MINI-REVIEW Nocardiopsis species as potential sources of diverse and novel extracellular enzymes Tahsin Bennur & Ameeta Ravi Kumar & Smita Zinjarde

Appl Microbiol Biotechnol (2014) 98:9173–9185 DOI 10.1007/s00253-014-6111-y

MINI-REVIEW

Nocardiopsis species as potential sources of diverse and novel extracellular enzymes Tahsin Bennur & Ameeta Ravi Kumar & Smita Zinjarde & Vaishali Javdekar

Received: 1 July 2014 / Revised: 18 September 2014 / Accepted: 19 September 2014 / Published online: 30 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Members of the genus Nocardiopsis are generally encountered in locations that are inherently extreme. They are present in frozen soils, desert sand, compost, saline or hypersaline habitats (marine systems, salterns and soils) and alkaline places (slag dumps, lake soils and sediments). In order to survive under these severe conditions, they produce novel and diverse enzymes that allow them to utilize the available nutrients and to thrive. The members of this genus are multifaceted and release an assortment of extracellular hydrolytic enzymes. They produce enzymes that are cold-adapted (α-amylases), thermotolerant (α-amylases and xylanases), thermoalkalotolerant (cellulases, β-1,3-glucanases), alkalitolerant thermostable (inulinases), acid-stable (keratinase) and alkalophilic (serine proteases). Some of the enzymes derived from Nocardiopsis species act on insoluble polymers such as glucans (pachyman and curdlan), keratin (feathers and prion proteins) and polyhydroxyalkanoates. Extreme tolerance exhibited by proteases has been attributed to the presence of some amino acids (Asn and Pro) in loop structures, relocation of multiple salt bridges to outer regions of the protein or the presence of a distinct polyproline II helix. The range of novel enzymes is projected to increase in the forthcoming years, as new isolates are being continually reported, and the development of processes involving such enzymes is envisaged in the future.

T. Bennur : A. R. Kumar : S. Zinjarde (*) Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, India e-mail: [email protected] V. Javdekar (*) Department of Biotechnology, Abasaheb Garware College, Pune 411004, India e-mail: [email protected]

Keywords Nocardiopsis . Carbohydrases . PHB depolymerases . Proteases

Introduction Actinomycetes are a major group of Gram-positive filamentous bacteria with high guanine and cytosine contents. They are undoubtedly most significant with respect to synthesis of antibiotics and other bioactive compounds. However, they also produce a variety of extracellular hydrolytic enzymes that mediate degradation and recycling of natural biopolymers (Olano et al. 2009). Several actinomycetes, including Nocardiopsis species, are isolated from different sites, and they inherently have a vast range of enzymatic abilities. Meena et al. (2013) have studied actinobacteria from a tropical marine hot spot (Andaman and Nicobar Islands) and have described them as potential sources for industrial and pharmaceutical products. Genera such as Saccharopolyspora, Streptomyces, Streptoverticillium, Microtetraspora, Actinopolyspora, Actinokineospora, Dactylosporangium and Nocardiopsis were obtained during this study. A majority of these isolates produced industrially important enzymes such as amylases, proteases, gelatinases, lipases, DNases, cellulases, ureases and phosphatases. On another occasion, Kumar et al. (2012) have isolated actinomycetes from earthworm castings and have screened them for antimicrobial activities and industrial enzymes. Streptomyces, Streptosporangium, Saccharopolyspora, Nocardia, Micromonospora, Actinomadura, Microbispora, Planobispora and Nocardiopsis were obtained. Several of these isolates produced amylases, caseinases, cellulases, gelatinases, xylanases and lipases. Tan et al. (2009) have screened the actinomycetes associated with goat faeces for their hydrolytic enzymes. Oerskovia, Streptomyces and Nocardiopsis species were the important genera that were obtained, and they produced enzymes such

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as amylases, proteases and galactosidases. On the basis of these reports, it is evident that members of the genus Nocardiopsis along with other actinomycetes are present in diverse environments, and they are important enzyme producers. Species of Nocardiopsis are strictly aerobic, Gram-positive, non-acid-fast actinomycetes (Grund and Kroppenstedt 1990). From the taxonomic point of view, members of the genus Nocardiopsis belong to phylum Actinobacteria, class Actinobacteria, order Actinomycetales and family Nocardiopsaceae (Rainey et al. 1996; Sun et al. 2010). A recent review indicates that there are 42 species included in this genus (Hamedi et al. 2013). In the years to come, this count is expected to increase. The organism is endowed with a variety of unusual physiological and metabolic features, and a large number of research laboratories have been using this as a model system for understanding fundamental phenomena related to enzymes and in developing applications as discussed in the following sections. Nocardiopsis species are intrinsically present in a vast range of ecological habitats. There are reports on the isolation of this actinomycete from a variety of harsh environments as summarized in Fig. 1. With respect to extreme temperatures, some species have been isolated from cold soils (Xu et al. 2014), and others have been obtained from desert sand dunes and compost soils, where temperatures are higher (Hozzein and Goodfellow 2008; Yamamura et al. 2010; Yan et al. 2011). A large plethora of species has been isolated from environments that are rich in salt. In this regard, Hamedi et al. (2013) in a recent review article have described Nocardiopsis species as the most abundant halophilic and halotolerant actinomycete. Among the 42 hitherto reported species, 13 are halophilic and six are halotolerant. They have been isolated from marine sediments (Fang et al. 2011; Meena et al. 2013), hypersaline solar salterns (Chun et al. 2000; Jose and Jebakumar 2012), saline soils (Al-Tai and Ruan 1994; Li et al. 2003; Li et al. 2006; Chen et al. 2010) and marine biological forms (Chen et al. 2009; Li et al. 2012). In addition, some species are alkali-tolerant being inherently present in Fig. 1 Summary of the extreme environmental conditions where Nocardiopsis species are prevalent

