Author’s Accepted Manuscript

Collagen and collagenolytic proteases: A review

Prashant K. Bhagwat, Padma B. Dandge

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PII: S1878-8181(18)30214-7
DOI: https://doi.org/10.1016/j.bcab.2018.05.005
Reference: BCAB757
To appear in: Biocatalysis and Agricultural Biotechnology
Received date: 13 March 2018
Revised date: 1 May 2018
Accepted date: 11 May 2018
Cite this article as: Prashant K. Bhagwat and Padma B. Dandge, Collagen and
collagenolytic proteases: A review, Biocatalysis and Agricultural Biotechnology,
https://doi.org/10.1016/j.bcab.2018.05.005
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 Collagen and collagenolytic proteases: A review

Prashant K. Bhagwata and Padma B. Dandgeb*

a
Department of Microbiology, Shivaji University, Kolhapur, 416004 India
b
Department of Biochemistry, Shivaji University, Kolhapur, 416004 India

*
Corresponding author: Dr. (Mrs.) Padma B. Dandge, Department of Biochemistry, Shivaji University,
Kolhapur 416004, India Co. No.: +919921111722. E-mail: pbd_biochem@unishivaji.ac.in

1
Abstract
Despite of having enormous applications, the use of collagen is predominantly limited because of its
high cost. Most of the mammalian sources used for its production have major drawbacks. However, compared to
mammalian sources, fish waste can be utilized as cost-effective alternative to produce collagen. Around 75%
part of fish is discarded as a waste which contains high concentration of collagen. Fish collagen has multiple
advantages over mammalian collagen and hence can be a promising alternative for it. Proteases with
collagenolytic activities are also of immense importance because of their industrial as well as biological
applications. Microbial collagenolytic proteases are gaining huge attention in these days because of their lower
requirements and higher productivity. They perform important role in global recycling of collagenous waste.
This review gives recent information on collagen and collagenolytic proteases. Here, utilization of seafood by-
products is discussed to recover the collagen and its recent applications are summarized. In addition to this,
current review also highlights the recent status of collagenases in which present strategies and new technology
used for the isolation, screening, production optimization, purification, characterization and applications of
microbial collagenases are discussed.

Keywords: Collagen; Fish waste; Collagenolytic protease; Applications of microbial collagenase.

2
Introduction
In a world with 7.3 billion people, which is estimated to rise by another 2 billion by 2050; human race
have the huge challenge of feeding the planet while safeguarding its natural resources for future generations.
Fishery industry is one of the established food sectors which can supply ample amount of food to deliberately
growing population. Fish is one of the most-traded food commodities in global market. It is essentially
imperative for developing countries, sometimes worth half the total value of their traded commodities (FAO
2014). Marked growth in fisheries and aquaculture sector was observed with increasing population. Total world
fishery production in the year of 2007 was around 140 million tonnes which increased up to 167 million tonnes
in the year of 2014 (Table 1).
India’s worldwide share in fishery industry is increasing day by day. India stands at 7th position
worldwide in case of fish captures from marine sources and it is at 3rd position in fish captures from inland
sources as per the report of Food and Agriculture Organization (FAO 2016). Heightened growth is seen in both
marine as well as inland fish production in India (Table 2 and 3). Interestingly inclined growth has been
observed in the inland fish production showing necessity of fish as a food source in noncoastal regions of India.
Huge amount of fish production and its consumption results in the generation of waste in equal
quantities as that of final product. Various steps are involved in fish processing like stunning, grading, slime
removal, deheading, washing, scaling, gutting, cutting of fins, meat bone separation and steaks and fillets. These
steps generate 20-80% of waste depending upon the level of processing and type of fish (Ghaly et al. 2013).
Proper disposal system should be used for the large quantity of waste produced, but a good care cannot be taken
for all the waste produced. Current waste disposal systems are polluting our environment in a very rigid manner.
Landfilling and incineration are some of the frequently used methods for waste disposal, but they are not fruitful
as they are costly as well as require a good maintenance (Kim & Venkatesan 2014). Fish waste possesses a huge
concentration of collagen protein, which is having very high market value although it is not utilized properly.
Fruitful results can be obtained if this collagenous waste is treated in an ecofriendly manner using a
biotechnological way.
This review provides pertinent information related to recovery of collagen from seafood by-products as
well as collagenolytic proteases from various microbial sources. First part of this review highlights the chemical
nature of collagen, its sources as well as its applications in various industries. In continuation to this later part
entails about collagenases, its sources and new techniques used for isolation and screening of collagenolytic
microorganisms. It is then followed by qualitative as well as quantitative analysis, production optimization,
purification, characterization and applications of microbial collagenases.

Collagen
Collagen is the foremost constituent of the extracellular matrix which is abundant fibrous structural
protein in all higher entities (Sweeney et al. 2008). It is mostly found in fibrous tissues such as skin, ligament
and tendon in the form of elongated fibrils and is also abundant in cornea, blood vessels, bone, cartilage,
intervertebral disc and the gut. These are the most abundant protein in mammals constituting over 30% of the
total proteins in animal body (Pati et al. 2010). The unique structure of collagen is responsible for its fibrous
nature which is very hard to degrade (Suzuki et al. 2006).

