Utilization of Chitinaceous Wastes for the Production of Chitinase

CHAPTER TWO

Utilization of Chitinaceous Wastes for the Production of Chitinase S. Das, D. Roy, R. Sen1 Indian Institute of Technology Kharagpur, Kharagpur, India 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Chitinaceous Wastes and Chitin 2.1 Chitin Structure and Complexes 2.2 Chitin Sources 3. Chitinase 4. Chitinase Production 4.1 Submerged Fermentation 4.2 Solid-State Fermentation 4.3 Fed-Batch Fermentation 4.4 Continuous Process 4.5 Biphasic Cell System 4.6 Recombinant Technology 5. Conclusions Acknowledgments References

28 28 29 31 32 34 35 37 38 40 40 41 41 42 42

Abstract Marine environment is the most abundant source of chitin. Several marine organisms possess chitin in their structural components. Hence, a huge amount of chitin wastes is deposited in marine environment when such organisms shed their outer skeleton and also after their demise. Waste chitins are potential nutrient source of certain microbes. These microbes produce chitinases that hydrolyze waste chitins. These organisms thus play an important role to remove the chitin wastes from marine environment. In connection with this, chitinases are found to be most important biocatalyst for the utilization of chitin wastes. Therefore, use of chitin for chitinase production is one of the useful tools for different types of bioprocesses.

Advances in Food and Nutrition Research, Volume 78 ISSN 1043-4526 http://dx.doi.org/10.1016/bs.afnr.2016.04.001

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2016 Elsevier Inc. All rights reserved.

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1. INTRODUCTION Chitin is a second most abundant natural biopolymer after cellulose. It is a homopolymer of N-acetyl-D-glucosamine (GlcNAc) linked by β-(1,4) bond. It is distributed in nature as structural body parts of fungi, marine diatoms, mollusks, arthropods, crustaceans, nematodes, etc. (Chen & Lee, 1995). Large amount of waste chitin is deposited in food processing and enzyme production industries; and removal of the waste chitin from different industries becomes a serious issue. Conventional method for chitin disposal by burning, land filling, and ocean dumping suffer from several environmental and economic issues (Suresh & Chandrasekaran, 1998). On the other hand, demands on chitin degrading enzyme, chitinase, production is increased from last few decades due to its potential biotechnological applications in pharmaceutical and biofuel industries (Dahiya, Tewari, & Hoondal, 2006). Microbial enzymes are observed to be advantageous over the enzyme from higher organisms due to low cost, variability in catalytic activity, and higher stability (Chandrasekaran, 1997). So, exploitation of waste chitin for microbial chitinase production by different bioprocesses is thought to be an important aspect for waste chitin utilization and bioremediation (Krishnaveni & Ragunathan, 2014).

2. CHITINACEOUS WASTES AND CHITIN It is estimated that out of total wastes generated in food processing industry around 20–50% are comprised of chitin (Wang & Chang, 1997). These are mostly generated during shell fish processing. Therefore, shellfish are treated as good source of chitin. But, there are certain limitations in chitin extraction from shellfish wastes—such as, seasonal harvesting of crustaceans, limited raw material supply, and generation of environmental hazardous material during deproteination of waste chitin. These are the reasons why the researchers are striving to search for new and sustainable sources of chitin. Fungal biomass may emerge as a potentially viable and alternative source for chitin production, since fungal cell wall is composed of mainly chitin biopolymer (Wang & Chang, 1997). Moreover, cost effective fungal cultivation in solid state and submerged cultures would be an added advantage to obtain required amount of chitin. On the other hand,

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chitin can be obtained from pupa silkworm as reported in recent past (Haga & Shirata, 1997). Following sections are dealt with chitin structure, complexes, and its availability from different sources.

2.1 Chitin Structure and Complexes Chitin is mainly composed of N-acetyl-D-glucosamine residues, which are attached by β-(1,4)-glycosidic linkage (Fig. 1). Important functional groups of chitin are listed in Table 1. Chitin is recognized as widely distributed in exoskeleton of invertebrates and cell wall of fungi. Chitin and cellulose are structurally analogous, where in the former the hydroxyl group of glucose moiety is replaced by the acetamide group. However, degree of acetylation

Fig. 1 Linear structure of chitin.

Table 1 List of Functional Groups Inferred from FTIR Inference from Spectral Response Functional Group (Mudasir, Tahir, & Wahyni, 2008)

dOH

dOH stretching (“a,” Fig. 1)

dCH3CONH

dNH stretching (“b,” Fig. 1) dCH stretching (“c,” Fig. 1) ]CO stretching (“d,” Fig. 1) dNH bending (“b,” Fig. 1) dCH bending (“c,” Fig. 1) dCN stretching (“e,” Fig. 1)

]CO of polysaccharides and glucosamine rings

]CO stretching (“f,” Fig. 1)

