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Extracellular Production of Amylase and Protease by Penicillium

Purpurogenum BKS9

Article · February 2017

DOI: 10.13189/bb.2017.050101

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DOI: 10.13189/bb.2017.050101

Extracellular Production of Amylase and Protease by

Penicillium Purpurogenum BKS9
Bijay Kumar Sethi1,*, Brahmananda Dikshit1, Santi Lata Sahoo2, Chinmay Pradhan2, Sangeeta Sena3,
Bikash Chandra Behera4

1
Department of Botany, KIIT Science College, Campus-8, KIIT University, Patia, India
2
Microbiology Research Laboratory, P.G. Department of Botany, Utkal University, India
3
Department of Biotechnology, MITS School of Biotechnology, Utkal University, India
4
Department of Biotechnology, North Orissa University, India

Copyright©2017 by authors, all rights reserved. Authors agree that this article remains permanently open access under the
terms of the Creative Commons Attribution License 4.0 International License

Abstract In the present study, Penicillium food, beverage, sugar, fermentation, detergent, textiles and
purpurogenum BKS9 was used with different agro waste paper industry [4]. These enzymes are found in animals
substrates i.e. starch, wheat bran, soya powder, boiled rice, (saliva, pancreas), plants (malt), bacteria and molds [5].
unboiled rice and milk powder for the production of both Sources of amylase and protease in yeast, bacteria and molds
amylase and protease by liquid static surface fermentation have been reported and their properties have been described
(LSSF). Among the various substrates tested, wheat bran [6]. Amylase and protease of fungal origin was found to be
(WB) was found to be the best substrate for maximum more stable than the bacterial enzymes on a commercial
(112.64 U/ml) amylase production whereas soya powder scale.
(121.23 U/ml) for production of protease. Immobilization Filamentous fungi exhibit numerous inimitable properties
study also revealed that the highest amylase was observed that put together them as exceptional candidates for
(137.6 U/ml) when wheat bran was used as substrate whereas industrial exploitations. Keeping in view of the credentials,
maximum protease production (130.73 U/ml) with soya seed many of them have been used for the production of bulk
powder. Maximum biomass production was observed (4.4 ± quantities of extracellular enzymes through fermentations [7].
0.2 g/50ml) when unboiled rice was taken as a substrate in The filamentous fungi generally used for the production of
comparison to other substrate. polymer-degrading enzymes are mostly of Aspergillus and
Keywords Biotechnological Agro Food Waste Penicillium [7, 8]. Fungi generally produce α-amylase
Utilization, Fungi Immobilization, Natural Substrates, (dextrinzing enzymes), beta-amylase (saccharifying
Wastes Fermentation enzymes), proteases and other enzymes to survive on this orb.
For that, they can utilize a variety of substrates. Among
different types of substrates, the agro-substrates are easily
available, cheap carbon sources required for the growth of
microorganisms, including filamentous fungi and for the
1. Introduction production of enzymes. In this context, unboiled rice, boiled
Exploitation of enzymes as bio-catalyst is becoming the rice, wheat bran and soya powders would be the rich source
best option which gradually replacing chemical catalysts in of both macro- and micronutrients essential for the growth of
many areas of industry especially in textile and detergent fungi.
industries [1]. Further, microbial enzymes are gaining Hence in the present study attempt has been made to
biotechnological importance for their technical and produce such industrially important amylase and protease
economical advantages [2]. Among all enzymes the fungal enzyme from different fungal sources using cheap and easily
amylase and protease are two most imperative enzymes in available agro waste like unboiled rice, boiled rice, wheat
contemporary biotechnology as they satisfy the industrial bran, soya powders etc.
needs and almost replaced the chemical hydrolysis of starch
and protease in food processing industries and have utmost
significance demand. Therefore they hold approximately 25%
2. Materials and Methods
of the total enzyme trading [3]. Amylase and protease as
extracellular enzymes have potential applications in a 2.1. Inoculums Development
number of biotechnological industrial processes such as in Penicillium purpurogenum BKS9 (GenBank accession No.
2 Extracellular Production of Amylase and Protease by Penicillium Purpurogenum BKS9

