B. BALKAN and F. ERTAN: Production of a-Amylase from P. chrysogenum, Food Technol. Biotechnol.

45 (4) 439–442 (2007) 439

ISSN 1330-9862 scientific note

(FTB-1726)

Production of a-Amylase from Penicillium chrysogenum

under Solid-State Fermentation by Using Some Agricultural
By-Products

Bilal Balkan and Figen Ertan*

Trakya University, Science and Art Faculty, Department of Biology, TR-22 080 Edirne, Turkey
Received: May 23, 2006
Accepted: November 6, 2006

Summary
Solid-state fermentation (SSF) was carried out using corncob leaf (CL), rye straw (RS),
wheat straw (WS) and wheat bran (WB) as substrates for a-amylase production by a fungal
culture of Penicillium chrysogenum. The effects of moisture level, particle size and inoculum
concentration on enzyme synthesis from P. chrysogenum were investigated. Optimal mois-
ture levels of substrates were 75, 65, 65 and 55 % for CL, WS, WB and RS substrates, re-
spectively. Optimal particle size and inoculum concentration for the production of a-amy-
lase were: >1 mm, 20 %; >1 mm, 20 %; 1 mm, 20 % and >1 mm, 30 % for CL, WS, WB and
RS, respectively. WB showed the highest enzyme production with 160 U/mL under opti-
mum conditions. The other enzyme activities were 28, 49 and 45 U/mL using CL, RS and
WS, respectively.

Key words: a-amylase, Penicillium chrysogenum, solid-state fermentation

Introduction pensive and uneconomical, so they need to be replaced

with more economically available agricultural by-pro-
The most widely used enzyme in the industry for ducts to reduce the costs. The use of SSF for enzyme
starch hydrolysis is a-amylase (EC 3.2.1.1, a-1,4-glucan- production has many advantages over SmF due to its
-4-glucanohydrolase), which catalyses the endocleavage simple technique, low capital investment, lower levels of
of the a-1,4-glycoside linkages and the release of short catabolite repression and better product recovery (4).
oligosaccharides and a-limit dextrin. This enzyme is used Bacteria, yeasts and fungi can grow on solid sub-
commercially for the production of sugar syrups from strates and find applications in SSF processes. Filamen-
starch which consist of glucose, maltose, and higher oli- tous fungi are the best adapted for SSF. The hyphal
gosaccharides (1). It is also extensively used in starch li- mode of fungal growth and their good tolerance to low
quefaction and paper, food, pharmaceutical and sugar water activity and high osmotic pressure conditions make
industries. To meet the demands of these industries, low fungi efficient and competitive in natural microflora for
cost medium is required for the fermentation of a-amy- bioconversion of solid substrates (5).
lase (2). The major factors that affect microbial synthesis of
Fungal a-amylases are produced by different fermen- enzymes in a SSF system include selection of a suitable
tation techniques. Production of these a-amylases has been substrate and microorganism, particle size of the sub-
investigated through submerged (SmF) and solid-state strate, inoculum concentration and moisture level of the
fermentation (SSF) (3). The production of a-amylase by substrate. In SSF, the selection of a suitable solid sub-
SmF using synthetic media has been studied by many strate for the fermentation process is a critical factor and
researchers. The contents of synthetic media are very ex- thus involves the screening of a number of agroindus-