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in the polysaccharide molecule depends on the source and chitin isolation process. At least, one out of six GlcNAc molecules remains deacetylated in native chitin (Trudel & Asselin, 1990). It is observed that length of the yeast chitin is only with 100 GlcNAc residues, whereas the crab chitin contains 5000–8000 GlcNAc residues. Chitin polymer is labile to alkali. It was observed that the length of the chitin chain was decreased upon hot alkali treatment during the deproteinization step of chitin isolation (Synowiecki & Al-Khateeb, 2003). Each polymeric chitin chain is associated with neighboring chain by hydrogen bond, where amino group (>NH) of one molecule makes bond with carbonyl (>C]O) group of the adjacent one. On the basis of nature of H-bond, chitin molecule can be designated as α, β, and γ chitin. In α-chitin, the chains are arranged in parallel fashion ("") and it is mostly found in arthropods and crustaceans; whereas, an antiparallel arrangement of chitin chain is observed in β-chitin ("#) and these are obtained from marine diatoms. In contrast, in γ chitin, the arrangement of the chitin chain is little complicated; where, out of each three chitin chain, two are arranged in parallel fashion and third one is arranged in antiparallel fashion (""#). However, the existence of γ chitin is a matter of controversy (Chanzy, 1997). The α-chitin is observed to be most stable form of chitin; while β-chitin can easily be converted to α-chitin by lithium thiocyanate treatment or formic acid precipitation (Hackman & Goldberg, 1965). Hardness, flexibility and permeability of the shell are determined by the ratio of α-chitin and β-chitin. Chitin cannot be melted in solid state due to the presence of high density of hydrogen bonds. However, it can be dissolved in concentrated acids, like hydrochloric acid, sulphuric acid or phosphoric acid, trichloroacetic acid, and formic acid. Greater solubilization rate is observed for β-chitin in comparison to the α-chitin. Besides this, chitin gets solubilized in hexafluoroisopropanol or hexafluoroacetone and some of the hot and concentrated neutral salts (Synowiecki & Al-Khateeb, 2003). Chitin microfibrils are generally resistant to the deacetylases due to the presence of hydrogen bonds. Deacetylases act on the newly synthesized chitin in chitosome prior to synthesis of chitin microfibrils (Kolodziejska & Sikorski, 1999). Several biomolecules like protein, other polysaccharides, and matrix of β-glucan molecules surround the chitin fibril in fungal cell wall to form alkali-insoluble complexes. In crustacean and insects, chitin forms a

Chitinase Production by Using Waste Chitin

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complex with proteins which are tanned by phenolic derivatives. Thus, a glycoprotein framework is constituted in these organisms. Finally, the presence of mineral salts, carotenoids, lipoproteins, and waxes surrounding the glycoprotein frame work, influence the elasticity, permeability, tensile strength, and hardness of the structure. Moreover, calcium carbonate and calcium phosphate, to some extent play a crucial role for the hardness of insect cuticle. Although, silica and iron oxide were also reportedly enhance the hardness of Rhizopods shell (Roberts, 1992).

2.2 Chitin Sources Chitin is widely available from two natural resources such as wastes from marine aqua animal shells and microbes which are described below. Recently, chitin is produced in large scale from the waste of crustacean harvesting industry. The data available on the amount of chitin resources indicate that approximately 0.7, 1.4, and 29.9 million tons of chitin are annually recovered from squid, oyster, and shellfish, respectively (Synowiecki & Al-Khateeb, 2000). Certain crustacean orders (Decapoda, Amphipoda, Copepoda, Anostraca, and Cladocera) are good sources of chitin as they have significant amount (2–12%) of chitin content in their total body mass (Johnson & Peniston, 1982). Crab and shrimp shells, on the other hand are rich source of chitin. In US around 39,000 tons of shell from crab (Cancer magister) and shrimp (Pandalus borealis) is being discarded annually (Knorr, 1991). Moreover, around 50,000 tons of shell is being recovered from the harvested crab (Chionoecetes opilio sp.) in the Canadian Atlantic regions; where, 80% of total weight is generally used to produce the by-products. Shrimp and krill wastes contain around 10% higher chitin than crab-processing wastes (Naczk, Synowiecki, & Sikorski, 1981). A similar amount of chitin is found in Louisiana crawfish (Procambarus clarkii). In crab, chitin is mainly deposited in leg, shoulder, and tips rather than other body parts (Shahidi & Synowiecki, 1991). Antarctic krill (Euphausia superba) is another important chitin source (Kolodziejska, Malesa-Cie´cwierz, Lerska, & Sikorski, 1999). However, fluoride and other minerals are found as a contaminant in krill shell, which occupies more than 10% of the weight (Kolodziejska et al., 1999). Fungal biomass is also known as an abundant source of chitin. It is observed that chitin content varies among the different strains of fungus (Muzzarelli, Ilari, Tarsi, Dubini, & Xia, 1994). The filamentous fungal strains such as Rhizopus, Penicillium, Aspergillus, Fusarium,

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Choanephora, Zygorhynchus are known to be used for chitin extraction and isolation (Muzzarelli et al., 1994). Along with chitin, chitosan and other polysaccharides are also present in the fungal cell wall (Knorr, Beaumont, & Pandya, 1989). In recent years, several attempts were made to isolate chitin from nonconventional fungal resources, because of consistent availability of fungal biomasses from different industries (Muzzarelli et al., 1994). The use of fungal chitin has sometimes advantageous over the shellfish chitin, for high growth rate of fungal strains, inexpensive cultivation in waste materials; absence of high amount of mineral salts and lower pretreatment cost. Besides, it is possible to control the fermentation, processing, and genetic modification of the fungal strains for improving the chitin yield. The chitin content varies from 2% to 60% on the basis of fungal cell wall dry weight. Overall 26–65% and 22–67% chitins and glucans, respectively, have been estimated from deproteinized cell wall of Basidiomycetes and Ascomycetes. For example, Agaricus bisporus, a very commonly known mushroom, is observed to possess proteins (22%), chitin (72%), cellulose (3%) along with microamount of glucosamine, and mineral salts in their cell wall. In mold mycelia, up to 25% protein, 3% nucleic acid, and 15% lipid are found to be present on the basis of their dry weight (Synowiecki & Al-Khateeb, 1997). High chitin content, up to 35% dry cell wall weight has been reported from Mucor rouxii (Bartnicki-Garcia & Nickerson, 1962; Synowiecki & Al-Khateeb, 1997). In addition to this, cell wall of Aphyllophorales is known to possess very high level of chitin content (almost 95%, w/w) in its cell wall (Gorovoj & Kosyakov, 1997). It has been found that it is necessary to optimize the growth medium, time, and other physical parameters for fungal cultivation to maximize the chitin content in fungal cell wall. However, this waste chitin is processed for chitin isolation and they are used for chitinase production.

