Cellulosome and cellulosome-like multienzyme complexes from anaerobic and aerobic microorganisms. (This table is adapted from Doi & Kosugi, 2004).
To develop a bio-based economy for sustainable economic growth, it is necessary to produce chemicals and fuels from renewable resources, such as plant biomass. Plant biomass contains a complex mixture of polysaccharides, mainly cellulose and hemicellulose (mainly xylan), and other polysaccharides (Aspinall, 1980). The hemicelluloses, as well as the aromatic polymer lignin, interact with the cellulose fibrils, creating a rigid structure strengthening the plant cell wall. Therefore, complete and rapid hydrolysis of these polysaccharides requires not only cellulolytic enzymes but also the cooperation of xylanolytic enzymes (Thomson, 1993). Many microorganisms that produce enzymes capable of degrading cellulose and hemicellulose have been reported and characterized. Two enzyme systems are known for their degradation of lignocellulose by microorganisms. In many aerobic fungi and bacteria, endoglucanase, exoglucanase, and ancillary enzymes are secreted individually and can act synergistically on lignocellulose. The most thoroughly studied enzymes are the glycosyl hydrolases of
1.1. Composition of lignocellulosic biomass
Lignocellulosic biomass is composed mainly of plant cell walls, with the structural carbohydrates, cellulose and hemicelluloses and heterogeneous phenolic polymer lignin as its primary components (Fig. 1). However, their proportions vary substantially, depending on the type, the species, and even the source of the biomass (Aspinall et al., 1980; Pérez et al., 2002; Pauly et al., 2008).
1.2. Biodegradation of lignocellulosic biomass
Several biological methods for lignocellulose recycling based on the enzymology of cellulose, hemicelluloses, and lignin degradation have been developed. To date, processes that use lignocellulolytic enzymes or microorganisms could lead to promising, environmentally friendly technologies. The relationship between cellulose and hemicellulose in the cell walls of higher plants is much more intimate than was previously thought. It is possible that molecules at the cellulose-hemicellulose boundaries, and those within the crystalline cellulose, require different enzymes for efficient hydrolysis.
Hemicellulase: Xylan is the main carbohydrate found in hemicelluloses. Its complete degradation requires the cooperative action of a variety of hydrolytic enzymes (Fig. 2B). Xylanases are frequently classified according to their action on distinct substrates: endo-1,4-β-xylanase (endoxylanase) (EC 18.104.22.168) generates xylooligosaccharides from the cleavage of xylan while 1,4-β-xylosidase (EC 22.214.171.124) produces xylose from xylobiose and short chain xylooligosaccharides. In addition, xylan degradation needs accessory enzymes, such as α-L-arabinofuranosidase (EC 126.96.36.199), α-4-O-methyl-D-glucuronidase (EC 188.8.131.52), acetyl xylan esterase (EC 184.108.40.206), ferulic acid esterase (EC 220.127.116.11), and p-coumaric acid esterase (EC 3.1.1.-), acting synergistically, to efficiently hydrolyze wood xylans. In the case of acetyl-4-O-methylglucuronoxylan, which is one of the most common hemicelluloses, four different enzymes are required for degradation: endo-1,4-β-xylanase, acetyl esterase (EC 18.104.22.168), α-glucuronidase, and β-xylosidase. The degradation of O-acetyl galactoglucomannan starts with the rupture of the polymer by endomannanase (EC 22.214.171.124). Acetylglucomannan esterase (EC 3.1.1.-) removes acetyl groups, and α-galactosidase (EC 126.96.36.199) eliminates galactose residues. Finally, β-mannosidase (EC 188.8.131.52) and β-glucosidase break down the endomannanase-generated oligomeric β-1,4 bonds (Thomson, 1993; Li et al., 2000; Pérez et al., 2002).
1.3. Multienzyme complex cellulosome
The enzyme systems for the lignocellulose degradation by microorganisms can be generally regarded as non-complexed or complexed enzymes (Lynd et al., 2002). In the case of aerobic fungi and bacteria, the cellulase enzymes are free and mostly secreted. In such organisms, by the very nature of the growth of the organisms, they are able to reach and penetrate the cellulosic substrate and, hence, the secreted cellulases are capable of hydrolyzing the substrate. The enzymes in these cases are not organized into high molecular weight complexes and are called non-complexed (Fig. 3A). The polysaccharide hydrolases of the aerobic fungi are largely described based on the examples from Trichoderma, Penicillum, Fusarium, Humicola, Phanerochaete, etc., where a large number of the cellulases are encountered (Dashtban et al., 2009; Sánchez, 2009). In contrast, various cellulases and hemicellulases from several anaerobic cellulolytic microorganisms, are tightly bound to a scaffolding protein, as core protein and organized to form structures on the cell surfaces; these systems are called complexed enzymes or cellulosomes (Fig. 3B). The cellulosome is thought to allow concerted enzyme activities in close proximity to the bacterial cell, enabling optimum synergism between the enzymes presented on the cellulosome. Concomitantly, the cellulosome also minimizes the distance over which hydrolysis products must diffuse, allowing efficient uptake of these oligosaccharides by the host cells (Bayer et al., 1994; Schwarz, 2001; Lynd et al., 2002).
Biotechnological applications in terms of hydrolysis efficiency for complexed enzyme systems might have an advantage over non-complexed enzyme systems. The high efficiency of the cellulosome has been attributed to (i) the correct ratio between catalytic domains that optimize synergism between them, (ii) appropriate spacing between the individual components to further favor synergism, (iii) the presence of different enzymatic activities (cellulolytic or hemicellulolytic enzymes) in the cellulosome that can remove “physical hindrances” of other polysaccharides in heterogeneous plant cell materials (Lynd et al., 2002), and (iv) the presence of carbohydrate-binding modules (CBMs) that can increase the rate of hydrolysis by bringing the cellulosome into intimate and prolonged association with its recalcitrant substrate (Shoseyov et al., 2006). Thus, the complexed enzyme system, cellulosome, may provide great potential for the degradation of plant biomass.
The cellulosome was first identified in 1983 from the anaerobic, thermophilic, spore-forming Clostridium thermocellum (Lamed et al., 1983). The cellulosome of C. thermocellum is commonly studied along with cellulosomes from the anaerobic mesophiles, C. cellulovorans (Doi et al., 2003), C. josui (Kakiuchi et al., 1998) and C. cellulolyticum (Gal et al., 1997). All cellulosomes share similar characteristics, they all contain a large distinct protein, referred to as the scaffoldin which allows binding of the whole complex to microcrystalline cellulose via CBM. Also, the cellulosome scaffoldin expresses type I cohesins which allow binding of a wide variety of cellulolytic and hemicellulolytic enzymes within the complex via the expression of complementary type I dockerins on enzymes. Similarly, at the C-terminal the scaffoldin expresses type II cohesins, which allow the binding of the cellulosome to the cell through type II dockerins on surface layer homology proteins (SLH) (Fig. 4).