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alkali slag dumps, lake soils and sediments (Schippers et al. 2002; Yang et al. 2008; Mwirichia et al. 2010). Li et al. (2013) on the basis of comparative genomic analysis have concluded that the high versatility and adaptability of Nocardiopsis species are due to their intrinsic genetic features. Since the members of genus Nocardiopsis are widespread in environmentally harsh conditions, it was deduced that the enzymes produced by this actinomycete would also be diverse and novel. It must be noted that extremophiles are of considerable significance with respect to (i) understanding structure-function relations of their constituent enzymes, (ii) studying the adaptability of such organisms to extreme environmental conditions and (iii) providing enzymes that can be used as advanced tools for a variety of applications (Niehaus et al. 1999; Vieille and Zeikus 2001; de Champdore et al. 2007; Gabani and Singh 2013; Elleuche et al. 2014). A thorough assessment of the available literature on the topic led to this exclusive review highlighting the production, biochemical properties, biophysical characterization and applications of the extracellular enzymes produced by this organism. To the best of our knowledge, there are no recent reviews on the enzymes produced by this biotechnologically relevant genus. In this review, current updates on the enzymes produced by this actinomycete have been broadly categorized on the basis of their substrates and on the type of environments from which the different strains producing these diverse enzymes have been isolated.

Carbohydrases obtained from Nocardiopsis species A range of carbohydrases is produced by Nocardiopsis species. Starch, cellulose, β-glucans, inulin, xylan and chitin are natural biopolymers that are degraded by extracellular amylases (EC 3.2.1.1), cellulases (EC 3.2.1.4), β-1,3-glucanases (EC 3.2.1.39), inulinases (EC 3.2.1.7), xylanases (EC 3.2.1.8) and chitinases (EC 3.2.1.14), respectively, produced by members of this genus. The details of carbohydrases produced by Nocardiopsis species are summarized in Table 1.

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Table 1 Summary of the carbohydrases produced by Nocardiopsis species Carbohydrase

Species/Location

Enzyme pH; temperature optima

Other features and applications

Reference

Cold-adapted α-amylase

Nocardiopsis strain 7326, Antarctica

8.0; 35 °C

Zhang and Zeng 2008

Cold-adapted α-amylase

N. aegyptia, Egypt

–

Thermotolerant α-amylase Thermostable α-amylase

Nocardiopsis species, Brazil Nocardiopsis sp. B2

5.0; 70 °C

Thermoalkalotolerant cellulase Alkalotolerant cellulase

Nocardiopsis sp. 5.0; 40 °C KNU, Korea Nocardiopsis sp. 8.0; 40 °C SES28, Argentina Nocardiopsis sp. F96, 9.0; 70 °C Japan

Stable between pH 5 and 10; inactivated above 45 °C; stimulated by Ca2+, Mn2+, Mg2+, Cu2+ and Co2+; inhibited by Rb2+, Hg2+ and EDTA Fermentation conditions optimized by Plackett-Burman statistical design; enhanced production after whole cell immobilization on luffa pulp Industrial applications involving high temperatures Maximum enzyme activity with 11 % (w/v) NaCl. Retained 75 and 69 % activity after incubation for 1 h at 75 and 85 °C, respectively. Effective immobilization in gellan gum microspheres. Generation of fermentable rice straw hydrolysates for ethanol production Application in the detergent industry

Thermoalkalotolerant β-1,3-glucanase

Alkali-tolerant thermostable inulinase Thermotolerant xylanases

9.0; 45 °C

Nocardiopsis species 8.0; 60 °C DN-K15, China N. dassonvillei subsp. X-I and X-II, 7.0; alba OPC-18, 60 °C X-III, Japan 50 °C

Abou-Elela et al. 2009

Stamford et al. 2001 Chakraborty et al. 2014

Saratale and Oh 2011 Walker et al. 2006

Hydrolyzed insoluble β-1,3-glucans; preferred β-1,3-1,4-glucans rather than β-1,3-glucans; cloning and expression; crystal structure; construction and characterization of chimeras Exoinulinase (fructose as hydrolysis product)

Masuda et al. 2003, 2006; Fibriansah et al. 2006, 2007; Koizumi et al. 2007, 2009

Xylan bioconversion into xylose for different products

Tsujibo et al. 1990a, 1991

Starch is an abundantly available natural polymer of Dglucose monomers linked via α(1→4)-glycosidic bonds. A variety of amylases act on the glucosidic linkages present in the biopolymer (Souza and Magalhães 2010). Nocardiopsis species inherently produce some α-amylases. The most important feature regarding these enzymes is their occurrence in species isolated from extreme environments with particular reference to temperature. As discussed in the “Introduction” section, some species of this genus have been isolated from cold regions, and they have been explored for the production of cold-active amylases. During an investigation on the isolation of psychrotrophic bacteria with cold-adaptive amylolytic, lipolytic or proteolytic activities, bacterial cultures were isolated from deep-sea sediment of Prydz Bay, Antarctica (Zhang and Zeng 2007). The amylase-producing strains belonged to genus Pseudomonas, Rhodococcus and Nocardiopsis. Nocardiopsis strain 7326 obtained from this region yielded a cold-adapted α-amylase optimally active at 35 °C under alkaline conditions (Zhang and Zeng 2008). The enzyme was inactivated at temperatures above 45 °C. This enzyme degraded starch to glucose, maltose and maltotriose. Other features of this enzyme are detailed in Table 1. Another cold-adapted