3. CHITINASE Chitinases (EC 3.2.1.14) are a group of hydrolytic enzymes that cleave the β-1,4-glycosidic bond between two consecutive N-acetyl-Dglucosamines (GlcNAc) of chitin chain (Xia et al., 2001). Wide ranges of organisms are known to produce chitinases, which play diverse role in different organisms which are listed in Table 2. Out of these different sources, microbial chitinases are known to possess potential industrial applications in agricultural and environmental sectors, such as, production of GlcNAc and

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Chitinase Production by Using Waste Chitin

Table 2 Chitinase-Producing Organism and Their Functions Chitinase Producer Requirement to Produce Chitinase References

Virus

Pathogenesis

Patil, Ghormade, and Deshpande (2000)

Fungus

Cell wall remodeling, morphogenesis, Adams (2004) and nutritional purposes

Bacteria

Chitin digestion for nutrition, energy Tsujibo et al. (1993), Park et al. source, and chitin recycling (1997), Svitil, Chadhain, Moore, and Kirchman (1997), and Wang and Chang (1997)

Insect

Postembryonic development and old Merzendorfer and Zimoch cuticle degradation (2003)

Plants

Defense mechanism against pathogen Graham and Sticklen (1994) and plant development

Human

Defense purposes

Boot et al. (2001, 2005) and van Eijk et al. (2005)

other chito-oligosaccharides for healthcare, isolation of fungal protoplasts for strain improvement (Dahiya, Tewari, Tiwari, & Hoondal, 2005), control of pathogenic fungi and mosquito by dissolving cell wall or shell chitin (De Marco, Lima, Desousa, & Felix, 2000; Mendonsa, Vartak, Rao, & Deshpande, 1996), and bioconversion of different chitinaceous wastes (Dahiya et al., 2006). Classification and nomenclature of chitinolytic enzyme are still not well defined. In a contemporary research, Graham and Sticklen proposed two major categories of chitinase—endochitinase and exochitinase (Graham & Sticklen, 1994). A group of chitinases that cleave randomly at internal sites of the chitin chain and produce low-molecular-weight oligomers of GlcNAc like chitotetraose, chitotriose, etc., are known as endochitinase (EC 3.2.1.14). On the other hand, the chitinases which cleave the chitin chain from terminal ends fall under exochitinases. According to the release of the product, exochitinases are again categorized into two subcategories— chitobiosidase and β-(1,4)-N-acetyl-glucosaminidase. An exochitinase, which releases only diacetylchitobioses from the nonreducing end of the chitin, is designated as chitobiosidases (EC 3.2.1.29). In contrast, β-(1,4)N-acetyl-glucosaminidases (EC 3.2.1.30) release only GlcNAc as sole product from nonreducing end of the chitin polysaccharide (Fig. 2).

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Fig. 2 Mode of action of different types of chitinases.

4. CHITINASE PRODUCTION Chitinase is produced by number of biotechnological methods, such as submerged fermentation, solid state fermentation, fed-batch fermentation, continuous fermentation, and biphasic cell system, etc. It is observed

Chitinase Production by Using Waste Chitin

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that expression of extracellular chitinases is induced by external chitin (Dahiya et al., 2006). The organisms producing extracellular chitinases consume chitin as potential carbon and nitrogen source. In this regard, chitin from crab or shrimp wastes could be used in chitinase production media. Chitinase production is found to be greatly affected upon physical and chemical changes (Dahiya et al., 2006). Presence of magnesium sulfate (MgSO4 7H20) and potassium dihydrogen phosphate (KH2PO4) in media has significant effects on the chitinase production (Khan, Hamid, Ahmad, Abdin, & Javed, 2010). However, certain organic supplements and sugar molecules exhibit contradicting effect on chitinase production in different organisms. Yeast extract shows antagonistic effect on chitinase production in Stenotrophomonas maltophilia (Khan et al., 2010); but the same component shows enhancement of chitinase production in Trichoderma harzianum (Nampoothiri et al., 2004). Same observation is found in case of glucose also. Chitinases production increases when glucose is added as supplement with chitin in production media of Enterobacter sp. (Dahiya et al., 2005). However the same glucose imparts a negative impact on chitinase production of Streptomyces lividans as observed by Miyashita, Fujii, and Sawada (1991). Besides, several agroindustrial wastes, such as rice bran and wheat bran serve as nutrient supplements in the media (Dahiya et al., 2005). Addition of amino acid like tryptophan, tyrosine, glutamine, and arginine in medium are shown to enhance the chitinase production of Bacillus sp. as reported previously (Dahiya et al., 2006). Along with chemical parameters, some physical parameters like pH of the production medium, aeration, inoculum size, temperature, and time of incubation also play a crucial role in the chitinase production (Dahiya et al., 2006). Discussion on different methods of chitinase production is a helpful tool for better understanding of the process.

4.1 Submerged Fermentation Selection of cultivation process is an important step for chitinase production. In most of the cases extracellular chitinases are produced in medium at low concentration. Handling of low amount of enzyme in large volume of media is a problem; which complicates further purification steps (Stoykov, Pavlov, & Krastanov, 2015). On the other hand, submerged fermentation (SMF) technique have certain features, such as well-controlled process parameters, increased mass transfer, and enhancement of oxygen delivery system, etc., which help it gaining added advantages over liquid culture (Stoykov et al., 2015). Besides, coimmobilization method has been

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developed to enhance chitinase production. Earlier, calcium alginate was used to coimmobilize the microbial cells and chitin for the production of chitinase from Micromonospora chalcea (O’Riordan, McHale, Gallagher, & McHal, 1989). In this case, chitinase was separately produced by free cells and coimmobilized cells in a production medium with 2% chitin. Coimmobilization helped increase chitinase production by 0.3 U as shown in earlier literature (O’Riordan et al., 1989). Several microbial strains studied for chitinase production by SMF are listed below (Table 3). Table 3 Microbial Chitinase Production by SMF Medium pH, Incubation Substrate Temperature (°C), Microorganism Habitat Used Chitinase Activity (U) References

