Characterization of highly active keratinase procuder Bacillus cereus KK69 for biological degradation of feather waste

Journal of Environmental Treatment Techniques

2019, Volume 4, Issue 3, Pages: 357-363

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Characterization of Highly Active Keratinase

Procuder Bacillus cereus KK69 for Biological

Degradation of Feather Waste

Kerem Kaya, Emrah Nikerel*

Genetics and Bioengineering Department, Faculty of Engineering, Yeditepe University, İstanbul, Turkey

Received: 20/07/2019

Accepted: 05/08/2019

Published: 01/12/2019

Abstract

A Bacillus cereus species, with highly active keratinase have been isolated from chicken feather waste. The keratinase is used

to valorise feather containing approximately 90% protein, mostly keratin which is hard-to-degrade and insoluble in water. Although

there are many old methods for decomposing feather such as incineration, burying, and chemically, enzymes have shown that are

to be useful. This study focuses on characterizing the microorganism and its keratinase enzyme where the microorganism that

produces enzyme can degrade more than 90% of the initial chicken feather in 2 days. Optimum working conditions of the enzyme

is determined. Enzyme shows maximum activity at pH 9 and 50ºC. Bioinformatic analysis of Bacillus cereus KK69 genome

revealed that there are many possible proteases for degradation of feather. Comparing to literature, this microorganism have

displayed that produces highly active keratinase. Beside proteases, industrially important other enzymes also have been screened

from annotations.

Keywords: Bacillus cereus, industrial biotechnology, chicken feather degradation, protease, keratinase enzyme

equipped with genetic material encoding the enzyme of

Introduction

interest using well-established protocols of recombinant

DNA technology. Complementary to this, natural producers,

isolated usually from extreme environments are important to

unravel the genetic diversity present in nature, are highly

specific to its substrate and their enzyme production is

relatively inexpensive. Isolates either should possess desired

properties of production hosts or these should provide

unique and useful genetic material. Industrial workhorses

should grow fast, be safe and “easy-to-cultivate”, produce

fast with minimal by-products, sustain to harsh bioprocess

conditions and should not produce toxic side products during

fermentation. Especially with the advent of genome editing

technologies, the efficiency of bioprocesses greatly

improved [6, 7]. Additionally, using advanced approaches

such as directed evolution [8] potentially increases product

yield of natural hosts.

Enzymes are the Swiss-army-knives of (industrial)

biotechnology for sustainable bio-economy. They are used

for various applications such as food, feed, waste

management, detergents, pulp and paper, dairy, textile, etc.

[

1]. For this, industrially important enzymes represent a

significant-market, nearly $4.6 billion in 2014, $4.9 billion

in 2015, and $5.0 billion in 2016 and by 2021 the market is

expected to reach $6.3 billion [2]. In several applications,

enzymes have shown to be an “alternative technology” in

industry. For instance, in detergent industry enzymes

(

protease, lipase, amylase, and cellulase) have had been used

to place surfactants (ethoxylated alcohol, linear alkyl

benzene sulfonate and sodium soap), resulting saving energy

due to low temperature operation and being environmentally

benign [3]. Green(er) industry concepts, wide industrial

application areas are using enzymes as an alternative [4, 5],

and economic concerns lead global enzyme market to grow

every year.

Microbial production of enzymes is performed using

either natural hosts or available industrial host cells, also

referred as “workhorses” e.g. Escherichia coli,

Saccharomyces cerevisiae, Aspergillus niger, typically

World population and nutritional demand is increasing

day-by-day. As one result of this, poultry consumption

increases every year, resulting further in poultry waste. Due

to Food and Agriculture Organization statistical database

(

FAOSTAT, [9]), in last 55 years world live poultry amount

of stock increased around 20.47 billion per annum

*Corresponding Author: Genetics and Bioengineering Department, Faculty of Engineering, Yeditepe University, İstanbul,

Turkey. E-mail: emrah.nikerel@yeditepe.edu.tr.

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Journal of Environmental Treatment Techniques

2019, Volume 4, Issue 3, Pages: 357-363

worldwide (1961, 4.35 billion; 2016, 24.82 billion) Feather

waste is hard-to-dispose, becomes a significant pollution

problem, since it potentially contains pathogenic

microorganisms, and has effluvium for local area [10].