Lu et al. 2014

α-amylase was obtained from Nocardiopsis aegyptia initially isolated from marine sediment in Egypt (Abou-Elela et al. 2009). The α-amylase was optimally produced at a relatively lower temperature of 25 °C under acidic conditions. From these reports, it is evident that Nocardiopsis species living under cold conditions have evolved to produce cold-adapted enzymes that enable their survival under such adverse conditions. It must be noted that such cold-active or cold-adaptive enzymes are not only significant with respect to fundamental scientific studies but are also relevant in industrial settings (Cavicchioli et al. 2011; Feller 2013). Like other cold-adapted starch-hydrolyzing enzymes, α-amylases obtained from Nocardiopsis species could find applications in bread making, textiles, brewing and detergents. In contrast to the aforementioned cold-adapted α-amylases, a thermotolerant variant of the enzyme has been obtained from an endophytic species of Nocardiopsis from Brazil (Stamford et al. 2001). The thermostability of the enzyme was evident from the fact that it was completely unaltered at 70 °C and retained 50 % of the activity when incubated at 90 °C for 10 min. This thermostable enzyme could be put to use for preparing high-fructose syrups. Such syrups are extensively

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used in the beverage industry as sweeteners for making soft drinks (Van der Maarel et al. 2002). Another recent paper describes the isolation and immobilization of a thermostable α-amylase derived from a haloalkaliphilic marine isolate referred to as Nocardiopsis sp. B2 (Chakraborty et al. 2014). The enzyme immobilized in gellan gum beads could be released in a sustained manner. This property was highlighted to be valuable in the pharmaceutical industries particularly for preparing digestive formulations wherein continued release of the enzyme over a period of time is desirable. Cellulose, the most abundant organic polymer on Earth, is composed of D-glucose units linked through β(1→4)-glycosidic bonds, and cellulases are hydrolytic enzymes acting on this polymer (Lynd et al. 2002). In a recent report, Anderson et al. (2012) have identified putative cellulose-degrading enzymes in the genomes of some actinobacteria. Among the 11 organisms evaluated, eight including Nocardiopsis dassonvillei IMRU 509 produced cellulases. Cellulase activity was observed when azurine cross-linked hydroxyethyl cellulose was used, and increased levels of the enzymes were particularly demonstrated in the presence of cellobiose. N. dassonvillei genomes showed sequences for six predicted cellulases. Apart from this report on the predicted cellulases in N. dassonvillei IMRU 509, another isolate, Nocardiopsis sp. (KNU) obtained from a Korean soil sample produced a variety of thermoalkalotolerant carbohydrases including cellulases (Saratale and Oh 2011). This strain was strongly cellulolytic and utilized carboxymethyl cellulose (CMC), avicel, cellobiose, filter paper and rice straw effectively. The organism grew optimally (at 37 °C and pH 6.5) under static conditions and produced cellulases that were thermotolerant and alkalotolerant. The authors have also reported that rice straw could be effectively converted into hydrolysates by using these enzymes. Such hydrolysates were further used as substrates by a strain of Saccharomyces cerevisiae for ethanol fermentation. Another isolate from Argentina, Nocardiopsis sp. SES28 produced cellulases when grown in the presence of CMC (Walker et al. 2006). The culture produced the enzyme when it was grown at pH 8.0. The enzyme retained most of its activity even at pH 10.0, and therefore, its suitability for application in the detergent industry was proposed. Cellulose-degrading enzymes are important in laundry operations, in fabric industries and in the production of biofuels from lignocellulosic biomass (Kuhad et al. 2011). On account of these varied applications, further studies on cellulases from different Nocardiopsis species are warranted. β-Glucans (polysaccharides of D-glucose linked by βglycosidic bonds) are a diverse group of polymers. They show a great diversity with respect to molecular mass, viscosity, solubility and three-dimensional configuration (Laroche and Michaud 2007). They are mainly encountered in the cell walls of certain fungi and the bran of cereal grains (Manzi and Pizzoferrato 2000; Kerckhoffs et al. 2003). β-1,3-

