Kinetics of improved 1,4-alpha-D-glucan glucohydrolase biosynthesis from a newly isolated Aspergillus oryzae IIB-6 and parameter significance analysis by 2-factorial design
© Fatima and Ali; licensee Springer. 2012
Received: 14 June 2012
Accepted: 2 October 2012
Published: 6 October 2012
Sixteen different mould cultures viz. Aspergillus, Alternaria, Arthroderma, Trichoderma, Fusarium, Penicillium, Rhizopus and Chochliobolus were isolated from the soil samples of Qatar by serial dilution method. The preliminary screening of isolates was done by selecting initial colonies showing relatively bigger zones of starch hydrolysis on nutrient agar plates. The isolates were then subjected to secondary screening by submerged fermentation (SmF). The 1,4-α-D-glucan glucohydrolase (GGH) activity ranged from 1.906-12.675 U/ml/min. The product yield was analysed in dependence of mycelial morphology, biomass level and protein content. The isolate Aspergillus oryzae llB-6 which gave maximum enzyme production was incubated in M3 medium containing 20 g/l starch, 10 g/l lactose, 8.5 g/l yeast extract, 6 g/l corn steep liquor (CSL), 1.2 g/l MgSO4.7H2O, 1.3 g/l NH4Cl, 0.6 g/l CaCl2.2H2O, pH 5 at 30±2°C and 200 rpm. On the basis of kinetic variables, notably Qp (0.058±0.01a U/g/h), Yp/s (0.308±0.03ab U/g) and qp (0.210±0.032abc U/g fungal biomass/h), A. oryzae IIB-6 was found to be a hyper producer of GGH (LSD 0.0345) compared to A. kawachii IIB-2. A noticeable enhancement in enzyme activity of over 30% was observed (13.917±1.01 U/ml/min) when the process parameters viz. cultural conditions (pH 5, incubation period 72 h) and nutritional requirements (6 g/l CSL, 9.5 g/l yeast extract, 10 g/l starch, 20 g/l lactose) were further optimized using a 2-factorial Plackett-Burman design. The model terms were found to be highly significant (HS, p≤0.05), indicating the potential utility of the culture (dof~3).
KeywordsAspergillus oryzae Batch-culture 2-factorial design Glucoamylase Kinetics Mould culture
The enzyme 1,4-α-D-glucan glucohydrolase (GGH, EC 18.104.22.168) is an exo-amylase which cleaves both α-1,4 and α-1,6 glycosidic bonds, yielding β-D-glucose from the non-reducing end of starch polymer chain. GGH degrades starch to glucose in theoretically 100% yields. The reaction rate decreases with the decreasing chain length of the dextrin substrate. The enzyme is also capable of catalysing a reverse of the normal hydrolysis reaction to produce mainly maltose and isomaltose (Rangabhashiyam et al. 2011). It has wide range of applications in industries for the production of dextrose, high-fructose corn syrup (HFCS) and ethanol (Rubinder et al. 2002). With the advent of new frontiers in biotechnology, the spectrum of enzyme applications has widened in many other fields, such as clinical, medicinal and analytical chemistries, and in textile, food, detergent, paper, backing, wine, brewing, distilling and fine chemical industries. Earlier, it was considered that plant and animal materials were the best sources of enzymes. Nowadays, however, microbial enzymes are becoming increasingly important for their technical and economic advantages (Kelly and Fogarty 1976; Kumar and Satyanarayana 2009). A diverse group of microorganisms has been reported to produce the enzyme. However, commercial enzyme has traditionally been produced by employing filamentous fungi, since they secrete large quantities of extracellular enzyme. The production of an active enzyme depends on the selection of a suitable mould for the purpose. Fungal GGH contains both starch binding and catalytic binding domains, the former being responsible for activity on raw starch (Karaoglu and Ulker 2006). The soil is known to be a repository of fungal amylase producers; Aspergillus, Penicillium, Trichoderma, Fusarium and Rhizopus spp. have been isolated and characterized. However, not even a single comprehensive report has appeared in the available literature dealing with the growth kinetics, mycelial morphology and parametric significance analysis through statistical factorial design. Furthermore, it is also imperative to screen useful fungi for manufacturing of desired product (Oshoma et al. 2010).