KT 222270) was used for all the experiments. After min, and the absorbance of the TCA-soluble fraction was
receiving, the fungal culture was maintained as pure cultures estimated as per Lowry et al. [14]. One unit (U) of protease
on Sabouraud’s dextrose agar (SDA) medium for further activity was defined as the amount of enzyme that releases 1
experiments. The differential streaking procedures was µg of tyrosine per min.
performed according to Dubey and Maheswari [9]. The
isolated fungal species were also sub-cultured on SDA slants 2.5. Separation and Measurement of Dry Cell Biomass
at regular intervals during the entire study period. Spore
suspension (1 ml) having spore concentration of about 1 × The biomass content was determined by measuring the dry
107 cells ml-1 from 7 days old culture was used as inoculum weight of the fungal sample. The sample was centrifuged at
in the subsequent experiments [10]. 10,000 rpm, at 4°C for 10 min and the fungal biomass was
rinsed with sterilized double distilled water and filtered out
2.2. Liquid Static Surface Fermentation for Production using Whatman No.1 filter paper. The biomass obtained was
of Amylase and Protease dried overnight inside the hot air oven at 80°C till the
constant weight was attained. Finally, the dry weight of the
Liquid static surface fermentation (LSSF) was carried out
fungal mycelia was weighed and calculated.
in triplicate in 150 ml Erlenmeyer flasks containing 50 ml of
Weight of biomass = weight of dry filter paper with fungal
sterilized fermentation medium having individually starch,
biomass - weight of blank and dry filter paper
milk powder (amul), wheat bran, soya powder, unboiled rice
[Dry weight of biomass= dry weight of fungus]
and boiled rice as substrate. The fermentation medium
containing theese substrate was inoculated with 1.5 × 107
cells ml-1 culture of P. purpurogenum BKS9 in triplicate and 2.6. Immobilization of P. Purpurogenum
incubated at 30 ± 2°C static conditions for 96 h. After
completion of fermentation, enzymes were extracted from The alginate entrapment of cells was performed according
the fermented media as per Alva et al. [11] with slight method of Sharma and Satyanarayana [15]. Sodium alginate
modifications. Harvested culture of the fermented broth was solution (3%) was prepared by dissolving sodium alginate in
centrifuged at 10,000 rpm for 30 min at 4 °C and the cell free 100 ml boiling water and autoclaved at 121°C for 15 minutes.
supernatant obtained was used as crude amylase and protease Both alginate slurry and cell suspension were mixed well i.e.
enzyme for further assay. in a ratio of 1:1 for 10 minutes to get a uniform mixture. The
slurry was taken into a sterile syringe and added drop wise
into chilled 0.2 M CaCl2 solution from 5-cm height and kept
2.3. Amylase Assay
for curing at 4°C for 1 h. The cured beads were washed with
Amylase activity was measured in triplicate following the sterile distilled water 3 to 4 times. When the beads were not
method of Bernfeld [12]. The reaction mixture containing being used, they were preserved in 0.9% sodium chloride
0.5 ml of 1% soluble starch solution prepared in 0.2 M solution in the refrigerator. All operations were carried out
acetate buffer (pH 4.0) and 0.5 ml of suitably diluted enzyme aseptically under laminar flow unit.
solution was incubated at 50°C for 10 min. Enzyme and Conidia immobilized in beads of calcium alginate were
reagent blanks were incubated maintaining the same inoculated in fermentation medium and incubated at 30°C
condition simultaneously. After 10 min of incubation the for 96 h. After successful fermentation, the beads were
reaction was terminated by adding 1.0 ml of 3, 5 drained and rinsed with sterile saline solution (0.8% NaCl)
dinitrosalicylic acid (DNS) solution (1 g of DNS dissolved in equal to twice the volume of culture liquor. The beads were
20 ml of 2 M NaOH, to which 30 g of sodium potassium then again ready for use in the production of amylase and
tartarate and water were added to make it 100 ml). The protease.
reaction mixture was boiled for 15 min and after cooling O.D.
was taken in spectrophotometer at 540 nm. One unit of 2.7. Production of Amylase and Protease by Repeated
enzyme activity was defined as the amount of enzyme Batch Process
release 1 µmol of reducing sugar per min.
The reusability of immobilized P. purpurogenum cells in
2.4. Protease Assay alginate was examined. Immobilized cells were aseptically
inoculated to sterilized fermentation broth of the above agro
Extracellular protease activity was determined in triplicate waste and incubated at 30°C for 96 h. Finally, the fermented
according to the method of van den Hombergh et al. [13]. media were filtered on Whatman No. 1 paper and beads were
Each 450 µl sample was incubated with 50 µl 1% (w/v) BSA washed twice with sterile saline solution (0.8% NaCl) and
(Fraction V, Sigma) in 0.1 M sodium acetate buffer (pH 4.0) kept in phosphate buffer (1 M, pH 7.0). Then, the beads were
at 37°C. After 30 min of incubation, the reaction was again introduced into the fresh medium. After attaining
terminated with 500 µl of 10% (w/v) trichloroacetic acid maximum enzyme production, the spent medium was
(TCA). After incubation at 0°C for 30 min, the precipitated replaced with fresh production medium (50 ml) and the
proteins were removed by centrifugation at 6000 rpm for 5 process was repeated for five batches until the beads/blocks
 Bioengineering and Bioscience 5(1): 1-6, 2017 3

