Collagen and collagenolytic proteases: A review

Author’s Accepted Manuscript Collagen and collagenolytic proteases: A review Prashant K. Bhagwat, Padma B. Dandge

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S1878-8181(18)30214-7 https://doi.org/10.1016/j.bcab.2018.05.005 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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: [email protected]

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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 byproducts 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.

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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

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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 filmforming materials (Gómez-Guillén et al. 2011; Herpandi et al. 2011).

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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; SilvaWeiss 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

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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-AspSer (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

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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

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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).

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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

2+

in the presence of Ca ions while; activity is inhibited by Fe2+ and Hg2+ metal ions. 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).

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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

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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

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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.

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25

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

(Million tonnes)

Production 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

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

Aquaculture

Note: Totals may not match due to rounding

(Source: FAO 2014; FAO 2016)

31

Table 2: Marine capture fisheries 2014

Country

Ranking

Average

2013

2014

Variation

2003-2012

(2003-2012) - 2014 (Tonnes)

1

2013-2014

(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

10.8

0.0

7

India

3,085,311

3,418,821

3,418,821

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

FAO estimate

(Source: FAO 2016)

32

Table 3: Inland waters capture 2014

Country

Ranking

Average

2013

2014

Variation

2003-2012

(2003-2012) - 2014 (Tonnes)

1

1

China

2

Myanmar

2013-2014

(Percentage)

2,215,351

2,307,162

2,295,157

3.6

-0.5

772,522

1,302,970

1,381,030

78.8

6.0

1

34.2

6.0

3

India

968,411

1,226,361

1,300,000

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

FAO estimate

(Source: FAO 2016)

33

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

Method

Yield (%)

ASC

0.09

PSC

0.29

ASC

0.16

PSC

0.39

ASC

4.13

PSC

7.26

Tilapia scales

ASC

3.2

Tilapia skin

ASC

27.2

SSC

2.18

ASC

27.04

PSC

55.92

ASC

5.8

PSC

7.2

ASC

3.9

PSC

5.6

ASC

6.7

PSC

8.8

ASC

4.7

PSC

6.5

ASC

3.2

PSC

5.1

ASC

5.7

PSC

7.7

ASC

63.40

PSC

69.53

ASC

46.13

PSC

64.94

ASC

9.79

ASC

25.5

PSC

19.8

ASC

0.7

PSC

3.5

ASC

16.7

PSC

16.1

ASC

50

PSC

70

Jellyfish bell

Jellyfish oral arms

Golden carp

Amur sturgeon cartilage

Catla skin

Catla scales

Catla fins

Mrigala skin

Mrigala scales

Mrigala fins

Catla skin

Rohu skin Carp scales Grass carp skin

Grass carp bone

Grass carp scale Trash fish skin

Reference

Khong et al. 2017

Ali et al. 2018

Chen et al. 2016

Liang et al. 2014

Mahboob 2015

Pal et al. 2015

Bhagwat & Dandge 2016

Wang et al. 2014

Muralidharan et al. 2013

34

Trash fish bone

ASC

50

PSC

67

ASC

48

PSC

64

ASC

30.5

PSC

45.1

ASC

27.6

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

Bighead carp bones

PSC

2.9

Bighead carp swim bladders

PSC

59.0

ASC

43.6

UASC

50.7

PSC

2.4

UPSC

6.2

Trash fish muscle

horse mackerel bone

Croaker bone

soft-shelled turtle

Cattle tendon

Kumar et al. 2012

Liu et al. 2012

Zou et al. 2017

Ran & Wang 2014

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

Organism

Mass

pH

(kDa)

optima

116

-

116

Temp. activation

inhibition

-

-

-

-

-

-

-

120

7.2

42

82

-

-

-

-

45

-

-

-

-

37

3.9

60

-

Fe2+, Hg2+

37.8

-

-

Ca2+

Zn2+, Fe2+

Bacillus cereus

87

-

-

-

-

Bacillus cereus

42.8

5.4-8.2

-

-

-

27.6

10

50

K+, Na+

Zn2+

45

7.4

45

-

-

125

9

50

-

-

-

-

-

-

8

40

7

50

Ca2+, Cu2+

7.5

45

Ca2+, Mg2+

Clostridium histolyticum Clostridium histolyticum Clostridium perfringens Vibrio alginolyticus Vibrio vulnificus Alicyclobacillus sendaiensis Porphyromonas gingivalis

Bacillus subtilis CN2 Pseudomonas sp. Bacillus subtilis FS 2 Bacillus cereus

106 &

R75E

95

Bacillus cereus MBL13 Bacillus licheniformis F11.4 Bacillus pumilus Col-J Pseudomonas marinoglutinosa

38

124 & 26

58.64

Optima (°C)

Zn2+, Ca2+, Mg

2+

Ca2+, Zn2+, Mg2+

Yoshihara et al. 1994 Matsushita et al. 1999

Zn2 (≥100 µM)

7.6

38

Ca2+

Matsushita et al. 1994 Takeuchi et al. 1992 Miyoshi et al. 1998 Tsuruoka et al. 2003 Kato et al. 1992 Makinen & Makinene 1987 Sela et al. 1998 Uchida et al. 2004 Hisano et al. 1989 Nagano et al. 2000 Zhang et al. 2015a

Cu2+, Fe2+

Liu et al. 2010

Fe2+, Mg2+,

Baehaki et al.

2+

Mn , Co

2+

Mn2+, Pb2+ Hg2+, Pb2+,

74

Reference

Zn2+, Ni2+, Fe2+

2012

Wu et al. 2010

Hanada et al. 1973

36

Fe2+, Hg2+, Rathayibacter sp.

72

7

30

Ca2+, Mn2+

Cu2+, Zn2+, Pb2+

Pseudomonas sp.

Zn2+, Ba2+, Ca2+

Fe2+, Hg2+

Labadie et al. 1997 Bhagwat et al.

58.6

8

60

50

9.0-9.5

60-65

ND

8.2

45

-

-

Lima et al. 2009

39.16

8

45

-

-

Lima et al. 2013

Fe2+, Hg2+

Hamdy 2008

SUK Thermoactinomyc es sp. E21 Candida albicans URM 3622

Mg2+, Ca2+, Co

Fe2+, Cu2+

2+

2016 Petrova et al. 2001

Penicillium aurantiogriseum URM4622 Ca2+, Co2+, Rhizoctonia solani

66

5

40

Cu2+, Mg2+, 2+

+

+

Zn , K , Na -

: Not defined.

37