Nocardia orientalis

Terrestrial Colloidal chitin

5, 28, 1.278

Usui, Hayashia, Nanjob, Sakaib, and Ishido (1987)

Serratia liquefaciens

Terrestrial Chitin

7.5, 30, 15.1

Joshi, Kozlowski, Richens, and Comberbach (1989)

Myrothecium verbucaria

Terrestrial Sclerotium 5.5, 28, 0.24 rolfsii mycelium

Vyas and Deshpande (1989)

Bacillus licheniformis

Terrestrial Collidal chitin

7, 50, 0.014

Takayanagi, Ajisaka, Takiguchi, and Shimahara (1991)

Talaromyces emersonii

Terrestrial Chitin

5, 45, 0.45

James, Hackett, Tuohy, and Coughlan (1991)

Streptomyces cinereoruber

Terrestrial Aspergillus 6.8, 30,17 niger cell wall

Tagawa and Okazaki (1991)

Vibrio alginolyticus

Terrestrial Squid chitin

7, 37, 5.8

Ohishi et al. (1996)

Monascus purpureus

Terrestrial Shrimp and crab shell powder

7, 25, 191

Wang, Hsiao, and Chang (2002)

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Table 3 Microbial Chitinase Production by SMF—cont'd Medium pH, Incubation Substrate Temperature (°C), Microorganism Habitat Used Chitinase Activity (U) References

Trichoderma harzianum

Terrestrial Chitin

5.5, 30, 14.7

Sandhya et al. (2004)

Aeromonas sp.

Marine

5,40, 1.1

Kuk et al. (2005)

Trichothecium roseum

Terrestrial Crab shell 6, 28, 0.78 chitin

Li, Zhang, Liu, and Lu (2004)

Aspergillus fumigatus

Terrestrial Colloidal chitosan

7, 37, 5.86

Jung, Kuk, Kim, Jung, and Park (2006)

Aspergillus terreus

Terrestrial Fish scale waste

6, 30, 4.31

Ghanem, Al-Garni, and Al-Makishah (2010)

7, 10, 0.095

Velmurugan, Kalpana, Hoon, Jung, and Yang (2011)

Plectosphaerella Marine sp.

Swollen chitin

Colloidal chitin

Trichoderma aureoviridae

Terrestrial Colloidal chitin

4.7, 28, 0.036

Agrawal and Kotasthane (2012)

Aspergillus terreus

Marine

6, 30, 4.7

Krishnaveni and Ragunathan (2014)

Shrimp waste

Along with the indigenous chitinase production by microorganisms, statistical methods are employed to optimize production medium constituents and enhancement of chitinase production. Khan et al. in 2010 validated the Design Expert prediction tool to enhance chitinase production in Stenotrophomonas maltophilia. Several reports on chitinase production enhancement by using statistical methodology are listed in Table 4.

4.2 Solid-State Fermentation Use of solid support or matrix during microbial growth and production of metabolites is known as solid state fermentation (SSF). It is commonly used for microbial enzyme production in different sectors especially for fungal chitinase production. Although, SSF experiences some drawbacks, such as

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Table 4 Microbial Chitinase Production by SMF and Statistical Optimization Increase in Chitinase Microorganism Habitat Statistical Method Used Production References

Alcaligenes xylosoxidans

Marine

Plackett–Burman design 2.42-fold and Box–Behnken response surface methodology

Vaidya, Vyas, and Chhatpar (2003)

29%, 9.3%, and 28%

Nawani and Kapadnis (2005)

Pantoea dispersa Marine

Plackett–Burman design 4.21-fold

Gohel, Chaudhary, Vyas, and Chhatpar (2006)

Streptomyces sp. Marine Da11

Plackett–Burman design 39.2-fold and Box–Behnken response surface methodology

Han, Li, Miao, and Zhang (2008)

Streptomyces sp. Terrestrial Response surface methodology and NK1057, numerical optimization NK528, and NK951

uncontrolled process parameters for maintenance of pH, temperature, substrate sterilization, culture purity, and process length; but, the use of cost effective matrices and easy operation of the process make it advantageous over the SMF (Karthik, Akanksha, & Pandey, 2014). In nature, chitin is present as solid and insoluble substrate. Thus, use of chitin from natural resources becomes an additional advantage for chitinase production by SSF. In several literatures different organisms are shown to produce chitinase by SSF. These are listed in Table 5.

4.3 Fed-Batch Fermentation Fed-batch fermentation is alternatively known as semi-batch culture where nutrient or substrates are supplied to the bioreactor according to the required amount. Thus, fed-substrate concentration can be controlled easily (Yamane & Shimizu, 2005). Earlier, in N-acetyl-D-glucosamine production Serratia marcescens QM B1466 was used to express chitinase in fed-batch fermentation (Kim, Creagh, & Haynes, 1998). The culture was grown in 10 L bioreactor for 7 days by using chitinaceous wastes (crab/shrimp chitin) in the media. The pH was set at 8.5 and temperature at 30°C with 3 h feeding time

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Table 5 Microbial Chitinase Production by SSF

Microorganism Habitat

Substrate Used

Medium pH, Incubation Temperature (°C), Chitinase Activity (U/gds)

Beauveria bassiana

Marine

Trichoderma harzianum

Terrestrial Wheat bran 4.5, 30, 3.14 and colloidal chitin

Prawn waste 9.5, 27, 248

Enterobacter sp. Terrestrial Wheat bran and flake chitin

8, 30, 1475

References

Suresh and Chandrasekaran (1998) Nampoothiri et al. (2004)

Dahiya et al. (2005)

Verticillium lecanii

Terrestrial Shrimp 6, 25, 1674 waste silage and sugar cane baggase

Matsumoto, SaucedoCastan˜eda, Revah, and Shirai (2004)

