Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

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Biocatalysis and Agricultural Biotechnology

journal homepage: http://www.elsevier.com/locate/bab

Enhanced production of alkaline protease by Neocosmospora sp. N1 using

custard apple seed powder as inducer and its application for stain removal
and dehairing
Fatema Matkawala a, Sadhana Nighojkar b, Anil Kumar a, Anand Nighojkar c, *
a
School of Biotechnology, Devi Ahilya University, Khandwa Road, Indore, 452001, India
b
Mata Gujri College of Professional Studies, A.B. Road, Indore, 452001, India
c
Maharaja Ranjit Singh College of Professional Sciences, Khandwa Rd., Indore, 452001, India

A R T I C L E I N F O A B S T R A C T

Keywords: Alkaline proteases find extensive applications ranging from detergent industries to therapeutics due to their
Protease production broad alkaline range activity. The present work describes the first report on optimization of alkaline protease
Statistical optimization production from Neocosmospora sp. N1 and explores its utility in detergent and leather industries. Wheat bran
Stain removal
was found to be a suitable substrate for protease production under solid state fermentation amongst several low
Dehairing
Histological staining
cost agro-based materials used in this study. Protease production was enhanced when wheat bran was supple­
FTIR mented with a novel inducer, custard apple seed powder in the ratio of 4:1 which increased enzyme production
up to 1.88 fold. One factor at a time approach was used to select parameters important for production and
response surface methodology was used for further optimization. The optimum level was attained at 123 h
fermentation time, 63% moisture content, 1 � 108 spores/ml inoculum size and 1.4 mm particle size which
resulted in 3.12 fold increase in protease production. The partially purified enzyme exhibited maximum activity
at 60 � C and was active over a wide pH range of 8–12. Protease was compatible with various laundry detergents
viz. Tide, Surf Excel, Ariel, Wheel etc. showing more than 80% stability even after 3 h of incubation and was
efficient in removing blood stain from the cotton cloth. The enzyme was also more efficient in dehairing goat skin
as compared to conventional leather processing treatment.

bleach and shelf life in detergent formulations (Vojcic et al., 2015).

Advances in improving performance of detergent proteases are ongoing
1. Introduction in terms of cost effectiveness, superior washing performance with fabric
care and improved stability. Hence, spotlight on subtilisin like proteases
Protease enzymes are ubiquitous in nature catalysing hydrolysis of derived from fungal species having robust properties have gained much
protein molecules into peptides and amino acids (Sumantha et al., interest in recent years. Fungi are known to secrete extracellular en­
2006). In fact, proteases make up the largest single family of enzymes zymes in large amounts and are a preferred choice for enzyme produc­
engrossing several bioengineering and biotechnological applications. tion due to ease in downstream processing. Recently, alkaline proteases
Alkaline proteases from the array of proteases, account for almost 40% have also gained prime importance in leather industries for their ability
of the total worldwide sales of enzymes owing to their activity and to dehair animal skin in a safe and eco-friendly manner (Sujitha et al.,
stability under harsh operational conditions (Wahab and Ahmed, 2018). 2018). The foremost step in conventional leather processing makes use
Ever since the advent of biotechnology, alkaline proteases, primarily of lime and sulphide for removing hairs from animal skin. This process
subtilisin derived from Bacillus species have served as an essential discharges considerable amount of harmful effluent causing severe
ingredient of modern laundry detergents (Maurer, 2004). The perfor­ impact on water and soil. Additionally, the inability to have precise
mance of a high grade detergent protease depends upon several pa­ control over the chemical reaction can cause skin damage and loss of
rameters such as degradation of protein stain, compatibility with hair and wool (Khandelwal et al., 2015). Therefore, proteases may serve
detergent components like surfactants, complex agents, perfumes and as a greener alternative, replacing the use of toxic chemicals in leather
other enzymes, stability in the presence of oxidizing agents such as

* Corresponding author.,
E-mail address: [email protected] (A. Nighojkar).

https://doi.org/10.1016/j.bcab.2019.101310
Received 26 June 2019; Received in revised form 16 August 2019; Accepted 26 August 2019
Available online 27 August 2019
1878-8181/© 2019 Elsevier Ltd. All rights reserved.
F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

approach and statistically optimised by response surface methodology

Abbreviations using Box-Behnken Design (BBD) to achieve maximum level of alkaline
protease production. The partially purified enzyme was thermostable
WB Wheat bran and active at alkaline pH. The enzyme was explored to check its effec­
CASP Custard apple seed powder tiveness in industrial applications like stain removal and dehairing of
SSF Solid state fermentation skin. The exceptional properties of protease from Neocosmospora sp. N1
BBD Box-Behnken Design studied in an earlier report (Matkawala et al., 2019) allow researchers to
explore more insights about the enzyme and utilize it commercially.