Collagen structure

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 Ramachandran (Ramachandran & Kartha 1955) proposed a three dimensional structure for collagen by
using fibre diffraction pattern of kangaroo tail tendon which is also known as Madras model. Later in the same
year with more stringent stereo chemical criteria, Rich (Rich & Crick 1955) refined the triple helical structure.
Both the models states that, a coiled coil conformation is formed by the three polypeptide chains. Triple helix, a
unique tertiary structure is the most prominent feature of the collagen molecule. The distinct structure of
collagen is formed by three identical or non-identical polypeptide chains. Each chain is composed of around
1000 amino acids or more in length in some collagen types. Super coiling of three polypeptide chains in a left
handed manner around a common axis, with staggering of one residue between the adjacent chains leads to a
single extended right-handed triple helical conformation. Glycine is the only amino acid which can be
accommodated in the interior part of the triple helix without chain distortion. The close packing of three chains
around a common axis leads to a steric constraint on every third residue. N, C-telopeptides are the non-helical
terminals of triple helix which perform a significant role in the formation of micro-fibril and fibril (Figure 1).
The arrangement of amino acids in a unique fashion leads to formation of triple-helical structure of
collagen. Glycine is having the smallest side group and is repeated at every third location in the order; it permits
close packaging of the chains into a helix and leaves very minute space for residues in the core. In the repeating
unit of Gly-X-Y, around 35% of the non-glycine positions are engaged by proline which is almost exclusively
found in the X-position while Y-positions are predominantly occupied by 4-hydroxyproline. Prolyl hydroxylase
converts proline in to hydroxyproline by post-translational hydroxylation (Kucharz 1992). Hydroxyproline
comprises around 10% of the amino acid composition of collagen which can be readily used for the
quantification of collagen or its degraded products in the presence of other proteins (Woessner 1961). Along
with hydroxyproline collagen also have the presence of unusual amino acid hydroxylysine. Hydroxylysine is
formed from lysine by enzymatic hydroxylation through lysyl hydroxylase; which is exactly similar to the
conversion of proline to hydroxyproline. Hydroxylysyl residues provides the attachment of sugar components
which is very vital for the formation of triple-helical structure of the collagen molecule (Piez 1984).
Around twenty eight diverse types of collagen have been identified in vertebrates which are composed
of at least forty six distinct polypeptide chains (Heino 2007; Shoulders & Raines 2009). Most abundant
collagens are of type I, II, and III which provides the scaffolding and guide cells to migrate, proliferate and
differentiate (Gullberg et al. 1992; Tuckwell et al. 1996; Farndale et al. 2008).

Sources of collagen
Collagen and gelatin are different forms of the same macromolecule. Gelatin is a soluble protein
obtained by partial hydrolysis of collagen. In recent times applications of collagen and gelatin in the field of
food, cosmetic, photographic, medicine and cell cultures have increased. Most of the times the collagen and
gelatin used in the industrial products are obtained from mammalian sources (bovine and porcine) whereas;
production of collagen and gelatin from the fish waste has received considerable attention in recent years
(Bhagwat & Dandge 2016).
On the other hand collagen and gelatin from mammalian sources are facing problems due to allergic
reactions induced by them as well as risk of transmissible diseases like ovine and caprine scrapie, bovine
spongiform encephalopathy (mad cow disease), foot/mouth disease and other zoonoses (Bhagwat & Dandge
2016). Moreover, certain religions forbid the use of bovine and porcine products. In comparison to this, fish

4
grade collagen and gelatin have the lower risk of transferring pathogens, and these products do not refute any
religious sensitivity (Herpandi et al. 2011).
Significant amount of waste is produced during fish processing which is around 75% of the fish weight
and it consists of head, skin, bones and scales. These by-products are a rich source of collagen having
bioactive properties (Silva et al. 2014). Various methods reported for extraction of the collagen in which
extracted collagen is attributed based on the difference in extraction processes as UAC (ultrasound assist
collagen), PSC (pepsin soluble collagen), ASC (acid soluble collagen) and SSC (salt soluble collagen) (Pal
& Suresh 2016b). Previously collagen has been extracted from the body parts of variety of fish like
Lagocephalus gloveri, Sepiella inermis, Lutjanus vitta, Magalaspis cordyla, Trachurus japonicus, Cypselurus
melanurus, Dentex tumifrons, Mugil cephalis, Sardinella longiceps, Otolithes ruber, Parupeneus heptacanthus,
Mystus macropterus, Evenchelys macrura, Saurida spp., Priacanthus tayenus, Syngnathus schlegeli etc.
(Venkatesan et al. 2017). Extraction of collagen from various sources using different extraction conditions is
given in Table 4. The extracted collagen is closely similar to mammalian collagen and hence can be used as a
captivating alternative for it (Bhagwat & Dandge 2016).

Applications of collagen
Collagens have tremendous industrial applications; majorly of which lies in pharmaceutical and food
industries (Figure 2). Collagen has been considered as an excellent biomaterial for the development of wound
dressing systems and tissue engineering constructs due to its exceptional biocompatibility, low antigenic and
high direct cell adhesion ability (Bilek & Bayram 2015; Hu et al. 2017; Pal et al. 2015; Pati et al. 2010). For
medical applications; collagens are reported to be processed into various forms such as sheets (Alberti et al.
2014), scaffolds (Campbell et al. 2017), tubes (Fujimaki et al. 2017), films (Wang et al. 2017), sponges
(Konstantelias et al. 2016), membranes (Nakahara et al. 2017), composites (Fu et al. 2017), fleeces (Zirk et al.
2016), injectable solutions (Moreira et al. 2016) and dispersions (Mottahedi & Han 2016).
Various efforts have been made in order to apply these systems for delivery of the drug in numerous
applications such as ophthalmology (Agban et al. 2016; Calderon-Colon et al. 2012; Deng et al. 2010), wound
and burn dressing (Held et al. 2016; Kondo & Kuroyanagi 2012; Muthukumar 2014), tumor treatment
(Wolinsky et al. 2012; Yip & Cho 2013) and tissue engineering (Grover et al. 2012; Su et al. 2012; Xia et al.
2013). Collagen is reported to contribute for the cell growth promotion, differentiation and regulation of various
cell functions (Yamada et al. 2014). It also plays a role in the formation of cell expression, tissues and organs
(Ferreira et al. 2012).
Supplementation of collagen in food enhances their nutritive as well as functional property which
ultimately results in improved health benefits (Bilek & Bayram, 2015). Synthesis of collagen decreases with
aging which can be gained by consuming collagen supplemented food products. The metabolites of collagen
attract fibroblasts and they help in the synthesis of new collagen which then assembles bone, skin and ligaments
(King’ori 2011). Collagen supplements helps to fulfill the collagen requirement of the body. Hence, the food
products supplemented with collagen may have tremendous potential and health benefits (Antoniewski &
Barringer, 2010; Hashim et al. 2015; Pal & Suresh 2016a). Recently in the food industry they are extensively
used in products as foaming agents, emulsifiers, stabilizers, microencapsulating agents and biodegradable film-
forming materials (Gómez-Guillén et al. 2011; Herpandi et al. 2011).

