T.A. Sathya and Mahejibin Khan

Traditional use of enzymes for food processing and production of food ingredients resulted in fast-growing enzyme industries world over. The advances in technologies gave rise to exploring newer enzymes and/or modified enzymes for specific application. Search for novel enzymes that can augment catalytic efficiency and advances in molecular biology techniques including sequencing has targeted microbial diversity through metagenomic approaches for sourcing enzymes from difficult to culture organisms. Such mining studies have received more attention in characterizing hydrolases, their prevalence, broad substrate specificities, stability, and independence of cofactors. The focus on glycosyl hydrolases from metagenome for their application in food sector is reviewed.

Abstract:

Keywords: functional food, glycosyl hydrolase, metagenomics, oligosaccharides

Introduction Carbohydrates, the main constituent of food products, occur in a variety of forms. Most common and abundant form of carbohydrates in food are starches, depolymerized starch, sucrose, glucose, fructose, sorbitol, digestible raffinose, stachyose, pectin, cellulose, hemicelluloses, pentosans, fructans, chitin, and seaweed polysaccharides. Dietary fiber present in food and food products consist of cellulose, hemicellulose, pectic substances, gums, mucilages, and resistant oligosaccharides (Poli and others 2011). Of the above cellulose, the least complex carbohydrate, made of linear β-1,4-linked D-glucose residues, is used as a bulking agent in food due to its absorbing ability and low solubility. Other carbohydrates used as bulking agents in foods are hemicellulose, a heteropolysaccharide classified based on the main sugar present in the backbone, xylan (β-1,4-linked D-xylose), mannan (β-1,4linked D-mannose), or xyloglucan (β-1,4-linked D-glucose) and pectin made of α-1,4-linked D-galacturonic acid residues methylesterified or substituted with acetyl groups, occurring as homo- or heterogalacturon units. Rhamnogalacturonan (RG) I and II, the hairy regions of pectin, consist of repeating disaccharide 4-α-Dgalacturonic acid-(1,2) and -α-L-rhamnose carrying side chains of neutral sugars and homogalacturonans with complex side chains. Although food enzyme industry over world is growing fast due to the use of an array of enzymes for various food-processing applications (Table 1), available biocatalysts remain insufficient for meeting all the demands. Still various processes, such as extraction of carbohydrates from plant biomass (Maness 2010), conversion of complex polysaccharides into simple sugars, and synthesis of oligosaccharides depend on chemical pretreatments of plant biomass prior to enzyme treatment (Vazquez and others 2000; Akpinar and others 2009; Kumar and Wyman 2009). Furthermore, consumers’ concept of “preventive health” and growing demand for functional foods have resulted in the need for multifunctional biocatalysts with high catalytic efficiency and stability

for transformation of complex polysaccharide into bioactive compounds. In the search for new bioactive compounds from natural genetic biodiversity, metagenomics (Figure 1 shows the steps involved in construction and screening of metagenomic library) can offer the result since it makes use of existing knowledge of molecular biology, specifically mining for genes that code for enzymes from the large genetic stock available in the difficult to culture or unculturable microorganisms (Schloss and Handelsman 2003; Daniel 2004).

Glycosyl Hydrolase (GH) Enzyme Diversity The enzymes that hydrolyze glycosidic bonds between 2 or more sugars or a sugar and a nonsugar moiety within carbohydrates or oligosaccharide are known as GHs or glycosidases (Lehninger 2005). The enzymes of this family, widely distributed across prokaryotic, eukaryotic, and archaea (Henrissat 1991; Henrissat and Bairoch 1993; Cantarel and others 2009; http/CAZY.com), have interesting functional diversity and are variable in copy number among organisms. Based on their modes of actions and amino acid sequence, 115 GH families have been recognized. In the recent years, protein structure and function understanding have realized that classification of GH enzymes based only on substrate specificities is inappropriate, since the same protein fold often harbor several types of specificities. Therefore, Cantarel and others (2009) proposed a more appropriate classification scheme based on the consequence of protein folding dictated by the amino acid sequence. The 115 different families of GH provide insight into the comparative structural features within a family, their evolutionary relationships with other family members, and mechanisms of action.

α-Amylase Family

Amylolytic enzymes are among the most important enzymes in the food industry. As a large family of enzymes, they act on a wide range of substrates to give rise to diverse products inMS 20140632 Submitted 4/15/2014, Accepted 8/18/2014. Authors are with Academy of Scientific and Innovative Research, New Delhi, India and with CSIR- cluding dextrins and progressively smaller polymers composed of Central Food Technological Research Institute, Mysore-20, Karnataka, India. Direct glucose units. α-Amylases, the representative of the family, catalysis inquiries to author Khan (E-mail: [email protected]). the hydrolysis of starch and related oligosaccharides and polysaccharides at the α-1,4-glucosidic linkages sometimes causing

R  C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12677 Further reproduction without permission is prohibited

Vol. 79, Nr. 11, 2014 r Journal of Food Science R2149

R: Concise Reviews in Food Science

Diversity of Glycosyl Hydrolase Enzymes from Metagenome and Their Application in Food Industry

R: Concise Reviews in Food Science

Diversity of glycosyl hydrolase enzymes . . . Table 1–Applications of important GH enzymes in food industry. GH family

Enzymes

Function in food industry

GH-13

α-Amylases Pullulanase Cyclomaltodextrinase α-Glucosidase Oligo1,6glucosidase Isoamylase Maltohexaose-forming α-amylase Neopullulanase Sucrose phosphorylase Dextran glucosidase Amylosucrase Maltotetraose-forming α-amylase Glucoamylases Heat-stable α-amylases Maltogenic amylase 4-α-Glucanotransferase

Liquefaction of starch Saccharification of starch in baking and preparation of maltose and high fructose syrup Production of glucose syrup, high fructose corn syrup, and alcohol Conversion of starch to sugar syrups, and the production of cyclodextrins for the pharmaceutical industry In bakery industry, amylases act on damaged starch granules in dough and help in releasing free water molecule which softens the dough and makes it more mobile Bacterial α-amylase are used to retard staling of dough, it hydrolyses the amylopectin into smaller units, and maintains softness and extends shelf-life of dough