Fusarium oxysporum

Terrestrial Wheat bran and flake chitin

6, 30, 23.6

Gkargkas et al. (2004)

Penicillium aculeatum

Terrestrial Wheat bran and flake chitin

5, 30, 12.53

Binod, Chandran, Pradeep, George, and Ashok (2007)

8.6, 32, 311.84

Suresh, Anil Kumar, and Sachindra (2011) and Suresh, Sachindra, and Bhaskar (2011)

Penicillium Terrestrial Wheat bran monoverticillium and shrimp shell chitin waste Oerskovia xanthineolytica

Terrestrial Wheat bran 7.5, 45, 148 and colloidal chitin

Waghmare, Kulkarni, and Ghosh (2011)

(Kim et al., 1998). Recently, Rao, Inman, Holmes, and Lalitha (2013) also reported the use of fed-batch fermentation for chitinase production in a mixed culture of Vibrio harveyi and Vibrio alginolyticus. In this study, a 10 L bioreactor was used with daily addition of 2% colloidal chitin (w/v) at 30°C, with 20% dissolved oxygen and 150 rpm agitation. With fed-batch fermentation up to threefold chitinase activity can be achieved. Thus, fed-batch fermentation is known to be effective process for chitinase production.

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4.4 Continuous Process Feeding of inputs (raw material, auxillary materials, energy) in a system and extraction of output from the system simultaneously at constant rate and ratio is known as continuous process. Attempts are made by researchers for continuous chitinase production. Fenice, Giambattista, Raetz, Leuba, and Federici (1998) reported continuous chitinase production by immobilized conidiophore of Penicillium janthinellum P9. In this report, polyurethane sponge and chemically modified macroporous cellulose are used as matrix for conidiophore immobilization; where modified cellulose is observed to be better carrier in terms of negligible amount of cell leakage (Fenice et al., 1998). Here, rapid chitinase production is observed by cell-immobilized repeated batch process compare to free cell-based process. Additionally, continuous increase in biomass in the same bioreactor results in the increase of chitinase production in repeated batches. In general, the continuous process is maintained at 168 h. Continuous processes for chitinase production was reported in 1 L fluidized bed reactor where immobilized P. janthinellum P9 were used (Fenice et al., 1998). Unlike repeated batch culture the continuous process generates more chitinase. It was observed that the amount of chitinase from repeated batch culture was 338 U/L, whereas with continuous process the amount of chitinase was increased to 450 U/L. Another mode of continuous chitinase production reported earlier is a membrane-based fermentation process (Kao, Huang, Chang, & Liu, 2007). A strain of Paenibacillus sp. CHE-N1 is used in this study. A membrane outer recycling loop is equipped with the bioreactor; where the effect of membrane pore size is evaluated for different process-related parameters like chitinase recovery, fouling, cellretention efficiency, etc. Here, 300 kDa pores sized M 9 microfiltration column is used for chitinase production. In previous report, colloidal chitin from crab shell wastes was used to feed every 3–4 days which resulted 78% increase in total chitinase activity (42.8 U) compared to batch mode (Kao et al., 2007).

4.5 Biphasic Cell System A PEG/dextran aqueous two-phase system (ATPS) was developed by Chen and Lee (1995) for the chitinase production enhancement by Serratia marcescens. In this process, the production media contains 2% PEG and 5% dextran ATPs. The swollen chitin is used as inducer for chitinase production. A polymer free reference system is used to compare the results

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Table 6 Chitinase Production by Recombinant Microbial Strains Substrate Medium pH, Incubation Temperature (°C), Used/ Chitinase Activity (U) References Inducer Microorganism

Serratia marcescens Chitin designated as BL40

7.2, 30, 184

Kole and Altosaar (1985)

Serratia marcescens SJ101

7.5, 30, 15 Cane molasses and chitin

Joshi et al. (1989)

Recombinant Escherichia coli gene from Aeromonas hydrophila

IPTG

7, 37, 0.903

Chen, Chang, and Cheng (1997)

Self-fusion of protoplasts of Trichoderma harzianum

Chitin

6.5, 25, 9.6

Prabavathy, Mathivanan, Sagadevan, Murugesan, and Lalithakumari (2006)

of fermentation. Here, 2.5%, 1.9%, and 3.1% increase in chitinase activity can be achieved in dextran solution, PEG solution, and ATPS.

4.6 Recombinant Technology Number of wild-type microbial strains is known to produce chitinase. But, hyper-chitinase producer are highly desirable for its different industrial applications. Several fermentation processes are reported to increase productivity by the use of recombinant strains (Karthik et al., 2014). In this regards, some of the recombinant strains are also reported to produce high level of chitinase. Reports on enhanced chitinase production by recombinant strains are listed Table 6.

5. CONCLUSIONS Marine environment consists of three fourth part of the earth surface and it is the richest source of chitinaceous wastes. These are recycled by the action of different microbial enzymes like chitinase. Thus, exploitation of chitin waste is found to be best option for microbial chitinase production. Unfortunately, very few marine microorganisms are reported to utilize waste chitin for chitinase production, therefore discovery marine

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microorganisms is necessary for chitinase production by the utilization of waste chitin in future. Besides, research on large-scale chitinase production is scanty. Detailed investigation on kinetic studies for fermentation, designing of bioreactor, influence of reactor parameters like agitation, aeration is also required for large-scale chitinase production. These investigations may be brought under consideration for future scope for chitinase production research.

ACKNOWLEDGMENTS SD is thankful to National Jute Board (Govt. of India) and HRDG (Govt. of India) for financial support for this study.