5
 Applications of collagen were also suggested in functional food (Lordan et al. 2011), drinks (Bilek &
Bayram, 2015), dietary supplements (Clark et al. 2008), confectionery (Cai et al. 2017) and desserts (Li et al.
2015). It has also been used as a food additive which subsequently showed the improvement in rheological
properties of foodstuffs (Baziwane & He 2003). Collagen films or coatings help to extend the shelf-life of the
products and also function as carriers of active substances (Bonilla et al. 2012; Galus & Kadzinska 2015; Silva-
Weiss et al. 2013). The collagen mediated delivery systems in the form of mini pellets and tablets are used for
drug delivery (Jeevithan et al. 2013; Lee et al. 2001).
Due to astonishing attributes of collagen, they are exploited in various other sectors like in cosmetic
industries (Yun 2005) as well as in removal of oil from oil spill (Thanikaivelan et al. 2012).

Collagenases
Proteases are the large group of hydrolytic enzymes which can cleave the peptide bonds of protein
molecules and subsequently degrade them into small peptides and amino acids. Having variety of applications in
various industries proteases comprises for about 60% of total worldwide enzyme trades, which are extracted
from animal, plant and microbial sources (Rao et al. 1998). So, studies are intensified on the discovery and
characterization of the novel and naturally occurring proteases.
Collagenases are the proteases having ability to degrade the various types of collagens. Due to rigid
triple helical structure of collagens; they are resistant to the common proteases but can be readily cleaved by site
specific action of collagenase enzyme (Figure 3) (Pal & Suresh 2016a). Collagenases are predominantly found
in various sources like animals, plants and microorganisms which differ in substrate specificities. Collagenase
activity from eukaryotic sources have very specific recognition site whereas prokaryotic bacterial collagenases
have broad specificity which helps them to dissociate both water insoluble collagens and water soluble ones
(Adhikari et al. 2012; Baehaki et al. 2012).
Due to its distinct activity, microbial collagenases have enormous industrial applications; potential
restorative applications include healing of wounds (Erdeve et al. 2007), treatment of sciatica in herniated
intervertebral discs (Chu 1987), preparation of intact mammalian cells (Kim et al. 2007), treatment of retained
placenta (Eiler & Hopkins 1993), and preclinical therapeutic studies on various types of destructive fibrosis,
such as liver cirrhosis (Jin et al. 2005), Dupuytren's contracture (Chen et al. 2011) and Peyronie’s disease
(Jordan 2008). With these profound applications in medical field collagenases also have immense importance in
various other fields like food industries, fish processing, brewing, clarification and stabilization of beer, meat
processing, animal tissue culture as well as in other scientific research (Bhagwat et al. 2016; Pal & Suresh
2016a).

Animal collagenase
Animal collagenase (EC 3.4.24.7) cleaves the collagen at specific site of the triple helix of collagen
(Pal & Suresh 2016a). Though the distribution of collagen is widely seen in vertebrates, the degradation of
insoluble or soluble native forms of collagen totally depends on the particular species as well as type of collagen
(Adhikari et al. 2012; Fasciglione et al. 2000). The extraction, purification and characterization of collagenase
from animal origin are frequently reported. Most of the reports are from viscera of fish (Murado et al. 2009;
Sovik & Rustad 2006; Villamil et al. 2017) whereas they are also reported from other animals (Ghamari et al.
2014; Glyantsev et al. 1997). Instead of their site specific action there is restriction on the use of collagenases

6
from animal system because of their complex system which subsequently increases the cost of purification
(Jhample et al. 2015).

Plant collagenase
As per the earlier reports most of the collagenases are reported from animal as well as microbial origin.
Very few reports are present for plant collagenases (Kim et al. 2007; Raskovic et al. 2014). As like the animal
collagenases plant collagenases also have site specific action on native collagen. The ability to produce
collagenase in plants has a significant role of defense against the pests (TR Gomes et al. 2011). Isolation and
characterization of collagenase from fig (Ficus carica) and ginger (Zingiber officinale) has been reported (Kim
et al. 2007; Raskovic et al. 2014). Plant system is as complicated as animal system which further restricts
production of collagenase from plants on high scale (Jhample et al. 2015).

Microbial collagenase
Microbial collagenases (EC 3.4.24.7) have broad substrate specificity which helps them to degrade
both water insoluble and water soluble collagens in their triple helical regions at X-Gly bonds (Adhikari et al.
2012; Baehaki et al. 2012; Pal & Suresh 2016a). The first microbial production of collagenase is obtained from
Clostridium sp. which is pathogenic in nature (Pal & Suresh 2016a). Similarly, production of collagenase has
been reported from some other pathogenic microorganisms (Houle et al. 2003; Jung et al. 1999; Miyoshi et al.
2008).
Microbial collagenases include some metalloproteases from M9 family and serine proteases from the
S1, S8, and S53 families (Figure 4). In addition to this, some of the members of U32 family are also reported to
be collagenases. M9 family members Clostridium and Vibrio collagenases contains a conserved zinc-binding
motif either (HEY/FTH) or (HEYT/VH) which functions as a catalytic domain (Eckhard et al. 2013). The S1
family has His-Asp-Ser as their catalytic residues whereas S8 family is characterized by an Asp-His-Ser
catalytic triad (Rawlings & Barrett 2004). Members of the S53 family have a unique catalytic triad as Glu-Asp-
Ser (Tsuruoka et al. 2003). The U32 family has numerous collagenases of unknown catalytic type (Rawlings et
al. 2010).
Currently Clostridium sp. is mostly used for the production of collagenase at industrial level. But
pathogenic and anaerobic nature of this microorganism moreover, its toxin producing ability limits the
application of its collagenase. Hence, the possibilities of outbreak of such microorganism and increasing cost of
enzyme production due to its such kind of nature directed researcher’s thirst to find the alternate sources of
microorganisms which are non-pathogenic or less pathogenic and would be able to produce this enzyme in cost
effective manner (Bhagwat et al. 2015). Hence, screening and isolation of high enzyme yielding
microorganisms; development and optimization of medium components for the cost-effective production of
collagenolytic enzymes with newer and novel applications are being currently pursued by various scientists
(Bhagwat et al. 2015; Bhagwat et al. 2016; Ferreira et al. 2017).
Microbial production is always superior to the animal or plant cells due to their limited requirements
and higher productivity (Jhample et al. 2015). Till now, collagenases have been purified and characterized from
very few microbial species. However there is scope for many other microbial collagenases which have not yet
been purified or characterized at structural level (Pal & Suresh 2016a).