2. Materials and methods

industries.
The cost of enzyme plays a critical role, delimiting the use of alkaline 2.1. Microorganism
proteases for various applications. A large part of this cost is accounted
by the production media and steps involved in the downstream pro­ The present study delineates alkaline protease production using a
cessing of the enzyme (Meena et al., 2013). Recent years have witnessed novel fungus isolated from soil of a local fruit market in Indore, MP,
new and innovative biotechnological processes for production of pro­ India. Molecular identification of the strain was carried out by
teases involving solid state fermentation (SSF) as a promising technol­ sequencing 18S rRNA region. Sequence data was aligned with publicly
ogy. SSF mimics the natural habitat of microorganisms utilizing available sequences and submitted to GenBank under accession number
agro-industrial wastes as solid substrates for supporting the growth MK417797. The isolated strain was designated as Neocosmospora sp. N1.
and metabolism of the microorganisms (Pandey, 2003). In addition, a The fungus was periodically sub cultured and maintained on potato
number of physio-chemical factors affect the growth of microorganisms dextrose agar slants.
like temperature, pH, aeration, water activity, bed properties, nature of
solid substrate and its particle size (Thomas et al., 2013). Response 2.2. Inoculum preparation
surface methodology proved to be a valuable tool for studying the effect
of several factors on enzyme production by varying them simultaneously Inoculum was prepared by harvesting spores from a 5 day old fungal
in a limited number of experiments, thus saving time and cost of opti­ culture using sterile distilled water, filtered through sterile glass wool
mising production processes. Each microorganism is unique in itself and counted using a counting chamber. Spore suspension of
having distinctive requirements for growth and production of extracel­ 1 � 107spores/ml was used for inoculation.
lular enzymes, hence the criteria for achieving optimised production
varies markedly. 2.3. Selection of solid substrate
SSF has proved to be more economic technology as compared to
submerged process especially in countries having abundant biomass and Agro-industrial residues namely, wheat bran, soy bran, corn bran,
agro-industrial waste available all year round. Moreover, it offers rice bran, orange peel, papaya peel, soybean husk, wheat husk, peanut
several advantages over submerged process including high volumetric shell were screened for alkaline protease production under SSF. All the
productivity, relatively high concentration of products, less effluent substrates were dried and sieved to obtain particle size of 0.5–1 mm. SSF
generation and requires simple fermentation equipments (Pandey et al., was carried out by taking 10 gm dried substrate in 250 ml Erlenmeyer
1999; Patidar et al., 2016). Over the years, fungi remain a preferred flask, moistened with 5 ml distilled water (50% v/w) having pH 8. All
choice while using SSF for enzyme production. Till date, several reports the flasks were autoclaved at 121 � C, 15 lbs for 20 min. 1 ml of spore
on optimization of protease production in submerged fermentation have suspension was inoculated and the flasks were incubated at 30 � C for
been published. In a study, Mechri et al. (2017) optimised cultural pa­ 96 h. Enzyme assay was performed in triplicate.
rameters for alkaline protease production in submerged fermentation
from Lysinibacillus fusiformis. Similarly, Mishra (2016) optimised pro­ 2.4. Effect of custard apple seed powder
duction using Brevibacillus brevis and Sathishkumar et al. (2015) re­
ported optimization of production using Bacillus subtilis for alkaline The effect of CASP on enzyme production was studied by combining
protease in submerged fermentation. Several fungal species like Asper­ wheat bran (WB) and CASP in different ratio (5:1, 4:1, 3:1, 2:1, 1:1, 1:0,
gillus (de Castro et al., 2015a), Penicillium (Agrawal et al., 2004), Fusa­ 0:1, 1:2, 1:3, 1:4 and 1:5) and carrying out the fermentation process.
rium (Ali and Vidhale, 2013) have been exploited for alkaline protease
production in SSF. To the best of authors’ knowledge, this is the first 2.5. Enzyme extraction
report on optimization of alkaline protease production using Neo­
cosmospora species. The fermented medium was thoroughly mixed with 50 mM Tris-HCl
Enzymes required for various industrial purposes are sought on the buffer, pH 8.5 (1:20 dilution, w/v) and incubated at 30 � C on a shaker at
basis of their characteristic properties like stability at specific temper­ 120 rpm for 1 h. The slurry was centrifuged at 10,000�g for 15 min at
ature and pH, activity in the presence of metal ions, organic solvents, 4 � C. The supernatant obtained was used as crude enzyme extract.
surfactants etc. Hence, novel microorganisms producing enzyme with
attractive properties are explored. Recently, filamentous fungi produc­ 2.6. Determination of alkaline protease activity
ing alkaline protease have gained much attention due to their rich
biochemical diversity, ease in growth on inexpensive medium and cost Alkaline protease activity was determined using alkali soluble casein
effective purification of enzyme from the medium (Abidi et al., 2008). as substrate according to the method of Charles et al. (2008) with slight
Thus, the present work describes the optimization of production of modifications. The reaction mixture consisted of 1 ml of 2% (w/v) casein
alkaline protease from a newly discovered Ascomycete, Neocosmospora prepared in Tris-HCl buffer, pH 8.5 with 0.1 ml of the enzyme and
sp. N1 isolated from soil. The composition of media significantly affects incubated for 30 min at 60 � C. The reaction was stopped by adding 2 ml
the cost of production and extracellular protease synthesis in microor­ of 0.4 M TCA to precipitate undigested protein. The tubes were centri­
ganisms. Hence, agro-based material, wheat bran was selected as sub­ fuged at 5000�g for 5 min to remove all the undigested proteins. A
strate for solid state fermentation and custard apple seed powder was 0.5 ml aliquot from the supernatant was withdrawn and mixed with 5 ml
used as a novel inducer in the system to enhance protease production. of 0.4 M Na2CO3. In this tube, 1 ml of 0.5 N Folin Ciocalteau reagent was
Further, the production parameters were standardised using OFAT added to visualise the reaction. Absorbance was read at 660 nm using

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F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

UV–visible spectrophotometer. One unit of enzyme activity (U) was 2.10. Validation of the experimental model
defined as the amount of enzyme required to liberate 1 μmole of tyrosine
equivalent per min under standard assay conditions. All the experiments The validation of model equation was conducted in triplicates for
were performed in triplicate and standard error was calculated. alkaline protease production under optimised conditions and the results
obtained empirically were compared with the response predicted by the
2.7. Scanning Electron Microscopy model. Growth kinetics and protease production in the optimised me­
dium were also determined. The fungal growth in the medium was
Detailed structural analysis of uninoculated substrate and the fer­ estimated by measuring N-acetyl-D-glucosamine (NAGA) contents ac­
mented medium was performed by drying the samples and analysing cording to the method described by Sakurai et al. (1977).
them by Scanning Electron Microscopy (SEM) type JEOL JSM 5600 at
UGC DAE Consortium, Indore. 2.11. Partial purification and characterization of protease