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Glucanases are a class of enzymes that can degrade these biopolymers. Decomposition of β-1,3-glucan is an important process because there are industrial requirements for β-1,3glucan hydrolysates (Laroche and Michaud 2007). These enzymes are also important in biocontrol of phytopathogenic fungi (Masih and Paul 2002). β-1,3-Glucan is a constituent of fungal cell walls, and its hydrolysis by β-1,3-glucanases can weaken or damage the fungal cell wall. An alkaliphilic Nocardiopsis sp. (F96) isolated in Japan produced three endo-β-1,3-glucanase isozymes with varying molecular masses referred to as BglF1, BglF2 and BglF3 (Masuda et al. 2006). The three isozymes shared N-terminal amino acid sequences and were thus deduced to be products of a single gene (bglF). This gene was cloned, and the expressed BglF protein showed highest amino acid sequence homology with the GHF16 family of endo-β-1,3-glucanases (Masuda et al. 2003). The enzyme was thermoalkalotolerant (optimum pH and temperature: pH 9.0 and 70 °C, respectively). The hydrolysis of different substrates such as laminarin, pachyman, curdlan, lichenan, CMC and avicel was evaluated. The enzyme efficiently hydrolyzed insoluble β-1,3-glucans such as pachyman and curdlan and the fairly soluble laminarin. However, it displayed highest activity towards lichenan, a β-1,3-1,4-glucan. These results indicated BglF to be a novel endo-β-1,3-glucanase. Studies involving mutational analysis suggested that Glu 123 and Glu 128 could be the catalytic residues of BglF (Masuda et al. 2006). This enzyme was successfully crystallized by the hanging-drop vapour diffusion method (Fibriansah et al. 2006). In a later report, the authors have presented the first crystal structure of this endo-β-1,3glucanase at 1.3-A° resolution and have compared it with the other homologous structures to analyze its substrate preference. Results of these studies confirmed that the enzyme had a catalytic centre comprising of Glu 123 as the putative nucleophile and Glu 128 as the acid-base catalyst. Trp 118 was found to be extremely important in substrate binding (Fibriansah et al. 2007). The β-1,3-glucanase was shown to have a single catalytic domain. In order to enhance the enzymatic properties of BglF, additional carbohydrate-binding domains were introduced, chimera proteins were created, and they were characterized (Koizumi et al. 2007, 2009). Four chimeras containing BglF along with the following carbohydrate-binding modules (i) Cterminal additional domain (CAD) of β-1,3-glucanase H from Bacillus circulans IAM1165, (ii) N-terminal additional domain (NAD) of β-1,3-glucanase H from B. circulans IAM1165, (iii) both CAD and NAD and (iv) chitin-binding domain (ChBD) of chitinase from alkaliphilic Bacillus sp. J813 were constructed. These chimeras respectively referred to as (i) BglF-CAD, (ii) NAD-BglF, (iii) NAD-BglF-CAD and (iv) BglF-ChBD were further characterized with regard to their binding with insoluble β-1,3-glucans and their hydrolytic behaviour towards these polymers. BglF-CAD chimeras

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displayed better binding abilities and hydrolytic activities towards these insoluble glucans. In particular, the hydrolytic activities of this chimera under alkaline conditions (pH 9.0– 10.0) and higher temperatures (50–70 °C) were better than the native protein. The chimera BglF-ChBD showed enhanced binding and hydrolysis of the substrate pachyman, and relative activities of this chimera around 50–70 °C were also higher than those of original BglF. Wild-type and recombinant strains over-expressing native and chimeric β-1,3-glucanases generally find applications in the biocontrol of fungi, in the production of yeast extract and in the clarification of wines (Cheng et al. 2013). The β-1,3glucanases derived from Nocardiopsis were unusual since they acted on insoluble glucans. Inulin is a natural carbohydrate reserve mainly observed in the roots and tubers of plants such as Jerusalem artichoke, chicory and dahlia (Kango and Jain 2011). This polymer consists of linear chains of β-2,1-linked D-fructofuranose molecules that are terminated with glucose residues through a sucrose-type linkage at the reducing end. Acid- or inulinasehydrolyzed inulin is used for the production of fructose syrups, inulo-oligosaccharides and bioethanol (Negro et al. 2006; Singh et al. 2007). Since acidic hydrolysis of inulin often results in the formation of undesirable colour and byproducts, the use of inulinases has become popular (Singh et al. 2006). Recently, an exoinulinase (β- D -fructan fructohydrolase) has been reported from Nocardiopsis species DN-K15 isolated from marine sediments in China (Lu et al. 2014). The enzyme was alkali tolerant and thermostable and was active over a wide range of pH (5.0–11.0). It retained more than 81 % of the activity after incubation at 60 °C for 1 h. Like other inulinases, this enzyme could also be used to obtain high fructose syrups and inulinosaccharides. Xylans are polysaccharides that mainly contain β-D-xylose units linked via β(1→4)-glycosidic bonds (Bastawde 1992). They are ubiquitous in nature, and several microorganisms including Nocardiopsis species produce xylanases, the enzymes that catalyze the breakdown of this biopolymer. In this regard, N. dassonvillei OPC-18 is reported to produce three types of xylanases. These enzymes named as X-I, X-II and XIII were purified and characterized (Tsujibo et al. 1990a). Although these enzymes were optimally active at pH 7.0, enzyme X-III retained almost 63 % of its activity even at pH 11.0. Other features associated with these enzymes are shown in Table 1. The authors later deduced and compared the amino acid compositions and partial N-terminal sequences of the enzymes (Tsujibo et al. 1991). The whole genomes of N. dassonvillei and Nocardiopsis alba are sequenced. The N. dassonvillei genome shows the existence of four predicted coding sequences (CDS) for xylanases: Ndas_2218, Ndas_2447, Ndas_2710 and Ndas_3986 (Sun et al. 2010). The N. alba genome shows the presence of one xylanases coding sequence, B005_4612

