Fusion of carbohydrate binding modules from <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis> with a family 10 xylanase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> S7

Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)₆ tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.     

Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant ceil wall hydroiases

Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non-catalytic cellulose-binding domain (CBD). To establish whether CBDs are common features of plant cell-wall hydroiases from C. fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of M_r 68000. The deduced primary sequence of XYLD was confirmed by the size (64kDa) and N-terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endo-acting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an /V-terminal signal peptide followed by a 190-residue domain that exhibited significant homology to Family-G xylanases. Truncated derivatives of xynD, encoding the W-terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190-residue N-terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90-residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197-amino-acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/prolineor novel glycine-rich linker regions. Although full-length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C-terminal CBD hydrolysed xylan but did not bind to cellulose. Fusion of the C-terminal domain to glutathione-Stransferase generated hybrid proteins that bound to crystalline cellulose, but not to amorphous cellulose or xylan. The location of CBDs in a C. fimi xylanase indicates that domains of this type are not restricted to cellulases, but are widely distributed between hemicellutases also, and therefore play a pivotal role in the activity of the whole repertoire of plant cell-wall hydrolases. The role of the NodB homologue in XYLD is less certain.     

Molecular and Biochemical Characterization of Two Xylanase-Encoding Genes from Cellulomonas pachnodae

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.     

Increased Production of Xylanase by Expression of a Truncated Version of the xyn11A Gene from Nonomuraea flexuosa in Trichoderma reesei

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3′ to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter⁻¹ of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.     

Characterization of modular bifunctional processive endoglucanase Cel5 from Hahella chejuensis KCTC 2396

Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K _m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca²⁺ greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.     

High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus Trichoderma reesei Requires a Carrier Polypeptide with an Intact Domain Structure

A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides. By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures. The carriers tested were the T. reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T. reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof. The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide. The recombinant Xyn11A produced had properties similar to those of the native xylanase. It constituted 6 to 10% of the total proteins secreted by the transformants. About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed. Even in the best Xyn11A producers, the levels of the fusion mRNAs were only ~10% of the level of cel7A (cbh1) mRNA in the untransformed host strain.     

In silico studies on bacterial xylanase enzyme: Structural and functional insight

Xylans are the second most abundant form of hemicelluloses and are the second most abundant polysaccharide in nature after cellulose. To degrade xylan, microbes produce mainly xylanase enzyme. Wide range of microorganisms like fungi, bacteria, yeast, marine algae etc. are capable of producing xylanase. Main source of xylanase is fungi but industrial production of bacterial xylanase is low cost, easy downstream process and high production rate. To understand primary, secondary and tertiary structure of xylanase, in silico composition of amino acids, basic physiological characteristics; viz., pI, molecular weight, instability index, GRAVY, molar extinction coefficient, secondary structure, presence of functional domain and motifs, phylogenetic tree, salt bridge compositions are determined. In silico study of xylanase focused on 36 different bacterial sources are performed by retrieving FASTA and PDB sequences using RCSB PDB. FASTA and PDB files are proceed further in ExPASy-ProtParam, RAMPAGE, QMEAN, MEME, PSIPRED, InterProScan, MOTIF scan, ERRAT, Peptide cutter, ESBRI and MEGA 7. The instability index range (16.90–38.78) clearly indicates that the protein is highly stable. α-helix mean value (27.11%) infers the protein is dominated by α-helix region. The aliphatic index (39.80–90.68) gives information that the protein is highly thermostable, prevalence by alanine amino acid in aliphatic side chain. No transmembrane domain was found in the protein which confirms the enzyme is extracellular in nature. Ancestor chart analysis confirmed that it is a part of carbohydrate metabolic process and more specifically a member of glycoside hydrolase super family.     

Characterization of EngF from Clostridium cellulovorans and Identification of a Novel Cellulose Binding Domain

The physical and enzymatic properties of noncellulosomal endoglucanase F (EngF) from Clostridium cellulovorans were studied. Binding studies revealed that the K_d and the maximum amount of protein bound for acid-swollen cellulose were 1.8 μM and 7.1 μmol/g of cellulose, respectively. The presence of cellobiose but not glucose or maltose could dissociate EngF from cellulose. N- and C-terminally truncated enzymes showed that binding activity was located at some site between amino acid residues 356 and 557 and that enzyme activity was still present when 20 amino acids but not 45 amino acids were removed from the N terminus and when 32 amino acids were removed from the C terminus; when 57 amino acids were removed from the C terminus, all activity was lost. EngF showed low endoglucanase activity and could hydrolyze cellotetraose and cellopentaose but not cellotriose. Activity studies suggested that EngF plays a role as an endoglucanase during cellulose degradation. Comparative sequence analyses indicated strongly that the cellulose binding domain (CBD) is different from previously reported CBDs.     