Degradation of cellulose into simple sugars Used in improving the malting of barley in beer and wine industry

GH-57

Cellulases Endoglucanase β-Mannanase Exo-1,3-glucanase Endo-1,6-glucanase Endoglycoceramidase Salt-tolerant cellulase Bacterial cellulases (subfamily E1)Nonbacterial cellulases (subfamily E2) Endo-β-1, 4-glucanase Chitinase Endoprocessivecellulase Cellobiohydrolase Glucoamylases

GH-17

Endoglucanase

GH-44 GH-11

Xyloglucanases Xylanases Endoxylanase Galactosidasesβ-Galactosidases

GH-5

GH-9 GH-48

GH-1 GH-28

Agarases GH-16 GH-50 GH-86 GH-96

Pectinases Endoglycosidases Exoglycosidases Thermostable pectinaseLow temperatures active PGs

Used in animal feed to improve the digestibility of cereal-based feed and nutritive quality of forages Used for the production of food nectars, purees, extraction of olive oil, fruit and vegetable juices, carotenoid used as food-coloring agents Production of glucose syrup, high fructosecorn syrup, and in whole grain and starch hydrolysis for alcohol production Hydrolyze longer cellooligosaccharides faster than shorter cellooligosaccharides Domestication of food crops Fiber degradation and the synthesis of xylooligosaccharides Production of low-lactose milk and production of galacto-oligosaccharides Clarification of fruit juicesSynthesis of pectic oligosaccharides Retention of flavor and color components in wine making

α-Agarases

Used as low-calorie additives to improve qualities of food

β-Agarases

Important food additive

transglycosylation reactions. Widely distributed throughout species of animals, plants, and microorganisms, they are a substantial portion of all glycoside hydrolases and in CAZY, the enzyme has been classified in GH family 13. Based on similarities and differences in their primary structure and substrate specificity, some amylolytic enzymes have been clustered into 14, 15, and 31 family of CAZY enzymes. In the GH family 57 (Henrissat and Bairoch 1996), availability of a large number of new sequences resulted in the division of the family into 40 subfamilies to bring into fold α-amylase, pullulanase, cyclo malto dextrinase, α-glucosidase, oligo-1,6-glucosidase, isoamylase, malto hexaoseforming α-amylase, neopullulanase, sucrose phosphorylase, dextran glucosidase, amylosucrase, maltotetraose-forming α-amylase, and so on. Amylases are predominantly used in food industry for liquefaction of starch, its saccharification, and for the preparation of

R2150 Journal of Food Science r Vol. 79, Nr. 11, 2014

maltose and high fructose syrup (Ravi-Kumar and Umesh-Kumar 2006). Many studies have reported the use of α-amylase in baking to improve the texture, flavor, aroma, general qualities, and to reduce the rates and degrees of firming in baked food. Amylases act on damaged starch granules in dough and help in releasing free water molecule that softens the dough. Bacterial α-amylase are used to retard staling of dough, it hydrolyses the amylopectin into smaller units, maintains softness, and extends shelf-life of dough. According to Duran and others (2001) and Martin and Hoseney (1991), the antistaling effect of amylase was due to the production of malto oligosaccharides that inhibited the cross-linkages between starch and gluten. Akers and Hoseney (1994) found that source of amylsase also affect the rate and degree of crumb firming. Considerable reports have been published contrasting the role of amylase as antistaling agent in bakery (Gray and Bemiller 2003).

Many studies focused on the discovery of new amylases originating from bacteria, and more recently from metagenome. These investigations allow the detection of novel amylases with interesting and unique characteristics, such as multifunctional amylases, those active in low temperatures, high temperature, high salt concentration, and so on. To isolate useful microbial enzymes from uncultured soil microorganisms, Yun and others (2004) constructed and screened a metagenomic library for amylase activity. One clone pSA4, from among approximately 30000 recombinant Escherichia coli clones, showed amylase activity. Further studies revealed that the pS2A4-derived AmyM amylase was a multifunctional enzyme that exhibited properties of several different amylases such as α-amylase, maltogenic amylase (or neo pullulanase), and 4-α-glucanotransferase belonging to the GH 13 family. The unique property of Amy M amylase was due to the presence of transglycosylation activity. Therefore, it was considered an intermediate to the maltogenic amylase, α-amylase, and α-4-glucanotransferase.

Cyclodextrin and maltodetrin degrading enzyme cda I 3A, and thermophillic pullulan hydrolyzing enzyme Nph 193A, isolated from Thailand hot spring sediment metagenome were optimally active at temperature 55 and 75 °C at pH 6 to7 (Tang and others 2008). In another study, Labes and others (2008) enriched the hot spring water with 0.1% starch for the isolation of cyclodextrinase and neopullulanase metagenomes using GH 13 consensus primers and expressed in E. coli. Sharma and others (2010) screened 909 bp amylase encoding gene (pAMY) related to uncultured bacteria from a soil metagenomic library. The enzyme used soluble starch, amylose, glycogen, and maltose as substrates and retained 90% of its activity at low temperature with all the substrates. Wang and others (2011), Ballschmiter and others (2006), and Zhang and Zeng (2011) reported on amylases from many different extreme environments. In an effort to understand the mechanism of action of GH 57 amylase family, in relation to structure, Blesak and Janecek (2013) studied 2 partially characterized GH 57 nonspecified amylases from an uncultured bacterium by bioinformatics

Metagenomic Library creation

Environmental Samples

DNA isolation

DNA Fragmentation

Ligation with vector

Metagenomic library Transformation into host

Screening

Screening of clones (selective plate, indicator plate etc)

Sequence

Identification of new genes/ sequences

Comparative biodiversity analysis

Identification of metabolic pathways

Expression of genes

Sequencing of clones of interest Enzyme purification

Sequence deposition in database

Novel biotechnological applications

Figure 1–Schematic representation of construction and screening of metagenomic library from different environmental sample. Vol. 79, Nr. 11, 2014 r Journal of Food Science R2151

R: Concise Reviews in Food Science

Diversity of glycosyl hydrolase enzymes . . .