Cellulosomes are produced mainly by anaerobic bacteria, mostly from the class clostridia, and some anaerobic fungi such as genus Neocallimastix (Dalrymple et al., 1997), Piromyces (Teunissen et al., 1991) and Orpinomyces (Li et al., 1997). However, evidence suggests the presence of cellulosomes or cellulosome-like multienzyme complexes in a few aerobic microorganisms (Table 1). It is speculated that several other cellulolytic bacteria may also produce cellulosomes not yet described.
|Sewage||Ding et al., 1999||Potato starch granules||Kim and Kim, 1993|
|Soil||Watthanalarm-lort et al., 2012||Bioreactor||van Dyk et al., 2009|
|Sewage||Ding et al., 2000||Anaerobic digester||Pason et al., 2006b|
|Anaerobic digester||Ponpium et al., 2000||Soil||Hou, et al., 2006|
|Rumen||Berger et al., 1990|
|Soil||Sabathé et al.,2002|
|Rumen||Lamed et al., 1987|
|Compost||Pagès et al., 1997||Soil||Jiang et al., 2004|
|Fermenter||Sleat et al., 1984|
|Compost||Kakiuchi et al., 1998|
|Paper mill||Pohlschröder et al., 1994||Rotted wood||Ohtsuki et al., 2005|
|Sewage soil||Lamed et al., 1983|
|Rumen||Blair and Anderson, 1999b|
|Rumen||Ohara et al., 2000|
|Rumen||Ding et al., 2001|
|Soil||Phitsuwan et al., 2010|
|Soil||Chimtong et al., 2011|
|Rumen||Dalrymple et al., 1997|
|Rumen||Qiu et al., 2000|
|Rumen||Borneman et al., 1989|
|Rumen||Teunissen et al., 1991|
|Faeces||Teunissen et al., 1991|
2. Novel multienzyme complex system from P. curdlanolyticus strain B-6
Efficient enzymatic degradation of lignocellulosic biomass requires a tight interaction between the enzymes and their substrates, and the cooperation of multiple enzymes to enhance the hydrolysis due to the complex structure. Multienzyme complexes, cellulosomes from anaerobic cellulolytic microorganisms, are dedicated to hydrolyzing lignocellulosic substances efficiently because of a large variety of cellulases and hemicellulases in complexes, useful enzymatic properties, and binding ability to insoluble cellulose and/or xylan via CBMs (Bayer et al., 2004; Doi and Kosugi, 2004; Schwarz et al., 2001; Shoham et al., 1999). When compared with aerobic enzymes, they produce several individual enzymes, but microorganisms are not binding to insoluble substrates. However, P. curdlanolyticus B-6 was found to produce a multienzyme complex under aerobic conditions (Pason et al., 2006a, 2006b). Little information has been reported on cellulosome-like multienzyme complexes produced by aerobic bacterium (Kim & Kim, 1993; Jiang et al., 2004; van Dyk et al., 2009). Therefore, the multienzyme complex produced by strain B-6 is critical for improving plant biomass degradation.
2.1. Selection of multienzyme complex-producing bacteria under aerobic cultivation
Among several Bacillus strains, isolated from various sources and cultivated under aerobic conditions, P. curdlanolyticus strain B-6 shows important evidences for multienzyme complex producing bacterium (Pason et al., 2006a) as follows: high production of cellulase and xylanase, presence of CBMs that have ability to bind to insoluble substances, adhesion of bacterial cells to insoluble substances, and production of multiple cellulases and xylanases in the form of a high molecular weight complex. Thus, strain B-6 exhibits great promise bacterium in the production of multienzyme complex under aerobic conditions. Some properties of bacterial cells and cellulase and xylanase from strain B-6 compared with other Bacillus spp. are shown in Table 2.
|Strain (||Specific activity (U/mg protein)||Enzyme binding ability to insoluble substances (%)||Adhesion of cells to insoluble substances (%)||Zymogram analysis|
|CMCase||Xylanase||Avicel||Xylan||Avicel||Xylan||CMCase band||Xylanase band|
|1. Strain B-6|
|2. Strain H-4|
|3. Strain S-1|
|4. Strain X-11|
|5. Strain X-24|
|6. Stain X-26|
P. curdlanolyticus strain B-6 was a facultative, spore-forming, Gram-positive, motile, rod-shaped organism and produced catalase. Thus, this bacterium was identified as a member of the genus Bacillus according to Bergey’s Manual of Systematic Bacteriology (Sneath, 1986). The bacterium was also identified by 16S rRNA gene sequence analysis. The use of a specific PCR primer designed for differentiating the genus Paenibacillus from other members of the Bacillaceae showed that this strain had the same amplified 16S rRNA gene fragment as a member of the genus Paenibacillus. Based on these observations, it is reckoned that this strain was transferred to the genus Paenibacillus (Shida et al., 1997). The 16S rDNA sequence of this strain had 1,424 base pairs and 97% similarity with Paenibacillus curdlanolyticus (Innis & Gelfand, 1990).
2.2. Characteristics of P. curdlanolyticus B-6 multienzyme complex
During growth of P. curdlanolyticus B-6 on Berg’s mineral salt medium containing 0.5% xylan as carbon sources, the protein concentration in the medium was low up to the late stationary growth phase. CMCase and xylanase activities could be detected in the culture medium after the late exponential phase (Pason et al., 2006b). At the declining growth phase, the extracellular xylanase and CMCase rapidly increased due to the release of enzymes from the cell surfaces into the culture medium. These phenomena were different from the growth patterns of other aerobic bacteria, which grew and produced extracellular enzymes into culture supernatant immediately, but similar to those of the anaerobic bacteria which produced multienzyme complexes (cellulosomes) around the cell surfaces and adhered to these substrates and secreted into culture supernatant later (Bayer & Lamed, 1986; Lamed & Bayer, 1988). The observation of cell surfaces at the late exponential growth phase by scanning electron microscopy (SEM) revealed that the cells adhered to xylan (Fig. 5A), similar to the cells of the cellulosome producing anaerobic bacterium, C. thermocellum, which is a cell associated entity that mediates the adhesion of the bacterium to cellulose (Lamed et al., 1987; Mayer et al., 1987), whereas the surface of the cells of strain B-6 at the late stationary growth phase lacked such structures because the multienzyme complex was released into the medium from the cell surfaces (Fig. 5B). In addition, the pattern of multienzyme complex in the culture medium at the late stationary growth phase was determined. Native-polyacrylamide gel electrophoresis (native-PAGE) exhibited a high molecular weight band at the top of the gel (Fig. 6, lane 1). This protein band was dissociated into major and minor components through treatment by boiling in sodium dodecyl sulphate (SDS) solution, showing at least 18 proteins with molecular masses in the range of 29 to 280 kDa (Fig. 6, lane 2). Among those protein bands, at least 15 bands showed xylanase activities (Fig. 6, lane 3) and at least 9 bands showed CMCase activities (Fig. 6, lane 4) on zymograms. These multiple cellulases and xylanases are assembled into the high molecular weight complexes and released from the cell surfaces into medium at the late stationary growth phase. In C. thermocellum, the cellulosome consisted of many different types of glycosyl hydrolases, including cellulases, hemicellulases, and carbohydrate esterases, which served to promote their synergistic action (Lamed et al., 1983). These evidences confirm that the strain B-6 can produce xylanolytic-cellulolytic enzyme system that exists as multienzyme complex under aerobic conditions.