Isolation and screening of collagenolytic microorganisms

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 Several methods are reported in order to isolate the high collagenase producing microorganisms. But
there is huge controversy among the similar terminologies used such as collagenase, collagenolytic protease and
gelatinase (Watanabe 2004). Due to high cost of acid soluble collagens most frequently gelatin which is
denatured collagen is used for assay techniques (Bhagwat et al. 2015; Suphatharaprateep et al. 2011).
Collagenases can directly hydrolyze the collagen as well as gelatin molecules while gelatinase are the enzymes
which can only hydrolyze the gelatin and not the collagen (Watanabe 2004). Whereas, in line with collagenases;
collagenolytic proteases are the enzymes which can degrade both collagen as well as gelatin substrates
(Bhagwat et al. 2015; Bhagwat et al. 2016; Suphatharaprateep et al. 2011). Hence, gelatinase differ from
collagenases and collagenolytic proteases depending on the substrate specificity. For qualitative and quantitative
measurement of collagenase producers various techniques are reported.

Qualitative analysis of collagenase

Qualitative analysis of collagenase activity can be carried out by using gelatin hydrolysis test. As per
the previous reports gelatin is used in different growth medium like nutrient agar, potato dextrose agar and
trypticase soy agar to isolate microorganisms with extracellular collagenase producing ability (Pal & Suresh
2016a). Collagenase producers can be identified depending on the zone of hydrolysis. In order to get clear zone
of hydrolysis plates are flooded with trichloroacetic acid (TCA) which precipitate the proteins and sharpen the
zone of hydrolysis (Bhagwat et al. 2015; Medina & Baresi 2007). Quantitative ability of the microorganism
(most preferably bacteria) can also be detected using the same method where ratio of zone of hydrolysis to the
colony diameter is taken in to consideration (Bhagwat et al. 2015). But this technique may give variable results
depending on type of microorganism and media used. In another method cell free extracts were used for
quantitative analysis where the plates supplemented with gelatin are bored with borer and extracts were placed
in the wells. The transparent zones around the wells are then used to quantitate the enzyme activity (Lima et al.
2009). Soluble collagen can also be used for the plate assay technique but as they cannot be easily sterilized as
well as are very costly so, very rarely used (Pal & Suresh 2016a).

Quantitative measurement of collagenase

Quantitative measurement of collagenase is highly influenced by the type of substrates used and
technique of end product determination. Mostly substrates used for the determination of collagenase activity are
insoluble collagen, soluble collagen and gelatin as well (Bhagwat et al. 2016; Suphatharaprateep et al. 2011;
Tran & Nagano 2002). In this assay technique appropriate concentration of substrate is incubated with enzyme
and activity of collagenase is further calculated by measuring the free amino acids released due to the action of
enzyme. Unit activity of collagenase is expressed as µmols of L-leucine/glycine equivalents released per minute
per ml of enzyme (Moore & Stein 1954). Molecular weight of glycine (75 Da) is very low as compared to
leucine (131 Da) whereas, weight of mean amino acid residue (110 Da) is close to the leucine than glycine.
Proline (115 Da) and serine (105 Da); these two amino acids are in close proximity to the weight of mean amino
acid residue and theoretically they could give more precise results. It could be better to express the activity of
collagenase in terms of serine equivalents than proline which is an imino acid.
In another method, collagen impregnated with azo dye is used as a substrate where unit activity of
collagenase is measured as the amount of enzyme per ml which produces an increase in the OD of 0.1 at 520 nm
(Lima et al. 2009). Use of other synthetic peptides such as FALGPA and Pz-peptide are also reported but they

8
are very rarely used. In all these techniques use of acid soluble collagen and gelatin gives faster results than
other sources which required higher incubation time (3-18 h) (Pal & Suresh 2016a).

Production otimization of microbial collagenase

Optimization of medium components is very essential for higher production of collagenase. In most of
the earlier reports, production of collagenase was carried out by using submerged fermentation (SmF) technique
(Baehaki et al. 2012; Bhagwat et al. 2015; Lima et al. 2009; Liu et al. 2010). Various types of components have
been used for the production of collagenase such as purified substrates like native collagen, gelatin, fish
collagen and crude substrates like fish skin, fish scale, bovine and marine byproducts (Bhagwat et al. 2015; Pal
& Suresh 2016a).

Optimization through One Factor At a Time method (OFAT method)

Most of the earlier reports were foccused on purification and characterization of collagenase using
either arbitary medium composition or previously reported medium composition instead of the optimization of
medium components for higher production (Baehaki et al. 2012; Kessler et al. 1977; Matsushita et al. 1994;
Murakawa et al. 1987). Further some of the reports were studied optimization of medium components using
One Factor At a Time method (OFAT method); which is very labourious and time consuming (Hamdy 2008;
Suphatharaprateep et al. 2011). The collagenase production from Bacillus cereus MBL13 strain showed
maximum 35 U/ml of activity (Liu et al. 2010). Similarly Bacillus cereus and Klebsiella pneumonie showed
23.07 and 10.53 U/ml of activity (Suphatharaprateep et al. 2011).

