《生物技术与人类》课程教学资源（经典阅读）2013-Ligninolytic enzymes from Ganoderma spp Current status

Critical Reviews in Microbiology,2012;Early Online:1-11 2012 Informa Healthcare USA,Inc. informa IS5N 1040-841X print/ISSN 1549-7828 online healthcare D0:103109/1040841X2012.722606 REVIEW Ligninolytic enzymes from Ganoderma spp:Current status and potential applications Xuan-Wei Zhou2,Wei-Ran Cong2,Kai-Qi Su2,and Yong-Ming Zhang2 21026010 Shanghai Normal University,College of Life and Environment Science,Shanghai 200234,China and2Shanghai Jiao Tong University,School of Agriculture and Biology,Shanghai 200240,China Abstract White-rot fungal species belonging to Ganoderma have long been used as medicinal mushrooms in many Asian countries.In recent years,however,attention is not just being paid to their pharmacological properties,but to their other potentially valuable features as well,including their secretion of enzymes which decompose lignin.The current B literature regarding lignin-modifying enzymes from the genus Ganoderma,their potential uses,and the components, structures and processes of lignocellulose degradation are discussed.The ligninolytic enzymes from the genus Ganoderma,as well as the number of additional enzymes that participate in lignin degradation,are summarized; further,the potential applications of these enzymes are analyzed and probed in this article.This review will provide insight on the valuable applications of Ganoderma spp.and will serve as a useful reference on the use of lignocellulose degradation as a means of environmental protection. Kjuo asn uosiad o Keywords:degradation,environmental protection,enzyme system,Ganoderma spp.,lignocellulose single copy for p Introduction gnocellulose.also known as woody celluloses during processing and handling,are mainly discarded, burned in the fields,or buried in the soil by plowing, close association of lignin and cellulose,which is the all of which cause significant environmental problems. primary component of plant cell walls.Woody cellulose Therefore,much attention is being paid to exploring and also contains a number of other polysaccharides,such inventing new ways of utilizing existing lignocellulose, as hemicelluloses.The lignocellulose from plant roots, not only as a source of bioenergy,but also as a means of stems and leaves is the major constituent of the waste eliminating environmental pollution and maintaining products derived from crops and trees.These biomass the ecological balance (Blanchette,1991;Hakala et al., wastes are abundant in nature,and only a few of them 2004;Mendonca et al.,2008). are exploited by humans.Crop residues contain 30-45% In nature,lignocellulose is mainly degraded by fungi cellulose and 3-13%lignin,and there is an even higher and bacteria.There are mainly three groups of fungi that proportion in tree wastes,where they represent 45-60 can degrade lignocellulose:white-rot fungi,brown-rot and 18-30%,respectively (Boerjan et al.,2003;Whetten fungi and soft-rot fungi.Among these fungi,white-rot and Sederoff,1995).There are about 15 x 109 tons of fungi have the ability to secrete extracellular ligninolytic these wastes produced around the world every year,and enzymes with the best ability to degrade lignocellulose only about 11%of them are productively exploited by biomass,and therefore have the best prospects for humans as feed for livestock,along with a small amount development and utilization.White-rot fungi are of lignocellulose that is used in the paper industry.Other saprophytic filamentous fungi that generally colonize lignocellulose biomass waste,such as the parts of plants wood in nature.They release lignin-degrading enzymes not suitable for eating and straws and residues produced and invade wood cells through the decomposition of Address for Correspondence:Xuan-Wei Zhou or Yong-Ming Zhang,Shanghai Normal University,College of Life and Environment Science, Shanghai 200234,China.E-mail:xuanweizhou@sjtu.edu.cn or zhym@shnu.edu.cn (Received 04 May 2012;revised 07 August 2012;accepted 16 August 2012) RIGHTS LI N K

1 Introduction Lignocellulose, also known as woody cellulose, is the close association of lignin and cellulose, which is the primary component of plant cell walls. Woody cellulose also contains a number of other polysaccharides, such as hemicelluloses. The lignocellulose from plant roots, stems and leaves is the major constituent of the waste products derived from crops and trees. These biomass wastes are abundant in nature, and only a few of them are exploited by humans. Crop residues contain 30–45% cellulose and 3–13% lignin, and there is an even higher proportion in tree wastes, where they represent 45–60 and 18–30%, respectively (Boerjan et al., 2003; Whetten and Sederoff, 1995). There are about 15 × 109 tons of these wastes produced around the world every year, and only about 11% of them are productively exploited by humans as feed for livestock, along with a small amount of lignocellulose that is used in the paper industry. Other lignocellulose biomass waste, such as the parts of plants not suitable for eating and straws and residues produced during processing and handling, are mainly discarded, burned in the fields, or buried in the soil by plowing, all of which cause significant environmental problems. Therefore, much attention is being paid to exploring and inventing new ways of utilizing existing lignocellulose, not only as a source of bioenergy, but also as a means of eliminating environmental pollution and maintaining the ecological balance (Blanchette, 1991; Hakala et al., 2004; Mendonça et al., 2008). In nature, lignocellulose is mainly degraded by fungi and bacteria. There are mainly three groups of fungi that can degrade lignocellulose: white-rot fungi, brown-rot fungi and soft-rot fungi. Among these fungi, white-rot fungi have the ability to secrete extracellular ligninolytic enzymes with the best ability to degrade lignocellulose biomass, and therefore have the best prospects for development and utilization. White-rot fungi are saprophytic filamentous fungi that generally colonize wood in nature. They release lignin-degrading enzymes and invade wood cells through the decomposition of Review Ligninolytic enzymes from Ganoderma spp: Current status and potential applications Xuan-Wei Zhou1,2, Wei-Ran Cong2 , Kai-Qi Su2 , and Yong-Ming Zhang2 1 Shanghai Normal University, College of Life and Environment Science, Shanghai 200234, China and 2 Shanghai Jiao Tong University, School of Agriculture and Biology, Shanghai 200240, China Abstract White-rot fungal species