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(Qiao et al. 2012). It must be noted that xylanases are important in the breakdown of xylan into xylose which in turn can be used for producing different types of biofuels (Taherzadeh and Karimi 2008). Chitin is an abundantly available natural polysaccharide that mainly acts as the supporting material of crustaceans, insects and fungi (Rinaudo 2006). It consists of 2acetamido-2-deoxy-β- D -glucose monomers linked via β(1→4) linkages, and a group of enzymes, the chitinases, hydrolyzes this polymer (Ravi Kumar 2000). During a study on the presence of chitin degraders from different sites, a variety of bacteria including those belonging to the genus Nocardiopsis were obtained (Nawani and Kapadnis 2003). During the same period, Tsujibo et al. (2003) isolated an alkaliphilic Nocardiopsis prasina strain OPC-131 in Japan that secreted three types of chitinases (ChiA, ChiB and ChiB Delta). The genes encoding the first two types of chitinases (ChiA and ChiB) were cloned and sequenced. While ChiA had a molecular mass of around 35 kDa and a catalytic domain homologous with family 18 chitinases, ChiB was a 32-kDa protein that contained the type 3 chitin-binding domain (ChtBD type 3) and a catalytic domain. The 32-kDa protein showed considerable similarity with the Streptomyces family 19 chitinases. ChiB Delta, the third enzyme, was established to be the truncated form of ChiB (it lacked the ChtBD type 3 domain). ChiB was more efficient in hydrolyzing chitin and demonstrated better antifungal activity than ChiB Delta. This suggested that the ChtBD type 3 domain of ChiB played a significant function in the effective hydrolysis of chitin and its antifungal activity. During another study, Apichaisataienchote et al. (2005) introduced ChiB gene in Streptomyces fradiae (an antagonist of Fusarium moniliforme). While the wild-type strain produced only two chitinases, the recombinant secreted three chitinases including ChiB. The supernatant of the recombinant strain effectively inhibited hyphal growth of F. moniliforme. The chitinase F1 (ChiF1) has been obtained from another alkaliphilic Nocardiopsis sp. (strain F96) from Japan, and its crystal structure has been elucidated (Matsui et al. 2004). The enzyme belongs to family 18 chitinases. The genome of N. dassonvillei shows the presence of three predicted coding sequences for chitinases, namely, Ndas_0360, Ndas_0763 and Ndas_1329 (Sun et al. 2010). Moreover, B005_3231, B005_3232 and B005_3655 are the sequences for chitinases in N. alba (Qiao et al. 2012). Chitindegrading enzymes and chitinolytic microorganisms in recent years have gained importance (Brzezinska et al. 2014). In the future, chitinases are projected to be safe and sustainable alternatives for controlling pathogenic fungi. These hydrolytic enzymes also mediate efficient degradation of chitin in nature. The importance of Nocardiopsis chitinases has been recently demonstrated by following a metaproteomic approach

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(Johnson-Rollings et al. 2014). The authors found Nocardiopsis-like chitinases to be the main enzymes responsible for chitinolytic activity in the soil that was being investigated.

Polyhydroxybutyrate depolymerase Poly[(R)-3-hydroxyalkanoic acids] or poly(HAs) or PHAs are a class of storage compounds that are produced by many bacteria during unbalanced growth. Several types of hydroxyalkanoic acids have been identified as constituents of PHA (Steinbüchel and Valentin 1995). They are projected as eco-friendly alternatives to petroleum-derived plastics (Suriyamongkol et al. 2007). These compounds are biodegradable because some bacteria have the enzymatic abilities of breaking down these polymers. In this regard, Ghanem et al. (2005) isolated a strain of N. aegyptia from marine seashore sediments in Egypt. The strain used 3-hydroxybutyrate (PHB) or its copolymers with 3-hydroxyvalerate (PHV) in different proportions P(3HB-co10–20 % HV) as the sole source of carbon. This was possible due to its ability to produce extracellular PHB depolymerases (EC 3.1.1.75). The organism was more effective in hydrolyzing copolymers than the homopolymer. A statistical experimental (Plackett-Burman) strategy was employed to standardize the conditions for maximum activity. The main factors that affected the process positively were the content of sodium gluconate, volume of medium/flask and age of inoculum. The organism degraded polymeric films as evidenced by the numerous irregular erosion pits seen in scanning electron micrographs. The thermoplastic properties and biodegradability of PHAs have made them important from the industrial point of interest, and bacteriologically produced PHB and its co-polymers are commercially available over the past decade. This subsequently highlights the requirement for microorganisms producing enzymes that degrade PHB and its co-polymers, in order to facilitate their degradation in an environment-friendly process. Nocardiopsis species producing PHB depolymerases are, therefore, very important in this regard. Although the production of PHB depolymerase has been experimentally validated in N. aegyptia, the genomes of N. dassonvillei DSM 4311 and N. alba show the presence of predicted coding sequences (CDS) for such enzymes. In N. dassonvillei, Ndas_0048 and Ndas_2245 and, in N. alba, B005_1932 are the predicted coding sequences for PHB esterases (Sun et al. 2010; Qiao et al. 2012). There is a need to investigate such species for depolymerase activities and optimize parameters for their production or genetically manipulate them if inherent levels are low.