Optimization through Response Surface Methodology (RSM)

Heightened production of collagenase is much important in order to meet the current commercial
requirements. Media optimization by classical method is very laborious, time consuming and fails to include the
interactive effects of the variables under study. Use of RSM can overcome the limitations of classical medium
optimization technique. RSM is a technique in which statistical and mathematical systems are collectively
applied in order to optimize the processes having several variables which can influence the response of interest
and the only objective is to optimize this response (Bas & Boyaci 2007). Collagenase production employing
statistical methods has been earlier reported (Lima et al. 2009; Bhagwat et al. 2015).
Extracellular collagenase production by Penicillium aurantiogriseum URM4622 with a 24 full factorial
system was employed to recognize the main interactive effects of the soybean flour concentration, initial
medium pH, temperature and speed of agitation. Maximum production of collagenase (164 U/ml) was observed
when the production conditions were kept at 0.75% soybean flour, pH 8.0, 28ºC and 200 rpm (Lima et al.
2011b).
In another study a full two-level design was used to identify the critical components affecting
collagenase production with three factors; soybean flour; initial medium pH, and temperature. The critical
components identified as soybean flour and initial medium pH were further optimized with the help of central
composite design (CCD). The maximum collagenase activity (283.36 U/ml) was found in the medium
containing 1.645% soybean flour at pH 7.21. As compared to initial response; statistical optimization by
response surface methodology upsurges collagenase yield by 5-fold in this study (Lima et al. 2011a). Hence
response surface methodological optimization is potentially useful for industrial applications as appropriate

9
substrate concentrations can be deduced by this, which can make the whole process economically more feasible
(Bhagwat et al. 2015).

Molecular cloning and protein engineering of collagenase

Very few numbers of purified collagenase and their respective gene sequences have been cloned in
previous studies. Alicyclobacillus sendaiensis strain NTAP-1 collagenase gene was cloned in Escherichia
coli and heterologous expression of respective enzyme was purified to homogeneity. Purified enzyme exhibited
the highest reactivity toward type I collagen (Tsuruoka et al. 2003). Escherichia coli expression system was
used for series of constructs of collagenase G and H from Clostridium histolyticum and collagenase T
from Clostridium tetani, the expressed collagenases were purified by various chromatographic techniques
which yielded at least 10 mg of highly purified collagenase per liter of culture medium (Ducka et al. 2009).
In another study, a 120 kDa collagenase A gene from Clostridium perfringens type C NCIB 10662
was cloned in Escherichia coli (Matsushita et al. 1994). They also performed cloning and expression of
collagenase H from Clostridium histolyticum (Yoshihara et al. 1994). Collagenase gene
from Grimontia (Vibrio) hollisae 1706B was cloned in the Brevibacillus expression system and its complete
nucleotide sequence was determined. The purified recombinant enzyme was shown very high specific activity
which was 4 fold greater than that of Clostridium histolyticum collagenase (Teramura et al. 2011). Most of the
molecular cloning research has been carried out on collagenase from clostridium. Hence, there is wide scope in
the study of cloning, structural and functional characterization of numerous other microorganisms with
collagenolytic activity.

Purification and characterization of microbial collagenase

Purification is very important step in order to study the biochemical, biophysical and structural
characteristics as well as applications of the specific collagenases. Very few reports are available on microbial
collagenase purification where, collagenases from Bacillus licheniformis F11.4, Pseudomonas sp. SUK,
Rhizoctonia solani, Pseudomonas marinoglutinosa, Porphyromonas gingivalis, Rathayibacter sp., Penicillium
aurantiogriseum URM4622, Bacillus cereus MBL13, Clostridium histolyticum, Vibrio vulnificus,
Thermoactinomyces sp. E21, Alicyclobacillus sendaiensis, Bacillus pumilus Col-J were purified and
characterized (Table 5) (Baehaki et al. 2012; Bhagwat et al. 2016; Hamdy 2008; Hanada et al. 1973; Kato et al.
1992; Labadie et al. 1997; Lima et al. 2013; Liu et al. 2010; Matsushita et al. 1999; Miyoshi et al. 1998; Petrova
et al. 2001; Tsuruoka et al. 2003; Wu et al. 2010). Numerous procedures have been used for the purification of
microbial collagenases such as ammonium sulfate precipitation, dialysis, ultrafiltration, ion exchange
chromatography, immobilized metal affinity chromatography, gel filtration chromatography, amylose affinity
chromatography and removal of N-terminal tag (Bhagwat et al. 2016; Ducka et al. 2009).
Production and purification of recombinant collagenase is reported from Clostridium histolyticum,
Clostridium tetani, Alicyclobacillus sendaiensis strain NTAP-1, Grimontia (Vibrio) hollisae 1706B and
Bacillus cereus (Tsuruoka et al. 2003; Ducka et al. 2009; Teramura et al. 2011; Lund & Granum 1999).
Collagenolytic proteases from Pseudomonas sp. SUK and Bacillus cereus were purified with 15.40 and 20.4
purification fold. Whereas, thermostable collagenase was purified from Thermoactinomyces sp. E21 with 101
fold purification (Petrova et al. 2001).

10
 Biochemical characterization of purified collagenases is also very important in order to determine its
applicatory use with respect to different sectors in which they are going to be exploited. Table 5 explains the
biochemical characteristics of microbial collagenases from different sources. Molecular mass of collagenases
ranges from 30-120 kDa. The optimum pH range of these collagenases varies from 4 to 10 while, optimum
temperature is found to be in the range of 30-65°C. In most of the cases, activity of collagenases is enhanced
in the presence of Ca ions while; activity is inhibited by Fe2+ and Hg2+ metal ions.
2+

Applications of microbial collagenase

Microbial collagenases are very well studied from various sources (Table 5) having practical
applications worldwide. Microbial collagenases are having immense importance due to their wide application in
various industries like pharmaceutical, food, meat, tannery, cosmetic and bio-restoration of frescoes (Pal &
Suresh 2016a; Ranalli et al. 2005; Watanabe 2004). Microbial collagenases have broad substrate specificity
which helps them to degrade collagens in their triple helical regions at X-Gly bonds (Adhikari et al. 2012;
Baehaki et al. 2012; Pal & Suresh 2016a). Reaction products formed by the action of microbial collagenase have
various properties which can be exploited in multiple ways. The applications of collagenase can be summarized
as follows (Figure 5).