R: Concise Reviews in Food Science

Diversity of glycosyl hydrolase enzymes . . . analysis. Though the study found the enzyme hydrolyzing soluble starch, specificities and the reaction products could not be determined. A unique enzyme RA 04, belonging to α-amylase family, was cloned and expressed from rumen fluid metagenomic DNA. The enzyme hydrolyzed α-D-(1,4) bonds 13-fold faster than α-D(1,6) bonds to yield maltose and glucose as products. It also exhibited transglycosylation activity. Amino acid sequence similarity and biochemical properties characterized it as cyclomaltodextrinase, However, structural analysis revealed unusual characteristics such as, presence of shorter N-terminal domain and absence of well-conserved N-terminal Trp reported in other wellcharacterized cyclomaltodextrinase (Ferrer and others 2005, 2007). The findings of Delavat and others (2012) on the 2 genes from acid mine drainage shed more light on the application of unconventional methods for bioprospecting gene diversity from incongruous environments. Even though expression and purification of cloned protein of these genes confirmed amylolytic activity by degrading AZCL-amylose and starch, exhibiting endoacting amylase activity in PACE experiments, the genes did not exhibit sequence similarity with known amylase or glycoside hydrolase, nor possessing any known enzyme domains.

Cellulases Cellulases are referred to as a group of complex enzymes made of endo-β-1, 4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. The enzymes work together to degrade cellulose into simple sugars. Most of the cellulases are produced by wide range of bacteria, fungi, and protozoa. However, termites and their intestinal microbial symbionts serve as potential sources for cellulase. Based on their catalytic mechanisms, cellulases have been classified into GH family 5, 9, 17, and 48. GH family 5 comprised of endoglucanase, β-mannanase, exo-1,3-glucanase, endo-1,6-glucanase, xylanase, and endoglycolceramidase. All these enzymes possessed conserved glutamic acid residue that was potentially involved in the catalytic mechanism (Py and others 1991). GH 9, the 2nd largest cellulase family was divided into 2 subgroups, E1 and E 11. While the group E1 contained only aerobic and anaerobic bacterial cellulases, E 11 included those cellulases from nonbacterial and bacterial origin (Tomme and others 1995). The GH 48 cellulase included endo-β-1, 4-glucanase, chitinase, endoprocessive cellulose, and cellobiohydrolase. Most of the GH 48 cellulases preferred amorphous or crystalline cellulose as substrate over carboxymethylcellulose (CMC) and cellobiose negatively affected the activity. A combination of cellulases, hemicellulases, and pectinases, collectively known as macerating enzymes, is widely used in food industry for the production of food nectars, purees, extraction of olive oil, fruit and vegetable juices, carotenoids used as food coloring agents (Grassin and Fauquembergue 1996; Cinar 2005; Venkatesh and Umesh-Kumar 2006; de Carvalho and others 2008). Glucanases are used for improving the malting of barley in beer and wine industry (Bamforth 2009). Cellulases are also used in animal feed to improve digestibility of cereal-based feed and nutritive quality of forages (Himmel and others 1999; Dhiman and others 2002). Despite many reports characterizing cellulases from various sources (Feng and others 2007), growing demand for newer enzymes suggest the necessity to find a wide range of cellulases with varying stability, substrate affinity, and activity at a range of pH and temperature. Pottkamper and others (2009) identified new cellulose active clones, from metagenomic library, stable in presence of R2152 Journal of Food Science r Vol. 79, Nr. 11, 2014

1-butyl-1-methyl-pyrrolidinium trifluro methane sulfonate. The genes belonged to GH 5 family and showed similarity with known cellulases of Vibrio japonicas and an unknown salt-tolerant microorganism. Further analysis of the amino acid sequence of the clones revealed that resistance in ionic liquids (ILs) resided in the N-terminal cellulose-binding domain. To discover new cellulase enzyme with better adaptability in ILs, Ilmberger and others (2012) constructed metagenomic libraries from 3 hydrolytic communities and screened 2 halo- and thermotolerant cellulases with IL resistance. These results also suggested relatedness of IL tolerance with thermophilicity and halotolerance of the cellulase. Recent studies on biogas digester metagenomic library identified 341, 246, and 386 endo-β-1,4-glucanase, β-glucosidase, and endo-β1,4-xylanase positive clones, and 9 GH genes were expressed and purified to characterize activities with 4 kinds of substrates (Yan and others 2013). Berlemont and others (2011) screened a metagenomic library PP1 obtained from Antarctic soil samples and found 14 amylase, 3 proteases, and 11 cellulase producing clones with apparent maximum activities around 35 °C. Screening metagenomic library from a biogas digester fed with pig manure and rice straw for unique property to enhance the efficiency of Tricoderma reesei cellulase identified a novel cellulase gene exo 2b from a fosmid clone (Geng and others 2012). The protein showed high specific activity toward both carboxy methyl cellulose (260 U/mg proteins) and β-D-glucan from barley (849 U/mg). The enzyme was stable at a wide pH range (5.5 to 9.0) and was thermostable at 60 °C in the presence of 60 mM cysteine. Voget and others (2006) overexpressed and characterized a halotolerant endoglucanse from soil metagenome. In presence of various salts, the enzyme was stable at 40 °C for up to 11 d and displayed activity at pH 5.5 and 9.0. Endoglucanase belonging to GH family 17 that initiated cellulose degradation pathway (Tomme and others 1995) primarily contained endo-1, 4-glucanases (Ballschmiter and others 2006). A gene (designated as cen 219) was isolated from Bursaphelenchus xylophilus metagenomic library. Sequence analysis revealed that cen 219 encoded a 367 amino acids endogluconase enzyme. This enzyme hydrolyzed a wide range of β-1,3-, and β-1,4-linked polysaccharides, with varying activities. The optimum temperature and pH were 50 °C and 6.0, respectively. The enzyme was also stable from 30 to 50 °C and from pH 4.0 to 7.0 (Zhang and others 2013a). Functional screening of large-size mangrove soil metagenomic library identified an endoglucanase gene mgce l44. The gene encoded a 648-aa protein with a catalytic domain of GH 44 family was characterized as an organic solvent and salt-tolerant enzyme with a potential for industrial applications (Mai and others 2014). Ruminant gut microbial diversity that include bacteria, fungi, archaea, and protozoa produce a wide array of enzymes that have important roles in the efficient degradation of plant biomass and detoxification of secondary compounds. Limitations in microbial culture techniques that affected studies concerning ruminant microbial diversity have largely been offset recently, by cultureindependent methods. Metagenome analysis of the gut microbiomes of the wood-degrading higher termites (Warnecke and others 2007; Pope and others 2010) and cow rumen metagenome (Brulc and others 2009; Hess and others 2011) have analyzed the mechanisms of cellulose degradation in uncultured organisms and microbial communities. Duan and others (2009) screened ruminant fosmids libraries of buffalo and identified 61 clones expressing cellulase activities. Protein structure analysis revealed that of