2.3. Effect of carbon sources on the induction of multienzyme complex in P. curdlanolyticus B-6
The effect of polymeric substances such as cellulose, xylan, corn hull, and sugarcane bagasse, and of soluble sugars such as L-arabinose, D-galactose, D-glucose, D-xylose, and cellobiose on the induction of multienzyme complexes in a facultatively anaerobic bacterium, P. curdlanolyticus B-6, was investigated under aerobic conditions (Waeonukul et al., 2008; 2009b). Cells grown on each carbon source adhered to cellulose. Hence strain B-6 cells from all carbon sources must have an essential component responsible for anchoring the cells to the substrate surfaces. Native–PAGE, SDS–PAGE, zymograms analysis, and enzymatic assays revealed that many proteins having xylanolytic and cellulolytic activities from P. curdlanolyticus B-6 grown on each carbon source were produced as multienzyme complex into the culture supernatants. These results indicated that strain B-6 produced multienzyme complexes when grown on both polymeric substances and soluble sugars. However, the subunits expressed in the multienzyme complex of strain B-6 depended on the carbon sources. These observations are consistent with previous reports that the enzymatic activities and enzyme compositions of the cellulosomes of C. thermocellum (Bayer et al., 1985; Bhat et al., 1993; Nochur et al., 1993), C. cellulolyticum (Mohand-Oussaid et al., 1999), and C. cellulovorans (Kosugi et al., 2001; Han et al., 2004; 2005) and the xylanosome of S. olivaceoviridis E-86 (Jiang et al., 2004) were affected by carbon sources in the media.
Many investigators have reported that the synthesis of cellulosome assemblies requires the presence of crystalline cellulose under anaerobic conditions, and that synthesis hardly occurs in growth on glucose or other soluble carbohydrates (Nochur et al., 1992; Blair & Anderson; 1999a; Bayer 2004; Doi & Kosugi, 2004). Some strains of C. thermocellum (Bayer et al., 1985; Bhat et al., 1993), however, can induce cellulosome synthesis when grown on cellobiose. P. curdlanolyticus B-6 differs from most cellulosome-producing microorganisms in that it produces multienzyme complex when grown on both polymeric substances and soluble sugars under aerobic conditions. Therefore, the mechanism of multienzyme complex formation by strain B-6 must be different from that of other microorganisms.
3. The feature of P. curdlanolyticus B-6 multienzyme complex
Recently, the structures and mechanisms for assembly of multienzyme complexes, cellulosomes, in anaerobic cellulolytic microorganisms are clear (Bayer et al., 2004, 2007; Doi & Kosugi, 2004). Generally, the key feature of the cellulosome is a scaffoldin that integrates the various catalytic subunits into the complex by self-assembly by cohesion-dockerin interaction. However, the structure and mechanism of the multienzyme complex produced by a facultatively anaerobic bacterium, such as P. curdlanolyticus B-6 is still unknown. In order to describe features of the multienzyme complex system produced by strain B-6, the multienzyme complex was purified by four kinds of chromatography (cellulose affinity, gel filtration, anion-exchange and hydrophobic-interaction chromatographys) (Fig. 7).
The multienzyme complex of P. curdlanolyticus strain B-6 with molecular mass of 1,450 (G1) was isolated from culture supernatant at the late stationary growth phase through cellulose affinity and Sephacryl S-300 gel filtration chromatographys (Pason et al., 2006b). Basically, the individual cellulosomes from anaerobic bacteria show 600 kDa to 2.1 MDa complexes size and show cohesion-dockerin domain as a signature protein (Bayer et al., 2004; Doi & Kosugi, 2004). While, multienzyme complexes from aerobic microorganisms, were range in mass from about 468 kDa to 2 MDa (with contained 5-12 protein subunits) (Table 3) and has no report of cohesion-dockerin domain. Here, the multienzyme complex produced by strain B-6 under aerobic conditions was the first report on characterization.
|Multienzyme complex||Mol. Mass (kDa)||Protein subunits||Ref.|
|1450||11||Pason et al.,2006b|
|669||7||Kim and Kim, 1993|
|2000||12||van Dyk et al., 2009|
|1000-2000||10||Hou et al., 2006|
|1200||5||Jiang et al., 2004|
|468||12||Ohtsuki et al., 2005|
|665||11||Sabathé et al.,2002|
|600||14||Gal et al., 1997|
|900||10||Shoseyov & Doi 1990|
|700||14||Kakiuchi et al., 1998|
|600||15||Pohlschröder et al., 1994|
|2100||14||Lamed et al., 1983|
|1500||15||Ohara et al., 2000|
Elucidation of the purified multienzyme feature of P. curdlanolyticus strain B-6 was followed by anion-exchange and hydrophobic-interaction chromatographys (Pason et al., 2010). The complex G1 from gel filtration chromatography (1,450 kDa) was purified by anion-exchange chromatography and showed at least five large protein complexes or aggregates, namely F1-F5. Among the fractions obtained from anion-exchange chromatography, F1 was apparently the most suited fraction to study on the organization and function of the multienzyme system of strain B-6 because F1 formed one clear band on the top of native PAGE, had the highest xylanase activity, and its subunit composition was clearly shown on SDS-PAGE. In the final step, complex F1 was separated to one major complex (H1) and two minor protein components (H2 and H3) by hydrophobic-interaction chromatography. The multienzyme complex (H1) was composed of a 280 kDa protein with xylanase activity, a 260 kDa protein that is a truncated form on the C-terminal side of the 280 kDa protein, two xylanases of 40 and 48 kDa, and 60 and 65 kDa proteins having both xylanase and CMCase activities (Fig. 8). The two components (280 and 40 kDa) of the multienzyme complex has characteristics similar to the cellulosome of C. thermocellum in that it is composed of a scaffolding protein and a catalytic subunit (Bayer et al., 1998; Demain et al., 2005). The 280 kDa protein resembled the scaffolding proteins of the multienzyme complex based on its migratory behavior in polyacrylamide gels and as a glycoprotein. The 280 kDa protein and a 40 kDa major xylanase subunit are the key components of multienzyme complex of the strain B-6.