Medical field
Collagenases are having significant application in medical sector. Cell migration and remodeling of
collagen during tissue repair and regeneration is important step in wound healing process where collagenase
play key role (Agren et al. 1992). Similarly to improve the healing process, clostridial collagenase ointment are
used which carry out the enzymatic debridement and potentially facilitates the process of epithelialization
during debriding (McCallon et al. 2014). Other potential applications include treatment of sciatica (Chu 1987),
treatment of retained placenta (Eiler & Hopkins 1993), and in preclinical therapeutic studies like liver cirrhosis
(Jin et al. 2005), Dupuytren's contracture (Chen et al. 2011) and Peyronie’s disease (Jordan 2008).
Dupuytren's contracture and Peyronie’s disease are originated due to over accumulation of fibrous
plaques in particular body organs. Very early reports showed that these diseases were operated by surgical
procedures which is very difficult task and required highly skilled surgeons having knowledge of specialized
corrective surgical techniques (Bulstrode et al. 2005; Levine & Lenting 1997). Application of clostridial
collagenase is boon for such diseases where enzyme is injected in the respective organ which subsequently
degrades the excessive deposition of collagen (Chen et al. 2011; Jordan 2008).

Animal Tissue Culture (ATC)

Cell culture is an essential tool having wide application in the field of molecular biology,
biotechnology as well as pharmaceutical industries. Microbial collagenase can be widely exploited in ATC
techniques. Cells are surrounded by extracellular matrix containing high concentration of collagen proteins;
hence in order to use the cells tissue disaggregation is important. Trypsin which is widely used for dissociation
of animal tissues; but it is very sensitive to temperature and acts in a narrow pH range of 7.2-7.4 (Manjusha et
al. 2013). In contrast to this microbial collagenase are active in broad pH and temperature range, so they are the
valuable source of enzyme in the fields of ATC. Numerous studies reported the use of microbial collagenase in
cell culture as a cell dislodging agent (Bhagwat et al. 2016; Manjusha et al. 2013).

11
Meat tenderization
Meat is extensively used as a food. A higher concentration of skeletal muscles is the reason of
toughness of meat which is substantially responsible for the unacceptability of meat products (Kemp et al.
2010). Mechanical tenderization is a process reported for meat tenderization which often promotes the
contamination of Escherichia coli as well as Salmonella typhimurium in meat products (Echeverry et al. 2009).
Another method reported for meat tenderization is use of protease from plant as well as microbial
origin. The proteases used in this method have broad specificities towards proteins present in the meat which
might result in development of undesired sensory characteristics in processed meat products (Pal & Suresh
2016a). Use of microbial collagenase having specific ability to degrade collagen is a pragmatic solution for
tenderization of meat products. Collagenase from Clostridium histolyticum and Vibrio B-30 has been reported
for its meat tenderization ability (Cronlund & Woychik 1987). But pathogenic nature of these organisms limits
the use of their collagenases. Some of the reports showed the use of collagenase from Pseudoalteromonas sp.
SM9913 and Pseudomonas sp. SUK which are non-pathogenic in nature and active at very low temperatures.
Using these types of enzymes meat can be tenderized at refrigeration temperature; lowering the chances of
microbial contamination (Bhagwat et al. 2016; Zhao et al. 2012).

Leather industry
Microbial collagenase potentially can be exploited in leather industries. Leather treated with
collagenase after tanning process will results into the opening of its fibrous network improving the diffusion of
dyes in leather. This process not only improves uptake of dye in leather but also responsible for enhancement of
leather softness, smoothness and general appearance (Kanth et al. 2008).

Bio-restoration of frescoes
A fresco is an ancient technique of painting. Due to increased pollution the surfaces of such
monuments were prone to black crusts, sulphation, nitration, deposition of hydrocarbons and dust. In addition to
this, stonework is also altered by organic matter which is applied during restoration but not removed completely
thereafter. The notable quantity of compounds such as glue and casein (organic compounds) acts as a good
substrate for the growth of microorganisms as well as mycetes which ultimately destroy the surface of the
painting. Collagenase from Clostridium histolyticum showed the highest removal efficiency of organic residues
as compared to other tested proteases (Ranalli et al. 2005). Hence microbial collagenase can be potentially used
for the bio-restoration of frescoes.

Extraction of collagen
Extraction of collagen has been reported from various sources (Baiano 2014). In this extraction process
generally acidic solution is used. After acid extraction the remaining biomass is still having the ample amount of
collagen remained in the biomass (Bhagwat & Dandge 2016). Microbial collagenase can be used to recover this
collagen in a cost effective way. Previously collagenases from Bacillus cereus and Klebsiella pneumonie have
been used to recover collagen from salmon skin after acidic extraction. The combination of collagenase with
that of acid extraction yielded higher collagen recovery as compared to using an acid acid extraction alone
(Suphatharaprateep et al. 2011).

Preparation of collagen peptides

12
 Collagen peptides are usually prepared by carrying out proteolysis of collagen. Numerous proteases
were reported for collagen hydrolysis which yields collagen peptides of 0.5 to 20 kDa (Pal & Suresh 2016a).
Formed collagen peptides have been approved by Center for Food Safety and Nutrition, US Food and Drug
Administration for consumption (Bilek & Bayram 2015). The collagen hydrolysates are bioactive in nature
having antioxidant, antimicrobial, antifatigue, anticancer, immunomodulatory, neuroactive, mineral and
hormonal regulating properties and angiotensin I converting enzyme (ACE) inhibitory characteristics. It can also
be exploited by pharmaceutical, food as well as cosmetic industries due to their variety of attributes such as
water holding capacity, fat binding capacity, foam stabilization, swelling, solubility and emulsifying properties
(Halim et al. 2016; Pal & Suresh 2016b). In addition to this, collagen peptides prevent arthritis, osteoporosis,
gastric ulceration and hypertension (Ku et al. 1993; Khare et al. 1995). Therefore, collagen peptides are of
immense importance in food and beverage industries.
In earlier reports, collagen hydrolysates formed by the action of collagenase from Penicillium
aurantiogriseum URM4622 showed radical scavenging properties and antimicrobial activity (Lima et al. 2015).
Whereas, collagen hydrolysates from squid skin showed antioxidant, anti-hyaluronidase and anti-tyrosinase
activities (Nakchum & Kim 2016).