2.8. Chemical composition of substrates The optimised medium was used for alkaline protease production in
SSF. The crude extract obtained (described under 2.5) was subjected to
Carbohydrate, protein, lipid, moisture and ash contents of WB and ammonium sulphate precipitation at 0–90% saturation and placed for
CASP were determined according to the methods described by the As­ overnight incubation at 0–4 � C. The protein precipitate was collected by
sociation of Official Analytical Chemists (AOAC, 2010). All tests were centrifugation at 9000�g for 15 min and was re-suspended in 25 mM
performed in triplicate and results were expressed as mean � standard Tris-HCl buffer, pH 8.5. Desalting was performed using Sephadex G-25
deviation. column chromatography using the same buffer. Optimum temperature
of the enzyme was determined by calculating enzyme activity in the
2.9. Statistical optimization of process parameters range of 10–90 � C and pH optima of the enzyme was determined by
measuring enzyme activity in the pH range of 6–12 using casein as a
Process parameters for alkaline protease production were studied by substrate.
one factor at a time (OFAT) approach and four variables namely;
fermentation time, inoculum size, moisture content and particle size of 2.12. Evaluation of washing performance of protease
CASP were identified to play a significant role in the fermentation
process. The optimum levels and interactions between significant pa­ 2.12.1. Compatibility and stability with laundry detergents
rameters were investigated by response surface methodology using Box- The stability and compatibility of the protease was studied using
Behnken Design, (Box and Behnken, 1960). Design-Expert 11.0 software various commercial detergents viz. Surf Excel (Hindustan Unilever
(Stat-Ease Inc., Minneapolis, MN, USA) was used to setup experimental Limited), Ariel (Procter & Gamble), Tide (Procter & Gamble), Wheel
range and levels of the four independent variables used in Box-Behnken (Hindustan Unilever Limited) and other locally available detergents viz.
Design are given in Table 1. The number of experiments (N) required for Chameli (Burhani Enterprises, Indore) and Badshah (Saify Hardware
the development of BBD was defined by the following equation: Stores, Indore). These detergents were diluted in water at the final
concentration of 7 mg/ml. The endogenous enzymes present in com­
N ¼ 2k (k-1) þ CO
mercial laundry detergents were inactivated by boiling at 100 � C for
Where, k is the number of variables and CO is the number of central 15 min. The partially purified protease was mixed with detergent solu­
points. tion in a ratio 1:1 (v/v) and incubated at 40 � C for 3 h (Wahab and
This equation was used to develop mathematical correlation be­ Ahmed, 2018). Aliquots were withdrawn after every 30 min time in­
tween four variables on the production of alkaline protease by experi­ tervals and residual enzyme activity was measured. The crude enzyme
mentally performing 29 runs with five replicates at central point. The activity of a control sample (without any detergent), incubated under
response data was used to fit the following quadratic polynomial model similar conditions, was taken as 100%.
equation:
2.12.2. Blood stain removal
Y ¼ β0 þ Σ βi Xi þ Σ βii X2i þ Σ βij Xij The stain removal ability of the protease was evaluated using
4 cm � 4 cm pieces of white cotton cloth. The cloth pieces were evenly
Where, Y represents predicted response; Xi represents independent
stained with 0.5 ml of animal blood followed by air drying for 18 h and
variables; Xij are variables interacting with each other; β0 is the coeffi­
subsequently washing with distilled water to remove excess blood.
cient of fitted response; βi is linear coefficient; βii is quadratic coefficient
These cloth pieces were again air dried and subjected to different wash
and βij represents interaction coefficient.
treatments. A stained cloth piece was treated with a local laundry
Table 2 represents the experimental design of BBD in coded levels of
detergent devoid of any endogenous enzyme. Another piece was treated
the four variables. Temperature 30 � C and pH 8 were maintained for all
with the protease used in this study. Finally, one of the stained cloth
the experimental runs. Flasks were analysed for alkaline protease ac­
piece was washed with a mixture of detergent and protease. All the
tivity at specific time intervals of 48, 96 and 144 h as planned in BBD.
above washing treatments were given at 40 � C for 15 min followed by air
drying. The cloth pieces were visually analysed for checking the effi­
ciency of stain removal. Untreated stained cloth piece was considered as
control.

2.12.3. Fourier-transform infrared spectroscopic analysis

Table 1
Levels of independent variables used in the experimental design. The structural changes on the cotton cloth fibres after stain removal
by enzyme and detergent washing treatments were analysed using
Variable Name of factor Unit Range and Levels
Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy
1 0 þ1 (ATR-FTIR) at School of Chemical Sciences, Devi Ahilya University,
A Fermentation time h 48 96 144 Indore. The spectra were performed using a Frontier PerkinElmer FTIR-
B Moisture content % (v/w) 30 50 70 SP10 spectrophotometer with a resolution of 4 cm 1 and scanning a
C Inoculum size 1 � 10� spores/ml 6 7 8 wavelength range from 500 to 4000 cm 1. For each sample, the dia­
D Particle size of CASP mm 0.5 1 1.5
mond crystal of an ATR apparatus was used and the applied torque was

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F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

Table 2
Experimental design and results for optimization of process parameters for alkaline protease production using Box-Behnken Method.
Std Run Fermentation Time (h) Moisture Content% (v/w) Inoculum Size 1 � 10X Particle Size (mm) Observed Response (U/ml) Predicted Response (U/ml)

Coded Decoded Coded Decoded Coded Decoded Coded Decoded

11 1 1 48 0 50 0 7 1 1.5 223.1 217.7

7 2 0 96 0 50 1 6 1 1.5 224.3 231.14
20 3 1 144 0 50 1 8 0 1 357.2 349.85
15 4 0 96 1 30 1 8 0 1 87.3 93.48
14 5 0 96 1 70 1 6 0 1 254.6 151.11
8 6 0 96 0 50 1 8 1 1.5 307.12 309.95
3 7 1 48 1 70 0 7 0 1 201 199.70
26 8 0 96 0 50 0 7 0 1 280.4 285.99
28 9 0 96 0 50 0 7 0 1 283.1 284.62
2 10 1 144 1 30 0 7 0 1 75.2 74.06
17 11 1 48 0 50 1 6 0 1 239.5 246.53
18 12 1 144 0 50 1 6 0 1 221.65 217.43
4 13 1 144 1 70 0 7 0 1 315.3 312.63
24 14 0 96 1 70 0 7 1 1.5 319 315.23
19 15 1 48 0 50 1 8 0 1 181.6 185.05
29 16 0 96 0 50 0 7 0 1 284.2 282.43
23 17 0 96 1 30 0 7 1 1.5 53.1 45.26
9 18 1 48 0 50 0 7 1 0.5 198 193.4
6 19 0 96 0 50 1 8 1 0.5 284.5 275.27
22 20 0 96 1 70 0 7 1 0.5 239.5 241.65
21 21 0 96 1 30 0 7 1 0.5 126.9 130.34
1 22 1 48 1 30 0 7 0 1 51.4 51.68
13 23 0 96 1 30 1 6 0 1 104 103.04
25 24 0 96 0 50 0 7 0 1 285.88 285.99
12 25 1 144 0 50 0 7 1 1.5 245.3 252.59
16 26 0 96 1 70 1 8 0 1 328.4 232.05
10 27 1 144 0 50 0 7 1 0.5 285.7 293.77
5 28 0 96 0 50 1 6 1 0.5 287.9 282.68
27 29 0 96 0 50 0 7 0 1 296.4 285.99

kept constant to maintain uniform pressure.