These apparently propose that P. curdlanolyticus B-6 produced multienzyme complex, which consisted of many subunit compositions. The large protein (280 kDa) may function as a scaffoldin-like protein that allowed the enzyme subunits, majority is 40 kDa, binding to form a multienzyme complex. The key components, 280 and 40 kDa, are identified in the next topic.
4. Molecular structure of important xylanases
P. curdlanolyticus B-6 produces an extracellular xylanolytic-cellulolytic multienzyme complex mainly comprised of xylanases under aerobic conditions. To understand the xylanase system, a genomic library of the strain B-6 was constructed and screened for high xylanase activity. Recently, six xylanase genes, S1 (Pason et al., 2010), xyn10A (Waeonukul et al., 2009a), xyn10B (Sudo et al., 2010), xyn10C (unpublished data), xyn10D (Sakka et al., 2011) and xyn11A (Pason et al., 2010) were cloned, and the translated products were characterized (Table 4).
|Enzyme||Modular structure||GH family||Mol. Mass (kDa)||GenBank accession No.|
S1 protein: From the early research, the 280 kDa subunit (S1) plays a role of scaffoldin in assembling the enzyme complex and shows xylanase activity (Pason et al., 2010). The S1 gene consists of 2,589 nucleotides and encodes 863 amino acids with a molecular weight of 91,000 Da, indicating that the 280 kDa subunit is highly glycosylated. Sequence analysis revealed that S1 did not have significant homology with any proteins in the databases except for two surface layer homology (SLH) domains in its N-terminal region. Surprisingly, the recombinant S1 exhibits xylanase activity, and cellulose- and xylan-binding ability, suggesting that the S1 should be a novel xylanase and CBM(s) with new functions (unpublished data).
Xylanase Xyn10A: The xyn10A gene consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular weight of 142,726 Da. Xyn10A is a multidomain enzyme comprised of nine domains in the following order: three family-22 CBMs, a family-10 catalytic domain of glycosyl hydrolases (GH), a family-9 CBM, a glycine-rich region, and three SLH domains. Xyn10A can effectively hydrolyze insoluble xylan and natural biomass without pretreatment such as sugarcane bagasse, corn hull, rice bran, rice husk and rice straw. Xyn10A binds to various insoluble polysaccharides such as cellulose, xylan and chitin. The SLH domains functioned in Xyn10A by anchoring this enzyme to the cell surfaces of P. curdlanolyticus B-6. Removal of the CBMs from Xyn10A strongly reduced the ability of binding and plant cell wall hydrolysis. Therefore, the CBMs of Xyn10A play an important role in the hydrolysis of native biomass materials (Waeonukul et al., 2009a).
Xylanase Xyn10B: The xyn10B gene consists of 1,047 nucleotides encoding a protein of 349 amino acids with a predicted molecular weight of 40,480 Da. Xyn10B consists of only a family-10 catalytic of GH. Xyn10B is an intracellular endoxylanase (Sudo et al., 2010).
Xylanase Xyn10C: The xyn10C gene consists of 957 nucleotides and encodes 318 amino acid residues with a predicted molecular weight of 35,123 Da. Xyn10C is a single module enzyme consisting of a signal peptide and a family-10 catalytic module of GH (unpublished data).
Xylanase Xyn10D: The xyn10D gene consists of 1,734 nucleotides and encodes 577 amino acid residues with a calculated molecular weight of 61,811 Da. Xylanase Xyn10D is a modular enzyme consisting of a family-10 catalytic module of the GH, a fibronectin type-3 homology (Fn3) module, and family-3 CBM, in that order, from the N terminus. The CBM3 in Xyn10D has an affinity for cellulose and xylan, and plays an important role in hydrolysis of arabinoxylan and native biomass materials (Sakka et al., 2011).
Xylanase Xyn11A: The xyn11A gene consists of 1,150 bp and encodes a protein of 385 amino acids with a molecular weight of 40,000 Da. Xyn11A is composed of two major functional domains, a catalytic domain belonging to family-11 GH and a CBM classified as family-36. A glycine- and asparagine-repeated sequences existed between the two domains. Xyn11A has been identified to be one of the major xylanase subunit in the multienzyme complex of strain B-6 (Pason et al., 2010).
Based on both biochemical and molecular biological findings, a simplistic schematic view of the enzyme system from P. curdlanolyticus B-6 and its interaction with substrate and cell surface was created and presented in Fig. 9. In this assessment, the S1 protein did not have significant homology with any proteins in the databases except for two S-layer homology domains in its N-terminal region. However, the S1 protein that exhibits xylanase activity and cellulose- and xylan-binding ability, and contains cell anchoring function, seems remarkable. The multifunctional protein S1 is also responsible for forming the enzyme subunits into the complex and anchoring the complex into cell surface via the SLH domain. The interaction between S1 protein and enzyme subunits should be a mechanism distinct from the cohesion-dockerin interaction known in cellulosome of anaerobic microorganisms, since cohesion- or dockerin– like sequences were not observed in the S1 protein or the major xylanase subunit, Xyn11A. In addition, strain B-6 also produces cell bound multimodular xylanase Xyn10A that contains the numerous CBMs and SLH domains. Xyn10A can bind to the plant cell wall through CBM, whereas the catalytic module (GH10) is able to access its target substrate. Thus, the CBM greatly increases the concentration of the enzyme in the vicinity of the substrate, leading to the observed increase in polysaccharide hydrolysis. Besides, the presence of the functional CBMs and SLH domains in Xyn10A allows the cells to attach to substrate. Although, the overall structure of the enzyme complex system of the strain B-6 is not entirely clear, the enzyme complex has unique characteristics distinct from multienzyme complex cellulosome of anaerobic microorganisms. However, the mechanism for complex formation, interaction between the S1 protein as scaffoldin and enzyme subunits, needs to be further investigated.