Global nitrogen cycle

Worldwide marine waste is produced in very huge quantities; which is gradually increasing with
respect to population as well as fish production (FAO 2014). But as like chitinous waste; collagenous waste is
not much accumulated in ocean sediments or the nearby territory as it is degraded by naturally occurring
microorganisms with collagenolytic potential (Pal & Suresh 2016a; Zhang et al. 2015b). This microbial
mediated degradation of collagenous waste helps to complete the global nitrogen cycle which is much important
for the perpetuation of life on earth (Zhang et al. 2015b).

Conclusion
With increasing population there is tremendous increase in quantity of waste produced on earth which
is a great concern for living beings. Fish processing waste which in other ways cause serious environmental
pollution is a potent source of collagen. Collagen from mammalian sources is very costly and in addition to this,
they pose the threat of allergic reactions as well as various zoonotic diseases; which can be overcome by the
collagen from fish sources. Huge quantities of fish waste can be processed in a biotechnological way to extract
valuable collagen having high market value. Currently food and pharmaceuticals are witnessing increasing
demand for collagen; whereas fish collagen is distinctly similar to that of mammalian collagen and hence can be
potentially utilized as a substitute to it.
Proteases with collagenolytic activity are having wide industrial applications. Recent studies show their
increasing applications in pharmaceutical, food and various other industries. Microbial collagenases are of
current interest due to their easy availability. Though earlier research was much focused on collagenase from
pathogenic and anaerobic microbial sources which develops certain safety concerns, newer research reports the
collagenase production from non-pathogenic and aerobic microbial sources. Besides this, there is lack of
understanding about qualitative as well as quantitative analysis of collagenolytic microorganisms and
collagenases. Most of the studied collagenases are characterized only up to biochemical level whereas very few
are structurally characterized and it would be interesting to study it in order to achieve better understanding

13
about its specificity which may lead to develop newer applications. Future research must be directed in order to
utilize collagen from fish waste in food and pharmaceutical industries; moreover preparation of bioactive
collagen peptides/hydrolysate with the help of collagenase and their utilization in pharmaceutical and food
industries would be interesting to study.

Acknowledgement
Prashant K. Bhagwat is thankful to UGC for awarding BSR meritorious fellowship for doctoral
research.

14
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Figure 1: Collagen type I chemical structure. (a) Sequence of amino acids - primary structure, (b) left handed
helix - secondary structure; right handed triple-helix - tertiary structure and (c) staggered - quaternary structure
[Friess 1998].

26
Figure 2: Applications of collagen

27
Figure 3: Degradation of collagen

28
Figure 4: Structures of ColG (M9), ColH (M9), MCP-01 (S8), kumamolisin-As (S53) and trypsin (S1). For
ColG, the activator domain and the peptidase domain of the collagenase unit (PDB accession number 2Y50) are
colored in purple and gray, respectively, and the Polycystic Kidney Disease [PKD] (PDB accession number
4AQO) and the Collagen Binding Domain [CBD] (PDB: 1NQD) are colored in yellow and orange, respectively.
For ColH, the peptidase domain (PDB: 4AR1), the PKD (PDB accession number 4U7K), and the CBD (PDB:
3JQW) are colored in gray, yellow, and orange, respectively. The catalytic domains of both MCP-01 (PDB:
3VV3) and kumamolisin-As (PDB: 1SN7) are shown in gray. The catalytic triads of serine collagenolytic
proteases MCP-01 (Asp49, His104, and Ser269) and kumamolisin-As (Ser278, Glu78, and Asp82) are shown in
stick representation. The Zn2+ in ColG and ColH is shown in orange, and Ca2+ is colored in green for all
enzymes [Zhang et al. 2015b]. Ribbon representation of trypsin (PDB ID: 2PTN), the prototypic example of
serine protease from family S1 in clan PA; colored according to the spectrum from the N-terminus (violet) to the
C-terminus (red). The catalytic triad (sticks) is hosted at the interface of two similar β-barrels [Di Cera E, 2009].

29
Figure 5: Applications of microbial collagenase

30
 Table 1: World fish production
2007 2008 2009 2010 2011 2012 2013 2014
Production (Million tonnes)
Capture
Inland 10.1 10.3 10.5 11.3 11.1 11.6 11.7 11.9
Marine 80.7 79.9 79.7 77.9 82.6 79.7 81.0 81.5
Total capture 90.8 90.2 90.2 89.1 93.7 91.3 92.7 93.4
Aquaculture
Inland 29.9 32.4 34.3 36.9 38.6 42.0 44.8 47.1
Marine 20.0 20.5 21.4 22.1 23.2 24.4 25.5 26.7
Total aquaculture 49.9 52.9 55.7 59.0 61.8 66.5 70.3 73.8
Total world fisheries 140.7 143.1 145.9 148.1 155.5 157.8 162.9 167.2
Note: Totals may not match due to rounding (Source: FAO 2014; FAO 2016)

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 Table 2: Marine capture fisheries
2014 Country Average 2013 2014 Variation
Ranking 2003-2012 (2003-2012) - 2014 2013-2014
(Tonnes) (Percentage)
1 China 12,759,922 13,967,764 14,811,390 16.1 6.0
2 Indonesia 4,745,727 5,624,594 6,016,525 26.8 7.0
3 USA 4,734,500 5,115,493 4,954,467 4.6 -3.1
4 Russia 3,376,162 4,086,332 4,000,702 18.5 -2.1
5 Japan 4,146,622 3,621,899 3,630,364 -12.5 0.2
6 Peru 7,063,261 5,827,046 3,548,689 -49.8 -39.1
1
7 India 3,085,311 3,418,821 3,418,821 10.8 0.0
8 Viet Nam 1,994,927 2,607,000 2,711,100 35.9 4.0
9 Myanmar 1,643,642 2,483,870 2,702,240 64.4 8.8
10 Norway 2,417,348 2,079,004 2,301,288 -4.8 10.7
1
FAO estimate (Source: FAO 2016)