*Corresponding author; Phone: ++90 284 235 2824; Fax: ++90 284 235 4010; E-mail: fertan@trakya.edu.tr
440 B. BALKAN and F. ERTAN: Production of a-Amylase from P. chrysogenum, Food Technol. Biotechnol. 45 (4) 439–442 (2007)

trial materials for microbial growth and product forma- hour at 30 °C on a rotary shaker at 220 rpm. The slurry
tion (6,7). was squeezed through muslin cloth. The extract was fil-
Wheat bran (WB) was obtained from a local flour tered through a Whatman No. 1 filter paper and the fil-
mill. Corncob leaf (CL), rye straw (RS) and wheat straw trate was used as the crude enzyme.
(WS) were collected from fields. These substrates are
used as cattle feed. Enzyme assay
The present work represents an investigation into Soluble starch (0.8 %) was dissolved in boiling 0.1
a-amylase production by SSF with WB, CL, RS, and WS M acetate buffer (pH=5.0) and then cooled to 30 °C. Fresh
as substrates and the determination of optimized pro- iodine reagent was prepared by diluting 1.0 mL of stock
duction conditions. solution (0.5 % I2 in 5.0 % KI) in 500 mL of distilled wa-
ter containing 5 mL of 5 M HCl. For the assay, 0.1 mL of
enzyme solution and 0.2 mL of soluble starch solution
Materials and Methods were incubated at 30 oC for 5 min. The reaction was stop-
ped by adding 5 mL of iodine reagent. The absorbance
Fungus was measured at 620 nm against a blank (10). One unit
Penicillium chrysogenum used in this study was iso- of a-amylase is defined as the amount of enzyme which
lated from the air in Edirne city and identified by Asan hydrolyses 0.1 mg of starch in a minute at 30 °C when
et al. (8). It was found to be a good a-amylase producer 1.6 mg of starch is present.
and its enzyme property was investigated in our previ-
ous study (9). It was maintained on potato dextrose agar Results and Discussion
slants at 4 °C.
Fig. 1 shows the effect of moisture level on enzyme
Inoculum preparation production for CL, RS, WS and WB substrates. Optimal
A volume of 7 mL of sterile distilled water was moisture levels of substrates were found to be 75, 55, 65
transferred to a sporulated (7-day-old) PDA slant cul- and 65 % for CL, RS, WS and WB, respectively. Enzyme
ture. The spores were dislodged using the inoculation productions were 20, 34, 40 and 127 U/mL with CL, RS,
needle under aseptic conditions and the suspension, WS and WB, respectively. Moisture content is a critical
with appropriate dilution, was used as inoculum. A vol- factor for SSF processes because this variable has influ-
ume of 1 mL of spore suspension contained about 5·106 ence on growth and biosynthesis and secretion of differ-
spores. ent metabolites. Lower moisture content causes reduc-
tion in solubility of the nutrients of the substrate, low
Solid-state fermentation degree of swelling and high water tension. On the other
hand, higher moisture levels can cause a reduction in
Masses of 5 g of dry substrates were taken into enzyme yield due to steric hindrance of the growth of
250-mL Erlenmeyer flasks. To adjust moisture levels (% strain by reduction in porosity (interparticle spaces) of
by mass per volume), 0.1 M acetate buffer (pH=5.0) was the solid substrate, thus interfering with oxygen transfer
added. The contents of the flasks were mixed thor- (11). The moisture levels in SSF processes vary between
oughly and autoclaved at 121 °C for 20 min. The flasks 30 and 85 %. The optimum moisture content for growth
were incubated at 28 °C for 7 days. and substrate utilization depends on the organism and
the substrate used for cultivation. Cultivation of Asper-
Optimization of process parameters gillus niger on starchy substrates such as cassava and
Various process parameters affecting enzyme pro- wheat bran requires considerably lower moisture levels
duction during SSF were optimized. The strategy was to than on coffee pulp or sugarcane bagasse. This is proba-
optimize each parameter independently of the others
and subsequently optimal conditions were employed in
all experiments. 140
In a sequential order, the various process parame-
120
Enzyme production/(U/mL)

ters were optimized for maximal enzyme production by

using different solid substrates such as WB, CL, RS and 100 45 %

WS. The tested process parameters were initial moisture 55 %

80 65 %
content (45, 55, 65 and 75 %, by mass per volume), parti-
75 %
cle size (>1, 1, 0.85 and 0.60 mm) and inoculum concen- 60
tration (10, 20, 30, 40 and 50 %, by volume per mass).
40