5. Biotechnological uses of P. curdlanolyticus B-6 multienzyme complex
Biological conversion of lignocellulosic materials has been proposed as a renewable and sustainable route for the production of value-added products (Bayer et al., 2007, Doi et al., 2003). There is much interest in exploiting the properties of multienzyme complexes for practical purposes. The facultative bacterium, P. curdlanolyticus strain B-6 produces a unique extracellular multienzyme system under aerobic conditions that effectively degrade cellulose and hemicellulose by gaining access through the protective matrix surrounding the cellulose microfibrils of plant cell walls. Therefore, the multienzyme complex from strain B-6 is a promising enzyme which can potentially be used in many applications, such as enhancing extraction and production of value-added bioproducts by saccharification of cell wall components and application for construction of the modular enzymes creation (Fig. 10).
Biological treatment and saccharification using microorganisms and their enzymes selectively for degradation of lignocellulosic residues has the advantages of low energy consumption, minimal waste production, and environmental friendliness (Schwarz, 2001). The catalytic components of the multienzyme complex release soluble sugars, simple 5- and 6-carbon, from lignocellulose providing the primary carbon substrates, which can be subsequently converted into fuels by microorganisms. For enzyme saccharification, the close proximity between cellulolytic and xylanolytic enzymes is key to concerted degradation of the substrate, whereby the activities of the different enzymes facilitate the activities of their counterparts by promoting access to appropriated portions of the rigid insoluble substrates, since the release of sugar products was high. The synergistic action of the combination of enzymes by different modes of actions (xylanases and cellulases) and the presence of xylan- or cellulose-binding ability on lignocellulose enhanced soluble sugars released from the plant cell walls. In practicality, the multienzyme complex produced from P. curdlanolyticus B-6 allows access to lignocellulosic substrate and produces reducing sugar more than non-complexed enzymes from fungi (T. viride and Aspergillus niger) when the same cellulase activity (0.1 unit) was applied for degradation of corn hull and rice straw residues (unpublished data). In addition, the multienzyme complex of strain B-6 has been used to improve the extraction of plant food such as making low-cyanide-cassava starch by using multienzyme complex to enhance linamarin released by allowing more contact between linamarase and linamarin (Sornyotha et. al., 2009). Also, extraction of volatile compounds such as sea food-like flavor from seaweed, served for food supplement. Consequently, enzymatic treatment has advantages for the preparation of β-glucan and acidic α-glucan-protein complex from the fruiting body of mushroom, Pleurotus sajor-caju because the specificity of the multienzyme complex and gentle conditions allow for the recovery of high purity glucans in their native forms with minimal degradation (Satitmanwiwat et al., 2012a,b).
Typically, most plant cell wall degrading enzymes are composed of a series of separate modules (modular enzymes). These domains may fold and function in an independent manner and are normally separated by short linker. P. curdlanolyticus B-6, produces a number of glycosyl hydrolase (GHs) families and CBM families which have different substrates recognition affinity and increase amorphous regions of cellulose by H-bond elimination. Interestingly, modular architecture created by chimeric proteins creation with various tandem CBMs, GHs, and SLH-specific, should make it possible to construct effective lignocellulosic degrading enzymes, strongly binding, targeting enzyme to their substrates and bacterial cell surfaces for enhancing a variety of substrates hydrolysis. The strong carbohydrate-binding property of the cellulose-binding domain and xylan-binding domain, specific degradative activities exhibit important properties of the lignocellulosic material degrading enzymes that can be used in biotechnology.
A facultatively anaerobic bacterium P. curdlanolyticus strain B-6, isolated from an anaerobic digester fed with pineapple wastes, is unique in that it produces extracellular xylanolytic-cellulolytic multienzyme complex capable of efficient degradation of plant biomass materials under aerobic conditions. The production of strain B-6 multienzyme complex under aerobic conditions has several advantages: (i) a simple process, (ii) low price of medium, (iii) high growth rate, (iv) large quantities of extracellular enzymes yields, and (v) safe use with regard to health and environmental aspects. Thus, strain B-6 and its multienzyme complex is a promising tool for an industrial process employing direct hydrolysis for the bioconversion of cellulose as well as hemicellulose in biomass. This review shows that strain B-6 multienzyme complex is a novel enzymatic system known at the biochemical, genetic, and mechanism level. It also stresses that some points still need to be further investigated, mainly (i) the elucidation of scaffolding protein functions, (ii) the characterization of others key enzyme subunits, (iii) the assembly mechanism of the multienzyme complex, (iv) improvement of the efficiency in degradation of biomass of the multienzyme complex, and (v) improvement of the production of the multienzyme complex. The latter will certainly represent a challenge for future research.
Aro N. Pakula T. Penttilä M. 2005 Transcriptional Regulation of Plant Cell Wall Degradation by Filamentous Fungi. 29 4September, 2005), 719 739 0168-6445