Feather contains up to 90% by weight keratin [11] which is

a structural protein present in several life forms for

protection and covering via skin, wool, fur, baleen, hair,

spines, quills; defence and aggregations via horns, claws,

nails, beaks, teeth, slimes; motions via hooves and feathers

300 µL samples were taken from the saline solution and

transferred to feather containing agar plates. The feather

plates were prepared by using (in g/L): NaCl (0.5), KH

2 4

(1.4), K HPO (0.7), MgSO (0.1), chicken feather (10), and

agar (10) at pH 7.2. After two days of incubation at 37°C,

colonies were streaked to Tryptic Soybean Agar (TSA)

plates for observing single colonies. Liquid media to produce

the keratinase enzyme, as well for degradation of feather in

broth was prepared similar to feather agar, except omitting

agar. Inocula of isolated selected bacteria for culture media

were grown in Tryptic Soybean Broth (TSB, HiMedia

M011). All chemicals used in this work are either from

Sigma-Aldrich or Merck unless otherwise stated. In all

fermentations for feather degradation, temperature, initial

pH and rotation of shaker (New Brunswick™ Innova® 44)

kept at 37°C, 7.2 and 150 rpm, respectively.

[

12].

Conventional feather decomposition is carried out using

either incineration or burying. Beside these methods, thermal

13], chemical, and enzymatic hydrolysis is performed [14]

[

to process feather and reclaim its nutritional value.

Keratinases or keratinolytic proteases are enzymes

dominantly noted as serine or metallo-proteases that can

degrade keratin containing substrates [15]. In its essence,

keratinases constitutes

sub-group of proteases or

Table 1: Bacillus sp. literature on feather degradation

peptidases, which in turn are enzymes that hydrolyse

proteins or peptides. Keratinases is often extracellularly

produced but also cell-wall bound [16, 17] and intracellular

Degradation

Percentage

Microorganism

Time Reference

Bacillus cereus B5esz

[16, 18] types have been reported. Microbial sources for

72.1%

10 days

[24]

[40]

[37]

producing keratinases are generally isolated from poultry

wastes, soil, lakes or hot springs [11]. Several fungi are also

reported to produce keratinase: Aspergillus fumigatus [19],

Aspergillus oryzae [20], Doratomyces microsporum [21],

Trichophyton sp. [22]. Alternatively, bacterial keratinases

are also extensively studied: Bacillus licheniformis [23],

Bacillus subtilis [24], Bacillus megaterium [25],

Pseudomonas sp. [26], Thermoanaerobacter keratinophilus

Bacillus cereus KB043

Bacillus subtilis FDS15

78.16%

79.33 % 21 days

Bacillus sp.

FK 46

Bacillus pumilus FH9

85%

7.8%

5 days

[39]

[16]

72 hours

[27]. Keratinolytic activity is typically measured as percent

degradation of known initial feather over time. Table 1

summarizes previous reports on feather degradation (or

keratinase production) via Bacillus species.

The keratinase enzyme has been extensively studied for

its characteristic (e.g. molecular weight) as well as in terms

of its optimum working conditions, e.g. temperature, pH.

The reported range for molecular weight is between 18 to

2.2. Screening of Proteolytic or Keratinolytic Activity

Skim milk agar was used for primary screening

proteolytic activity of the isolates, following Alnahdi, 2012

[30] with minor modifications. Skim milk (100 g/L) and agar

(20 g/L) are separately prepared, mixed at equal volume and

autoclaved at 121°C for 5 minutes. The isolates were

transferred to skim milk agar and the proteolytic activity was

assessed by visually inspecting the clear zones on the plate.

Standard protease activity was also measured using Sigma

Aldrich’s Technical Bulletin [31] based on determination of

tyrosine released from casein upon hydrolysis with protease.

130 µL 0.65% Casein solution dissolved in Glycine buffer

(pH 9) and 25 µL enzyme containing sample is mixed and

incubated for 10 minutes at 37ºC. This is followed by

addition 130 µL 110 mM trichloroacetic acid (TCA) and

incubation of the mixture for 20 minutes at 37ºC. The

mixture is then centrifuged at 10,000 rpm for 5 minutes.