Xylanases are of interest to bakery, ramie fiber degumming, all cellulases, 14 belonged to GH family 5. The data predicting dominance of GH 5 family of cellulose (Feng and others 2007; feed additive, and poultry industries (Paloheimo and others 2010). Warnecke and others 2007) also supported the previously pub- They are also used to improve nutritional properties of agricultural silage (Khandeparkar and Bhosle 2007; Menon and others lished metagenomic data of Voget and others (2006). 2010; Woldesenbet and others 2012). In bread making, xylanases improve handling properties, stability of the dough and elasticXylanases Xylans, made of β-1,4 linked xylopyranoses as linear back- ity of the gluten network, crumb structure and bread volume bone with branches, constitute the 2nd most important group (Olse 1995; Butt and others 2008). By converting hemicellulose of polysaccharides in plant cell walls. Based on their structure into small water-soluble oligosaccharide that binds water in the and functions, xylanases have been classified into 6 GH families, dough, they decrease dough firmness, making crumbs more uniGH 5, GH 8, GH 10, GH 11, GH 30, and GH 43 in CAZY form (Wang and others 2004a; Monfort and others 2007). Although several xylanases have been reported from diverse midatabase (Collins and others 2005). Among these, family 10 and 11 xylanases are abundantly available and widely used in various crobiota (Sunna and others 1997; Kulkarni and others 1999; Sunna industrial applications. Most of the high molecular weight xy- and Bergquist 2003; Sharma and others 2007) and are being used lanases with versatile substrate specificity are included in GH 10 effectively in various industries, new xylanases with better catfamily, while GH 11 xylanases are considered as true xylanases be- alytic activity for a specified conditions are still in demand for cause of their high substrate specificity (Henrissat and others 1991; feed industries. Several authors have described xylanase clones Paes and others 2012). Some of the enzyme sequences classified from metagenomic libraries of soil (Hu and others 2008), termites in families 16, 52, and 62 also processes xylalolytic activities but (Warnecke and others 2007), and rice straw degrading enrichdue to the presence of 2 catalytic domains they are not considered ment culture (Ziemer 2013) and cow manure (Cheng and others 2012). A clone that produced an alkali and thermostable xylanases as true xylanases.

Construction of forest soil metagenomic library

Confirmation of positive clones

Screening of clones on various substrates for GH enzymes

• • •

Isolation of soil metagenomic DNA DNA digestion with Sau3AI. Ligation with pJAZZ vector and transformation into E.coil JM109

• •

Plasmid isolation Restriction digestion

Enzyme

Substrate used

α- Amylase

Starch

Cellulase

Carboxy methyl cellulose,

Xylanase

4-O-methyl-D-glucurono-D-xylanremazol brilliant blue R

Pectinase

Polygalacturonic acid

Agarase

Agar

Sequencing of positive clones & Insilico analysis

• • • •

Phylogenetic analysis Homology search Conserved domain analysis Function prediction

Sub-cloning of positive clone into expression vector

• • •

Amplification of gene by specific primers Ligation with QIAexpress vector Transformation into E.coli

Purification of enzyme and biochemical characterization

• • • •

Figure 2–Flow chart of cloning and screening of various GH enzyme from forest soil metagenome (Sathya and others 2014).

Ammonium sulphate precipitation Gel filtration chromatography Temperature, pH optimization Substrate specificity

Vol. 79, Nr. 11, 2014 r Journal of Food Science R2153

R: Concise Reviews in Food Science

Diversity of glycosyl hydrolase enzymes . . .

R: Concise Reviews in Food Science

Diversity of glycosyl hydrolase enzymes . . . Table 2–List of glycosyl hydrolase used in food industry identified through metagenomics. Enzymes from metagenomes

Glycosyl hydrolase (GH) families

Source

Reference

α-Amylases α-Amylases α-Amylases α-Amylases α-Amylases α-Amylases Heat-stable α-amylases Cellulases Cellulases Cellulases Cellulases Cellulases Endoglucanases Endoglucanases Endoglucanases Endoglucanases

GH-13 GH-13 GH-13 GH-13 GH-57 GH-57 GH-13

Soil Sediments of hot spring (Thailand) Hot spring soil Soil Rumen fluid Acid mine drainage Antarctic soil

Yun and others (2004) Tang and others (2008) Labes and others (2008) Sharma and others (2010) Blesak and Janecek (2013) Delavat and others (2012) Berlemont and others (2011)

GH-5 GH-5 GH-5 GH-5 GH-5 GH-17 GH-17 GH-17 GH-17

Pottkamper and others (2009) Ilmberger and others (2012) Yan and others (2013) Berlemont and others (2011) Geng and others (2012) Voget and others (2006) Zhang and others (2013a) Mai and others (2014) Pope and others (2010); Warnecke and others (2007)

Endoglucanases Endoglucanases Xylanases Xylanases Xylanases Xylanases Xylanases Xylanases Xylanases Xylanases

GH-17 GH-17 GH-11 GH-11 GH-11 GH-11 GH-11 GH-11 GH-11 GH-11

Soil Hydrolytic community Biogas digester Antarctic soil samples Biogas digester fed with pig manure and rice straw Soil Bursaphelenchus xylophilus Mangrove soil Gut microbiomes of the wood-degrading higher termites Cow rumen Buffalo rumen Soil Termites Rice straw degrading enrichment culture Holstein cattle rumen Compost soil Agroresidues Holstein cattle rumen Agricultural silage