Aspinall G. O. 1980Chemistry of Cell-Wall Polysaccharides, In: The Biochemistry of Plants. A Comprehensive Treatise, 3Preiss J., 473 500Academic Press, 0-12675-403-9York
Bayer E. A. Belaich J. P. Shoham Y. Lamed R. 2004 The Cellulosomes: Multienzyme Machines for Degradation of Plant Cell Wall Polysaccharides. 58October, 2004), 521 554 0066-4227
Bayer E. A. Chanzyt H. Lamed R. Shoham Y. 1998Cellulose, Cellulases and Cellulosomes. Current Opinion in Structural Biology, 8 5October, 1998), 548 557 0095-9440X
Bayer E. A. Lamed R. 1986 Ultrastructure of the Cell Surface Cellulosome of Clostridium thermocellum and Its Interaction with Cellulose.Journal of Bacteriology, 167 3September, 1986), 828 836 0021-9193
Bayer E. A. Lamed R. Himmel M. E. 2007The Potential of Cellulases and Cellulosomes for Cellulosic Waste Management 18 3June, 2007), 237 245 0958-1669
Bayer E. A. Morag E. Lamed R. 1994 The Cellulosome-a Treasure-Trove for Biotechnology. 12 9September, 1994), 379 386 0167-7799
Bayer E. A. Setter E. Lamed R. 1985 Organization and Distribution of the Cellulosome in Clostridium thermocellum.Journal of Bacteriology, 163 2August, 1985), 552 559 0021-9193
Berger E. Jones W. A. Jones D. T. Woods D. R. 1990 Sequencing and Expression of a Cellodextrinase (ced1) Gene from Butyrivibrio fibrisolvens H17c Cloned in Escherichia coli.Molecular and General Genetic, 223 2September, 1990), 310 318 0026-8925
Bhat S. Goodenough P. W. Owen E. Bhat M. K. 1993 Cellobiose: A True Inducer of Cellulosome in Different Strains of Clostridium thermocellum 111 1July, 1993), 73 78 0378-1097
Blair B. G. Anderson K. L. 1999a Regulation of Cellulose Inducible Structures of Clostridium cellulovorans. 45 3March, 1999), 242 249 0008-4166
Blair B. G. Anderson K. L. 1999bCellulose-Inducible Ultrastructural Protuberances and Cellulose-affinity Proteins of Eubacterium cellulosolvens. Anaerobe, 5 5October, 1999) 547 554 1075-9964
Borneman W. S. Akin D. E. Ljungdahl L. G. 1989Fermentation Products and Plant Cell Wall Degrading Enzymes Produced by Monocentric and Polycentric Anaerobic Ruminal Fungi. Applied and Environmental Microbiology, 55 5May, 1989), 1066 1073 0099-2240
Chimtong S. Tachaapaikoon C. Pason P. Kyu K. L. Kosugi A. Mori Y. Ratanakhanokchai K. 2011Isolation and Characterization of Endocellulase-Free Multienzyme Complex from Newly Isolated Thermoanaerobacterium thermosaccharolyticum Strain NOI-1. Journal of Microbiology and Biotechnology, 21 3March, 2011), 284 292 1017-7825
Dalrymple B. P. Cybinski D. H. Layton I. Mc Sweeney C. Xue G. P. Swadling Y. J. Lowry J. B. 1997Three Neocallimastix patriciarum Esterases Associated with the Degradation Complex Polysaccharides are Members of a New Family of Hydrolases. Microbiology, 143 8August, 1997) 2605 2614 1350-0872
Dashtban M. Schraft H. Qin W. 2009Fungal Bioconversion of Lignocellulosic Residues; Opportunities & Perspectives. International Journal of Biological Sciences, 5 6September, 2009), 578 595 1449-1907
Demain A. L. Newcomb M. Wu J. H. D. 2005Cellulase, Clostridium, and Ethanol. Microbiology and Molecular Biology Reviews, 69 1March, 2005), 124 154 1092-2172
Ding S. Y. Bayer E. A. Steiner D. Shoham Y. Lamed R. 1999A Novel Cellulosomal Scaffoldin from Acetivibrio cellulolyticus that Contains a Family 9 Glycosyl Hydrolase. Journal of Bacteriology, 181 21November, 1999), 6720 6729 0021-9193
Ding S. Y. Bayer E. A. Steiner D. Shoham Y. Lamed R. 2000A Scaffoldin of the Bacteroides cellulosolvens Cellulosome that Contains 11 Type II Cohesins. Journal of Bacteriology, 182 17September, 2000), 4915 4925 0021-9193
Ding S. Y. Rincon M. T. Lamed R. Martin J. C. Mccrae S. I. Aurilia V. Shoham Y. Bayer E. A. Flint H. J. 2001Cellulosomal Scaffoldin-Like Proteins from Ruminococcus flavefaciens. Journal of Bacteriology, 183 6March, 2001), 1945 1953 0021-9193
Doi R. H. Kosugi A. 2004Cellulosomes: Plant-Cell-Wall-Degrading Enzyme Complexes. Nature Reviews Microbiology, 2 7July, 2004), 541 551 1740-1526
Doi R. H. Kosugi A. Murashima K. Tamaru Y. Han S. O. 2003Cellulosomes from Mesophilic Bacteria. Journal of Bacteriology, 185 20October, 2003), 5907 5914 0021-9193
Gal L. Pages S. Gaudin C. Belaich A. Reverbel-Leroy C. Tardif C. Belaich J. P. 1997Characterization of the Cellulolytic Complex (Cellulosome) Produced by Clostridium cellulolyticum. Applied and Environmental Microbiology, 63 3March, 1997), 903 909 0099-2240
Goyal A. Ghosh B. Eveleig D. 1991Characterization of Fungal Cellulases. Bioresource Technology, 36 1Special Issue for Enzymatic Hydrolysis of Cellulose, 1991), 37 50 0960-8524
Han S. O. Cho H. Y. Yukawa H. Inui M. Doi R. H. 2004Regulation of Expression of Cellulosomes and Noncellulosomal (Hemi)Cellulolytic Enzymes in Clostridium cellulovorans During Growth on Different Carbon Sources. Journal of Bacteriology, 186 13July, 2004), 4218 4227 0021-9193
Han S. O. Yukawa H. Inui M. Doi R. H. 2005Effect of Carbon Source on the Cellulosomal Subpopulations of Clostridium cellulovorans. Microbiology, 151 5May, 2005), 1491 1497 1350-0872
Hou P. Li Y. Wu B. Yan Z. Yan B. Gao P. 2006Cellulolytic Complex Exists in Cellulolytic Myxobacterium Sorangium. Enzyme and Microbial Technology, 38 1-2January, 2006), 273 278 0141-0229
Innis M. A. Gelfand D. H. 1990Optimization of PCRs, In: PCR protocols: A Guide to Methods and Application, Innis, M.A.; Gelfand, D.H.; Sninsky, J.J. & White, T.J. 3 12Academic Press, 0-12372-180-6Diego
Jiang Z. Q. Deng W. Li L. T. Ding C. H. Kusakabe I. Tan S. S. 2004A Novel, Ultra-Large Xylanolytic Complex (Xylanosome) Secreted by Streptomyces olivaceoviridis. Biotechnology Letters, 26 5March, 2004), 431 436 0141-5492
Kakiuchi M. Isui A. Suzuki K. Fujino T. Fujino E. Kimura T. Karita S. Sakka K. Ohmiya K. 1998Cloning and DNA Sequencing of the Genes Encoding Clostridium josui Scaffolding Protein CipA and Cellulase CelD and Identification of Their Gene Products as Major Components of the Cellulosome. Journal of Bacteriology, 180 16August, 1998), 4303 4308 0021-9193