belonging to Ganoderma have long been used as medicinal mushrooms in many Asian countries. In recent years, however, attention is not just being paid to their pharmacological properties, but to their other potentially valuable features as well, including their secretion of enzymes which decompose lignin. The current literature regarding lignin-modifying enzymes from the genus Ganoderma, their potential uses, and the components, structures and processes of lignocellulose degradation are discussed. The ligninolytic enzymes from the genus Ganoderma, as well as the number of additional enzymes that participate in lignin degradation, are summarized; further, the potential applications of these enzymes are analyzed and probed in this article. This review will provide insight on the valuable applications of Ganoderma spp. and will serve as a useful reference on the use of lignocellulose degradation as a means of environmental protection. Keywords: degradation, environmental protection, enzyme system, Ganoderma spp., lignocellulose Address for Correspondence: Xuan-Wei Zhou or Yong-Ming Zhang, Shanghai Normal University, College of Life and Environment Science, Shanghai 200234, China. E-mail: xuanweizhou@sjtu.edu.cn or zhym@shnu.edu.cn (Received 04 May 2012; revised 07 August 2012; accepted 16 August 2012) Critical Reviews in Microbiology, 2012; Early Online: 1–11 © 2012 Informa Healthcare USA, Inc. ISSN 1040-841X print/ISSN 1549-7828 online DOI: 10.3109/1040841X.2012.722606 Critical Reviews in Microbiology 00 00 1 11 04May2012 07August2012 16August2012 1040-841X 1549-7828 © 2012 Informa Healthcare USA, Inc. 10.3109/1040841X.2012.722606 2012 Ligninolytic enzymes from Ganoderma spp. X.-W. Zhou et al. Critical Reviews in Microbiology Downloaded from informahealthcare.com by Fudan University on 09/20/12 For personal use only

2 X.-W.Zhou et al. lignocellulose,leading to a characteristic white spongy wood as substrate to grow and reproduce.These fungi clump called white rot.There are many kinds of white- can generally secrete various enzymes that decompose rot fungi,including many higher basidiomycetes,most of lignin,cellulose,and hemicellulose into simple carbo- which belong to Aphyllophorales,Homobasidiomycetes. hydrates as a source of carbon and energy (Tuomela The typical species of white-rot fungi is Phanerochaete et al.,2000;Chi Bao,2004).White-rot fungi,especially chrysosporium Burdsall,which mainly distributed in basidiomycetes,can entirely reduce lignin to CO.and North America.At present,thousands of white-rot H,O,and thus are considered to be one of the best means fungi have been characterized,but only more than of degrading lignin (Hatakka,1994).The decomposition ten of them can be utilized to degrade lignocellulose; process of lignin occurs at the stage of secondary metab- these fungi mainly belong to Trametes,Bjerkandera, olism.It generates enzymes requisite for the decompo- Phanerochaete,Pleurotus,Lentinula (Liu and Lai,2001), sition process only when some major nutrients,such as and Ganoderma.(Adaskaveg et al.,1990;D'Souza et al., nitrogen,carbon,and sulfur,are limited.The degradation 1999).Given the unique metabolism and extracellular of lignin by white-rot fungi is a chain reaction based on ligninolytic enzymes of white-rot fungi,they can the employment of free radicals.Under aerobic condi- potentially be employed to decompose large amounts tions,HO,activates catalase,which triggers the forma- 2060 of organic pollution;therefore,these fungi can likely be tion of highly active free radical intermediates,and then utilized in the future disposal of waste water,gas,solids, catalyzes the oxidation of the substrate with various free as well as the disposal and environmental remediation radicals produced by chain reactions.These reactions of lignocellulosic wastes (Pointing,2001).As early as are highly nonspecific and are not stereoselective,which 1980s,people began paying attention to the special means that the relationship between white-rot fungal ability of white-rot fungi to promote the degradation enzymes and the substrate to be degraded is not like that of lignocellulose (Adaskaveg and Gilbertson,1986; between a typical enzyme and substrate(Higuchi,2004; Blanchette,1984).For many years,researchers have Fan et al.,2009)The process by which basidiomycetes demonstrated that Ganoderma applanatu has the degrade lignin is shown in Figure 1. mooalonpepuoiu strongest ability to degrade lignocellulose,with a capacity that exceeds even Pleurotus ostreatus (Fr.) Enzyme system for lignin degradation Ajuo asn Kummer (Maeda et al,2001).As a result,G.spp.has The white-rot fungal enzymes that participate in lignin aroused extensive interest in their potential roles in degradation include lignin peroxidase(Lip)(EC1.11.1.14), environmental protection in addition to its use in the manganese-dependent peroxidase (MnP)(EC1.11.1.13), prevention and treatment of various diseases (Zhou laccases (Lac)and oxidases producing hydrogen perox- etal,2012) ide,such as glyoxal oxidase(GLOX)and aryl-alcohol oxi- dase;these proteins can be regarded as representative of Lignin degradation and related enzymes the ligninolytic enzyme system of white-rot fungi.Many researchers have shown that different white-rot fungi,or Lignin and the basic degradation process the same fungus under different conditions,may have a Lignin different key enzyme system(Hammel and Cullen,2008; As a major cell wall component,lignin,which is created Hatakka,1994:Krause et al.,2003). through the polymerization of phenolic precursors,pro- vides protection against attack by microorganisms and LiP mechanical pressure(Beguin et al.,1994;Li et al.,1995). LiP is an extracellular glycosylated protein with an iso- Lignin is the second most abundantorganic compound in electric point of 3.2-4.0 and a molecular weight (MW) nature,trailing only cellulose(Kishi et al.,1997).Not only of 38-43kDa.A heme group that is deeply buried in the does it have important impacts on agriculture,industry protein constitutes its activity center.Under aerobic con- and the environment,it is also a large untapped source ditions,with a low concentration of H,O,as an initiator, of bioenergy.Lignin is an amorphous polymer with a heme peroxidases nonenzymatically oxidize lignin to 3-dimensional reticular structure composed of phenyl- radical cation after a process of a two-electron oxidation propanoid units connected by carbon and ether bonds. and two subsequent one-electron reductions,which ini- The precursors of the phenylpropane units are three aro- tiates