β-Galactosidases β-Galactosidases β-Galactosidases Pectinases Pectinases Agarases

GH-1 GH-1 GH-1 GH-28 GH-28 GH-16

Soil Marine Soil samples of Turpan Basin Forest soil Soil Soil from unplanted field

Brulc and others ( 2009); Hess and others (2011) Duan and others (2009) Hu and others (2008) Warnecke and others (2007) Ziemer (2013) Cheng and others (2012) Son-Ng and others (2009) Verma and Satyanarayana (2012) Cheng and others (2012) Khandeparkar and Bhosle (2007); Menon and others (2010); Woldesenbet and others (2012) Wang and others (2010) Wierzbicka-Wos and others (2013) Zhang and others (2013b) Sathya and others (2014) Singh and others (2012) Voget and others (2013)

was identified from a metagenomic library made from DNA extracted of compost-soil samples (Son-Ng and others 2009). The Amino acid homology and hydrophobic cluster analysis categorized this high molecular weight xylanase to GH 11 family. This was based on an earlier study (Sakka and others 1993) classifying a high molecular mass xylanase of Clostridium stercorium to this family. From a rumen metagenomic library, Wang and others (2012) characterized a xylanase clone, HF94-B02, that encoded endoxylanase of the GH family 11 with a specific activity of 11500 U/mg. In another study, Verma and Satyanarayana (2012) obtained a highly alkali stable (pH 9.0) and thermostable (80 °C) xylanase from environmental samples by metagenomic approach. Cheng and others (2012) reportedly expressed a xylanase Xyln-SH1 from a Holstein cattle rumen metagenomic library. The enzyme was strictly specific to xylan from softwood and released xylooligosaccharides and ferulic acid from wheat straw. This activity suggested that Xyln-SH1 has prebiotic potential in food industry for the production of xylo oligosaccharides (Vazquez and others 2000).

market, their applications are limited due to low thermostability and product inhibition. Recently, a glycoside hydrolase family1 gene, designated as bgl MKg, was isolated through activity based screening of marine metagenomic library. The recombinant bgl MKg expressed in E. coli reportedly demonstrated high β-galactosidase, β-glucosidase, and β-fucosidase activities. The cold adaptation of the enzyme catalyzed a reverse transglycosylation reaction (Wierzbicka-Wos and others 2013). Another βgalactosidase gene zd410 isolated by screening a soil metagenomic library was regarded as a cold-adapted due to its optimal temperature of 38 °C and 54% residual activity at 20 °C (Wang and others 2010). Wide specificity and activity spectrum of β-galactosidases from different metagenomes make these enzymes potentially interesting for industrial sectors. Zhang and others (2013b) isolated a thermostable β-galactosidase (Gal308) from soil samples of Turpan Basin in China. The enzyme showed several novel enzymatic properties, such as high thermostability and tolerance to galactose and glucose. These properties make Gal 308 an obvious alternative to the existing galactose-sensitive commercial β-galactosidases.

β-Galactosidases

Pectinases

β-galactosidase belongs to GH family 42 and catalyzes the hydrolysis of the glycosidic bond in β-galactosides. It is mainly used in food industry for the production of galacto-oligosaccharides from lactose and for low-lactose milk and dairy products for lactose intolerant people. Although β-galactosidases are available in

R2154 Journal of Food Science r Vol. 79, Nr. 11, 2014

Pectin degrading enzymes belong to the family GH28 of the general classification system of glycoside hydrolases. This family comprises several enzymes that assist in pectin degradation and cleave different linkages in pectin. Polygalacturonase (PGase) break the GalA-GalA bond, rhamno galacturonase (RGase) hydrolyses

GalA-rhamnose linkage, and xylogalacturonase (XGase) digests bonds between Gal A and xylose. On the basis of location of the digested bond, PGase has been categorized as exo and endo (Khan and others 2013). Pectinases have been used for decades in the food and wine making industry for the processing of fruit juices. These are used to reduce the viscosity, to soften the peel of citrus fruits, and to increase fruit juice volume from banana, grapes, and apples (Baker and wicker 1996; Kaur and others 2004). A cocktail of pectinases with cellulase, arabinose, and xylanase is used to increase the pressing efficiency of the fruits for juice extraction (Gailing and others 2000). A relatively new application envisaged for pectinases is the production of functional oligosaccharide, which exhibits important physiological and biological activities beneficial to the human health (Khan and others 2013). Singh and others (2012) isolated a gene encoding for thermostable pectinase from soil metagenome. The enzyme had temperature and pH optima of 70 °C and 7.0, respectively and was also active over a broad temperature and pH range. In order to study the diversity of polysaccharide degrading enzyme, Sathya and others (2014) constructed a forest soil metagenomic library and found 9 pectinolytic positive clones by functional screening (Figure 2). Further characterization of pectin degrading gene (pg_4) revealed that it hydrolyzed both apple and citrus pectin and retained more that 80% activity at pH 5 to 9 and temperature 20 to 60 °C.

Agarases Agar is one of the important food additives. It comprises alternating residues of [O-3,6-α-anhydro-L-galacto pyranosyl (1,3) O-β-D-galacto pyranose] linked by β-1,4 bonds. Enzymes that liquefy agar by cleaving either the polymer’s α-L-(1,3) linkage or its β-D-(1,4) linkage are known as agarase. While α-agarase hydrolyzes α-1,3 of agarose, β-type hydrolyzes its β-1,4 linkages. Based on their amino acid sequence and catalytic domain α-agarases have been classified into GH family, GH 16, GH 50, and GH 86, and β-type into GH 96 family. Agarase belonging to the family GH 16 are the most abundant and yields neo agaro tetraose (DP4). Family GH 50 and GH 86 agarase end products are neoagaro biose (DP2), neo agaro tetraose (DP4), neo agaro octaose (DP8), and DP6 which are neoagaro hexaoses (Fu and Kim 2010; Liao and others 2011). Agarases are known to have wide applications in food as well as cosmetics industries. The oligosaccharides synthesized from agar hydrolysis has shown good antioxidative activities, inhibit lipid peroxidation (Wang and others 2004b), reduce bacterial growth, slow down the degradation of starch, and used as low-calorie additives to improve food qualities (Giordano and others 2006).Most of the agarases have been purified and characterized from seawater, marine sediments, marine algae, and marine molluscs. Recently few reports are also available for the isolation of agarase enzyme form metagenome. Voget and others (2013) discovered 6 agarase genes in a soil metagenome library from unplanted field. It yielded 2 clones with pectate lyase activity, and 1 clone with α 1,4-glucan branching enzyme activity. Table 2 lists the recently identified GH enzymes used in food industry through metagenomics.