The methods of cultivation greatly influence the production and properties of the enzyme. The most common methods of production involve either solid state (SSF) or submerged fermentation (SmF). Traditionally, GGH has been produced by the later processes as enzyme production was about 5 fold higher than with the former (Pandey et al. 2000). The conditions of fermentation such as growth period, temperature, pH, agitation and aeration and medium composition greatly affect the enzyme production under SmF. In SmF, the morphology of filamentous microorganisms varies between two extreme forms, pellets and free filaments, depending on culture conditions and the genotype of the strain. According to previous reports, mycelial morphology is crucial to the process of fermentation, not only in relation to the shape of the hyphae themselves and the aggregation into microscopic clumps (micro-morphology), but also in the pelleted form of growth (macro-morphology). However, reports on the preferred morphology are often contradictory since each one of the two extreme forms - pellets vs. filaments - has their own characteristics concerning cell physiology, growth kinetics, nutrient consumption and broth rheology, which can be regarded either as advantages or as drawbacks (Clementi and Rossi 1986; Papagianni 2004). The present study is concerned with screening of mould cultures, isolated from soil, for the production and optimization of cultural conditions for GGH being carried out aseptically from the selected species and their morphological changes. The 2-factorial Plackett-Burman experimental design was used to further identify the significant batch culture conditions influencing enzyme productivity.
Results and discussion
Screening of different mould cultures for GGH production in submerged fermentation*
Protein content (μg/ml)
Enzyme activity (U/ml/min)
Comparison of various kinetic parameters for GGH productivity by A. kawachii (IIB-2) and A. oryzae (IIB-6) at 72 h of fermentation*
Hyper producing fungal isolates
Specific growth rate
Enzyme production variables
qp (U/g fungal biomass/h)
Substrate consumption variables
Yx/s (g fungal biomass/g)
qs (g/g fungal biomass/h)
Qx (g fungal biomass/l/h)
Least significant difference (LSD)
Significance level <p>
Evaluation of fermentation medium for GGH production by A. oryzae IIB-6 in submerged fermentation*
Protein content (μg/ml)
Enzyme activity (U/ml/min)
0.01 N HCl
Sodium acetate buffer
The lactose concentrations (20 g/l) gave maximum enzyme production (13.917±1.012 U/ml/min, LSD~0.636). The mycelial morphology was observed as round pellets of intermediate sized. The protein content and DCM were 84.00±4.05 μg/ml (LSD~2.712) and 14.56±1.24 g/l (LSD~0.507), respectively. Other lactose concentrations (10 and 15 g/l) also induced relatively better enzyme production i.e., 13.461±1.511 U/ml/min and 13.573±0.502 U/ml/min, respectively. However in contrast to our study, Singh and Soni (2001) used 10 g/l lactose to stimulate enzyme production. Similar observations have also been made by Negi and Banerjee (2010) for amylase production by A. awamori. Some other workers (Vidya et al., 2012) used lactose at 30 g/l concentration and found to be the best source for maximum amylase production.
Application of Plackett-Burman design at various process parameters (designated by different captions) for GGH production by A. oryzae IIB-6*
Process parameters identified through 2-factorial design
Incubation period (h)A
CSL conc. (g/l)C
Yeast extract (g/l)D
Starch conc. (g/l)E
Lactose conc. (g/l)F
Statistical analysis of 2-factorial experimental design at various significant process parameters for GGH production by A. oryzae IIB-6*
Significant process parameters
Sum mean values
Degree of freedom
A soil-inhabited mould isolate A. oryzae IIB-6 was identified as a hyper producer of 1,4-α-D-glucan glucohydrolase (GGH) in submerged fermentation (SmF). M3 as a basal medium gave better GGH yield at 30±2°C (200 rpm). The cultural conditions such as pH 5 and incubation period (72 h) were also optimized. Among the carbon and nitrogen sources, lactose (20 g/l) and yeast extract (9.5 g/l) raised the enzyme activity to a maximal of 13.91 U/ml/min. The values of kinetic variables, notably Qp (0.058±0.011a U/ml/h), Yp/s (2.455±0.551a U/ml/g) and qp (0.210±0.032abc U/g fungal biomass/h) demonstrated that the isolated mould culture has a faster growth rate and subsequently a higher enzyme production capability (LSD~0.034). An overall improvement of more than 30% in terms of enzyme activity was accomplished when the significant process parameters were determined after Plackett-Burman design. The value of correlation (1.618E+0025 with dof~3) depicted that the model terms are highly significant (HS, p≤0.05). However, enzyme characterization is in progress prior to scale up studies.
The chemicals and reagents used in this study were of analytical grade and procured directly from Sigma (USA), BDH (UK) and Fluka (Switzerland).