nonenzymatic free radical chain reactions to real- matic alcohols(monolignols):p-coumaryl,coniferyl and ize the highly efficient and broad catabolism of complex sinapyl alcohols.The respective aromatic constituents organic molecules.The redox potential of LiP is high,and of these alcohols in the polymer are p-hydroxyphenyl it is the only key enzyme of white-rot fungi to degrade (H),guaiacyl(G)and syringyl(S)moieties(Buranov and nonphenolic lignin directly(Sarkanen et al.,1991). Mazza,2008;Lewis and Yamamoto,1990) Manganese peroxidase Process of lignin degradation MnP is found in white-rot fungi as a series of glycosylated Lignin is difficult to degrade naturally because of its isoenzymes.Its isoelectric point ranges from 4.2 to 4.5. complicated and irregular structure.However,through The MW of MnP ranges from 38 to 62.5kDa,with the MW the decomposition of lignin,some fungi still can utilize of most purified enzymes around 45kDa (Hofrichter, Critical Reviews in Microbiology RIGHTS LI N K

2 X.-W. Zhou et al. Critical Reviews in Microbiology lignocellulose, leading to a characteristic white spongy clump called white rot. There are many kinds of white￾rot fungi, including many higher basidiomycetes, most of which belong to Aphyllophorales, Homobasidiomycetes. The typical species of white-rot fungi is Phanerochaete chrysosporium Burdsall, which mainly distributed in North America. At present, thousands of white-rot fungi have been characterized, but only more than ten of them can be utilized to degrade lignocellulose; these fungi mainly belong to Trametes, Bjerkandera, Phanerochaete, Pleurotus, Lentinula (Liu and Lai, 2001), and Ganoderma. (Adaskaveg et al., 1990; D’Souza et al., 1999). Given the unique metabolism and extracellular ligninolytic enzymes of white-rot fungi, they can potentially be employed to decompose large amounts of organic pollution; therefore, these fungi can likely be utilized in the future disposal of waste water, gas, solids, as well as the disposal and environmental remediation of lignocellulosic wastes (Pointing, 2001). As early as 1980s, people began paying attention to the special ability of white-rot fungi to promote the degradation of lignocellulose (Adaskaveg and Gilbertson, 1986; Blanchette, 1984). For many years, researchers have demonstrated that Ganoderma applanatu has the strongest ability to degrade lignocellulose, with a capacity that exceeds even Pleurotus ostreatus (Fr.) Kummer (Maeda et al., 2001). As a result, G. spp. has aroused extensive interest in their potential roles in environmental protection in addition to its use in the prevention and treatment of various diseases (Zhou et al., 2012). Lignin degradation and related enzymes Lignin and the basic degradation process Lignin As a major cell wall component, lignin, which is created through the polymerization of phenolic precursors, pro￾vides protection against attack by microorganisms and mechanical pressure (Béguin et al., 1994; Li et al., 1995). Lignin is the second most abundant organic compound in nature, trailing only cellulose (Kishi et al., 1997). Not only does it have important impacts on agriculture, industry and the environment, it is also a large untapped source of bioenergy. Lignin is an amorphous polymer with a 3-dimensional reticular structure composed of phenyl￾propanoid units connected by carbon and ether bonds. The precursors of the phenylpropane units are three aro￾matic alcohols (monolignols): p-coumaryl, coniferyl and sinapyl alcohols. The respective aromatic constituents of these alcohols in the polymer are p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) moieties (Buranov and Mazza, 2008; Lewis and Yamamoto, 1990). Process of lignin degradation Lignin is difficult to degrade naturally because of its complicated and irregular structure. However, through the decomposition of lignin, some fungi still can utilize wood as substrate to grow and reproduce. These fungi can generally secrete various enzymes that decompose lignin, cellulose, and hemicellulose into simple carbo￾hydrates as a source of carbon and energy (Tuomela et al., 2000; Chi & Bao, 2004). White-rot fungi, especially basidiomycetes, can entirely reduce lignin to CO2 and H2 O, and thus are considered to be one of the best means of degrading lignin (Hatakka, 1994). The decomposition process of lignin occurs at the stage of secondary metab￾olism. It generates enzymes requisite for the decompo￾sition process only when some major nutrients, such as nitrogen, carbon, and sulfur, are limited. The degradation of lignin by white-rot fungi is a chain reaction based on the employment of free radicals. Under aerobic condi￾tions, H2 O2 activates catalase, which triggers the forma￾tion of highly active free radical intermediates, and then catalyzes the oxidation of the substrate with various free radicals produced by chain reactions. These reactions are highly nonspecific and are not stereoselective, which means that the relationship between white-rot fungal enzymes and the substrate to be degraded is not like that between a typical enzyme and substrate (Higuchi, 2004; Fan et al., 2009). The process by which basidiomycetes degrade lignin is shown in Figure 1. Enzyme system for lignin degradation The white-rot fungal enzymes that participate in lignin degradation include lignin peroxidase (LiP) (EC1.11.1.14), manganese-dependent peroxidase (MnP) (EC1.11.1.13), laccases (Lac) and oxidases producing hydrogen perox￾ide, such as glyoxal oxidase (GLOX) and aryl-alcohol oxi￾dase; these proteins can be regarded as representative of the ligninolytic enzyme system of white-rot fungi. Many researchers have shown that different white-rot fungi, or the same fungus under different conditions, may have a different key enzyme system (Hammel and Cullen, 2008; Hatakka, 1994; Krause et al., 2003). LiP LiP is an extracellular glycosylated protein with an iso￾electric point of 3.2–4.0 and a molecular weight (MW) of 38–43 kDa. A heme group that is deeply buried in the protein constitutes its activity center. Under aerobic con￾ditions, with a low concentration of H2 O2 as an initiator, heme peroxidases nonenzymatically oxidize lignin to radical cation after a process of a two-electron oxidation and two subsequent one-electron reductions, which ini￾tiates nonenzymatic free radical chain reactions to real￾ize the highly efficient and broad catabolism of complex organic molecules. The redox potential of LiP is high, and it is the only key enzyme of white-rot fungi to degrade nonphenolic lignin directly (Sarkanen et al., 1991). Manganese peroxidase MnP is found in white-rot fungi as a series of glycosylated isoenzymes. Its isoelectric point ranges from 4.2 to 4.5. The MW of MnP ranges from 38 to 62.5kDa, with the MW of most purified enzymes around 45kDa (Hofrichter, Critical Reviews in Microbiology Downloaded from informahealthcare.com by Fudan University on 09/20/12 For personal use only