Conclusions Though microorganisms in nature have the capacity to degrade crystalline polysaccharides, the metagenomic approach has not explained characterization of such enzymes that would benefit food industries tremendously. A focus toward the understanding of the

3-dimensional enzyme structures that the microbial diversity can provide, in relation to native plant polysaccharide, is important from the point of view of application. Hence, metagenome screening procedure should venture toward sophisticated biochemical analysis determining enzyme scaffolding of crystalline polysaccharides for glycosidic bond catalytic activities. Relating specificities of hydrolases to crystal packing substrate structures through bioinformatics approach will pave way for choosing the right enzyme for an envisaged product.

References Akers AA, Hoseney RC. 1994. Water-soluble dextrins from a-amylase-treated bread and their relationship to bread firming. Cereal Chem 71:223–6. Akpinar O, Erdogan K, Bostanci S. 2009. Production of xylooligosaccharides by controlled acid hydrolysis of lignocellulosic materials. Carbohyd Res 344(5):660–6. Baker RA, Wicker L. 1996.Current and potential application of enzyme infusion in the food Industry. Trends Food Sci Technol 7:279–84. Ballschmiter M, Futterer O, Liebl W. 2006. Identification and characterization of a novel intracellular alkaline a-amylase from the hyperthermophilic bacterium Thermotoga maritime MSB8. Appl Environ Microbiol 72:2206–11. Bamforth CW. 2009. Current perspectives on the role of enzymes in brewing. J Cereal Sci 50(3):353–7. Berlemont R, Pipers D, Delsaute M, Angiono F, Feller G, Galleni M, Power P. 2011. Exploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements. Rev Argent Microbiol 43(2):94–103. Blesak K, Janecek S.2013. Two potentially novel amylolytic enzyme specificities in the prokaryotic glycoside hydrolase α-amylase family GH57. Microbiology 159:2584–93. Brulc JM, Antonopoulos DA, Berg Miller ME, Wilson MK, Yannarell AC, Dinsdale EA, Edwards RE, Frank ED, Emerson JB, Wacklin P, Coutinho PM, Henrissat B, Nelson KE, White BA. 2009. Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases. Proc Natl Acad Sci USA 106:1948–53. Butt MS, Tahir-Nadeem M, Ahmad Z, Sultan MT. 2008.Xylanases and their applications in baking industry. Food Technol Biotechnol 46(1):22–31. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. 2009. The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37(Database issue):D233–8. Cheng F, Sheng J, Dong R, Men Y, Gan L, Shen L. 2012. Novel xylanase from a holstein cattle rumen metagenomic library and its application in xylooligosaccharide and ferulic acid production from wheat straw. J Agric Food Chem 60(51):12516–24. Cinar I. 2005. Effects of cellulase and pectinase concentrationson the colour yield of enzyme extracted plant carotenoids. Proc Biochem 40:945–9. Collins T, Gerday C, Feller G. 2005. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 29:3–23. Daniel R. 2004. The soil metagenome—a rich resource for the discovery of novel natural products. Curr Opin Biotechnol 15:199–204. de Carvalho LMJ, de Castro IM, da Silva CAB. 2008. A study of retention of sugars in the process of clarification of pineapple juice (Ananas comosus, L. Merril) bymicro- and ultrafiltration. J Food Engr 78(4):447–54. Delavat F, Lett MC, Li`evremont D. 2012. Novel and unexpected bacterial diversity in an arsenic rich ecosystem revealed by culture-dependent approaches. Biol Direct 7:28. Dhiman TR, Zaman MS, Gimenez RR,Walters JL, Treacher R. 2002. Performance of dairy cows fed forage treated with fibrolytic enzymes prior to feeding. Anim Feed Sci Tech 101:115– 25. Duan CJ, Xian L, Zhao GC, Feng Y, Pang H, Bai XL, Tang JL, Ma QS, Feng JX. 2009. Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens. J Appl Microbiol 107:245–56. Duran E, Leon A, Barber B, Benedito De Barber C. 2001. Effect of low molecular weight dextrins on gelatinization and retrogradation of starch. Eur. Food Res Technol 212: 203–7. Feng Y, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, Hu YL, Wei J, Tang JL, Feng JX. 2007. Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl Microbiol Biotechnol 75:319–28. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-Duarte D, Santos VA, Strompl C, Elborough K, Jarvis G, Neef A, Yakimov MM, Timmis KN, Golyshin PN. 2005. Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environ Microbiol 7(12):1996–2010. Ferrer M, Beloqui A, Golyshina OV, Plou FJ, Neef A, Chernikova TN, Fern´andez-Arrojo L, Ghazi I, Ballesteros A, Elborough K, Timmis KN, Golyshin PN. 2007. Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome. Biotechnol J 2:207–13. Fu XT, Kim SM. 2010. Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Mar Drugs 8:200–18. Gailing MF, Guibert A, Combes D. 2000. Fractional factorial designs applied to enzymatic sugar beet pulps pressing improvement. Biopro Engr 22:69–74. Geng A, Zou G, Yan X, Wang Q, Zhang J, Liu F, Zhu B, Zhou Z. 2012. Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei. Appl Microbiol Biotechnol 96:951–62. Giordano A, Andreotti G, Tramice A, Trincone A .2006. Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides. Biotechnol J 1:511–30. Grassin C, Fauquembergue P. 1996. Fruit juices. In: Godfrey T, West S, editors. Industrial enzymology. 2nd ed. London, UK: MacMillan Press. p 226–64. Gray JA, BeMiller JN. 2003. Bread staling: molecular basis and control. Compr Rev Food Sci F 2:1–21. Henrissat B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–16.