Probing the Functions of Carbohydrate Binding Modules in the CBEL Protein from the Oomycete Phytophthora parasitica

Oomycetes are microorganisms that are distantly related to true fungi and many members of this phylum are major plant pathogens. Oomycetes express proteins that are able to interact with plant cell wall polysaccharides, such as cellulose. This interaction is thought to be mediated by carbohydrate-binding modules that are classified into CBM family 1 in the CAZy database. In this study, the two CBMs (1–1 and 1–2) that form part of the cell wall glycoprotein, CBEL, from Phytophthora parasitica have been submitted to detailed characterization, first to better quantify their interaction with cellulose and second to determine whether these CBMs can be useful for biotechnological applications, such as biomass hydrolysis. A variety of biophysical techniques were used to study the interaction of the CBMs with various substrates and the data obtained indicate that CBEL’s CBM1-1 exhibits much greater cellulose binding ability than CBM1-2. Engineering of the family 11 xylanase from Talaromyces versatilis (TvXynB), an enzyme that naturally bears a fungal family 1 CBM, has produced two variants. The first one lacks its native CBM, whereas the second contains the CBEL CBM1-1. The study of these enzymes has revealed that wild type TvXynB binds to cellulose, via its CBM1, and that the substitution of its CBM by oomycetal CBM1-1 does not affect its activity on wheat straw. However, intriguingly the addition of CBEL during the hydrolysis of wheat straw actually potentiates the action of TvXynB variant lacking a CBM1. This suggests that the potentiating effect of CBM1-1 might not require the formation of a covalent linkage to TvXynB.     

Characterization of a Neocallimastix patriciarum xylanase gene and its product

A xylanase gene (xynC) isolated from the anaerobic ruminal fungus Neocallimastix patriciarum was characterized. The gene consists of an N-terminal catalytic domain that exhibited homology to family 11 of glycosyl hydrolases, a C-terminal cellulose binding domain (CBD) and a putative dockerin domain in between. Each domain was linked by a short linker domain rich in proline and alanine. Deletion analysis demonstrated that the CBD was essential for optimal xylanase activity of the enzyme, while the putative dockerin domain may not be required for enzyme function.     

A kinetic study of Trichoderma reesei Cel7B catalyzed cellulose hydrolysis

One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24 h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15–35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme’s capability in hydrolyzing the soluble substrate.     

Increase in Xylanase Production by Streptomyces lividans through Simultaneous Use of the Sec- and Tat-Dependent Protein Export Systems

Xylanase B1 (XlnB1) from Streptomyces lividans is a protein consisting of two discrete structural and functional units, an N-terminal catalytic domain and a C-terminal substrate binding domain. In the culture medium, two forms of xylanase B are present, namely, XlnB1 and XlnB2, the latter of which corresponds to the catalytic domain of XlnB1 deprived of the substrate binding domain. Both forms of the xylanase have the same activity on xylan. The enzyme is secreted through the Sec-dependent pathway with a better yield of XlnB1 than XlnB2. Interestingly, XlnB2 exhibits 80% identity with XlnC which is secreted exclusively through the Tat-dependent pathway. To demonstrate whether XlnB1 and XlnB2 could also be secreted through the Tat-dependent pathway, the Tat-targeting xlnC signal sequence was fused to the structural genes of xlnB1 and xlnB2. Both XlnB1 and XlnB2 were secreted through the Tat-dependent pathway, but XlnB2 was better produced than XlnB1. As XlnB1 and XlnB2 could be better secreted through the Sec- and Tat-dependent systems, respectively, a copy of the structural gene of xlnB1 fused to a Sec signal sequence and a copy of the structural gene of xlnB2 fused to a Tat signal sequence were inserted into the same plasmid under the control of the xlnA promoter. The transformant produced xylanase activity which corresponded approximately to the sum of activities of the individual strain producing xylanase by either the Sec- or Tat-dependent secretion system. This indicated that both secretion systems are functional and independent of each other in the recombinant strain. This is the first report on the efficient secretion of a protein using two different secretion systems at the same time. Assuming that the protein to be secreted could be properly folded prior to and after translocation via the Tat- and Sec-dependent pathways, respectively, the simultaneous use of the Sec- and Tat-dependent pathways provides an efficient means to increase the production of a given protein.     