Table 3
Screening of solid substrates for alkaline protease production by
2.13. Dehairing studies Neocosmospora sp. N1 in solid state fermentation.
Solid Substrate Enzyme Activity (U/ml)
2.13.1. Hair removal from goat skin Wheat bran 62.4 � 2.80
Dehairing property of the crude enzyme was studied using fresh goat Corn bran 34.46 � 1.87
skin pieces (4 � 4 cm) with hair. Enzymatic treatment was performed by Soy bran 32.86 � 1.72
soaking skin piece in 50 mM Tris-HCl buffer, pH 8.5 containing 500 U/ Rice bran 15.17 � 0.73
Soybean husk 12.45 � 0.64
ml of enzyme and incubating at 40 � C in a water bath for 24 h. A skin
Wheat husk 35.6 � 1.76
piece soaked in the same buffer and incubated under similar conditions Peanut shell 8.99 � 0.41
was considered as control. Chemical hair removal was carried out by Orange peel 10.4 � 0.45
soaking the skin in a mixture of 6% lime and 3.5% sodium sulphide Papaya Peel 12.5 � 0.61
incubated under the same conditions. After incubation, hairs were
gently scratched from the surface of skin pieces using a spatula. The hair
cost of production and enhance environment sustainability (Show et al.,
and skin pieces obtained from chemical and enzymatic treatments were
2015). Therefore, WB was selected as a suitable substrate to carry out
gradually dehydrated and analysed by SEM.
the fermentation process. The production of enzymes in SSF is governed
by the composition of substrates and process parameters (de Castro
2.13.2. Histological examination of dehaired skin
et al., 2015a). Selecting a support material which can also supply nu­
Dehaired pelts obtained from the enzyme and chemical treatment on
trients in the medium is a characteristic attribute influencing the growth
goat skin were fixed in 10% formalin solution for 24 h. The fixed tissues
of microorganism and formation of desired product in SSF (Trivedi et al.,
were embedded in paraffin blocks and 4 μm thin sections were cut using
2012). Several studies have been reported illustrating the use of WB as a
microtome. These sections were stained using hematoxylin-eosin stain
powerful solid material for production of alkaline protease (Mechri
and observed under light microscope for histological examination (Rao
et al., 2017; Meena et al., 2013). Furthermore, WB has been employed as
et al., 2009).
a universal substrate for production of many industrially important
enzymes like cellulase and xylanase acting as a complete nutritious feed
3. Results and discussion for microorganisms (Limkar et al., 2019).
It is well established in literature that the production of extracellular
3.1. Selection of substrates for alkaline protease production proteases by microorganisms can be induced by adding complex protein
in the medium. Also, the requirement of nitrogen varies with the type of
This study aimed to screen several agro-based materials for pro­ microorganism (Kumar and Takagi, 1999). Using this background, WB
duction of alkaline protease using the Ascomycete, Neocosmospora sp. was combined with CASP (having good amount of protein) in different
N1. WB was found to be a superior solid substrate for alkaline protease ratio to study its effect on alkaline protease production (Fig. 1A).
production exhibiting 62.4 U/ml activity as compared to other agro- Interestingly, a 1.88 fold increase in enzyme production was observed
based materials used in this study (Table 3). The use of inexpensive when WB and CASP were combined in the ratio of 4:1. Fig. 1B shows the
substrates in the production medium is preferred in order to reduce the

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F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

Fig. 1. (A) Effect of CASP on alkaline protease production, when combined with wheat bran (WB) in different ratio. (B) SEM images of (a) dried CASP (1000x) (b)
dried wheat bran (250x) (c) growth of Neocosmospora sp. N1 on fermented medium (650x).

SEM images of dried substrates and the fermented medium. The

Table 4
microscopic structures of CASP and WB define their porous nature with
Proximate analysis of substrates used for production of alkaline protease in SSF.
efficient adsorption capacity. These can be easily approached by the
fungus for obtaining nutrients required for the growth and product Chemical components (%) WB CASP WB:CASP (4:1)
formation. Scientists have used various materials as inducers for prote­ Carbohydrate 62.73 � 3.1 29.23 � 1.44 59.12 � 2.43
ase production. Bhattacharya et al. (2012) utilised the extract of Mahua Protein 27.12 � 1.2 57.61 � 2.38 37.43 � 1.54
flowers to enhance protease production from Aeromonas species in Lipid 3.15 � 0.17 7.06 � 0.31 1.56 � 0.056
Moisture 4.78 � 0.21 2.34 � 0.11 1.71 � 0.058
submerged fermentation. In another study, Abidi et al. (2008) used Ash 2.22 � 0.12 3.76 � 0.14 1.74 � 0.062
Spirulina (algae) as an inducer for production of alkaline protease by C:N ratio 2.31 � 0.13 0.51 � 0.02 1.58 � 0.057
Botrytis cinerea while Agrawal et al. (2004) supplemented WB with soy
protein isolate for enhancing alkaline protease production by Aspergillus
oryzae and Beauveria felina. than the carbon/nitrogen ratio of WB alone, hence it favoured protease
Proximate analysis of both the substrates was performed which production. de Castro et al. (2015b) demonstrated a negative correlation
revealed that WB and CASP comprised of 27.12% and 57.61% protein, between C/N ratio with protease production and used a quaternary
respectively. The carbon/nitrogen ratio of substrates as well as their mixture of WB, soybean meal, cotton seed meal and orange peel having a
optimised combination was calculated and given in Table 4. The com­ C: N ratio of 2.35 for protease production from Aspergillus niger. In
bination of WB: CASP (4:1) has a ratio of 1.58 which was found to be less another study for acidic protease production by Aspergillus foetidus,

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F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

Souza et al. (2017) also justified that low C/N ratio of the substrate fa­ the model was found to be 0.996 while the adjusted and predicted R2
vours protease production. Hence, the combination of WB: CASP (4:1) values were calculated as 0.992 and 0.980. This indicated that only
was used for further optimization of protease production from Neo­ 0.04% of total variations cannot be explained by this model. Thus, the
cosmospora sp. N1. present R2 value reflected a good fit between observed and predicted
responses and implied that the model is reliable for predicting alkaline
3.2. Statistical optimization of process parameters protease production.