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Features of proteases produced by Nocardiopsis species Several microorganisms produce extracellular proteases that facilitate breakdown of complex proteins to simpler peptides, thereby providing a more accessible source of nutrition (Turk 2006). Different species of Nocardiopsis produce a variety of proteases, some with unique biochemical properties, structural features and a range of applications as detailed in Table 2. Most of the Nocardiopsis species produce extracellular alkaline serine proteases (EC 3.4.21). For example, a Japanese group studied an alkalophilic strain of N. dassonvillei subspecies prasina OPC-210 with respect to the purification and characterization of two types of extracellular alkaline serine proteases, designated as NDP-I and NDPII (Tsujibo et al. 1990b). Both the enzymes displayed an optimum pH in the alkaline range, and optimum temperatures were 70 and 60 °C, respectively, for NDP-I and NDP-II. NDPI was stable between a pH range of 4.0 to 8.0, and NDP-II was more alkali stable (pH 6.0 to 12.0). On the basis of amino acid compositions and N-terminal partial sequence similarity studies, NDP-I was classified as a chymotrypsin-like serine protease, and NDP-II was classified as an aqualysin-like serine protease (Tsujibo et al. 1990c). Nocardiopsis NCIM 5124 isolated from an oil-polluted tropical marine environment near Mumbai, India (Dixit and Pant 2000a) and recently identified as N. dassonvillei on the basis of 16 s rDNA sequence similarities (unpublished results) also produced two types of alkaline serine endopeptidases referred to as proteases I and II (Dixit and Pant 2000b). Both the alkaline proteases demonstrated collagenolytic and fibrinolytic activities, and protease I could also degrade elastin. Proteases with such unusual substrate specificities are very important in medical and other biotechnological applications. It must be noted that thromboses can lead to fatal conditions such as myocardial infarction. Fibrinolytic agents of clinical significance are mostly plasminogen activators. However, such activators display certain undesired side effects, exhibit low specificity for fibrin and are relatively expensive (Kim et al. 2006). Fibrinolytic enzymes are being regarded as important alternatives in thrombolytic therapy (Phan et al. 2011). Collagenases have potential applications in the leather industry in enhancing dye penetration and also in the environmentfriendly bioconversion of collagen wastes (Kanth et al. 2008; Duarte et al. 2014). The ability to degrade elastin, however, is suggestive of pathogenic properties, as actinomycetes with this ability can cause mycetoma (Lacey and Goodfellow 1975). Proteases I and II derived from NCIM 5124 were classified as alkaline serine endopeptidases, apparently belonging to the group of chymotrypsin-like serine proteases characterized by broad S1 subsite specificity as discussed by Perona and Craik (1997). Both proteases required extended substrate binding for efficient catalysis. Although the two proteases were similar in their overall properties and

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Table 2 Summary of the proteases produced by Nocardiopsis species Enzyme produced Species/Origin

Molecular mass (kDa); pH; temperature optima

Alkaline serine proteases

N. dassonvillei Protease I, 21; 10.0 subspecies to 12.0; 70 °C prasina OPCand protease II, 210, Japan 36; 10.5; 60 °C

Alkaline serine proteases

N. dassonvillei NCIM 5124, India

Alkaline proteases N. prasina HA-4, India Alkaline serine N. alba OK-5, protease India

Protease I, 21 Protease II 23; 10.0–11.0; 60 °C

Methods of purification

Other features and applications

Reference

Acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75, phenyl-Toyopearl 650 M column chromatography Protease I: on CMSephadex chromatography Protease II: DEAEcellulose, Sephadex G-50, phenyl Sepharose, hydroxyapatite chromatography Not reported

Isoelectric points (pI): protease I 6.4 and protease II 3.8 Protease I: chymotrypsinlike Protease II aqualysin-like

Tsujibo et al. 1990b, c

pI protease I (NprotI), 8.3

Dixit and Pant 2000a; Rohamare et al. 2013

Not reported; 7.0 and 10.0; 55 °C 20 kDa; 10.0; 80 °C Hydrophobic interaction chromatography

Alkaline serine protease Milk-clotting protease

Nocardiopsis Not reported; 10.5; species, Brazil 50 °C Nocardiopsis Not reported; 7.5; species, Brazil 55 °C

Keratinase and protease

Nocardiopsis species SD5, India

30 and 60; 9.0; 50 °C

Not reported Ammonium sulphate precipitation and DEAE-cellulose chromatography Not reported

secondary structure characteristics, the results of substrate specificity studies suggested that they could differ with respect to their active site geometry. More recently, structure-function studies on protease I (designated as NprotI) have revealed that the polyproline II (PPII) fold, an unusual structural element, was responsible for imparting kinetic stability to the protein (Rohamare et al. 2013). This is the first report on a nonstructural protein of microbial origin displaying the PPII helix (the details regarding this are discussed later in this review). There are two more reports on proteases from Nocardiopsis species of Indian origin. An isolate obtained from a limestone quarry in Northeast India identified as N. prasina HA-4 produced alkaline proteases (Ningthoujam et al. 2009). An alkaline protease from a salt-tolerant alkaliphilic N. alba strain (OK-5) of Indian origin has been reported (Gohel and Singh 2012). In addition to the features listed in Table 2, the enzyme displayed a shift in temperature optimum from 70 to 80 °C in the presence of 4 M sodium chloride and 30 % sodium glutamate. The presence of sodium glutamate, H2O2, βmercaptoethanol and different surfactants enhanced the activity. Both these reports (Ningthoujam et al. 2009; Gohel and Singh 2012) have suggested the use of these Nocardiopsis-derived proteases as detergent additives.

Protease II, 7.0 NprotI displayed unique polyproline II helix (PPII) Suitable as detergent Ningthoujam et al. 2009 additive Gohel and Singh 2012 Enhanced activity in the presence of Na-glutamate, H2O2, BME and surfactants Suitable as detergent additive Moreira et al. 2002 Milk clotting proteases

Cavalcanti et al. 2004

Suitable in feather waste management

Saha et al. 2013

Some Nocardiopsis species of Brazilian origin are also reported to produce proteases with distinctive features. Moreira et al. (2002) have described the production of an alkaline serine protease with features listed in Table 2. The enzyme derived from this species was marginally inhibited by the surfactant sodium cholate, unaffected by Tween 80 and stimulated by saponin, sodium dodecyl sulphate (SDS) and Tween 20. This serine protease was bleach-stable and retained activity in the presence of high concentrations (10 %v/v) of H2O2. The protease also maintained 50 to 60 % of its activity in the presence of commercial detergents. The authors have suggested the application of this Nocardiopsis-derived protease as a detergent additive. An extracellular proteolytic enzyme preparation from another Nocardiopsis species isolated from a Northeastern Brazilian soil sample displayed milkclotting properties (Cavalcanti et al. 2004). It must be noted that microbial enzymes acting on milk proteins may provide attractive alternatives to the conventionally used enzyme chymosin (Hashem 1999; Jacob et al. 2011). The milkclotting enzyme from Nocardiopsis species was partially purified, and it displayed optimal milk-clotting activity at pH 7.5 and 55 °C. Further, the authors have optimized conditions for maximum production of the enzyme (Cavalcanti et al. 2005).