Isolation and preliminary screening of mould cultures
The soil samples were collected from various localities of Doha (Qatar) in sterilized polythene bags. Each sample was diluted by serial dilution method. One millilitre of appropriately diluted soil suspension (10− 5, 10− 6) was plated on starch agar medium (10 g/l raw corn starch, 1.496 g/l KH2PO4, 1 g/l MgSO4.7H2O, 1 g/l NaNO3, 20 g/l agar, pH 4.8 and sterilized at 121°C, 15 lbs/in2 pressure for 15 min) using pour plate method. The plates were incubated at 30±2°C for 72 h and subsequently flooded with iodine solution (2 g/l iodine, 4 g/l potassium iodide prepared in deionized water). The zone of clearance around the microbial growth indicated GGH activity. The initial colonies of mould cultures showing bigger zones (~2 mm2) of starch hydrolysis in the plates were picked up and transferred to potato dextrose agar (PDA) slopes (pH 5.6) aseptically and then incubated at 30±2°C for 4–6 days until optimal growth. The slant cultures were stored at 4°C in a mini cold lab (430D, Gallenkamp, London, UK) and renewed at least twice a month.
Identification of mould isolates
The fungal isolates were identified morphologically using a scotch tape of approximately 1cm in length. The sticky end was placed over the fungal culture to pick up mycelia and other reproductive structures of fungi as reported by Harris (2000). It was placed upwards on a microscope slide. A drop of 0.5 g/l trypan blue (prepared in lactophenol) was added. A coverslip was placed over the slide culture and then visualized at 40X under a compound microscope. The identified mould cultures were confirmed after (Onion et al. 1986).
A volume of 10 ml of sterilized 0.5 g/l di-acetyl ester of sodium sulpho succinic acid (monoxal OT) were aseptically transferred to a slant culture having optimal conidial growth. The clumps of spores were broken with the help of a sterile inoculating wire loop. A homogeneous suspension was made by gently shaking the tube. The spore count was made by a haemocytometer (130M, Neubyeur, Munich, Germany) and found to be 1.2×107 CFU/ml.
Fermentation procedure and critical phases
Shake flask fermentation technique was employed for 1,4-α-D-glucan glucohydrolase (GGH) production under submerged fermentation (SmF) technique. One milliliter spore suspension was transferred to the individual 250 ml Erlenmeyer flasks containing 50 ml sterilized (at 121°C, 15-lbs/in2 pressure for 15 min) M3 liquid medium (found optimal). The initial pH was adjusted to 5. All the microbial fermentations were carried out in a rotary shaking incubator at 30±2°C, 200 rpm for 72 h. The experiments were run parallel in a set of three replicates.
Following media were evaluated for GGH production during the course of study,
M1. 30 g/l wheat bran 30, 1-L 0.01 N HCl, pH 4.6.
M2. 10 g/l starch, 5 g/l lactose, 10 g/l nutrient broth, 2 g/l (NH4)2SO4, 2 g/l CaCl2.2H2O, 1-L deionized water, pH 5.5.
M3. 20 g/l starch, 10 g/l lactose, 8.5 g/l yeast extract, 6 g/l corn steep liquor, 1.2 g/l MgSO4.7H2O, 1.3 g/l NH4Cl, 0.6 g/l CaCl2.2H2O, 1-L distilled water, pH 5.
M4. 3 g/l yeast extract, 20 g/l peptone, 0.05 g/l MgSO4.7H2O, 0.2 g/l CaCl2.2H2O, 0.1 g/l FeSO4, 1-L phosphate buffer, pH 7.2.
M5. 10 g/l starch, 10 g/l nutrient broth, 2.4 g/l (NH4)2SO4, 5 g/l CaCl2.2H2O, 1-L sodium acetate buffer, pH 6.4.
GGH isolation from fermented mash culture
The fermented broth was filtered through an oven dried (at 102°C for 15 min) pre-weighed Whatman filter paper No. 1. The mycelial morphology was observed. The clear filtrate was used for enzyme and protein assay, while cell mass was used to calculate dry weight.
Determination of mycelial morphology
Mycelial morphology was observed at macro level (Onion et al. 1986). Mycelia were categorized on the basis of their form and size as follow: Fine pellets (round clumps with diameters between 1–1.5 mm); small pellets (round clumps with diameters between 3–3.5 mm); intermediate pellets (round with diameters between 4–4.5 mm); large pellets (round clumps with diameters between 6–6.5 mm); mixed (a mixture of all previous four forms); viscous (a thick mixture of small and fine pellets with some free filaments); gelatinous (gel like mixture of fine pellets and filaments) and dumpy mass (irregular single mass with variable mycelial sizes).