Released tyrosine from casein is quantified using Folin’s

reagent. 250 µL supernatant was added to solution that

contains 625 µL 500 mM Na CO and 125 µL Folin’s

Reagent and then incubated for 30 minutes at 37ºC. After

incubation samples were read at 660 nm in

spectrophotometer (Thermo Scientific Genesys 10S UV-

VIS). To correct for tyrosine originating from hydrolysed

feather, each sample have a read blank that TCA is included

before casein is added. In addition to the above proteolytic

activity, keratinolytic activity has been assessed by

quantifying degraded feather during fermentation relative to

initially present amount per volume per time.

40 kDa [28], and the optimum working conditions are

between 6 and 12.5 for pH and mesophilic to thermo stable

between 40-75°C) for temperature, while seldomly acidic

(

keratinases has also been reported [28].

The applications of keratinases is well beyond feather

decomposition, with various uses in e.g. feed, fertilizer,

detergent and leather industry, prion degradation, cosmetics,

and pharmaceutics [29]. Therefore, the isolation,

identification and characterization keratinase producers

represents both scientific and industrial significance.

In the light of the above, the aim of this paper is to report

firstly the isolation and genomic and biochemical

characterization of new microorganisms with keratinolytic

activity. Secondly, the produced enzyme is also

characterized in its optimum conditions as well as hydrolysis

kinetics.

. Material and Methods

.1. Isolation and Growth Media

Following Cai et al. (2009), isolation of microorganisms

was performed using 10 g/L of chicken feather added in

.9% (w/v) NaCl solution and incubated at 37°C for 1 week

24]. To screen microorganisms with keratinolytic activity,

[

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Journal of Environmental Treatment Techniques

2019, Volume 4, Issue 3, Pages: 357-363

.3. Determination of kinetic parameters for keratinolysis

3.1. Biochemical characterization of Bacillus cereus KK69

strain

푚푎푥

The kinetic parameters (퐾_푀and 푣

) of a standard

푚푎푥

Michaelis-Menten equation (푣 = 푣

푆/(푆 + 퐾 )) for

As first step, being the main feature of the new isolate, the

extent of feather degradation has been characterized (Figure

2 - Left). Feather degradation over time was measured to be

up to 82%, 92%, and 93% on 1st, 2nd, and 3rd day

respectively (Figure 2 - Right), which is far larger than the

mechanical effect for feather degradation, assessed by

incubating the feather at same conditions without inoculate,

as 17% on 3rd day.

푀

keratinolysis are estimated using kinetic data on feather

degradation with different initial feather levels. The

keratinolytic activity vs. initial feather concentration data

has been fit to the model, using non-linear optimization

tools, minimizing the squared error between the data and the

model, corrected for the standard deviation in the

experimental error, taking thereby the experimentally

measured variation into account. The initial value for the

minimization problem has been provided by the calculated

parameters of a Lineweaver-Burk plot [32].

2.4. SDS-PAGE for molecular weight determination

The molecular weight of enzyme’s is estimated by SDS-

PAGE [33]. Gel, prepared using polyacrylamide resolving

gel 12% (w/v) and polyacrylamide stocking gels 5% (w/v).

Coomassie Brilliant Blue R-250 used for staining. The

resulting bands on SDS-PAGE is used to determine the

molecular weight of the protein, which is estimated with

Pageruler™ Prestained Protein Ladder (Thermo Scientific

26617).

Figure 2: Typical feather degradation in two days. (Left) Conditions:

37°C, 150 rpm; 50 mL working volume, 20 g/L feather and (Right)

the extent of feather degradation as percentage of feather over days.

.5. Whole Genome Sequencing and Bioinformatics

analyses

Whole genome of isolated microorganism is obtained

Later, the effect of initial feather concentration as

substrate on the hydrolysis kinetics is assessed, which

yielded typical Michaelis-Menten type curve (Figure 3 -

Top). Using the resulting hydrolysis data (Figure 3 -

Bottom), the parameters of the irreversible Michaelis-

Menten equation are estimated as Km is 14.4 gFeather /L and

Vmax is 0.60 gFeather/L/hr.

The feather degradation experiments were followed by

standard protease activity assays to assess the effect of

hydrolysis time, pH and temperature on protease activity. In

doing so, we first define the standard enzyme production and

assay conditions as 48h fermentation at 37°C, 150 rpm and

initial pH 7.2 with 20 g/L initial feather concentration and

the protease assay as 10 minutes hydrolysis at pH 9 and

temperature 37°C using casein as substrate. Under these

conditions, we measured the protease activity to be

using High Pure PCR Template Preparation Kit (Roche, Cat.