Ligninolytic enzymes from Ganoderma spp.3 Lignin Y CHOH B CH Water-soluble CHOH lignin fragments Laccases and/or other peroxidases rom hemicelluloses Radicals Cooxidants e.g.ROO aturated fat acid,thioits?) Fungal hypha (biom ss) Organic acids (oxalate,malate) H202 Chelated Mn Mn Manganese peroxidase Figure 1.Model of lignin degradation by basidiomycetes white-rot fungi.White-rot fungi use polysaccharides and cellulose acetate B derived from hemicellulose as a source of carbon and energy,while at the same time the lignin is degraded(enzymatic combustion).The residual cellulose on rotting wood that has not been degraded is white(Hofrichter,2000).(See colour version of this figure online at www. informahealthcare.com/mby) 2002).Its active site is composed of a heme group,one [ABST),Lac can oxidize nonphenolic units without 'Ajuo asn Mn2+ion,and two Ca2+ions that stabilize the structure. H,O,,which is an advantage when degrading lignin The MnP protein is composed of ten long single B-strands (Wang,2009).The redox potential of Lac is low (300-400 and a short single B-strand.The amino acid sequences mV).It can degrade nonphenolic lignin only after form- of MnP and LiP are 43%similar.The major difference ing laccase mediator system together with a mediator between MnP and LiP is that the C-terminal end of LiP (Kahraman and Gurdal,2002).Besides three types of lies between two propionate groups of the heme,while enzymes mentioned above,white-rot fungi can also that of MnP is separated from the heme group.Also,LiP secrete some enzymes that assist in the degradation of has four disulfide bonds,while MnP has five disulfide complex organic molecules,such as glucose oxidase,cat- bonds:four of these are the same as Lip,and the fifth is at alase,pyranose oxidase,GLOX,methanol oxidase.The the C-terminal strand,which may be related to the Mn2+ H,O,produced by these enzymes in the aerobic environ- active site(Dong et al.,2005). ment is used to initiate the catalytic cycle of peroxidases. Not all white-rot fungi can secrete Lip,MnP and Lac at Laccase the same time,as some white-rot fungi can secrete only Lac,a glycoprotein member of the blue multi-copper one or two of them.Previous literature has shown that p oxidase family,is a copper-containing polyphenol oxi- chrysosporium can only produce Lac,and produces little dase(Hakulinen et al.,2002).Fungal lac is also glycopro- when metabolic reactions use glucose as carbon source. tein,and is composed of three parts:a peptide chain,a This shows that enzyme production is closely related to sugar chain,and a Cu2ion.Its isoelectric point is 2.8-4.3 the culture conditions(Srinivasan et al.,1995).All of the (Gao et al.,2011),and its MW ranges from 60 to 390kDa key enzymes mentioned above are extracellular enzymes (Wang and Li,2003).The peptide chain of Lac is com- secreted to the surface of the wood,where they can then posed of 500-550 amino acids (Zhang et al.,2003).The initiate a series of free radical reactions to break down oligosaccharide chain mainly contains hexosamine,glu- the organic matter(Reddy,1995).In the lignin degrada- cose,mannose,galactose,fucose and arabinose,which tion process,the chemical reactions belong to oxidation account for 10-80%of the glycoprotein's MW.The MW of catalysis with the mediator present in the vast extracel- Laccase varies greatly due to the varying size and compo- lular space.Therefore it is very likely to have strong sition of the sugar chain.The mechanism by which Lac degradative activity against a broad spectrum of organic degrades lignin involves the transfer ofelectrons received molecules. from phenolic substrates to molecular oxygen,thereby reducing molecular oxygen to water while oxidizing phe- Ligninolytic enzymes from Ganoderma nolic substrates to semiquinone radicals.In the presence Ganoderma spp.,a genus of basidiomycetes,belongs to of 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate Polyporaceae (or Ganodermaceae)of Aphyllophorales. 2012 Informa Healthcare USA,Inc. RIGH T S L I N K

Ligninolytic enzymes from Ganoderma spp. 3 © 2012 Informa Healthcare USA, Inc. 2002). Its active site is composed of a heme group, one Mn2+ ion, and two Ca2+ ions that stabilize the structure. The MnP protein is composed of ten long single β-strands and a short single β-strand. The amino acid sequences of MnP and LiP are 43% similar. The major difference between MnP and LiP is that the C-terminal end of LiP lies between two propionate groups of the heme, while that of MnP is separated from the heme group. Also, LiP has four disulfide bonds, while MnP has five disulfide bonds: four of these are the same as LiP, and the fifth is at the C-terminal strand, which may be related to the Mn2+ active site (Dong et al., 2005). Laccase Lac, a glycoprotein member of the blue multi-copper oxidase family, is a copper-containing polyphenol oxi￾dase (Hakulinen et al., 2002). Fungal lac is also glycopro￾tein, and is composed of three parts: a peptide chain, a sugar chain, and a Cu2+ ion. Its isoelectric point is 2.8–4.3 (Gao et al., 2011), and its MW ranges from 60 to 390kDa (Wang and Li, 2003). The peptide chain of Lac is com￾posed of 500–550 amino acids (Zhang et al., 2003). The oligosaccharide chain mainly contains hexosamine, glu￾cose, mannose, galactose, fucose and arabinose, which account for 10–80% of the glycoprotein’s MW. The MW of Laccase varies greatly due to the varying size and compo￾sition of the sugar chain. The mechanism by which Lac degrades lignin involves the transfer of electrons received from phenolic substrates to molecular oxygen, thereby reducing molecular oxygen to water while oxidizing phe￾nolic substrates to semiquinone radicals. In the presence of 2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate [ABST]), Lac can oxidize nonphenolic units without H2 O2 , which is an advantage when degrading lignin (Wang, 2009). The redox potential of Lac is low (300–400 mV). It can degrade nonphenolic lignin only after form￾ing laccase mediator system together with a mediator (Kahraman and Gurdal, 2002). Besides three types of enzymes mentioned above, white-rot fungi can also secrete some enzymes that assist in the degradation of complex