Determination of dry mass of substrates 20

Dry mass of substrates was determined by drying
0
them in an oven at 80 °C for 24 h. Corncob leaf Rye straw Wheat straw Wheat bran
Moisture level/% (by mass per volume)
Enzyme extraction
Fig. 1. Effect of initial moisture level (% by mass per volume)
Fermented substrates were mixed thoroughly with on a-amylase production in an SSF system. Inoculum fraction
0.1 M acetate buffer (pH=5.0, 50 mL) containing 0.1 % was 20 % (by volume per mass). Process conditions: tempera-
Tween 80 (3). Contents were mixed by shaking for one ture 28 °C, incubation time 7 days
B. BALKAN and F. ERTAN: Production of a-Amylase from P. chrysogenum, Food Technol. Biotechnol. 45 (4) 439–442 (2007) 441

bly because of the greater water holding capacity of the particles provide better respiration/aeration efficiency
former substrates (5). (due to increased interparticle space). In contrast, a small
As shown in Fig. 2, appropriate inoculum concen- substrate particle may result in substrate accumulation,
trations were 20 % for CL, WS and WB and 30 % for RS. which may interfere with microbial respiration/aeration
Enzyme productions were 25, 40, 45 and 152 U/mL with and therefore result in poor growth and enzyme produc-
CL, RS, WS and WB, respectively. Inoculum level was tion (13). The lower enzyme productions were obtained
an important factor for the production of a-amylase. at small particle sizes (0.6 and 0.85 mm). Krishna and
Higher inoculum concentration increased the moisture Chandrasekaran (14) reported that banana fruit stalk par-
content to a significant extent. The free excess liquid ticles of 400 mm favoured maximal a-amylase produc-
present in an unabsorbed form will therefore give rise to tion compared to larger particles. On the other hand,
an additional diffusional barrier together with that im- Baysal et al. (4) reported that WB and RH with particle
posed by the solid nature of the substrate and lead to a size of 1000 and 500 mm, respectively, favoured a-amy-
decrease in growth and enzyme production (4). Lower lase production.
inoculum level results in a lower number of cells in the
production medium. This requires a longer time to grow
to an optimum number to utilize the substrate and form Conclusions
the desired product (12).
The use of SSF for production of a-amylase using P.
chrysogenum is an economical process and is very simple
160 to apply. All the substrates supported biosynthesis of
140 a-amylase using P. chrysogenum under SSF. CL, WS, RS
Enzyme production/(U/mL)

120 can be used as substrates for enzyme synthesis in SSF.

However, these substrates did not cause enzyme pro-
100 corncob leaf
ductions as high as WB. Therefore, WB was superior to
80 rye straw
wheat straw
other substrates for synthesis of a-amylase from P. chry-
60
wheat bran sogenum by SSF.
40