The effect of different process parameters on alkaline protease pro­ 3.2.1. Response surface plot
duction was studied by OFAT approach, and four factors namely, The regression equation presented as 3-D response surface plots
fermentation time, moisture content, inoculum level and particle size of depicting the interaction among the variables (Fig. 2). Here, each
CASP were found to be significant. Response surface methodology using response surface plot represented the effect of two independent vari­
Box Behnken Design was employed to determine the optimum level of ables, holding the other variables at zero levels.
significant variables to enhance alkaline protease production by Neo­ A concurrent relationship was seen when effect of fermentation time
cosmospora sp. N1. and moisture content was plotted against the enzyme activity, hence
A set of 29 experiments was performed and the results of the ex­ increase in moisture content with increase in fermentation time favours
periments on effect of four independent variables studied at three levels response up to the optimum level (Fig. 2a). It was seen that protease
and five central points are presented in Table 2. The statistical signifi­ production was enhanced by increasing the moisture content of the
cance of the model equation was evaluated by analysis of variance medium up to 63% after which a decline in enzyme production was
(ANOVA) using Design-Expert 11.0 software and data is presented in observed. These results are consistent with earlier reports. Germano
Table 5. The generated response was fitted in a second-order polynomial et al. (2003) reported requirement of 55% moisture for protease pro­
equation: duction using Penicillium sp. in SSF. Similarly, de Castro et al. (2015a)
also used 50% moisture for protease production using Aspergillus niger.
Y ¼ 286 þ 33.81A þ 96.66B þ 17.85C-4.21Dþ22.63ABþ48.36AC- Microbial growth and enzyme production in SSF is governed by the
16.37ADþ22.62BCþ 38.33BDþ 21.55CD- 35.78A2 -90.68B2 –0.388C2 – initial moisture content of the medium. Low moisture reduces water
10.84D2 activity, diminishing growth of microorganism due to lack of availability
Where A is the fermentation time, B is the moisture content, C is the of nutrients. Whereas, a comparatively higher moisture level impairs
size of inoculum and D is the particle size of CASP. This equation was oxygen transfer and reduces porosity as the substrate particles tend to
used to correlate the impact of experimental variables with enzyme stick together (Uyar and Baysal, 2004). The response plot also showed
production. Model coefficients were estimated by multiple linear re­ that increasing the fermentation time till 123 h favoured optimum
gressions and those with P < 0.05 were considered significant. It was protease production from Neocosmospora sp. N1. Fermentation time is a
observed that the linear effects of A, B, C; quadratic effects of A, B, D and prime factor defining an industrial process. Less fermentation period
interaction effects of AB, AC, AD, BC, BD and CD were significant for leads to high productivity and lowers the risk of contamination (Mazutti
alkaline protease production and D, C2 were found insignificant et al., 2007). However, Novelli et al. (2016) suggested that protease
(P > 0.05). The highest protease activity in the experimental design was activity is associated with different fermentation times for different
found to be 349.85 U/ml at 50% moisture, fermentation time of 144 h, strains. Ali and Vidhale (2013) observed maximum protease production
inoculum level of 1 � 108 spores/ml and 1 mm particle size of CASP. in Fusarium oxysporum in 72 h while Shivakumar (2012) reported 120 h
These results displayed that the predicted and experimental values did fermentation time for optimum protease production from Aspergillus sp.
not show significant difference (P > 0.05) suggesting the suitability of Optimum fermentation time of 72 h was also reported for production of
model for maximizing alkaline protease production. protease from Penicillium sp. by Agrawal et al. (2004).
The model F-value of 250.32 implied that model is significant The response surface plots determining optimum level of inoculum
(P < 0.0001). The “Lack of Fit F-value” of 1.83 implies the Lack of Fit is size suggested that increasing the inoculum level enhanced enzyme
not significant relative to the pure error which is the desired attribute production and inoculum size of 1 � 108 spores/ml was found to be
and signifies that data fits the model. The goodness of fit of the model optimum for protease production (Fig. 2b, d, f). A positive correlation
was checked by determination coefficient (R2). In this study, R2 value for was seen when inoculum level interacted with moisture content

Table 5
Analysis of variance for the experimental results of Box-Behnken Design.
Source Sum of Squares df Mean Square F-value p-value

Model 2.108Eþ05 14 15056.55 250.32 <0.0001 significant

A-Fermentation Time 13719.42 1 13719.42 228.09 <0.0001
B-Moisture Content 1.121Eþ05 1 1.121Eþ05 1863.90 <0.0001
C-Inoculum Size 3822.40 1 3822.40 63.55 <0.0001
D-Particle Size (CASP) 213.19 1 213.19 3.54 0.0807
AB 2047.56 1 2047.56 34.04 <0.0001
AC 9355.73 1 9355.73 155.54 <0.0001
AD 1072.56 1 1072.56 17.83 0.0009
BC 2047.56 1 2047.56 34.04 <0.0001
BD 5875.22 1 5875.22 97.68 <0.0001
CD 1858.47 1 1858.47 30.90 <0.0001
A2 8303.13 1 8303.13 138.04 <0.0001
B2 53342.49 1 53342.49 886.82 <0.0001
C2 0.9765 1 0.9765 0.0162 0.9004
D2 762.80 1 762.80 12.68 0.0031
Residual 842.10 14 60.15
Lack of Fit 690.92 10 69.09 1.83 0.2945 not significant
Pure Error 151.18 4 37.80
Cor Total 2.116Eþ05 28

*R2 ¼ 0.996, R2 (pred) ¼ 0.980, R2 (adj) ¼ 0.992.