Kim C. H. Kim D. S. 1993Extracellular Cellulolytic Enzymes of Bacillus circulans Are Present as Two Multi-Protein Complexes. Applied Biochemistry and Biotechnology, 42 1July, 1993), 83 94 0885-4513
Klemm D. Heublein B. Fink H. P. 2005Cellulose: Fascinating Biopolymer and Sustainable Raw Material. Angewandte Chemie International, 44 22May, 2005), 3358 3393 1433-7851
Kosugi A. Murashima K. Doi R. H. 2001Characterization of Xylanolytic Enzymes in Clostridium cellulovorans: Expression of Xylanase Activity Dependent on Growth Substrates. Journal of Bacteriology, 183 11June, 2001), 7037 7043 0021-9193
Lamed R. Bayer E. A. 1988The Cellulosome of Clostridium thermocellum, In: Advances in Applied Microbiology, 33Laskin, A.I., 1 46Academic Press, 0-12002-633-3Diego
Lamed R. Naimark J. Morgenstern E. Bayer E. A. 1987Specialized Cell Surface Structure in Cellulolytic Bacteria. Journal of Bacteriology, 169 8August, 1987), 3792 3800 0021-9193
Lamed R. Setter E. Bayer E. A. 1983Characterization of a Cellulose-Binding Cellulase-Containing Complex in Clostridium thermocellum. Journal of Bacteriology, 156 2November, 1983), 828 836 0021-9193
Li X. L. Chen H. Ljungdahl L. G. 1997Two Cellulases, CelA and CelC, from the Polycentric Anaerobic Fungus Orpinomyces Strain PC-2 Contain N-terminal Docking Domains for a Cellulase-Hemicellulase Complex. Applied and Environmental Microbiology, 63 12December, 1997), 4721 4728 0099-2240
Li K. Azadi P. Collins R. Tolan J. Kim J. S. Eriksson K. E. L. 2000Relationship Between Activities of Xylanases and Xylan Structures. Enzyme and Microbial Technology, 27 1-2July, 2000), 89 94 0141-0229
Lynd L. R. Weimer P. J. van Zyl W. H. Pretorius I. S. 2002Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiology and Molecular Biology Reviews, 66 3September, 2002), 506 577 1092-2172
Mayer F. Coughlan M. P. Mori Y. Ljungdahl L. G. 1987Macromolecular Organization of the Cellulolytic Enzyme Complex of Clostridium thermocellum as Revealed by Electron Microscopy. Applied and Environmental Microbiology, 53 12December, 1987), 2785 2792 0099-2240
Mohand-Oussaid O. Payot S. Guedon E. Gelhaye E. Youyou A. Petitdemange H. 1999The Extracellular Xylan Degradative System in Clostridium cellulolitycum Cultivated on Xylan: Evidence for Cell-Free Cellulosome Production. Journal of Bacteriology, 181 13July, 1999), 4035 4040 0021-9193
Nochur S. V. Demain A. L. Roberts M. F. 1992Carbohydrate Utilization by Clostridium thermocellum: Importance of Internal pH in Regulating Growth. Enzyme and Microbial Technology, 14 5May, 1992), 338 349 0141-0229
Nochur S. V. Roberts M. F. Demain A. L. 1993True Cellulase Production by Clostridium thermocellum Grown on Different Carbon Sources. Biotechnology Letter, 15 6June, 1993), 641 646 0141-5492
Ohara H. Karita S. Kimura T. Sakka K. Ohmiya K. 2000Characterization of the Cellulolytic Complex (Cellulosome) from Ruminococcus albus. Bioscience, Biotechnology, and Biochemistry, 64 2February, 2000), 254 260 0916-8451
Ohtsuki T. Suyanto S. Yazaki S. U. Mimura A. 2005Production of Large Multienzyme Complex by Aerobic Thermophilic Fungus Chaetomium sp. nov. MS-017 Grown on Palm Oil Mill Fibre. Letters in Applied Microbiology, 40 2February, 2005), 111 116 0266-8254
Pagès S. Bélaïch A. Bélaïch J. P. Morag E. Lamed R. Shoham Y. Bayer E. A. 1997Species-Specificity of the Cohesin-Dockerin Interaction Between Clostridium thermocellum and Clostridium cellulolyticum: Prediction of Specificity Determinants of the Dockerin Domain. Proteins: Structure, Function, and Bioinformatics, 29 4December, 1997), 517 527 0887-3585
Pason P. Chon G. H. Ratanakhanokchai K. Kyu K. L. Jhee O. H. Kang J. Kim W. H. Choi K. M. Park G. S. Lee J. S. Park H. Rho M. S. Lee Y. S. 2006aSelection of Multienzyme Complex Producing Bacteria Under Aerobic Cultivation. Journal of Microbiology and Biotechnology, 16 8August, 2006), 1269 1275 1017-7825
Pason P. Kosugi A. Waeonukul R. Tachaapaikoon C. Ratanakhanokchai K. Arai T. Murata Y. Nakajima J. Mori Y. 2010Purification and Characterization of a Multienzyme Complex Produced by Paenibacillus curdlanolyticus B-6. Applied Microbiology and Biotechnology, 85 3January, 2010), 573 580 0175-7598
Pason P. Kyu K. L. Ratanakhanokchai K. 2006bPaenibacillus curdlanolyticus Strain B-6 Xylanolytic-Cellulolytic Enzyme System That Degrades Insoluble Polysaccharides. Applied and Environmental Microbiology, 72 4April, 2006), 2483 2490 0099-2240
Pauly M. Keegstra K. 2008Cell Wall Carbohydrates and Their Modification as a Resource for Biofuels. The Plant Journal, 54 4May, 2008), 559 568 0960-7412
Pérez J. Muñoz-Dorado J. De-la-Rubia T. Martínez J. 2002Biodegradation and Biological Treatments of Cellulose, Hemicellulose and Lignin: an Overview. International Microbiology, 5 2June, 2002), 53 63 1139-6709
Phitsuwan P. Tachaapaikoon C. Kosugi A. Mori Y. Kyu K. L. Ratanakhanokchai K. 2010A Cellulolytic and Xylanolytic Enzyme Complex from an Alkalothermoanaerobacterium, Tepidimicrobium xylanilyticum BT14. Journal of Microbiology and Biotechnology, 20 5May, 2010), 893 903 1017-7825
Pohlschröder M. D. Leschine S. B. Canale-Parola E. 1994Multicomplex Cellulase-Xylanase System of Clostridium papyrosolvens C7. Journal of Bacteriology, 176 1January, 1994), 70 76 0021-9193
Ponpium P. Ratanakhanokchai K. Kyu K. L. 2000Isolation and Properties of a Cellulosome-Type Multienzyme Complex of the Thermophilic Bacteroides sp. Strain P-1. Enzyme and Microbial Technology, 26 5-6March, 2000), 459 465 0141-0229
Qiu X. Selinger B. Yanke L. J. Cheng K. J. 2000Isolation and Analysis of Two Cellulase cDNAs from Orpinomyces joyonii. Gene, 245 1March, 2000), 119 126 0378-1119