Vol. 79, Nr. 11, 2014 r Journal of Food Science R2155

R: Concise Reviews in Food Science

Diversity of glycosyl hydrolase enzymes . . .

R: Concise Reviews in Food Science

Diversity of glycosyl hydrolase enzymes . . . Henrissat B, Bairoch A. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293:781–8. Henrissat B, Bairoch A.1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316:695–6. Hess M, Sczyrba A, Egan R, Kim TW, Chokhawala H, Schroth G, Luo S, Clark DS, Chen F, Zhang T, Mackie RI, Pennacchio LA, Tringe SG, Visel A, Woyke T, Wang Z, Rubin EM. 2011. Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Science 331:463–7. Himmel ME, Ruth MF, Wyman CE. 1999. Cellulase for commodity products from cellulosic biomass. Curr Opin Biotechnol 10:358–64. Hu Y, Zhang G, Li A, Chen J, Ma L. 2008. Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach. Appl Microbiol Biotechnol 80:823–30. Ilmberger N, Meske D, Juergensen J, Schulte M, Barthen P, Rabausch U, Angelov A, Mientus M, Liebl W, Schmitz RA, Streit WR. 2012. Metagenomic cellulases highly tolerant towards the presence of ionic liquids—linking thermostability and halotolerance. Appl Microbiol Biotechnol 95:135–46. Kaur G, Kumar S, Satyanarayana T. 2004. Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould sporotrichum thermophile Apinis. Bio Resour Technol 94:239–43. Khan M, Nakkeeran E, Umesh-Kumar S. 2013. Potential application of pectinase in developing functional foods. Ann Rev Fd Sci Technol 4:21–34. Khandeparkar R, Bhosle NB. 2007. Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp. Biores Technol 98:897–903. Kulkarni N, Shendye A, Rao M. 1999. Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23:411–56. Kumar R, Wyman CE. 2009. Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies. Biotechnol Prog 25(3):807–19. Labes A, Karlsson EN, Fridjonsson OH, Turner P, Hreggvidson GO, Kristjansson JK, Holst O, Schonheit P. 2008. Novel members of glycoside hydrolase family 13 derived from environmental DNA. Appl Environ Microbiol 74(6):1914–21. Lehninger AL. 2005. Carbohydrates and glycobiology. In: Lehninger AL, Nelson DL, Cox MM, editors. Lehninger principles of biochemistry, 4th ed. New York, NY: Macmillan. p 305–25. Liao L, Xu XW, Jiang XW, Cao Y, Yi N, Huo YY, Wu YH, Zhu XF, Zhang Xq, Wu M. 2011. Cloning, expression, and characterization of a new—agarase from vibrio sp. strain CN41. Appl Environ Microbiol 17:7077–9. Mai Z, Su H, Yang J, Huang S, Zhang S. 2014. Cloning and characterization of novel GH 44 family endoglucanase from mangrove soil metagenomic library. Biotechnol Lett 36(8):1701–9. Maness N. 2010. Extraction and analysis of soluble carbohydrates. Methods Mol Biol 639:341– 70. Martin ML, Hoseney RC. 1991. A mechanism of bread firming. II. Role of starch hydrolyzing enzymes. Cereal Chem 68:503–7. Menon V, Prakash G, Prabhune A, Rao M. 2010. Biocatalytic approach for the utilization of hemicellulose for ethanol production from agricultural residue using thermostable xylanase and thermotolerant yeast. Bio Res Technol 101:5366–73. Monfort A, Blasco A, Prieto JA, Sanz P. 1997. Construction of baker’s yeast strains that secrete different xylanolytic enzymes and their use in bread making. J Cereal Sci 26:195–9. Olse HS. 1995. Use of enzymes in food processing. In: Reed G, Nagodawithana T, editors. Enzymes, biomass, food and feed biotechnology. Weinheim, Germany: Wiley-VCH. p 663– 736. Paes G, Berrin JG, Beaugrand J. 2012. GH11 xylanases: structure/function/properties relationships and applications. Biotechnol Adv 30:564–92. Paloheimo M, Piironen J, Vehmaanpera J. 2010. Xylanases and cellulases as feed additives. In: Bedford MR, Partridge GG, editors. Enzymes in farm animal nutrition. United Kingdom: CAB Intl. p 1–12. Poli A, Anzelmo G, Fiorentino G, Nicolaus B, Tommonaro G, Donato PD. 2011. Polysaccharides from wastes of vegetable industrial processing: New opportunities for their eco-friendly re-use. In: Elnashar M, editor. Biotechnology of biopolymers. Europe: InTech Publisher. p 33–56. Pope PB, Denman SE, Jones M, Tringe SG, Barry K, Malfatti SA, McHardy AC, Cheng JF, Hugenholtz P, McSweeney CS, Morrison M. 2010. Adaptation to herbivore by the Tammar Walla by includes bacterial and glycoside hydrolases profiles different to other herbivores. Proc Natl Acad Sci 107(33):14793–8. Pottkamper J, Barthen P, Ilmberger N, Schwaneberg U, Schenk A, Schulte M, Ignatiev N, Streit WR. 2009. Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids. Green Chem 11:957–65. Py B, Bortoli-German I, Haiech J, Chippaux M, Barras F. 1991. Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis. Protein Engr 4(3):325–33. Ravi-Kumar K, Umesh-Kumar S. 2006. Bioprocessing of starch using enzyme technology. In: Shetty K, Paliyath G, Pometto A, Levin RE, editors. Food biotechnology. Boca Raton, Fla.: Taylor and Francis Group. p 709–20. Sakka K, Kojima Y, Kondo T, Karita S, Ohmiya K. 1993. Nucleotide sequence of Clostridium stercorariumxyn a gene encoding xylanase a: identification of catalytic and cellulose binding domains. Biosci Biotechnol Biochem 57:273–7. Sathya TA, Jacob AM, Khan M. 2014. Cloning and molecular modeling of pectin degrading glycosyl hydrolase of family 28 from soil metagenomic library. Mol Biol Rep 41:2645–56. Schloss PD, Handelsman J. 2003. Biotechnological prospects from metagenomics. Curr Opin Biotechnol 14:303–10