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F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

Fig. 2. Response surface plots showing interaction effects of (a) fermentation time and moisture content (b) fermentation time and inoculum size (c) fermentation
time and particle size (d) moisture content and inoculum size (e) moisture content and particle size (f) inoculum size and particle size.

7
F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

(Fig. 2d). Earlier reports demonstrated that size of inoculum has a pro­ predicted and observed experimental value verifying that the model
found effect on growth of microorganism and enzyme production since a prediction was in good agreement with the experimental data, vali­
lower inoculum size may result in insufficient biomass for optimum dating the model design. Statistical optimization of culture parameters
product formation (Pandey et al., 2000) while a higher inoculum level resulted in 3.12 fold increase in protease production over unoptimised
increases competition between cells resulting in reduced mass transfer conditions.
capacity thus decreasing enzyme production (Limkar et al., 2019; Reddy Numerous studies portray the use of response surface methodology
et al., 2008). for optimization of medium components and physiochemical parame­
Fig. 2c and f represents the horizontal surface plots obtained between ters as it is more effective than the conventional methods of optimiza­
particle size of CASP interacting with fermentation time and inoculum tion, saves time and cost invested in attaining optimised conditions.
level, respectively. The graph reflects minimal interaction of particle Mishra (2016) used Box-Behnken Design to optimize medium and
size with these variables while a slight inclined curve was observed fermentation conditions for Brevibacillus brevis and attained 1.5 fold
between the particle size of CASP and the moisture content (Fig. 2e) enhancement in alkaline protease production. Wahab and Ahmed
suggesting significant interaction amongst them. Thus, increasing the (2018) achieved a 3.6 fold hike in enzyme production using Central
particle size of CASP decreased protease production and optimum level Composite Design (CCD) for optimization of protease production by
was predicted at 1.4 mm. The particle size of substrates used in SSF Aspergillus niger. Patel et al. (2019) optimised medium for Bacillus subtilis
largely affects enzyme production. Smaller substrate particles provide a using CCD and obtained up to 4 fold increase in protease production
larger surface area facilitating growth of microbes; however, too small over basal medium. Every organism has a unique requirement for these
particles may result in substrate agglomeration resulting in retarded parameters therefore each one has to be considered individually in order
growth. Alternatively, larger particles limit microbial growth due to to maximize enzyme production. (Suberu et al., 2019; Mechri et al.,
reduction in surface area, poor aeration and mass transfer (Pandey et al., 2017). To the best of authors’ knowledge, this is the first ever contri­
2000). Ali and Vidhale (2013) used rice bran with 0.85 mm mesh size bution towards use of CASP as an inducer for production of protease by
while Prakasham et al. (2006) used 1.4–1.0 mm particle size of green using Neocosmospora sp. N1 and its statistical optimization.
gram husk for protease production.
3.3. Partial purification and characterization of protease
3.2.2. Validation of the model
The validation of the experimental model was conducted by per­ The protease enzyme was purified from crude extract by using
forming fermentation under the predicted conditions. The optimised 0–90% ammonium sulphate fractionation and desalted over Sephadex
values of four variables taken under consideration are fermentation time G-25 chromatography column. It was concentrated by reverse dialysis
123 h, moisture content 63%, inoculum size 1 � 108 spores/ml and against solid sucrose having protease activity of 2030.6 U/ml and spe­
particle size 1.4 mm. The predicted response was calculated to be 338.9 cific activity of 59.09 U/mg. The partially purified enzyme was opti­
U/ml. The growth kinetics and protease production of the culture were mally active over pH 8 to12 and temperature 60 � C (Fig. 4). Jain et al.
studied using this optimised medium (Fig. 3). Protease production (2012) reported an alkaline protease from a Bacillus sp. having optimum
increased with the rate of growth and maximum production was temperature of 60 � C and pH 10. Similar results were reported by
observed towards the onset of stationary phase after which it remained Thakrar and Singh (2019) for a protease produced from Nocardiopsis sp.
constant throughout the study. Microbial growth associated with The partially purified alkaline protease obtained in this study was used
biosynthesis of protease has long been debated. Soares et al. (2005) further in application studies.
proposed that protease production is growth associated and maximum
enzyme production is observed at mid exponential phase. On the con­ 3.4. Wash performance studies
trary, Gupta et al. (2002) suggested that proteases are known to be
associated with the onset of stationary phase, which is marked by the 3.4.1. Compatibility with laundry detergents
transition from vegetative growth to sporulation stage in spore-formers. The suitability of an enzyme as a detergent additive depends on its
This study supported the fact that extracellular protease production is a compatibility with various laundry detergents. The data presented in
manifestation of nutrient limitation at the onset of stationary phase. Fig. 5A showed that the protease used in this study was extremely stable
Maximum protease activity of 364.4 U/ml was achieved using the in the presence of detergents like Ariel, Tide, Chameli and Badshah
standardised conditions. There was a high degree of similarity between retaining more than 90% activity even after 3 h of incubation. The ac­
tivity of protease slightly diminished when incubated with Surf Excel
and Wheel, where enzyme retained 80% activity after incubation under
similar conditions. Hammami et al. (2018) reported a protease from
Bacillus mojavensis which was stable in detergents like Ariel, Tex’tile and
Carrefour, retaining 100% activity when incubated for 1 h at 40 � C.
Benmrad et al. (2018) isolated a protease from Penicillium sp. which
retained 100% activity in Tide, Ariel and OMO, while Germano et al.
(2003) reported only 60% activity of protease from Penicillium in OMO
detergent when incubated for 1 h at 28 � C. A protease from Aspergillus
niger was stable for 1 h at 40 � C in Ariel, Tide and Lange detergents
(Wahab and Ahmed, 2018). The performance of a protease in detergent
depends on number of factors, including the detergent compounds, thus
the stability of this protease also varied with each laundry detergent.
The above result suggested that protease produced from Neocosmospora
sp. N1 can find utility as a detergent additive.