No: 11 796 828 001). Sequencing is performed by

microbesNG. (https://microbesng.uk). de novo sequence

assembly is done using velvet and CLC. Annotation is

performed servers

using

RAST

(

http://rast.nmpdr.org/rast.cgi, [34]). Upon annotation, the

resulting gene list is filtered for possible keratinase coding

genes, including peptidases, proteases, together with the

corresponding nucleotide sequence. The sequence has been

translated into amino acid sequence and expected molecular

weight of the enzyme is calculated taking into account the

molecular weights of the amino acids and the chain length.

Results and Discussion

Chicken feather has been incubated in saline solution

and cultures from this solution have been spread to feather-

agar, whereby isolated were selected and screened for their

activities on skim milk agar (Figure 1). Selected isolates

based o clear-zone assay performance, a single colony was

selected and tested for its feather degradation performance

in liquid medium. Preliminary identification of the colony

via Fatty Acid Methyl Ester (FAME) analysis (data not

shown) pointed that the isolate was Bacillus cereus.

0.17±0.02 U/mL. We compare the effect of each process

variable to this base-case scenario.

.2. Effect of initial feather concentration for protease

activity

Protease activity is measured over time on different

starting feather concentrations. At 80 g/L highest activity

was measured as 0.909 U/mL (49 h), at 40 g/L 0.669 U/mL

(

32 h), 20 g/L 0.289 (24 h). Here, it should be noted that,

available feather consisting mainly of keratin, would result

in free tyrosine in the medium, upon keratinolytic activity

from the microorganism. This might interfere with protease

activity measurements, which are themselves based on

quantification of tyrosine released from casein upon

proteolytic activity. Therefore, extra care must be taken to

take this into account by correctly selecting the blank during

protease assay.

Figure 1: Protease activity on skim milk agar. a) 29 b) 44 c) 67 d)

9 numbered isolates

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2019, Volume 4, Issue 3, Pages: 357-363

Figure 3: (Top) Feather degradation at different initial feather

concentrations and (Bottom) overall keratinase activity on feather

degradation, inoculum size 10%, 37°C 150 rpm, 2 days

.3. Effect of Temperature and pH on protease activity

To assess the effect of pH and temperature on the

protease activity, a series of standard protease assays were

performed. Temperatures (20°C, 40°C, 50ºC, 60°C, and

0ºC) were performed. The enzyme exhibited highest

Figure 4: The effect of process parameters on the proteolytic

activity. (Top) Initial feather concentration on feather degradation,

activity at 50ºC (Figure 4) and at 80ºC, no activity was

measured which shows that it is a mesophilic enzyme. As for

the pH (7, 8, 9, 10, and 11), highest activity was obtained at

pH 9 which shows that it is an alkaline protease.

(Middle) protease assay on temperature and (Bottom) protease assay

on pH.

For various DNA segments (including the sequence

.4. Genomic characterization of the keratin degrading

bacteria

The isolate has been further characterized by Whole

coding 16S rRNA), the best match was always various B.

cereus strains, confirming earlier FAME results. Noting the

absence of perfect match between the DNA sequence of the

isolate and the ones at available databases, we conclude that

the feather degrading isolate was Bacillus cereus is new

isolate and further name it as Bacillus cereus KK69 strain.

Lastly, we used RAST server to find compare the genome of

KK69 strain with previously studied organisms. This further

confirmed that the isolate is a Bacillus strain, close to

Bacillus cereus Q1 strain.

Genome Sequencing. Genomic DNA was isolated and the

DNA has been sequenced as a service using Illumina HiSeq

2500 infrastructure. Resulting raw reads (submitted to NCBI

database with SRA number: SRR7691678) has the average

GC content of 35.3% and have been de novo assembled

using CLC Genomics Workbench (with final genome size,

N50 and L50 numbers being 5.5 MB, 431108 and 3

respectively) and the resulting 143 contigs have been aligned

to NCBI database.