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In particular, soybean flour favoured the induction, synthesis and secretion of the protease. In a later report, this group has developed liquid-liquid extraction methods involving reversed micelle and aqueous two-phase systems for recovering the aforementioned milk-clotting protease (Porto et al. 2005). It must be understood that reversed micelles are typically aggregates of surfactants in organic solvents wherein the aliphatic chains are directed towards the exterior and the polar heads towards the interior. The resultant polar nuclei containing an aqueous microphase can be used to extract and solubilize hydrophilic compounds including proteins that can be back-extracted during a later step (Lazarova and Tonova 1999; Krishna et al. 2002). Microbial keratinases are proteolytic enzymes degrading recalcitrant and insoluble structural proteins present in feathers, hair and nails. Some keratinases can also degrade prion proteins (Gupta et al. 2013a, b). They are generally used in the dehairing of hides, keratin waste management, pharmaceuticals and prion decontamination. There are a few reports on some Nocardiopsis species producing keratinolytic enzymes. An alkaliphilic N. alba strain (TOA-1) isolated from a tile joint in Japan produced a variety of alkaline hydrolytic enzymes (Mitsuiki et al. 2002). Among these, NAPase (N. alba protease A), an acid-resistant, kinetically stable protease, was studied in detail. The novel feature of this enzyme was that it was stable under acidic conditions. The role of keratinase such as KerA derived from Bacillus licheniformis PWD-1 in the degradation of prion proteins was first demonstrated by Langveld et al. (2003). NAPases could also effectively degrade the scrapie prion PrPSc without any prior chemical or physical treatment (Mitsuiki et al. 2006). NAPase when incubated with the prion protein at 60 °C and at pH >10.0 brought about its degradation within 3 min. It must be noted that prion proteins are released into the environment through body fluids, disposal of cadavers, contaminated effluents from slaughterhouses, hospitals and research facilities, and they pose potential health hazards (Bartelt-Hunt and Bartz 2013). The chemical and physical methods available for their disinfection and sterilization are often harsh, energy-intensive and do not ensure complete loss of infectivity (Rutala and Weber 2010). In this regard, the use of enzymes such as NAPase may provide an eco-friendly alternative. The structural basis underlying the acid stability of this enzyme was determined by Kelch et al. (2007) and is discussed later. In a more recent report, a thermoalkaliphilic strain of Nocardiopsis sp. (SD5) was isolated from feather wastes in India (Saha et al. 2013). The crude preparations contained two proteases that exhibited keratinolytic and proteolytic activities as detailed in Table 2. The role of this strain in controlling feather waste pollution was suggested. It is well documented that feathers are a major contributor towards poultry wastes, and these can be treated with keratinolytic enzymes to yield keratin hydrolysates that are rich in nitrogen content and

Appl Microbiol Biotechnol (2014) 98:9173–9185

hydrophobic amino acids (Gupta and Ramnani 2006; Brandelli 2008; Brandelli et al. 2010). Keratin hydrolysates are useful products that can be used in diverse fields (Gupta et al. 2013a, b). The potential for developing commercially viable products from Nocardiopsis species (particularly with respect to proteases) is evident from some patents that have been granted to different investigators. There are patents on the use of these enzymes in making yeast extracts and casein hydrolysates (Kalum 2008; Lynglev and Nielsen 2009). There is also one permitting their recombinant production and use in animal feed and detergents (Lassen et al. 2010).

Structural features of stable enzymes produced by Nocardiopsis species Some of the enzymes produced by Nocardiopsis species display unusual stabilities under different conditions, and the structural features responsible for such behaviour have been detailed. Most of these studies are related to proteases. In two cases, proteases derived from Nocardiopsis species are kinetically stable proteins (Kelch et al. 2007; Rohamare et al. 2013). It must be noted that in such proteins, stability is determined only on the basis of kinetic barriers and not by thermodynamic equilibria. Such proteins have high free energy kinetic barriers separating the folded and unfolded states, and they tend to exist in the folded state even under harsh conditions. Kelch et al. (2007) have described the unfolding characteristics of the acid-resistant, kinetically stable protease A (NAPase) derived from N. alba as detailed in this paragraph. The authors have made most comparisons of this enzyme with a neutrophilic homolog (α-lytic protease, alphaLP). Although both the enzymes (NAPase and alphaLP) had a similar number of acid-titratable residues, on the basis of kinetic studies, it was concluded that the height of the unfolding free energy barrier for NAPase was less sensitive to acid than that of its neutrophilic counterpart (alphaLP). This attribute preserved the activity of NAPase under acidic conditions. Further assessment of the structural details of the two enzymes identified the relocation of multiple salt bridges (inherently present in the domain interface of alphaLP) to outer regions in NAPase. An acid-stable form of alphaLP (wherein a single interdomain salt bridge was replaced with a corresponding NAPase-like intradomain salt bridge) displayed an exceptional (>15-fold) increase in acid resistance. This study based on NAPase, as an example, has highlighted the significance of kinetic stability as an evolutionary means of overcoming extreme conditions. More recently, NprotI, the protease derived from N. dassonvillei NCIM 5124, was shown to display unusual features (Rohamare et al. 2013). Structural and functional transitions of NprotI in the presence of chaotropic agents,