3.4.2. Blood stain removal

Stain removal ability of the protease was further evaluated using
blood stained cloth pieces. The results of cloth pieces treated variedly
Fig. 3. Growth kinetics and protease production of Neocosmospora sp. N1 in are displayed in Fig. 5B. A limited washing performance was observed
optimised medium. with detergent treatment as compared to enzyme treatment alone,

8
F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

Fig. 4. Effect of (A) temperature and (B) pH on enzyme activity of partially

purified protease.

while, the stain was completely removed when the cloth piece was
treated with the mixture of detergent and enzyme. Supplementation of
this protease in detergent improves its washing performance sustaining
its utility in future industrial applications as a bioadditive in detergent
formulations. The washing performance of alkaline proteases from Ba­
Fig. 5. (A) Relative activity of alkaline protease in presence of various laundry
cillus sp. is well documented (Rao et al., 2009). However, only a few
detergents (7 mg/ml) at different time intervals. Enzyme activity in absence of
alkaline proteases produced from fungal species are reported for their
detergent was considered as control. (B) Wash performance analysis of prote­
stain removal ability. Benmrad et al. (2018) reported a protease from ase. (a) original cotton cloth piece (b) blood stained cloth; stained cloth pieces
Penicillium chrysogenium which could remove blood stain in 1 h when washed with (c) water, (d) commercial detergent (7 mg/ml), (e) protease (500
incubated along with detergent at 40 � C. An alkaline protease from U/ml) and (f) mixture of detergent (7 mg/ml) þ protease (500 U/ml). (C) FTIR
Botrytis cinerea was able to remove blood stain when incubated at 60 � C spectrogram of original cloth (black), stained cloth washed with detergent (red)
for 15 min along with detergent (Abidi et al., 2008). and stained cloth washed with enzyme (blue). (For interpretation of the ref­
erences to colour in this figure legend, the reader is referred to the Web version
3.4.3. Fourier-transform infrared spectroscopy analysis of this article.)
FTIR was used to analyse the effect of detergent and enzyme treat­
ment on cotton fibre after stain removal. The data comparing different in % transmission of detergent treated cloth spectrum when compared to
spectrograms is depicted in Fig. 5C (the red spectrum defines cotton the control spectrum. Laundry detergents are composed of harsh
treated with detergent, blue spectrum shows cotton treated with pro­ chemicals which interact with cloth fibres during washing and eventu­
tease and black spectrum represents the original fabric). A number of ally weaken them. This is well justified in the spectra obtained from our
peaks, characteristic of cotton cloth were assigned in the spectrum. FTIR analysis. The increase in peak intensity of detergent treated cloth
Intense peaks of 3300.72 cm 1 and 1711.62 cm 1 indicated the presence spectrum accounts for lesser number of bonds. Therefore, the fabric can
of hydroxyl group (OH) and carbonyl group (CO). Absorption around lose its strength by repeated washing with detergent. The outstanding
1630–1700 cm 1 showed protonation of the ionized carboxylate group capability of our protease to remove stain without making structural
(COO) and the peaks between 1400 and 1420 cm 1 are associated with changes on the fabric amplifies its efficacy in detergent industries. In a
HCH and OCH stretching vibration. Another peak at 1338.53 cm 1 similar study, stained cloth washed with protease produced from Peni­
indicated the presence of halogenated derivatives (CF) (Fan et al., cillium chrysogenium did not impart structural change on the fabric
2012). The overall spectrum of control cloth and enzyme treated cloth (Benmrad et al., 2018).
did not show much difference; however, there was a significant increase

9
F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

3.5. Dehairing studies structure in papillary dermis was completely damaged as a result of
chemical treatment on skin. The result was further supported by SEM
3.5.1. Removal of hairs from goat skin images revealing the structural change in upper dermal layer of treated
Leather industries have started adapting the use of enzymatic skin (Fig. 6C). Saleem et al. (2012) described the use of an alkaline
dehairing of animal hides for reducing environment pollution. In this protease from Bacillus cereus in dehairing of animal hides with absence
study, goat skin pieces were dehaired using protease produced from the of collagenase activity. George et al. (2014) used alkaline protease from
fungal isolate, Neocosmospora sp. N1 and compared with the chemical Vibrio metschnikovii for dehairing of goat skin without having collage­
treatment (lime and sulphide) used as conventional method in leather nase activity. The inactivity of protease on skin collagen is one of the
industries (Fig. 6A). Hairs were easily removed after 24 h of incubation prerequisite for its application in dehairing as damage to the collagen
in both enzymatic and chemically treated skin as compared to control. fibres of the grain layer imparts unfavourable properties to finished
Upon visual assessment, the enzyme treated skin had cleaner pores, leather (Haddar et al., 2011).
smooth grain structure, white and softer appearance in contrast to the
chemical treated skin which turned black, harder with residual hair 4. Conclusion
visible in some of the hair pores. Earlier scientific reports have addressed
the applicability of Bacillus derived alkaline protease for dehairing of Microbial proteases dominate the enzyme industry, contributing two
animal skin (Hammami et al., 2018; Briki et al., 2016; Tiwary and third of the total enzyme sales. The rising demand of proteases neces­
Gupta, 2010). The protease produced from Neocosmospora sp. N1 was sitates the search for novel proteases and allows researchers to develop
comparable to Bacillus derived proteases and can find utility in leather effective technologies which can circumvent the major cost associated
industries. with enzyme production. This study focuses on optimising the produc­
tion of alkaline protease using a novel fungus, Neocosmospora sp. N1.
3.5.2. Histological staining and scanning electron microscopy The enzyme was optimally active at 60 � C and a broad range of pH 8 to
Histological sections of untreated goat skin and dehaired pelts from 12. The use of readily available agro-waste material WB in the
enzymatic and chemical treatments are shown in Fig. 6B after staining fermentation process could possibly reduce major cost associated with
with hematoxylin-eosin stain. The control skin showed the presence of the enzyme production in industries. The production of protease was
intact epidermis and collagen fibres. The absence of epidermis and enhanced up to 1.88 fold using another agro-waste CASP as a novel
empty follicles appearance was evident in the treated skin. The skin inducer in the medium. Further, the process parameters were statisti­
piece treated with enzyme had little effect on collagen fibres indicating cally optimised to attain maximum alkaline protease production of
that the enzyme was devoid of collagenase activity while the collagen 364.4 U/ml. The cost effective production with appreciable yield of

Fig. 6. (A) Goat skin pieces treated with (a) buffer

(b) protease (500 U/ml) in buffer solution and (c) 6%
lime and 3.5% sulphide solution, incubated for 24 h
at 40 � C. (B) SEM image of (a) control skin, (b)
enzyme treated skin and (c) chemical treated skin at
250x magnification. (C) Histological staining of tis­
sue section obtained from (a) control skin (b) enzyme
treated skin and (c) chemical treated skin. Micro­
graphs were taken at 10x magnification using a light
microscope. IE, PD, PD*, ICF and TCF represents
intact epidermis, papillary dermis, damaged papil­
lary dermis, intact collagen fibres and treated
collagen fibres, respectively.