The cell mass left in the pre-weighed filter paper was washed twice with distilled water and oven dried at 102°C for 2 h. The dry cell mass (DCM) was calculated by subtracting the weight of filter paper from the final weight and converted to g/l. GGH was assayed according to the method of Caldwell et al. (1968). One millilitre of enzyme (diluted to 10−3 times) and 1 ml of substrate (50 g/l Litner’s soluble starch solution in 0.05 M sodium acetate buffer, pH 5) was incubated at 60°C for 60 min with a constant stirring speed of 100 rpm. The amount of reducing sugar liberated was determined using 3, 5-dinitrosalicylic acid (DNS) reagent by measuring A546nm on a spectrophotometer against glucose as the standard. “One unit of GGH activity was the amount of enzyme that liberates 1 mg of reducing sugar (as glucose) under the specified assay conditions”. The sugar released was then converted into U/ml/min. Protein concentration was estimated in the filtrate by the method of Bradford (1976) with crystalline bovine serum albumin as the standard. The protein content was monitored by measuring A595nm.
Secondary screening of isolated mould cultures by SmF technique
Sixteen different mould cultures (coded as IIB-1 to IIB-16) of genera Aspergillus, Alternaria, Arthroderma, Fusarium, Trichoderma, Penicillium, Rhizopus and Chochlobolus spp. were screened for GGH production (Table 1). All the cultures were tested in triplicates by incubating the fermentation medium (M3 optimal) at pH 5, 30±2°C, 200 rpm for 72 h.
Parametric analysis by kinetic study
Kinetic variables were studied according to the procedure of (Pirt 1975). The values for specific growth rate i.e., μ (h-1) were calculated from the plots of ln(X) versus time of fermentation. The growth yield coefficient (Yx/s) was calculated as the dry cell mass divided by the amount of saccharide utilized during the course of fermentation. The product yield coefficients namely Yp/s and Yp/x were determined by using the relationships Yp/s=dP/dS and Yp/x=dP/dX, respectively. The volumetric rates for substrate utilization (Qs) and product formation (Qp) were determined from the maximum slopes in plots of substrate utilized and GGH produced versus the time of fermentation (dt). The volumetric rate for biomass formation (Qx) was calculated from the maximum slope in a plot of cell mass formation versus incubation time period. The specific rate constants for product formation (qp) and substrate utilization (qs) were determined by the equations qp=μ×Yp/x and qs=μ×Ys/x, respectively. Further, the specific rate for cell mass formation (qx) was, calculated by multiplying the specific growth rate (μ) with the growth yield coefficient (Yx/s).
Determination of significant batch culture conditions
Different fermentation media (M1, M2, M3, M4, M5) were evaluated for GGH production by A. oryzae IIB-6 (Table 3). All media were incubated at 30±2°C, 200 rpm for 72 h. The time course profile for GGH production in shake flasks was studied by incubating M3 medium. Incubation was carried out for 12–108 h at 30±2°C (200 rpm). The optimal initial pH for enzyme production was measured by incubating the fermentation medium for 72 h under a narrow pH range (4–6.5). The optimum concentrations of nitrogen and carbon sources were also investigated. The effects of different concentrations of CSL (2–10 g/l) and yeast extract (6.5-10 g/l) as nitrogen sources on GGH production were measured and compared with the control (run parallel). Different concentrations of starch (10–40 g/l) and lactose (5–25 g/l) as carbon sources were employed to study their effects on enzyme production. The experiments of C/N sources were conducted separately in triplicates at pH 5 for 72 h.
Statistical analysis and application of Plackett-Burman experimental design
In Eq. I, Eο is the effect of first parameter under study while M+ and M− are responses of enzyme by the selected fungal isolate. N is the total number of optimizations. In Eq. II, E is the significant parameter, β1 is the linear coefficient, β2 the quadratic coefficient while β3 is the interaction coefficient among significant process parameters.
Institute of Industrial Biotechnology
Corn steep liquor
Highly significant, dof, degree of freedom
Potato dextrose agar
Dry cell mass
Analysis of variance.
We are extremely grateful to the Director IIB and Vice Chancellor for their contributions to promote research culture in the University.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
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