organic molecules, such as glucose oxidase, cat￾alase, pyranose oxidase, GLOX, methanol oxidase. The H2 O2 produced by these enzymes in the aerobic environ￾ment is used to initiate the catalytic cycle of peroxidases. Not all white-rot fungi can secrete LiP, MnP and Lac at the same time, as some white-rot fungi can secrete only one or two of them. Previous literature has shown that P. chrysosporium can only produce Lac, and produces little when metabolic reactions use glucose as carbon source. This shows that enzyme production is closely related to the culture conditions (Srinivasan et al., 1995). All of the key enzymes mentioned above are extracellular enzymes secreted to the surface of the wood, where they can then initiate a series of free radical reactions to break down the organic matter (Reddy, 1995). In the lignin degrada￾tion process, the chemical reactions belong to oxidation catalysis with the mediator present in the vast extracel￾lular space. Therefore it is very likely to have strong degradative activity against a broad spectrum of organic molecules. Ligninolytic enzymes from Ganoderma Ganoderma spp., a genus of basidiomycetes, belongs to Polyporaceae (or Ganodermaceae) of Aphyllophorales. Figure 1. Model of lignin degradation by basidiomycetes white-rot fungi. White-rot fungi use polysaccharides and cellulose acetate derived from hemicellulose as a source of carbon and energy, while at the same time the lignin is degraded (enzymatic combustion). The residual cellulose on rotting wood that has not been degraded is white (Hofrichter, 2000). (See colour version of this figure online at www. informahealthcare.com/mby) Critical Reviews in Microbiology Downloaded from informahealthcare.com by Fudan University on 09/20/12 For personal use only

4 X.-W.Zhou et al. These fungi cannot carry out photosynthesis;rather, Investigating the same phenomena,De Souza Silva they can only absorb nutrients such as lignin,cellulose, and his colleagues quantified the LME production of hemicellulose,and organic nitrogen from degraded four strains of Ganoderma(spp.CB364,GASI3.4,CCB209 wood and other substrates.Therefore,members of this and GASI2)and found that both strains GASI3.4 and genus are classified as obligate saprophytes.Generally CCB209 can produce LiP,MnP and Lac simultaneously, Ganoderma spp.are solitary or clustered in the withered while strains CB364 and GASI2 can only produce Lac(De wood tissues around the broad-leaved trees (most are Souza Silva et al.,2005).In addition,to investigate the Fagaceae)and break the wood tissues down to absorb production of ligninolytic enzymes,Asgher et al.used the nutrients;thus,they are also known as wood-rotting waste materials,such as wheat straw,rice straw,banana fungi.Wild Ganoderma spp.mainly grow on trees in stem,bagasse,corn cobs and corn straw,as the basic broad-leaved forests,including members of Fagaceae, substrates for solid-state fermentation of Ganoderma. Hamamelidaceae,Elaeocarpus,and Carpinus.Of the Despite the fact that different types and compositions of white-rot fungi,wild G.applanatum possess the stron- culture media can significantly influence the production gest ability to decompose lignin (Maeda et al.,2001). of ligninolytic enzymes,the results showed that all of the Most of Ganoderma spp.can be cultivated in either lig- selected substrates could generate Lip,MnP and Lac.In Z1/0Z/60 uid-state culture or solid-state culture with crop residues their experiments,the maximum enzyme activities with employed as culture media so it has the broad substrates rice straw as a substrate were recorded:LiP,2185 IU/ml: for lignin degradation(Zhou et al.,2012). MnP,1972 IU/ml;and laccase,338 IU/ml (Asgher et al., 2010).In addition,the ability of Ganoderma to produce LMEs enzymes is influenced by the developmental stage.For Ganoderma spp.can secrete a variety of enzymes that example,Liu et al.studied the variation of extracellular uepn can hydrolyze lignin into monosaccharides,such as enzymes secreted by fungi at different developmental 6 arabinose,xylose,galactose,fructose and glucose,as well stages through the substituted cultivation of Ganoderma as disaccharides of small molecules such as arabinose, on cottonseed hulls.The results showed that extracel- xylose,galactose,fructose and glucose,which serve as lular lignin-decomposing enzymes Lac and MnP were carbon and energy sources.The enzymes produced by generated,but there was no LiP that was made during Ajuo asn Ganoderma are referred to as lignin-modifying enzymes the process.Lac and MnP appeared to peak during the (LME)and include LiP.MnP and Lac.However,not all bud development stage simultaneously.They thought Ganoderma strains can produce these enzymes at the that at the appearance of pileus,the store of nutrients in same time,and some Ganoderma strains can only secrete the mycelial growth stage had been largely consumed, one or two of them.Further,the types of enzymes pro- and in order to provide enough nutrients,mycelia began duced are influenced by the type of Ganoderma strains, to accelerate the secretion of extracellular lignin-decom- medium composition and culture conditions (D'Souza posing enzymes.While in the prophase,the higher Lac et al.,1996;D'Souza et al.,1999;De Souza Silva et al., secretion and lower MnP peroxidase activity might be 2005;Varela et al.,2000) related to the existence of free gossypol in the cottonseed Adaskaveg et al.have isolated six species of hulls substrate,which acts as a kind of inducer.With the Ganoderma,including G.colossum (RGL15829, gossypol being resolved under the laccase action,the JEA529),G.meredithiae (JEA395-399),G.oregonense bioactivity of Lac decreased(Liu and Lan,2009). (RLG15851,JEA398),G.zonatum (JEA346,357),and Some studies have attempted to use other methods Ganoderma spp.