10
F. Matkawala et al. Biocatalysis and Agricultural Biotechnology 21 (2019) 101310

protease from Neocosmospora sp. offers a great deal of advantage to in­ Fan, M., Dai, D., Huang, B., 2012. Fourier Transform infrared spectroscopy for natural
fibres. In: Salih Salih (Ed.), Fourier Transform - Materials Analysis, ISBN 978-953-51-
dustries, therefore, further work on large scale production can be carried
0594-7.
out in future. The efficiency of crude protease was evaluated for in­ George, N., Sondhi, S., Soni, S.K., Gupta, N., 2014. Lime and sulphide-free dehairing of
dustrial applicability. The enzyme showed excellent compatibility with animal skin using collagenase-free alkaline protease from Vibrio metschnikovii
commercial detergents and was potent in removing blood stain from NG155. Indian J. Microbiol. 54, 139–142.
Germano, S., Pandey, A., Osaku, C.A., Rocha, S.N., Soccol, C.R., 2003. Characterization
cotton fabric. The protease also exhibited outstanding dehairing prop­ and stability of proteases from Penicillium sp. produced by solid-state fermentation.
erty and proved better than the chemical treatment used in leather in­ Enzym. Microb. Technol. 32, 246–251.
dustries. Owing to the thermostability and alkaline nature, this protease Gupta, R., Beg, Q.K., Khan, S., Chauhan, B., 2002. An overview on fermentation,
downstream processing and properties of microbial alkaline proteases. Appl.
can serve as a promising tool in detergent, leather, food and textile in­ Microbiol. Biotechnol. 60, 381–395.
dustries. However, the enzyme has restricted use at low temperature and Haddar, A., Hmidet, N., Ghorbel-Bellaaj, O., Fakhfakh-Zouari, N., Sellami-Kamoun, A.,
acidic environment. Nasri, M., 2011. Alkaline proteases produced by Bacillus licheniformis RP1 grown on
shrimp wastes: application in chitin extraction, chicken feather degradation and as a
dehairing agent. Biotechnol. Bioproc. Eng. 16, 669–678.
Funding information Hammami, A., Fakhfakh, N., Abdelhedi, O., Nasri, M., Bayoudh, A., 2018. Proteolytic
and amylolytic enzymes from a newly isolated Bacillus mojavensis SA:
characterization and applications as laundry detergent additive and in leather
This research did not receive any specific grant from any funding processing. Int. J. Biol. Macromol. 108, 56–68.
agencies in the public, commercial, or not-for-profit sectors. Jain, D., Pancha, I., Mishra, S.K., Shrivastav, A., Mishra, S., 2012. Purification and
characterization of haloalkaline thermoactive, solvent stable and SDS-induced
protease from Bacillus sp.: a potential additive for laundry detergents. Bioresour.
Declaration of interest
Technol. 115, 228–236.
Khandelwal, H.B., More, S.V., Kalal, K.M., Laxman, R.S., 2015. Eco-friendly enzymatic
None. dehairing of skins and hides by C. brefeldianus protease. Clean Technol. Environ.
Policy 17, 393–405.
Kumar, C.G., Takagi, H., 1999. Microbial alkaline proteases: from a bioindustrial
Acknowledgements viewpoint. Biotechnol. Adv. 17 (7), 561–594.
Limkar, M.B., Pawar, S.V., Rathod, V.K., 2019. Statistical optimization of xylanase and
The authors acknowledge the facilities provided by the Department alkaline protease co-production by Bacillus spp using Box-Behnken Design under
submerged fermentation using wheat bran as a substrate. Biocatal. Agric. Biotechnol.
of Biotechnology, Ministry of Science and Technology, Government of 17, 455–464.
India, New Delhi (DBT) in School of Biotechnology under M.Sc. Matkawala, F., Nighojkar, S., Kumar, A., Nighojkar, A., 2019. A novel thiol-dependent
Biotechnology Program and Bioinformatics sub-center. The authors serine protease from Neocosmospora sp. N1. Heliyon 5 (8), e02246.
Maurer, K.H., 2004. Detergent proteases. Curr. Opin. Biotechnol. 15, 330–334.
acknowledge UGC-DAE Consortium for Scientific Research for extend­ Mazutti, M., Ceni, G., Luccio, M.D., Treichel, H., 2007. Production of inulinase by solid-
ing the facility of Scanning Electron Microscope used in this work and state fermentation: effect of process parameters on production and preliminary
thank Dr. D.M. Phase, UGC-DAE Consortium for Scientific Research, characterization of enzyme preparations. Bioproc. Biosyst. Eng. 30, 297–304.
Mechri, S., Berrouina, M.B.E., Benmrad, M.O., Jaouadim, N.Z., Rekik, H., Moujehed, E.,
Indore. The authors acknowledge FTIR facility provided by Dr. (Prof.) Chebbi, A., Sayadi, S., Chamkha, M., Bejar, S., Jaouadi, B., 2017. Characterization of
Ashok Kumar, Head, School of Chemical Sciences, Devi Ahilya Univer­ a novel protease from Aeribacillus pallidus strainVP3 with potential biotechnological
sity, Indore. The authors also acknowledge the facilities of Department interest. Int. J. Biol. Macromol. 94, 221–232.
Meena, P., Tripathi, A.D., Srivastava, S.K., Jha, A., 2013. Utilization of agro-industrial
of Biosciences, Maharaja Ranjit Singh College of Professional Sciences,
waste (wheat bran) for alkaline protease production by Pseudomonas aeruginosa in
Indore used in the present study. SSF using Taguchi (DOE) methodology. Biocatal. Agric. Biotechnol. 2, 210–216.
Mishra, V.K., 2016. Optimization of thermotolerant alkaline protease production from
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