Perturbation of apoptosis upon binding of tRNA to the heme domain of cytochrome c

In response to apoptotic stimuli, cytochrome c, an inter-membrane space protein is released from mitochondria to activate the cascade of caspases that leads to apoptosis. Recent evidence suggests that cytochrome c interacts with tRNA in the cytoplasm and this interaction was shown to inhibit the caspase mediated apoptotic process. Interestingly, cytochrome c does not contain any putative RNA binding domain. In this report, we sought to define the structural component of cytochrome c that is involved in binding of tRNA. By using gel mobility shift assays, we show that holocytochrome c can interact with tRNA but not apocytochrome c that lacks the heme domain suggesting that heme is essential for the interaction of cytochrome c to tRNA. In addition, using in vitro cross linking and circular dichroism spectroscopic studies, we show that cytochrome c can undergo heme mediated oligomerization. Prevention of heme mediated oligomerization of cytochrome c by potassium ferricyanide treatment prevents the binding of tRNA and promotes caspase activation. Our studies provide a novel regulation of apoptosis by heme dependent tRNA interaction to cytochrome c.     

Functional characterization of a cellulose binding xylanase from Fusarium oxysporum

Summary An extracellular endoxylanase from Fusarium oxysporum binds onto crystalline cellulose. A small peptide (~ 2kDa) could be isolated after partial proteolysis of the native protein. It consists of 18 amino acids, is located in the C-terminal region of the protein and corresponds functionally to a cellulose binding domain (CBD), the first one to be reported in a fungal xylanase. The amino acid sequence of this peptide shows no homology with any known CBD.     

Multi-Mode Binding of Cellobiohydrolase Cel7A from Trichoderma reesei to Cellulose

Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-β-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was exclusively active site mediated and was consistent with Langmuir''s one binding site model with K _d and A _max values of 2.9 nM and 126 nmol/g BC, respectively. This is the strongest binding observed with non-complexed cellulases and apparently represents the productive binding of TrCel7A to cellulose chain ends on the hydrophobic face of BC microfibril. With increasing free TrCel7A concentrations the isotherm gradually deviated from the Langmuir''s one binding site model. This was caused by the increasing contribution of lower affinity binding modes that included both active site mediated binding and non-productive binding with active site free from cellulose chain. The binding of TrCel7A to BC was found to be only partially reversible. Furthermore, the isotherm was dependent on the concentration of BC with more efficient binding observed at lower BC concentrations. The phenomenon can be ascribed to the BC concentration dependent aggregation of BC microfibrils with concomitant reduction of specific surface area.     

Analyses of the gene and amino acid sequence of thePrevotella (bacteroides) ruminicola 23 xylanase reveals unexpected homology with endoglucanases from other genera of bacteria

The DNA sequence for the xylanase gene fromPrevotella (Bacteroides) ruminicola 23 was determined. The xylanase gene encoded for a protein with a molecular weight of 65,740. An apparent leader sequence of 22 amino acids was observed. The promoter region for expression of the xylanase gene inBacteroides species was identified with a promoterless chloramphenicol acetyltransferase gene. A region of high amino acid homology was found with the proposed catalytic domain of endoglucanases from several organisms, includingButyrivibrio fibrisolvens, Ruminococcus flavefaciens, andClostridium thermocellum. The cloned xylanase was found to exhibit endoglucanase activity against carboxymethyl cellulose. Analysis of the codon usage for the xylanase gene found a bias towards G and C in the third position in 16 of 18 amino acids with degenerate codons.     

Induction of membrane-bound xylosidase in a Streptomyces sp.

The xylosidase bound with mycelia of Streptomyces sp. was a typical inducible enzyme. Xylosidase production was induced by various xylooligosaccharides, xylan, and non-metabolizable β-xylosides. The xylooligasoccharides and xylan induced xylosidase production better than β-xylosides, which induced more xylanase production extracellularly. The induction pattern of xylosidase was considerably different that of xylanase.The crude xylosidase solubilized by toluene and Triton X-100 from mycelial fraction was vary unstable and active on xylooligosaccharides and β-phenylxyloside but was not active on xylan, starch, cellulose, maltose and cellobiose.     

EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.     

Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.