(JEA 615-625)from different hosts, to improve the level of Lac production in Ganoderma and came to the conclusion that different strains of strains.Wang et al.selected for Ganoderma strains with Ganoderma have varying abilities to generate lignino- high Lac productivity by protoplast mutagenesis.They lytic enzymes (Adaskaveg et al.,1990).In the specified mutagenized Ganoderma strains under high pressure medium without any other supplemental components, condition and discovered a mutant,termed G1502, Ganoderma spp.can only produce Lac,while low levels which displayed high-Lac productivity.The isolation of MnP were detected in the fermentation products of and purification of Lac from the fermentation broth a Ganoderma strain grown with sawdust of poplar and of mutant G1502 was carried out,and the enzymatic other woods in culture media,and no LiP was detected properties of Lac were further studied (Wang and Wang, in any of the media tested (D'Souza et al.,1999).With 2008;Wang et al.,2006).Xu and Lan studied the optimal P.chrysosporium and Coriolus versicolor serving as con- culture media and conditions in shaking flasks for Lac trols,Zhang et al.chose sixteen commonly used strains production by Ganoderma lucidum(Xu and Lan,2006). of Ganoderma and investigated the types of LMEs pro- Ouyang et al.cloned the gene and promoter of Lac from duced and their ability to degrade lignin.The results G.lucidum,and analyzed its sequence,thereby brining showed that ten of the selected sixteen Ganoderma Ganoderma Lac to the forefront of molecular biology strains could produce Lac and peroxidase,thereby (Ouyang et al.,2009).Lin and his colleagues purified demonstrating that different kinds of Ganoderma pos- crude laccase from G.lucidum by employing both sess different abilities to produce lignin-degrading Sephadex G-75 gel filtration and DEAE-Sepharose Fast enzymes(Zhang et al.,2005a). Flow ion exchange column chromatography;a final yield Critical Reviews in Microbiology RIGHTS LI N K

4 X.-W. Zhou et al. Critical Reviews in Microbiology These fungi cannot carry out photosynthesis; rather, they can only absorb nutrients such as lignin, cellulose, hemicellulose, and organic nitrogen from degraded wood and other substrates. Therefore, members of this genus are classified as obligate saprophytes. Generally Ganoderma spp. are solitary or clustered in the withered wood tissues around the broad-leaved trees (most are Fagaceae) and break the wood tissues down to absorb the nutrients; thus, they are also known as wood-rotting fungi. Wild Ganoderma spp. mainly grow on trees in broad-leaved forests, including members of Fagaceae, Hamamelidaceae, Elaeocarpus, and Carpinus. Of the white-rot fungi, wild G. applanatum possess the stron￾gest ability to decompose lignin (Maeda et al., 2001). Most of Ganoderma spp. can be cultivated in either liq￾uid-state culture or solid-state culture with crop residues employed as culture media so it has the broad substrates for lignin degradation (Zhou et al., 2012). LMEs Ganoderma spp. can secrete a variety of enzymes that can hydrolyze lignin into monosaccharides, such as arabinose, xylose, galactose, fructose and glucose, as well as disaccharides of small molecules such as arabinose, xylose, galactose, fructose and glucose, which serve as carbon and energy sources. The enzymes produced by Ganoderma are referred to as lignin-modifying enzymes (LME) and include LiP, MnP and Lac. However, not all Ganoderma strains can produce these enzymes at the same time, and some Ganoderma strains can only secrete one or two of them. Further, the types of enzymes pro￾duced are influenced by the type of Ganoderma strains, medium composition and culture conditions (D’Souza et al., 1996; D’Souza et al., 1999; De Souza Silva et al., 2005; Varela et al., 2000). Adaskaveg et al. have isolated six species of Ganoderma, including G. colossum (RGL15829, JEA529), G. meredithiae (JEA395–399), G. oregonense (RLG15851, JEA398), G. zonatum (JEA346, 357), and Ganoderma spp. (JEA 615–625) from different hosts, and came to the conclusion that different strains of Ganoderma have varying abilities to generate lignino￾lytic enzymes (Adaskaveg et al., 1990). In the specified medium without any other supplemental components, Ganoderma spp. can only produce Lac, while low levels of MnP were detected in the fermentation products of a Ganoderma strain grown with sawdust of poplar and other woods in culture media, and no LiP was detected in any of the media tested (D’Souza et al., 1999). With P. chrysosporium and Coriolus versicolor serving as con￾trols, Zhang et al. chose sixteen commonly used strains of Ganoderma and investigated the types of LMEs pro￾duced and their ability to degrade lignin. The results showed that ten of the selected sixteen Ganoderma strains could produce Lac and peroxidase, thereby demonstrating that different kinds of Ganoderma pos￾sess different abilities to produce lignin-degrading enzymes (Zhang et al., 2005a). Investigating the same phenomena, De Souza Silva and his colleagues quantified the LME production of four strains of Ganoderma (spp. CB364, GASI3.4, CCB209 and GASI2) and found that both strains GASI3.4 and CCB209 can produce LiP, MnP and Lac simultaneously, while strains CB364 and GASI2 can only produce Lac (De Souza Silva et al., 2005). In addition, to investigate the production of ligninolytic enzymes, Asgher et al. used waste materials, such as wheat straw, rice straw, banana stem, bagasse, corn cobs and corn straw, as the basic substrates for solid-state fermentation of Ganoderma. Despite the fact that different types and compositions of culture media can significantly influence the production of ligninolytic enzymes, the results showed that all of the selected substrates could generate LiP, MnP and Lac. In their experiments, the maximum enzyme activities with rice straw as a substrate were recorded: LiP, 2185 IU/ml; MnP, 1972 IU/ml; and laccase, 338 IU/ml (Asgher et al., 2010). In addition, the ability of Ganoderma to produce enzymes is influenced by the developmental stage. For example, Liu et al. studied the variation of extracellular enzymes secreted by fungi at different developmental stages through the substituted cultivation of Ganoderma on cottonseed hulls. The results showed that extracel￾lular lignin-decomposing enzymes Lac and MnP were generated, but there was no LiP that was made during the process. Lac and MnP appeared to peak during the bud development stage simultaneously. They thought that at the appearance of pileus, the store of nutrients in the mycelial growth stage had been largely consumed, and in order to provide enough nutrients, mycelia began to accelerate the secretion of extracellular lignin-decom￾posing enzymes. While in the prophase, the higher Lac secretion and lower MnP peroxidase activity might be related to the existence of free gossypol in the cottonseed hulls substrate, which acts as a kind of inducer. With the gossypol being resolved under the laccase action, the bioactivity of Lac decreased (Liu and Lan, 2009). Some studies have attempted to use other methods to improve the level of Lac production in Ganoderma strains. Wang et al. selected for Ganoderma strains with high Lac productivity by protoplast mutagenesis. They mutagenized Ganoderma strains under high pressure condition and discovered a mutant, termed G1502, which displayed high-Lac productivity. The isolation and purification of Lac from the fermentation broth of mutant G1502 was carried out, and the enzymatic properties of Lac were further studied (Wang and Wang, 2008; Wang et al., 2006). Xu and Lan studied the optimal culture media and conditions in shaking flasks for Lac production by Ganoderma lucidum (Xu and Lan, 2006). Ouyang et al. cloned the gene and promoter of Lac from G. lucidum, and analyzed its sequence, thereby brining Ganoderma Lac to the forefront of molecular biology (Ouyang et al., 2009). Lin and his colleagues purified crude laccase from G. lucidum by employing both Sephadex G-75 gel filtration and DEAE-Sepharose Fast Flow ion exchange column chromatography; a final yield Critical Reviews in Microbiology Downloaded from informahealthcare.com by Fudan University on 09/20/12 For personal use only

