglucosidase A107823 Enzeco Glucoamylase-L Liquid/Solid Glucoamylase Syder Brand Glucoamylase Glucoamylase Sunson GA-L, GA KDN-GE01TM Dextrozyme DEXTRO 300L Glucozyme Af6

DuPont Genencor Science, Wuxi, China Aladdin Industrial Co., Ltd., Shanghai, China Enzyme development Corporation Sichuan Shan Ye Bio-Tech Co., Ltd. Wuxi Syder Bioproducts Co., Ltd. Sunson Industry Group Co., Ltd. Qingdao Continent Industry Co., Ltd. Novozymes (Denmark) Advanced Enzyme Technologies Ltd. Amano Enzymes USA Co., Ltd.

Glucoamylase YY0515

Aspergillus niger A. niger A. niger e e A. niger A. niger A. niger e A. niger Rhizopus niveus Rhizopus delemar A. niger

Amyloglucosidase A7420 Glucoamylase Liquid 25 KG/Drum

A. niger e

Shanghai Yuanye Biological Technology Co., Ltd., Shanghai, China Sigma Aldrich, St. Louis, MO, USA Jinzhu Tibet Co., Ltd.

e, information about the source of enzyme is not available.

Glucoamylase; and Glucoamylase YY0515 from A. niger by Shanghai Yuanye Biological Technology Co., Ltd., Shanghai, China. Some other examples of commercially available GAs and their manufacturers are listed in Table 2.4.

2.8 Conclusion and Perspective The huge commercial demand for GA makes even a small improvement in production and catalytic efficiency lucrative. There are continuous efforts to synthesize improved strains with a wide pH range and thermostability and better catalytic efficiency to suit commercial starch processing. Advances in molecular biology and protein and genetic engineering are being used to synthesize improved GA-producing strains to enhance the yield and functional properties of the enzyme. To reduce the cost of starch saccharification, GAs with better thermostability, better yield, and wider range of pH stability are being produced using advanced techniques such as site-directed mutagenesis, recombinant DNA technology, and cloning. Cheap biomass requiring minimal additional nutrients is being explored to reduce the production cost. There is a lot of room to further improve the strains, design functional properties of GA to suit particular industrial processes, and optimize production and purification of GA using new techniques and developments in the areas of microbiology, molecular biology, protein engineering, fermentation technology, bioreactors, etc., so that industrial applications using GA can become more economical.

Chapter 2  Amylolytic Enzymes: Glucoamylases

41

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Chapter 2  Amylolytic Enzymes: Glucoamylases

45

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