REFERENCES Adams, D. J. (2004). Fungal cell wall chitinases and glucanases. Microbiology, 150, 2029–2035. Agrawal, T., & Kotasthane, A. S. (2012). Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India. SpringerPlus, 1, 73. Bartnicki-Garcia, S., & Nickerson, W. J. (1962). Nutrition, growth and morphogenesis of Mucor rouxii. Journal of Bacteriology, 84, 841–858. Binod, P., Chandran, S., Pradeep, S., George, S., & Ashok, P. (2007). Fungal biosynthesis of endo-chitinase and chitobiase in solid state fermentation and their application for the production of N-acetyl-D-glucosamine from colloidal chitin. Bioresource Technology, 98, 2742–2748. Boot, R. G., Blommaart, E. F. C., Swart, E., Van Der Vlugt, K. G., Bijl, N., Moe, C., et al. (2001). Identification of a novel acidic mammalian chitinase distinct from chitotriosidase. Journal of Biological Chemistry, 276, 6770–6778. Boot, R. G., Bussink, A. P., Verhoek, M., De Boer, P. A. J., Moorman, A. F. M., & Aerts, J. M. F. G. (2005). Marked differences in tissue-specific expression of chitinases in mouse and man. The Journal of Histochemistry and Cytochemistry, 53, 1283–1292. Chandrasekaran, M. (1997). Industrial enzymes from microorganisms: The Indian scenario. Journal of Marine Biotechnology, 5, 36–39. Chanzy, H. (1997). Chitin crystals. In A. Domard, G. A. F. Roberts, & K. M. Varun (Eds.), Advances in chitin science (pp. 11–21). Lyon, France: Jacques Andre Publisher. Chen, S. J., Chang, M. C., & Cheng, C. Y. (1997). Effect of induction conditions on production and excretion of Aeromonas hydrophila chitinase by recombinant Escherichia coli. Journal of Fermentation and Bioengineering, 84(6), 610–613. Chen, J. P., & Lee, M. S. (1995). Enhanced production of Serratia marcescens chitinase in PEG/dextran aqueous two-phase systems. Enzyme and Microbial Technology, 17, 1021–1027. Dahiya, N., Tewari, R., & Hoondal, G. S. (2006). Biotechnological aspects of chitinolytic enzymes: A review. Applied Microbiology and Biotechnology, 71, 773–782. Dahiya, N., Tewari, R., Tiwari, R. P., & Hoondal, G. S. (2005). Production of an antifungal chitinase from Enterobacter sp. NRG4 and its application in protoplast production. World Journal of Microbiology and Biotechnology, 21, 1611–1616. De Marco, J. L., Lima, C. L. H., Desousa, M. V., & Felix, C. R. (2000). A Trichoderma harzianum chitinase destroys the cell walls of the phytopathogen Crinipellis pernicosa, the causal agent of the witches broom disease of cocoa. World Journal of Microbiology and Biotechnology, 16, 383–386.

Chitinase Production by Using Waste Chitin

43

Fenice, M., Giambattista, R. D., Raetz, E., Leuba, J. L., & Federici, F. (1998). Repeatedbatch and continuous production of chitinolytic enzymes by Penicillium janthinellum immobilised on chemically-modified macroporous cellulose. Journal of Biotechnology, 62, 119–131. Ghanem, K. M., Al-Garni, S. M., & Al-Makishah, N. H. (2010). Statistical optimization of cultural conditions for chitinase production from fish scales waste by Aspergillus terreus. African Journal of Biotechnology, 9(32), 5135–5146. Gkargkas, K., Mamma, D., Nedev, G., Topakas, E., Christakopoulos, P., Kekos, D., et al. (2004). Studies on a N-acetyl-d-glucosaminidase produced by Fusarium oxysporum F3 grown in solid-state fermentation. Process Biochemistry, 39, 1599–1605. Gohel, V., Chaudhary, T., Vyas, P., & Chhatpar, H. S. (2006). Statistical screenings of medium components for the production of chitinase by the marine isolate Pantoea dispersa. Biochemical Engineering Journal, 28, 50–56. Gorovoj, L., & Kosyakov, V. (1997). Chitin and chitosan bio adsorbents for radionuclide and heavy metals. In A. Domard, G. A. F. Roberts, & K. M. Varum (Eds.), Advances in chitin science (pp. 858–863). Lyon: Jacques Andre Publisher. Graham, L. S., & Sticklen, M. B. (1994). Plant chitinases. Canadian Journal of Botany, 72, 1057–1083. Hackman, R. H., & Goldberg, M. (1965). Studies on chitin. VI. The nature of alpha- and beta-chitins. Australian Journal of Biological Sciences, 18(4), 935–946. Haga, A., & Shirata, A. (1997). Analysis of function of chitin prepated from silkworm Bombyx mori. In A. Domard, G. A. F. Roberts, & K. M. Varum (Eds.), Advances in chitin science (pp. 84–87). Lyon: Jacques Andree Publishers. Han, Y., Li, Z., Miao, X., & Zhang, F. (2008). Statistical optimization of medium components to improve the chitinase activity of Streptomyces sp. Da11 associated with the South China Sea sponge Craniella australiensis. Process Biochemistry, 43(10), 1088–1093. James, M. C., Hackett, T. J., Tuohy, M. G., & Coughlan, M. P. (1991). Chitinase production by Talaromyces emersonii. Biotechnology Letters, 13(9), 677–682. Johnson, E. L., & Peniston, Q. P. (1982). Utilization of shellfish waste for chitin and chitosan production. In R. E. Martin, G. J. Flick, C. E. Hobard, & D. R. Ward (Eds.), Chemistry and biochemistry of marine food products (p. 415). Westport, CT: Avi Publishing Company. Chapter 19. Joshi, S., Kozlowski, M., Richens, S., & Comberbach, D. M. (1989). Chitinase and chitobiase production during fermentation of genetically improved Serratia liquef aciens. Enzyme and Microbial Technology, 11, 289–296. Jung, W. J., Kuk, J. H., Kim, K. Y., Jung, K. C., & Park, R. D. (2006). Purification and characterization of exo-β-d-glucosaminidase from Aspergillus fumigatus S-26. Protein Expression and Purification, 45(1), 125–131. Kao, P. M., Huang, S. C., Chang, Y. C., & Liu, Y. C. (2007). Development of continuous chitinase production process in a membrane bioreactor by Paenibacillus sp. CHE-N. Process Biochemistry, 42, 606–611. Karthik, N., Akanksha, K., & Pandey, A. (2014). Production, purification and properties of fungal chitinases: A review. Indian Journal of Experimental Biology, 52(11), 1025–1035. Khan, M. A., Hamid, R., Ahmad, M., Abdin, M. Z., & Javed, S. (2010). Optimization of culture media for enhanced chitinase production from a novel strain of Stenotrophomonas maltophilia using response surface methodology. Journal of Microbiology and Biotechnology, 20, 597–1602. Kim, K. A., Creagh, L. A., & Haynes, C. A. (1998). Effective production of N-acetyl-βD-glucosamine by Serratia mareescens using chitinaceous waste. Biotechnology and Bioprocess Engineering, 3, 71–77. Knorr, D. (1991). Recovery and utilization of chitin and chitosan in food processing waste management. Food Technology, 45, 114–122.