20
References
0
10 20 30 40 50
Inoculum fraction/% (by volume per mass) 1. H. Hagihara, K. Igarashi, Y. Hayashi, K. Endo, K. Ikawa-
-Kitayama, K. Ozaki, S. Kawai, S. Ho, Novel a-amylase
Fig. 2. Effect of inoculum fraction (% by volume per mass) on that is highly resistant to chelating reagents and chemical
a-amylase production in an SSF system. Process conditions: ini- oxidants from the alkaliphilic Bacillus isolate KSM.K.38,
tial moisture levels 75, 55, 65 and 65 % for CL, RS, WS and WB Appl. Environ. Microbiol. 67 (2001) 1744–1750.
substrates, respectively, temperature 28 °C, incubation time 7 2. I. Haq, H. Ashraf, M.A. Qadeer, J. Iqbal, Production of al-
days pha-amylase by Bacillus licheniformis using an economical
medium, Bioresour. Technol. 87 (2003) 57–61.
3. S. Ramachandran, A.K. Patel, K.M. Nampoothiri, S. Chan-
The particle size (specific surface area) is also very
dran, G. Szakacs, C.R. Soccol, A. Pandey, Alpha amylase
important for SSF. Fig. 3 shows the effect of substrate from a fungal culture grown on oil cakes and its proper-
particle size (mm) on a-amylase production in an SSF. ties, Braz. Arch. Biol. Technol. 47 (2004) 309–317.
Enzyme productions were 28, 45, 49 and 160 U/mL with 4. Z. Baysal, F. Uyar, Ç. Aytekin, Solid state fermentation for
CL, RS, WS and WB substrates, respectively. Particle sizes production of a-amylase by a thermotolerant Bacillus sub-
of >1 and 1 mm favoured a-amylase production. Larger tilis from hot-spring water, Process Biochem. 38 (2003) 1665–
1668.
5. M. Raimbault, General and microbiological aspects of so-
lid substrate fermentation, Electron. J. Biotechnol. 1 (1998)
160 (http://www.ejbiotechnology.info).
6. A. Pandey, C.R. Soccol, P. Nigam, V.T. Soccol, L. Vanden-
Enzyme production/(U/mL)

140
>1 mm bergh, R. Mohan, Biotechnological potential of agro-indus-
120 trial residues II: cassava bagasse, Bioresour. Technol. 74 (2000)
1 mm
100
0.85 mm
81–87.
80 0.60 mm 7. H.K. Sodhi, K. Sharma, J.K. Gupta, S.K. Soni, Production
60
of a thermostable a-amylase from Bacillius sp. PS-7 by so-
lid state fermentation and its synergistic use in the hydro-
40 lysis of malt starch for alcohol production, Process Biochem.
20 40 (2005) 525–534.
0 8. A. Asan, B. Sen, S. Sarica, Airborne fungi in urban air of
Corncob leaf Rye straw Wheat straw Wheat bran Edirne city (Turkey), Biologia Bratislava, 57 (2002) 59–68.
Particle size/mm 9. B. Balkan, F. Ertan, Production and properties of a-amy-
Fig. 3. Effect of substrate particle size (mm) on a-amylase pro- lase from Penicillium chrysogenum and its application in starch
duction in an SSF system. Process conditions: initial moisture hydrolysis, Prep. Biochem. Biotechnol. 35 (2005) 169–178.
levels 75, 20, 55 and 30 % for CL, RS, WS and WB, respectively, 10. A.M. Abou-Zeid, Production, purification and characteri-
inoculum fraction 65, 20, 65 and 20 % for CL, RS, WS and WB, zation of an extra cellular amylase enzyme isolated from
respectively, temperature 28 °C, incubation time 7 days Aspergillus flavus, Microbios, 89 (1997) 55–66.
442 B. BALKAN and F. ERTAN: Production of a-Amylase from P. chrysogenum, Food Technol. Biotechnol. 45 (4) 439–442 (2007)

11. N. Perez-Guarre, A. Torrado-Agrasar, C. Lopez-Macias, L. 13. A. Pandey, P. Selvakumar, R.C. Soccol, P. Nigam, Solid
Pastrana, Main characteristics and application of solid sub- state fermentation for the production of industrial en-
strate fermentation, Electron. J. Environ. Agric. Food Chem. 2 zyme, Bioresour. Technol. 77 (1999) 149–162.
(2003) 243–350.
14. C. Krishna, M. Chandrasekaran, Banana waste as substrate
12. P. Kashyap, A. Sabu, A. Pandey, G. Szakas, C.R. Soccol,
Extracellular L-glutaminase production by Zygosaccharomy- for a-amylase production by Bacillus subtilis (CBTK 106)
ces rouxii under solid state fermentation, Process Biochem. under solid-state fermentation, Appl. Microbiol. Biotechnol.
38 (2002) 307–312. 46 (1996) 106–111.