32
 Table 3: Inland waters capture
2014 Country Average 2013 2014 Variation
Ranking 2003-2012 (2003-2012) - 2014 2013-2014
(Tonnes) (Percentage)
1 China 2,215,351 2,307,162 2,295,157 3.6 -0.5
2 Myanmar 772,522 1,302,970 1,381,030 78.8 6.0
1
3 India 968,411 1,226,361 1,300,000 34.2 6.0
4 Bangladesh 967,401 961,458 995,805 2.9 3.6
5 Cambodia 375,375 528,000 505,005 34.5 -4.4
6 Uganda 390,331 419,249 461,196 18.2 10.0
7 Indonesia 324,509 413,187 420,190 29.5 1.7
8 Nigeria 254,264 339,499 354,466 39.4 4.4
9 Tanzania 307,631 315,007 278,933 -9.3 -11.5
10 Egypt 259,006 250,196 236,992 -8.5 -5.3
1
FAO estimate (Source: FAO 2016)

33
Table 4: Extraction conditions and yield of collagens from various sources

Source Method Yield (%) Reference

ASC 0.09
Jellyfish bell
PSC 0.29
Khong et al. 2017
ASC 0.16
Jellyfish oral arms
PSC 0.39
ASC 4.13
Golden carp Ali et al. 2018
PSC 7.26
Tilapia scales ASC 3.2
Chen et al. 2016
Tilapia skin ASC 27.2
SSC 2.18
Amur sturgeon cartilage ASC 27.04 Liang et al. 2014
PSC 55.92
ASC 5.8
Catla skin
PSC 7.2
ASC 3.9
Catla scales
PSC 5.6
ASC 6.7
Catla fins
PSC 8.8
Mahboob 2015
ASC 4.7
Mrigala skin
PSC 6.5
ASC 3.2
Mrigala scales
PSC 5.1
ASC 5.7
Mrigala fins
PSC 7.7
ASC 63.40
Catla skin
PSC 69.53
Pal et al. 2015
ASC 46.13
Rohu skin
PSC 64.94
Carp scales ASC 9.79 Bhagwat & Dandge 2016
ASC 25.5
Grass carp skin
PSC 19.8
ASC 0.7
Grass carp bone Wang et al. 2014
PSC 3.5
ASC 16.7
Grass carp scale
PSC 16.1
Trash fish skin ASC 50
Muralidharan et al. 2013
PSC 70

34
 Trash fish bone ASC 50
PSC 67
Trash fish muscle ASC 48
PSC 64
ASC 30.5
horse mackerel bone
PSC 45.1
Kumar et al. 2012
ASC 27.6
Croaker bone
PSC 48.6
Bigeye snapper skin ASC 10.94 Kittiphattanabawon et al.
Bigeye snapper bone ASC 1.59 2005
Bighead carp fins PSC 5.1
Bighead carp scales PSC 2.7
Bighead carp skins PSC 60.3 Liu et al. 2012
Bighead carp bones PSC 2.9
Bighead carp swim bladders PSC 59.0
ASC 43.6
soft-shelled turtle Zou et al. 2017
UASC 50.7
PSC 2.4
Cattle tendon Ran & Wang 2014
UPSC 6.2

ASC = Acid soluble collagen; PSC = Pepsin soluble collagen; SSC = Salt soluble collagen; UASC =
Ultrasound assisted acid soluble collagen; UPSC = Ultrasound assisted pepsin soluble collagen.

35
Table 5: Biochemical characterization and effects of different metal ions on microbial collagenase
Temp.
Mass pH
Organism Optima activation inhibition Reference
(kDa) optima
(°C)
Clostridium Yoshihara et al.
116 - - - -
histolyticum 1994
Clostridium Matsushita et al.
116 - - - -
histolyticum 1999
Clostridium Zn2+, Ca2+, Matsushita et al.
120 7.2 42 2+
Zn2 (≥100 µM)
perfringens Mg 1994
Vibrio Takeuchi et al.
82 - - - -
alginolyticus 1992
Miyoshi et al.
Vibrio vulnificus 45 - - - -
1998
Alicyclobacillus Tsuruoka et al.
37 3.9 60 - Fe2+, Hg2+
sendaiensis 2003
Porphyromonas
37.8 - - Ca2+ Zn2+, Fe2+ Kato et al. 1992
gingivalis
Makinen &
Bacillus cereus 87 - - - -
Makinene 1987
Bacillus cereus 42.8 5.4-8.2 - - - Sela et al. 1998
Bacillus subtilis Uchida et al.
27.6 10 50 K+, Na+ Zn2+
CN2 2004
Hisano et al.
Pseudomonas sp. 45 7.4 45 - -
1989
Bacillus subtilis Nagano et al.
125 9 50 - -
FS 2 2000
Bacillus cereus 106 & Zhang et al.
- - - -
R75E 95 2015a
Bacillus cereus Ca2+, Zn2+,
38 8 40 Cu2+, Fe2+ Liu et al. 2010
MBL13 Mg2+
Bacillus
124 & Fe2+, Mg2+, Baehaki et al.
licheniformis 7 50 Ca2+, Cu2+ 2+ 2+
26 Mn , Co 2012
F11.4
Bacillus pumilus
58.64 7.5 45 Ca2+, Mg2+ Mn2+, Pb2+ Wu et al. 2010
Col-J
Hg2+, Pb2+,
Pseudomonas Hanada et al.
74 7.6 38 Ca2+ Zn2+, Ni2+,
marinoglutinosa 1973
Fe2+

36
 Fe2+, Hg2+,
Labadie et al.
Rathayibacter sp. 72 7 30 Ca2+, Mn2+ Cu2+, Zn2+,
1997
Pb2+
Pseudomonas sp. Bhagwat et al.
58.6 8 60 Zn2+, Ba2+, Ca2+ Fe2+, Hg2+
SUK 2016
Thermoactinomyc Mg2+, Ca2+, Petrova et al.
50 9.0-9.5 60-65 2+
Fe2+, Cu2+
es sp. E21 Co 2001
Candida albicans
ND 8.2 45 - - Lima et al. 2009
URM 3622
Penicillium
aurantiogriseum 39.16 8 45 - - Lima et al. 2013
URM4622
Ca2+, Co2+,
Rhizoctonia solani 66 5 40 Cu2+, Mg2+, Fe2+, Hg2+ Hamdy 2008
2+ + +
Zn , K , Na

- : Not defined.

37