started disintegrating. The enzyme titers and cell leakage of for amylase activity, wheat bran was found to be the best
each cycle were determined. Amylase activity was measured substrate for maximum (112.64 U/ml) amylase production
following the method described by Bernfeld [12] and (Fig. 1). Similarly, in case of production of protease soya
extracellular protease activity was determined according to powder (121.23 U/ml) was found to be the best substrate for
the method of van den Hombergh et al. [13]. P. purpurogenum (Fig. 1). Milk powder has also a high
potential as a substrate because both protease and amylase
2.8. Statistical Analysis activities were found to be very high when used as a
Statistical analysis was performed by SPSS, version 10 substrate.
for windows (SPSS Inc., Chicago, IL, USA).
3.2. Measurement of Dry Cell Biomass
3. Results Biomass production was found to be different for different
3.1. Liquid Static Surface Fermentation for Production substrates. Maximum biomass was obtained from P.
of Amylase and Protease purpurogenum when cultivated with rice (4.4 ± 0.2 g/50ml)
at 30°C for 96 h (Fig. 2).
In the present study, among the various substrates tested

Figure 1. Comparative analysis of amylase and protease production using LSSF by P. purpurogenum

Figure 2. Biomass produced by P. purpurogenum under liquid static surface fermentation

4 Extracellular Production of Amylase and Protease by Penicillium Purpurogenum BKS9

Figure 3. Calcium alginate immobilized spores of P. purpurogenum

Table 1. Production of amylase & protease (U/ml) using immobilized beads of P. purpurogenum

Batch Unboiled rice Boiled rice Wheat bran Soya powder

operation Amylase Protease Amylase Protease Amylase Protease Amylase Protease
First 68.84 73.51 91.56 63.61 119.92 89.91 97.91 121.95
Second 78.91 80.90 97.04 69.60 137.6 101.32 109.32 130.73
Third 56.86 64.93 93.9 63.73 115.94 94.85 100.25 119.91

3.3. Immobilization of P. Purpurogemum Spores for ability to degrade starch. Khan and Yadav [19] have isolated
Production of Amylase and Protease four fungi from soil and were screened for alpha amylase
production. Among these four fungi, Aspergillus niger was
The reusability of alginate immobilized spores for found to have best activity among all the four isolates.
amylase and protease production was studied. Beads In the present study, various agro-substrates were screened
entrapping P. purpurogenum spores (Fig.3) were for both amylase and protease production by LSSF. Among
successfully used in 3 repeated cycles of fermentation using the various substrates tested for amylase activity, wheat bran
unboiled rice, boiled rice, wheat bran and soya seed powder was found to be the best substrate for amylase production by
as the substrates. The production of amylase and protease P. purpurogenum. In the present study, due to significantly
gradually increase from first cycle to the second cycle and higher volumetric activity achieved with wheat bran, it was
decrease thereafter. Maximum production of amylase (137.6 used in the modified fermentation medium. It is important to
U/ml) was attained with wheat bran whereas the production note that this substrate is economic and could be employed
of protease (130.73 U/ml) with soya powder (Table 1). for industrial production of amylase. Biosynthesis of
manifold forms of glucoamylase (GA) and production of
acid protease with wheat bran as a substrate by different
4. Discussion fungal sp. were also reported earlier [20, 21, 22]. Sindhu et al.
[23] have studied the effect of incubation period on the
Bacterial amylases and proteases have long been used in production of protease using P. godlewskii SBSS 25 and
industry, but, now-a-days, they are replaced by fungal reported the highest production of enzyme on the fourth day
amylases as these enzymes can be effortlessly extracted and of cultivation in SmF which is at par with the present finding.
separated from fungal biomass [16]. Liquid static and/or Immobilization technique is generally employed for the
solid state fermentation can be used for the large-scale entrapment of microbial cells, biocatalysts and other
production of these enzymes. Numerous amylase-producing metabolites for the repeated use. But, recently, it has been
organisms have been documented [17, 18]. For example, extended to fungi. Immobilized fungal spore/cells were first
many species of genus Aspergillus are renowned for their applied for the biotransformation of cortex lone to
 Bioengineering and Bioscience 5(1): 1-6, 2017 5

hydrocortisone by Curvularia lunata and of glucose to amylase production by mixed culture of Aspergillus niger and
itaconic acid by A. terreus. At present, immobilization of Saccaromyces cerevisae grown on sorghum pomace, African
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