Ligninolytic enzymes from Ganoderma spp.5 of 83.9%and a specific activity of 81-fold were achieved. cloned and expressed(D'Souza et al.,1996;Varela et al., The results of native polyacrylamide gel electropho- 2000;Zhang et al.,2005b;Ouyang et al.,2009);and(3) resis (PAGE)combined with activity straining showed some researchers screened Ganoderma for strains with that there were three kinds of isoenzymes.The optimal high lignin-degrading activity,laying the foundation for pH value of purified Lac was between 2.2 and 2.6 with their application in industrial and agricultural produc- the peroxidase substrate ABTS (3-ethylbenzthiazoline- tion(Sun et al.,2012;Zhang et al.,2005a). 6-sulfonic acid),and the optimum temperature was 45C.When the temperature was below 45C and the pH value was in the range of 4.6-7.8,Lac exhibited maximal The application of LMEs from Ganoderma stability.Lac from G.lucidum were universally inhibited Transforming lignocellulosic biomass into high-value by metal and acid ions,and desalting facilitated the activ- animal forage ity (Lin et al.,2009a,2009b).These in-depth studies of Lac It is well known that lignin is the main limiting factor that from Ganoderma laid a solid foundation for the applica- influences the digestibility of crude fiber in feed,and tion of Ganoderma in environmental management and lignin in crop straw is connected to cellulose and hemi- remediation. celluloses by various chemical bonds;the association of Z1/0Z/60 these polymers gives rise to more complicated materials Other enzymes termed lignocelluloses.Because of the cross-linking and There are also other enzymes from Ganoderma spp.that mixture of lignin,hemicellulose and cellulose,biopoly- are relevant to the lignin degradation processes,includ- mers that are normally degraded by rumen microorgan- ing reductase,methylase,superoxide dismutase (SOD), isms,become difficult to degrade.Therefore,in order to cellobiose dehydrogenase,amylases,proteases and improving the nutritional value of crop straw we must uepn other enzymes.Some of these enzymes work in the lignin develop methods of reducing lignin content and destroy- 6 degradation process of Ganoderma directly or indirectly, ing the covalent bonds between structural units. and can play a very important role in the process of the White-rot fungi,with the ability of degrading lignocel- Ganoderma metabolic activities.There are also many luloses,can only generate enzymes to degrade cellulose, reports related to this area of investigation.For example, including cellulose cellobiohydrolase,endo(exo)-1,4- Ajuo asn manganese SOD(Mn-SOD)is an enzyme that commonly B-glucanase and B-glucan glucosidase,in the presence exists in Ganoderma.The MW of Mn-SOD is estimated to of it (Beguin et al.,1994).Researchers have shown that be 95kDa,while the isoelectric point is 5.6.This enzyme is most of the enzymes are present in Ganoderma(Table 1), tetramer composed of four 25kDa subunits of equal size and LMEs Lip,MnP and Lac are common in Ganoderma. 是 (Pan et al.,1997).Li et al.confirmed that both Mn-SOD Varela and Martinez cloned the gene that is closely related and Cu/Zn-SOD were present in Ganoderma,and they with Lip,and reached the conclusion that Ganoderma also studied the catalytic activity of the enzymes(Li et al., was a potential producer of LiP (Varela et al.,2000).In 1997).He et al.employed G.lucidum (G5)as material order to identify strains that effectively biodegrade lig- for the study of how different concentrations of La(NO) nin,Lin et al.conducted screening of lignin-degrading influence extracellular enzyme activity during the period fungus for the presence of cellulase.Eight fungal strains of Gnaoderma mycelia growth in liquid culture.The were selected by detecting growth status and enzyme results showed that among the various enzymes,includ- activity on media containing guaiacol,aniline and tan- ing cellulase,a-amylase,B-amylase and other protease, nic acid;G.applanatum WPl had the highest ability when the concentration of La"was 1mM,the cellulase degrade cellulose(14.7%),while others having the lowest and other protease had the highest activity,while when ability of 6.32%to degrade cellulose.(Lin et al.,2009b) the concentration La>was 0.005mM,the activity of Altogether,these experiments demonstrated the poten- B-amylase was the highest (He et al.,1998).In addition tial application of Ganoderma in degrading lignin,and to these LMEs in Ganoderma,other enzymes involved that it can be influenced by different culture conditions in the lignin degradation processes are summarized in and media types.Asgher et al.used various culture media Table 1. containing crop wastes as substrates for G.lucidum IBL- With the emergence of PAGE,molecular cloning,and 06 and confirmed this conclusion (Asgher et al.,2010).In other techniques in biotechnology,the enzymology of another white-rot fungi,the gene encoding LiP isozymes lignin degradation by Ganoderma spp.has made sig- was cloned,but the strain could not generate LiP,possi- nificant progress;current research ranges from in vivo bly because the activity of LiP was limited by culture con- functional studies to the in vitro study of the molecular ditions and methods(Dittmer et al.,1997;Reddy,1993). characteristics and functions of these enzymes.The great progress has mainly centered on three aspects: Biopulping and treatment of organic wastewater (1)enzymes from different species of Ganoderma were Biopulping is defined as the treatment of wood chips with purified and their physicochemical and biochemical lignin-degrading fungi,especially white-rot fungi,while properties were studied (Kim and Nho,2004;Ko et al., the selective breaking down of lignin in plant fiber prior 2001;Huie and Di,2004;Kumaran et al.,2011a;Tian and to pulping is known as biomechanical pulping.In fact, Zhang,2005);(2)some of the genes encoding LMEs were biopulping is the microbial pretreatment of mechanical 2012 Informa Healthcare USA,Inc. 