The assembled genome sequence was annotated using

RAST server. The annotation pipeline resulted in 5603

coding sequences, 44% being assigned to one of the 482

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2019, Volume 4, Issue 3, Pages: 357-363

subsystems. In particular, the subsystem protein degradation

contains 56 coding sequences, ranging from metalloendo

peptidases (EC3.4.24.-), aminopeptidases (EC3.4.11.-),

optimal temperature scale. As for the pH, standard protease

activity protocol enzyme have shown its highest activity at

pH 9, slightly over pH 8. This shows that enzyme is an

alkaline protease which keratinases are working mostly at

alkaline conditions. Future directions for this host include,

reactor optimizations (constant pH, aeration) for scale-up

studies, use in combination (either in co-culture or in

tandem) with already known enzyme workhorses, and

screening other potentially valuable industrial enzymes on

various applications and different keratin sources (feather,

hooves etc).

metallocarboxypeptidases (EC 3.4.17.-)

endopeptidase (EC 3.4.21.-) and omega peptidases (EC

.4.19.-), indicating the relatively large genomic portfolio of

to serine

the B.cereus KK69 for protease-like enzymes (Nearly 50

different protease-like enzymes are found in the annotation

file, data not shown). The sequences of each protease is

submitted

SecretomeP

2.0

Server

(

http://www.cbs.dtu.dk/services/SecretomeP) with the aim

to pinpoint the sequence of the gene(s) responsible for the

observed extracellular keratinolytic activity [35], which

yielded 46 out of 56 proteases being secreted with either

classical (signal peptide) or non-classical (e.g. secretion

signal is within the protein sequence) secretion.

The annotated genome has also been screened for

possible hydrolysis enzymes that can potentially be used in

industrial context. With around 200 EC 3.x.x.x enzymes

annotated, the genome of B. cereus KK69 indeed contains a

rich portfolio of hydrolysis enzymes. The list contains

various phosphatases (EC 3.1.3.x triphosphatase, alkaline

phosphatase, Phosphoglycolate phosphatase), carbohydrate

degrading enzymes (EC 3.2.1.x, alpha, beta-gluco or

galatosidases, pulluanases, chitinases), lipases (EC 3.1.x.x,

monoglyceride, triacylglycerol and Lysophospholipases) as

well as other proteases including microbial collegenase (EC

3.4.24.3). Considering that above 15% of the 1310 genes

assigned to an EC number is classified as hydrolase,

B.cereus KK69 can be considered as gene source for later

studies on industrially relevant enzymes.

The keratinolytic activity has been detected in culture

media, even in the absence of bacteria. To further probe the

secreted proteins, the supernatant of the culture media has

been loaded to SDS-PAGE gel for determination of

Molecular Weights of the secreted proteins. Two bands

between 15-25 kDa, two bands between 35-40 kDa, one

band between 55-70 kDa, one band between 70-100 kDa,

and one band between 100-130 kDa has been observed

(

Figure 5).

Possible proteases (both intracellular and extracellular)

are listed from the genome annotation along with their

molecular weight. 56 different possible proteases are

detected, their molecular weight ranges between 8-100 kDa,

in line with the SDS-PAGE results. One should note that,

SDS-PAGE results only monitors the approximate

molecular weight of the protein, yet the secreted proteins

might also have other, possibly hydrolysis activity.

Conclusions

A new bacteria with high keratinolytic activity has been

isolated based on both clear zone on skim milk agar and

feather degradation performance. This microorganism,

Bacillus cereus KK69, has been characterized in its

degradation kinetics as well as genomic features. The use of

bacteria allows processing keratinolytic substances,

allowing generation of high-value added products. The

bacteria grows optimally between 20-40°C and its enzyme

portfolio degrades feather up to 92.42% at 2nd day, which

ranks as highly active when compared to the reports in

literature. Expectedly, the activity decreases in either end of

Figure 5: SDS-PAGE analysis of supernatant from degraded

feather

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Journal of Environmental Treatment Techniques

2019, Volume 4, Issue 3, Pages: 357-363

1 Brandelli A., Bacterial keratinases: useful enzymes for

bioprocessing agroindustrial wastes and beyond. Food

and Bioprocess Technology, 2008. 1(2):105-116.

2 McKittrick J., et al., The structure, functions, and

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Acknowledgment

Journal editorial board thanks following reviewers to

review this article.

468.

Ethical issue

Authors are aware of, and comply with, best practice in

publication ethics specifically with regard to authorship

3 Brebu M., and Spiridon I., Thermal degradation of

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(

avoidance of guest authorship), dual submission,

manipulation of figures, competing interests and compliance

with policies on research ethics. Authors adhere to

publication requirements that submitted work is original and

has not been published elsewhere in any language.

Competing interests

The authors declare that there is no conflict of interest

that would prejudice the impartiality of this scientific work.

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