44

S. Das et al.

Knorr, D., Beaumont, M. D., & Pandya, Y. (1989). Potential of acid soluble and water soluble chitosan in biotechnology. In G. Skjak-Braek, T. Anthonsen, & P. Sandford (Eds.), Chitin and chitosan (pp. 101–118). Elsevier Applied Science. Kole, M. M., & Altosaar, I. (1985). Increased chitinase production by a non-pigmented mutant of Serratia marcescens. FEMS Microbiology Letters, 26(3), 265–269. Kolodziejska, I., Malesa-Cie´cwierz, M., Lerska, A., & Sikorski, Z. E. (1999). Properties of chitin deacetylase from crude extracts of Mucor rouxii. Journal of Food Biochemistry, 23, 45–57. Kolodziejska, E., & Sikorski, Z. E. (1999). Endogenous enzymes in Antarctic krill: Control of activity during storage and utilization. In Z. E. Sikorski (Ed.), Seafood enzymes (pp. 505–524). Marcel Dekker. Krishnaveni, B., & Ragunathan, R. (2014). Chitinase production from marine wastes by Aspergillus terreus and its application in degradation studies. International Journal of Current Microbiology and Applied Sciences, 3(1), 76–82. Kuk, J. H., Jung, W. J., Jo, G. H., Kim, Y. C., Kim, K. Y., & Park, R. D. (2005). Production of N-acetyl-β-D-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme. Applied Microbiology and Biotechnology, 68, 384–389. Li, D. C., Zhang, S. H., Liu, K. Q., & Lu, J. (2004). Purification and partial characterization of a chitinase from the mycoparasitic fungus Trichothecium roseum. The Journal of General and Applied Microbiology, 50(1), 35–39. Matsumoto, Y., Saucedo-Castan˜eda, G., Revah, S., & Shirai, K. (2004). Production of N-acetylhexosaminidase of Verticillium lecanii by solid state and submerged fermentations utilizing shrimp waste silage as substrate and inducer. Process Biochemistry, 39, 665–671. Mendonsa, E. S., Vartak, P. H., Rao, J. U., & Deshpande, M. V. (1996). An enzyme from Myrothecium verrucaria that degrades insect cuticle for biocontrol of Aedes aegypti mosquito. Biotechnology Letters, 18(4), 373–376. Merzendorfer, H., & Zimoch, L. (2003). Chitin metabolism in insects: Structure, function and regulation of chitin synthases and chitinases. Journal of Experimental Biology, 206, 4393–4412. Miyashita, K., Fujii, T., & Sawada, Y. (1991). Molecular cloning and characterization of chitinase gene from Streptomyces lividans. Journal of Microbiology, 137, 2065–2072. Mudasir, R. G., Tahir, I., & Wahyni, E. T. (2008). Immobilization of dithizone onto chitin isolated from prawn sea water shells (P. merguensis) and its preliminary study for the adsorption of Cd(II) ion. Journal of Physical Science, 19, 63–78. Muzzarelli, R. A. A., Ilari, P., Tarsi, R., Dubini, B., & Xia, W. (1994). Chitosan from Absidia coerulea. Carbohydrate Polymers, 25, 45–50. Naczk, M., Synowiecki, J., & Sikorski, Z. E. (1981). The gross chemical composition of Antarctic krill shell waste. Food Chemistry, 7, 175–179. Nampoothiri, K. M., Baiju, T. V., Sandhya, C., Sabu, A., Szakacs, G., & Pandey, A. (2004). Process optimization for antifungal chitinase production by Trichoderma harzianum. Process Biochemistry, 39, 1583–1590. Nawani, N. N., & Kapadnis, B. P. (2005). Optimization of chitinase production using statistics based experimental designs. Process Biochemistry, 40, 651–660. Ohishi, K., Yamagishi, M., Ohta, T., Suzuki, M., Izumida, H., Sano, H., et al. (1996). Purification and properties of two chitinases from Vibrio alginolyticus H-8. Journal of Fermentation and Bioengineering, 82(6), 598–600. O’Riordan, A., McHale, M. L., Gallagher, J., & McHal, A. P. (1989). Chitinase production following co-immobilization of Microspora chalcae with chitin in calcium alginate. Biotechnology Letters, 11, 735–738. Park, J. K., Morita, K., Fukumoto, I., Yamasaki, Y., Nakagawa, T., Kawamukai, M., et al. (1997). Purification and characterization of the chitinase (ChiA) from Enterobacter sp. G-1. Bioscience, Biotechnology, and Biochemistry, 61, 684–689.