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Ligninolytic enzymes from Ganoderma spp. 5 © 2012 Informa Healthcare USA, Inc. of 83.9% and a specific activity of 81-fold were achieved. The results of native polyacrylamide gel electropho￾resis (PAGE) combined with activity straining showed that there were three kinds of isoenzymes. The optimal pH value of purified Lac was between 2.2 and 2.6 with the peroxidase substrate ABTS (3-ethylbenzthiazoline- 6-sulfonic acid), and the optimum temperature was 45°C. When the temperature was below 45°C and the pH value was in the range of 4.6–7.8, Lac exhibited maximal stability. Lac from G. lucidum were universally inhibited by metal and acid ions, and desalting facilitated the activ￾ity (Lin et al., 2009a,2009b). These in-depth studies of Lac from Ganoderma laid a solid foundation for the applica￾tion of Ganoderma in environmental management and remediation. Other enzymes There are also other enzymes from Ganoderma spp. that are relevant to the lignin degradation processes, includ￾ing reductase, methylase, superoxide dismutase (SOD), cellobiose dehydrogenase, amylases, proteases and other enzymes. Some of these enzymes work in the lignin degradation process of Ganoderma directly or indirectly, and can play a very important role in the process of the Ganoderma metabolic activities. There are also many reports related to this area of investigation. For example, manganese SOD (Mn-SOD) is an enzyme that commonly exists in Ganoderma. The MW of Mn-SOD is estimated to be 95kDa, while the isoelectric point is 5.6. This enzyme is tetramer composed of four 25kDa subunits of equal size (Pan et al., 1997). Li et al. confirmed that both Mn-SOD and Cu/Zn-SOD were present in Ganoderma, and they also studied the catalytic activity of the enzymes (Li et al., 1997). He et al. employed G. lucidum (G5) as material for the study of how different concentrations of La(NO3 )3 influence extracellular enzyme activity during the period of Gnaoderma mycelia growth in liquid culture. The results showed that among the various enzymes, includ￾ing cellulase, α-amylase, β-amylase and other protease, when the concentration of La3+ was 1mM, the cellulase and other protease had the highest activity, while when the concentration La3+ was 0.005mM, the activity of β-amylase was the highest (He et al., 1998). In addition to these LMEs in Ganoderma, other enzymes involved in the lignin degradation processes are summarized in Table 1. With the emergence of PAGE, molecular cloning, and other techniques in biotechnology, the enzymology of lignin degradation by Ganoderma spp. has made sig￾nificant progress; current research ranges from in vivo functional studies to the in vitro study of the molecular characteristics and functions of these enzymes. The great progress has mainly centered on three aspects: (1) enzymes from different species of Ganoderma were purified and their physicochemical and biochemical properties were studied (Kim and Nho, 2004; Ko et al., 2001; Huie and Di, 2004; Kumaran et al., 2011a; Tian and Zhang, 2005); (2) some of the genes encoding LMEs were cloned and expressed (D’Souza et al., 1996; Varela et al., 2000; Zhang et al., 2005b; Ouyang et al., 2009); and (3) some researchers screened Ganoderma for strains with high lignin-degrading activity, laying the foundation for their application in industrial and agricultural produc￾tion (Sun et al., 2012; Zhang et al., 2005a). The application of LMEs from Ganoderma Transforming lignocellulosic biomass into high-value animal forage It is well known that lignin is the main limiting factor that influences the digestibility of crude fiber in feed, and lignin in crop straw is connected to cellulose and hemi￾celluloses by various chemical bonds; the association of these polymers gives rise to more complicated materials termed lignocelluloses. Because of the cross-linking and mixture of lignin, hemicellulose and cellulose, biopoly￾mers that are normally degraded by rumen microorgan￾isms, become difficult to degrade. Therefore, in order to improving the nutritional value of crop straw we must develop methods of reducing lignin content and destroy￾ing the covalent bonds between structural units. White-rot fungi, with the ability of degrading lignocel￾luloses, can only generate enzymes to degrade cellulose, including cellulose cellobiohydrolase, endo(exo)-1,4- β-glucanase and β-glucan glucosidase, in the presence of it (Béguin et al., 1994). Researchers have shown that most of the enzymes are present in Ganoderma (Table 1), and LMEs LiP, MnP and Lac are common in Ganoderma. Varela and Martinez cloned the gene that is closely related with LiP, and reached the conclusion that Ganoderma was a potential producer of LiP (Varela et al., 2000). In order to identify strains that effectively biodegrade lig￾nin, Lin et al. conducted screening of lignin-degrading fungus for the presence of cellulase. Eight fungal strains were selected by detecting growth status and enzyme activity on media containing guaiacol, aniline and tan￾nic acid; G. applanatum WP1 had the highest ability degrade cellulose (14.7%), while others having the lowest ability of 6.32% to degrade cellulose. (Lin et al., 2009b). Altogether, these experiments demonstrated the poten￾tial application of Ganoderma in degrading lignin, and that it can be influenced by different culture conditions and media types. Asgher et al. used various culture media containing crop wastes as substrates for G. lucidum IBL- 06 and confirmed this conclusion (Asgher et al., 2010). In another white-rot fungi, the gene encoding LiP isozymes was cloned, but the strain could not generate LiP, possi￾bly because the activity of LiP was limited by culture con￾ditions and methods (Dittmer et al., 1997; Reddy, 1993). Biopulping and treatment of organic wastewater Biopulping is defined as the treatment of wood chips with lignin-degrading fungi, especially white-rot fungi, while the selective breaking down of lignin in plant fiber prior to pulping is known as biomechanical pulping. In fact, biopulping is the microbial pretreatment of mechanical Critical Reviews in Microbiology Downloaded from informahealthcare.com by Fudan University on 09/20/12 For personal use only