Appl Microbiol Biotechnol (2014) 98:9173–9185

alcohols and proteases and at high temperatures were studied by employing a variety of biophysical and assay-based techniques. Circular dichroism spectra of the native protein, structural variations in presence of guanidine hydrochloride and a distinct isodichroic point under denaturing conditions indicated the presence of a distinctive polyproline II helix (PPII) in the protease. It must be noted that the polyproline II structure is encountered in proteins when sequential proline residues are present. This helix is mainly present in collagen and some segments or subunits of other proteins (RNA polymerase II, wheat glutenin, titin and dehydrins). As discussed above in the case of NAPase, kinetically stable proteins are not easily unfolded and resist denaturation by SDS and proteolytic digestion. The authors have discussed that the unusual stability of NprotI towards high concentrations of denaturing agents, organic solvents and proteolytic enzymes makes this enzyme an interesting candidate for structural investigations. The stability was attributed to the presence of PPII helix observed for the first time as a global conformation in a non-structural protein of microbial origin. The PPII fold of the enzyme was found to be more stable towards chemical denaturants and proteolytic enzymes than to physical denaturants like heat, which could be assigned to the fact that PPII helix does not have any internal hydrogen bonds for stabilization. Heat could as well be disrupting the other stabilizing non-covalent interactions. The chemical reagents on the other hand might be fulfilling the need of PPII helix for hydrogen bonding. This unusual stability of NProtI seems to be an example of a distinct attribute that nature has imparted on a Nocardiopsis species to enable its survival under harsh conditions. The molecular basis underlying the stability of serine protease derived from N. prasina has also been elucidated by analyzing 121 multiple point mutation-containing mutant enzyme clones (Farrell et al. 2012). Fast residual activity assays, a feature classification algorithm and structure-based energy calculation algorithms were used to understand the structural features. On the basis of multivariate regression analysis of the Table 3 Enzymatic abilities of some selected species of Nocardiopsis

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data obtained from such mutant clones, the significance of two residues (Asn 47 and Pro 124 situated in loop regions that confer stability to a highly homologous α-lytic protease) was understood. On the basis of the literature assessment on the subject and the detailed discussion in the previous sections, it is obvious that different species of Nocardiopsis are a potential source of diverse and novel enzymes. Among the few Nocardiopsis cultures that have been identified to the species level, it is evident that some produce more than one kind of novel enzymes as detailed in Table 3. Two strains of N. dassonvillei produce extracellular alkaline serine proteases, and one produces a thermotolerant xylanase. N. alba produces two types of proteases: an acid-stable keratinolytic enzyme and an alkaline protease. N. prasina strains are endowed with enzymes such as alkaline proteases and three types of chitinases. N. aegyptia strains produce two diverse novel enzymes, a cold-adapted α-amylase and PHB depolymerase. It is also seen that Nocardiopsis species producing a range of novel enzymes have been obtained from specific geographical locations. Particularly, such cultures have been isolated from South America (Argentina, Brazil), Asia (China, India, Japan and Korea), Africa (Egypt) and Antarctica. On the basis of their location of isolation, there is a difference in the features of the enzymes produced. For example, the isolate from Antarctica produces a cold-adapted α-amylase and those from tropical regions produce thermostable enzymes. The most prevalent feature shown by these enzymes is their alkali tolerance and thermostability. Cellulases, glucanases, chitinases and proteases are enzymes that show these features (Tables 1 and 2). The wide range of enzymes allows the organisms to survive under extreme environmental conditions. In some cases, the structural details responsible for the unusual stability of these enzymes have been elucidated. In conclusion, Nocardiopsis species with unique physiological properties have been isolated from diverse locales, and several research workers address issues related to enzymes

Strain

Enzymes

Reference

N. dassonvillei OPC-210

Alkaline serine proteases

Tsujibo et al. 1990b

N. dassonvillei NCIM 5124

Alkaline serine proteases

Dixit and Pant 2000a, b; Rohamare et al. 2013

N. dassonvillei subsp. alba OPC-18

Thermotolerant xylanases

Tsujibo et al. 1990a

N. alba

Acid-stable protease

Kelch et al. 2007

N. alba strain (OK-5) N. prasina

Alkaline protease Proteases

Gohel and Singh 2012 Farrell et al. 2012

N. prasina OPC-131

Chitinases

Tsujibo et al. 2003

N. prasina HA-4

Proteases

Ningthoujam et al. 2009

N. aegyptia

PHB depolymerase

Ghanem et al. 2005

N. aegyptia

Cold-adapted α-amylase

Abou-Elela et al. 2009

9182

associated with these isolates. The biochemical diversity of the genus is evident from the range of novel enzymes that it produces. The array of novel enzymes produced by this microbe may be responsible for its recurrent incidence in a range of habitats. The organism secretes carbohydrases such as amylases, cellulases and glucanases that act on glucosecontaining polymers. Inulinases and xylanases are the other carbohydrases obtained from this genus. Chitinases and PHB depolymerase, in turn, can have a wide range of biotechnologically relevant applications, and the variety of proteases it produces is commendable. Certain processes involving the members of this genus have the potential to be scaled-up, and patents on such methods have been granted. In recent years, several novel species have been reported from a variety of geographical locations, and investigations on the enzymatic capabilities of these isolates would allow the range of enzymes to be expanded in the years to come. Acknowledgments All authors thank the University Grants Commission for funding under UPE Phase II. TB thanks CSIR, India, for Junior Research Fellowship.

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