Chitinase Production by Using Waste Chitin

45

Patil, S. R., Ghormade, V., & Deshpande, M. V. (2000). Chitinolytic enzymes: An exploration. Enzyme and Microbial Technology, 26, 473–483. Prabavathy, V. R., Mathivanan, N., Sagadevan, E., Murugesan, K., & Lalithakumari, D. (2006). Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum. Bioresource Technology, 97(18), 2330–2334. Rao, B. M., Inman, F., Holmes, L., & Lalitha, K. V. (2013). Chitinase production in a fedbatch fermentation of colloidal chitin using a mixed culture of Vibrio harveyi and Vibrio alginolyticus. Fishery Technology, 50(1). Roberts, G. A. F. (1992). Chitin Chemistry. London: Mac Millan Press. Sandhya, C., Adapa, L. K. K., Nampoothiri, M., Binod, P., Szakacs, G., & Pandey, A. (2004). Extracellular chitinase production by Trichoderma harzianum in submerged fermentation. Journal of Basic Microbiology, 44(1), 49–58. Shahidi, F., & Synowiecki, J. (1991). Isolation and characterization of nutrients and valueadded products from snow crab (Cinoecetes opilio) and shrimp (Pandalus borealis) processing discards. Journal of Agricultural and Food Chemistry, 39, 1527–1532. Stoykov, Y. M., Pavlov, A. I., & Krastanov, A. I. (2015). Chitinase biotechnology: Production, purification, and application. Engineering in Life Science, 15, 30–38. Suresh, P. V., Anil Kumar, P. K., & Sachindra, N. M. (2011). Thermoactive β-N-acetyl hexosaminidase production by a soil isolate of Penicillium monoverticillium CFR 2 under solid state fermentation: Parameter optimization and application for N-acetyl chitooligosaccharides preparation from chitin. World Journal of Microbiology and Biotechnology, 27, 1435–1447. Suresh, P. V., & Chandrasekaran, M. (1998). Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation. World Journal of Microbiology and Biotechnology, 14, 655–660. Suresh, P. V., Sachindra, N. M., & Bhaskar, N. (2011). Solid state fermentation production of chitin deacetylase by Colletotrichum lindemuthianum ATCC 56676. Journal of Food Science and Technology, 48, 349–356. Svitil, A. L., Chadhain, S. M. N., Moore, J. A., & Kirchman, D. C. (1997). Chitin degradation proteins produced by the marine bacterium Vibrio harveyi growing on different forms of chitin. Applied and Environmental Microbiology, 63, 408–413. Synowiecki, J., & Al-Khateeb, N. (1997). Mycelia of Mucor rouxii as a source of chitin and chitosan. Food Chemistry, 60, 605–610. Synowiecki, J., & Al-Khateeb, N. (2000). The recovery of protein hydrolysate during enzymatic isolation of chitin from shrimp Crangon crangon processing discards. Food Chemistry, 68, 147–152. Synowiecki, J., & Al-Khateeb, N. (2003). Production, properties, and some new applications of chitin and its derivatives. Critical Reviews in Food Science and Nutrition, 43(2), 145–171. Tagawa, K., & Okazaki, K. (1991). Isolation and some cultural conditions of Streptomyces species which produce enzymes lysing Aspergillus niger cell wall. Journal of Fermentation and Bioengineering, 71, 230–236. Takayanagi, T., Ajisaka, K., Takiguchi, Y., & Shimahara, K. (1991). Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u. Biochimica et Biophysica Acta, 1078(3), 404–410. Trudel, J., & Asselin, A. (1990). Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis. Analytical Biochemistry, 189, 249–253. Tsujibo, H., Minoura, K., Miyamoto, K., Endo, H., Moriwaki, M., & Inamori, Y. (1993). Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520. Applied and Environmental Microbiology, 59, 620–622. Usui, T., Hayashia, Y., Nanjob, F., Sakaib, K., & Ishido, Y. (1987). Transglycosylation reaction of a chitinase purified from Nocardia orientalis. Biochimica et Biophysica Acta, 923(2), 302–309.

46

S. Das et al.

Vaidya, R., Vyas, P., & Chhatpar, H. S. (2003). Statistical optimization of medium components for the production of chitinase by Alcaligenes xylosoxydans. Enzyme and Microbial Technology, 33, 92–96. van Eijk, M., van Roomen, C. P. A. A., Renkema, G. H., Bussink, A. P., Andrews, L., Blommaart, E. F. C., et al. (2005). Characterization of human phagocyte-derived chitotriosidase, a component of innate immunity. International Immunology, 17, 1505–1512. Velmurugan, N., Kalpana, D., Hoon, H. J., Jung, C. H., & Yang, S. L. (2011). A novel low temperature chitinase from the marine fungus Plectosphaerella sp. strain MF 1. Botanica Marina, 54, 75–81. Vyas, P. R., & Deshpande, M. V. (1989). Chitinase production by Myrothecium verbucaria and its significance for fungal mycelium degradation. The Journal of General and Applied Microbiology, 35, 343–350. Waghmare, S. R., Kulkarni, S. S., & Ghosh, J. S. (2011). Chitinase production in solid-state fermentation from Oerskovia xanthineolytica NCIM 2839 and its application in fungal protoplasts formation. Current Microbiology, 63, 295–299. Wang, S. L., & Chang, W. T. (1997). Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell power medium. Applied and Environmental Microbiology, 63, 380–383. Wang, S., Hsiao, W., & Chang, W. (2002). Purification and characterization of an antimicrobial chitinase extracellularly produced by Monascus purpureus CCRC31499 in a shrimp and crab shell powder medium. Journal of Agricultural and Food Chemistry, 50(8), 2249–2255. Xia, G., Jin, C., Zhou, J., Yang, S., Zhang, S., & Jin, C. (2001). A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. European Journal of Biochemistry, 268, 4079–4085. Yamane, T., & Shimizu, S. (2005). Fed-batch techniques in microbial processes. Advances in Biochemical Engineering/Biotechnology, 30, 147–194.