R2156 Journal of Food Science r Vol. 79, Nr. 11, 2014

Sharma S, Adhikari S, Satyanarayana T. 2007. Alkali-thermostable and cellulose free xylanase production by an extreme thermophile Geobacillus thermoleovorans. W J Microbiol Biotechnol 23:483–90. Sharma S, Khan FG, Qazi GN. 2010. Molecular cloning and characterization of amylase from soil metagenomic library derived from north western Himalayas. Appl Microbiol Biotechnol 86(6):1821–8. Singh R, Dhawan S, Singh K, Kaur J. 2012. Cloning, expression and characterization of a metagenome derived thermoactive/thermostable pectinase. Mol Biol Rep 39(8): 8353–61. Son-Ng I, Li CW, Yeh Y, Chen PT, Chir JL. 2009. A novel endoglucanase from the thermophilic bacterium Geobacillus sp. 70PC53 with high activity and stability over a broad range of temperatures. Extremophiles 13:425–35. Sunna A, Bergquist PL .2003. A gene encoding a novel extremely thermostable 1, 4-b- xylanase isolated directly from an environmental DNA sample. Extremophiles 7:63–70. Sunna A, Moracci M, Rossi M, Antranikian G .1997. Glycosyl hydrolases from hyperthermophiles. Extremophiles 1:2–13. Tang H, Bowers J, Wang X, Ming R, Alam M, Paterson A. 2008. Synteny and collinearity in plant genomes. Science 320:486–8. Tomme P, Warren RA, Gilkes NR.1995. Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol 37:1–8. Vazquez MJ, Alonso JL, Dominguez H, Parajo JC. 2000. Xylo oligosaccharides: manufacture and applications. Trends Food Sci Technol 11:387–93. Venkatesh KS, Umesh-Kumar S. 2006. Production of pectinases and utilization in food processing. In: Shetty K, Paliyath G, Pometto A, Levin RE, editors. Food biotechnology. Boca Raton, Fla.: Taylor and Francis Group. p 329–44. Verma D, Satyanarayana T. 2012. Cloning, expression and applicability of thermo-alkali-stable xylanase of Geobacillus thermoleovorans in generating xylo oligosaccharides from agro-residues. Bio Res Technol 107:333–8. Voget S, Leggewie C, Uesbeck A, Raasch C, Jaeger KE, Streit WR. 2013. Prospecting for novel biocatalysts in a soil metagenome. Appl Environ Microbiol 69(10):6235–42. Voget S, Steele HL, Streit WR. 2006. Characterization of a metagenome-derived halotolerant cellulose. J Biotechnol 126:26–36. Wang JX, Jiang XL, Mou HJ, Guan HS. 2004. Anti-oxidation of agar oligosaccharides produced by agarase from a marine bacterium. J Appl Phycol 16:333–40. Wang M, van Vliet T, Hamer RJ. 2004. Evidence that pentosans/xylanase affects the re agglomeration of the gluten network. J Cereal Sci 39:341–9. Wang K, Li G, Yu SQ, Zhang CT, Liu YH. 2010. A novel metagenome-derived betagalactosidase: gene cloning, overexpression, purification and characterization. Appl Microbiol Biotechnol 88:155–65. Wang H, Gong Y, Xie W, Xiao W, Wang J, Zheng Y, Hu J, Liu Z. 2011. Identification and characterization of a novel thermostable GH-57 gene from metagenomic fosmid library of the Juan De Fuca ridge hydrothermal vent. Appl Biochem Biotechnol 164: 1323–38. Wang J, Sun Z, Zhou Y, Wang Q, Ye J, Chen Z, Liu J. 2012. Screening of a xylanase clone from a fosmid library of rumen microbiota in HU sheep. Anim Biotechnol 23: 156–73. Warnecke F, Luginbuhl P, Ivanova N, Ghassemian M, Richardson TH, Stege JT, Cayouette M, McHardy AC, Djordjevic G, Aboushadi N, Sorek R, Tringe SG, Podar M, Martin HG, Kunin V, Dalevi D, Madejska J, Kirton E, Platt D, Szeto E, Salamov A, Barry K, Mikhailova N, Kyrpides NC, Matson EG, Ottesen EA, Zhang X, Hernandez M, Murillo C, Acosta LG, Rigoutsos I, Tamayo G, Green BD, Chang C, Rubin EM, Mathur EJ, Robertson DE, Hugenholtz P, Leadbetter JR. 2007. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 450:560–5. Wierzbicka-Wos A, Bartasun P, Cieslinski H, Kur J. 2013. Cloning and characterization of a novel coldactive glycoside hydrolase family 1 enzyme with β-glucosidase, β-fucosidase and β-galactosidase activities. BMC Biotechnol 13:22–34. Woldesenbet F, Virk AP, Gupta N, Sharma P. 2012. Effect of microwave irradiation on xylanase production from wheat bran and bio bleaching of eucalyptus kraft pulp. Appl Biochem Biotechnol 167:100–8. Yan X, Geng A, Zhang J, Wei Y, Zhang L, Qian C, Wang Q, Wang S, Zhou Z. 2013. Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing. Appl Microbiol Biotechnol 97(18):8173–82. Yun J, Kang S, Park S, Yoon H, Kim MJ, Heu S, Ryu S. 2004. Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library. Appl Environ Microb 70:7229–35. Zhang J, Zeng R. 2011. Molecular cloning and expression of an extracellular α amylase gene from an Antarctic deep sea psychrotolerant Pseudomonas stutzeri strain 7193. W J Microb Biotech 27(4):841–50. Zhang L, Fan Y, Zheng H, Du F, Zhang K, Huang X, Wang L, Zhang M, Niu Q. 2013a. Isolation and characterization of a novel endoglucanase from a bursaphelenchus xylophilus metagenomic library. PLoS ONE 8(12):e82437. Zhang X, Li H, Li CJ, Ma T, Li G, Liu YH. 2013b. Metagenomic approach for the isolation of a thermostable β-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin. BMC Microbiol 13:237–47. Ziemer CJ. 2013. Newly cultured bacteria with broad diversity isolated from 8 week continuous culture enrichments of cow faeces on complex polysaccharides. App Env Microbiol 80:574– 85.