Rabinovich M. L. Melnik M. S. Bolobova A. V. 2002Microbial Cellulases (Review). Applied Biochemistry and Microbiology, 38 4July, 2002), 305 322 0003-6838
Sabathé F. Bélaïch A. Soucaille P. 2002Characterization of the Cellulolytic Complex (Cellulosome) of Clostridium acetobutylicum. FEMS Microbiology Letters, 217 1November, 2002), 15 22 0378-1097
Saha B. C. 2003Hemicellulose Bioconversion. Journal of Industrial Microbiology and Biotechnology, 30 5May, 2003), 279 291 1367-5435
Sakka M. Higashi Y. Kimura T. Ratanakhanokchai K. Sakka K. 2011Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a Xylanase That Contains a Family 3 Carbohydrate-Binding Module. Applied and Environmental Microbiology, 77 12June, 2011), 4260 4263 0099-2240
Sánchez C. 2009Lignocellulosic Residues: Biodegradation and Bioconversion by Fungi. Biotechnology Advances, 27 2March-April, 2009), 185 194 0734-9750
Satitmanwiwat S. Ratanakhanokchai K. Laohakunjit N. Chao L. K. Chen-T S. Pason P. Tachaapaikoon C. Kyu K. L. 2012aImproved Purity and Immunostimulatory Activity of β-(1-->3)(1-->6)-Glucan from Pleurotus sajor-caju Using Cell Wall-Degrading Enzymes. Journal of Agricultural and Food Chemistry, 60 21May, 2012), 5423 5430 0021-8561
Satitmanwiwat S. Ratanakhanokchai K. Laohakunjit N. Pason P. Tachaapaikoon C. Kyu K. L. 2012bPurification and Partial Characterization of an Acidic α-Glucan-Protein Complex from the Fruiting Body of Pleurotus sajor-caju and Its Effect on Macrophage Activation. Bioscience, Biotechnology, and Biochemitry, in press, 0916-8451 0916 8451
Scheller H. V. Ulvskov P. 2010Hemicelluloses. Annual Review of Plant Biology, 61June, 2010), 263 289 1543-5008
Schwarz W. H. 2001The Cellulosome and Cellulose Degradation by Anaerobic Bacteria. Applied Microbiology and Biotechnology, 56 5-6September, 2001), 634 649 0175-7598
Shida O. Takagi H. Kadowaki K. Nakamura L. K. Komagata K. 1997Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the Genus Paenibacillus and Emended Description of the Genus Paenibacillus. International Journal of Systematic and Evolutionary Microbiology, 47 2April, 1997), 289 298 1466-5026
Shoham Y. Lamed R. Bayer E. A. 1999The Cellulosome Concept as an Efficient Microbial Strategy for the Degradation of Insoluble Polysaccharides. Trends in Microbiology, 7 7July, 1999), 275 281 0096-6842X
Shoseyov O. Doi R. H. 1990Essential 170-kDa Subunit for Degradation of Crystalline Cellulose by Clostridium cellulovorans Cellulase. Proceedings of the National Academy of Sciences of the United States of America, 87 6March, 1990), 2192 2195 0027-8424
Shoseyov O. Shani Z. Levy I. 2006Carbohydrate Binding Modules: Biochemical Properties and Novel Applications. Microbiology and Molecular Biology Reviews, 70 2June, 2006), 283 295 1092-2172
Sleat R. Mah R. A. Robinson R. 1984Isolation and Characterization of an Anaerobic, Cellulolytic Bacterium, Clostridium cellulovorans sp. nov. Applied and Environmental Microbiology, 48 1July, 1984), 88 93 0099-2240
Sneath P. H. A. 1986Endospore-Forming Gram-Positive Rods and Cocci, In: Bergey’s Manual of Systematic Bacteriology, 2Sneath, P.H.A.; Mair, N.S.; Sharpe, M.E. & Holt, J. G., 1104 1207William & Wilkins, 0-68307-893-3
Sornyotha S. Kyu K. L. Ratanakhanokchai K. 2010An Efficient Treatment for Detoxification Process of Cassava Starch by Plant Cell Wall-Degrading Enzymes. Journal of Bioscience and Bioengineering. 109 1January, 2010), 9 14 1389-1723
Sudo M. Sakka M. Kimura T. Ratanakhanokchai K. Sakka K. 2010Characterization of Paenibacillus curdlanolyticus Intracellular Xylanase Xyn10B Encoded by the xyn10B gene. Bioscience, Biotechnology, and Biochemistry, 74 11November, 2010), 2358 2360 0916-8451
Teunissen M. J. Op den. Camp H. J. M. Orpin C. G. Huis in’t. Veld J. H. J. Vogels G. D. 1991Comparison of Growth Characteristics of Anaerobic Fungi Isolated from Ruminant and Non-Ruminant Herbivores During Cultivation in a Defined Medium. Journal of General Microbiology, 137 6June, 1991), 1401 1408 0022-1287
Thomson J. A. 1993Molecular Biology of Xylan Degradation. FEMS Microbiology Letters, 10 1-2January, 1993), 65 82 0378-1097
Tomme P. Warren R. A. J. Gilkes N. R. 1995Cellulose Hydrolysis by Bacteria and Fungi, In: Advances in Microbial Physiology, 37Poole, R.K., 1 81Academic Press, 0-12027-737-9
van Dyk J. S. Sakka M. Sakka K. Pletschke B. I. 2009The Cellulolytic and Hemi-Cellulolytic System of Bacillus licheniformis SVD1 and the Evidence for Production of a Large Multi-Enzyme Complex. Enzyme and Microbial Technology, 45 5November, 2009), 372 378 0141-0229
Waeonukul R. Kyu K. L. Sakka K. Ratanakhanokchai K. 2008Effect of Carbon Sources on the Induction of Xylanolytic-Cellulolytic Multienzyme Complexes in Paenibacillus curdlanolyticus Strain B-6. Bioscience, Biotechnology, and Biochemistry, 72 2February, 2008), 321 328 0916-8451
Waeonukul R. Pason P. Kyu K. L. Sakka K. Kosugi A. Mori Y. Ratanakhanokchai K. 2009aCloning, Sequencing, and Expression of the Gene Encoding a Multidomain Endo-β-1,4-Xylanase from Paenibacillus curdlanolyticus B-6, and Characterization of the Recombinant Enzyme. Journal of Microbiology and Biotechnology, 19 3March, 2009), 277 285 1017-7825
Waeonukul R. Kyu K. L. Sakka K. Ratanakhanokchai K. 2009bIsolation and Characterization of a Multienzyme Complex (Cellulosome) of the Paenibacillus curdlanolyticus B-6 Grown on Avicel Under Aerobic Conditions. Journal of Bioscience and Bioengineering, 107 6June, 2009), 610 614 1389-1723
Watthanalamloet A. Tachaapaikoon C. Lee Y. S. Kosugi A. Mori Y. Tanasupawat S. Kyu K. L. Ratanakhanokchai K. 2012Amorocellulobacter alkalithermophilum gen. nov., sp. nov. an Anaerobic Alkalithermophile, Cellulolytic-Xylanolytic Bacterium Isolated from Soil in a Brackish Area of a Coconut Garden. International Journal of Systematic and Evolutionary Microbiology, doi:ijs.0. 027854 0