High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2

Cyclophilins are an evolutionarily conserved family of peptidyl-prolyl cis-trans isomerases (PPIases). A cyclophilin B (cypB) gene from the anaerobic fungus Orpinomyces sp. strain PC-2 was cloned and overexpressed in Escherichia coli. It was expressed as an amino-terminal 6 x His-tagged recombinant protein to facilitate purification. Highly purified protein (26.5 kDa) was isolated by two chromatographic steps involving affinity and gel filtration for biochemical studies of the enzyme. The recombinant CypB displayed PPIase activity with a k(cat)/K(m) of 8.9 x 10(6) M(-1) s(-1) at 10 degrees C and pH 7.8. It was inhibited by cyclosporin A (CsA) with an IC(50) of 23.5 nM, similar to those of the native protein and other cyclophilin B enzymes from animals. Genomic DNA analysis of cypB revealed that it was present as a single copy in Orpinomyces PC-2 and contained two introns, indicating it has a eukaryotic origin. It is one of the most heavily interrupted genes with intron sequences found in anaerobic fungi. The three-dimensional model of Orpinomyces PC-2 CypB was predicted with a homology modeling approach using the Swiss-Model Protein Modeling Server and three dimensional structure of human CypB as a template. The overall architecture of the CypB molecule is very similar to that of human CypB.     

热解纤维素菌属F32中乙酰木聚糖酯酶Axe7的表征

【目的】从嗜热厌氧微生物热解纤维素菌属F32(Caldicellulosiruptor sp.F32)菌株中鉴定出可水解木聚糖侧链乙酰基团的脂酶。【方法】通过基因组序列注释、比对以及蛋白结构预测的方法,发现一个潜在的脂酶7家族的(CE-7)乙酰木聚糖脂酶Axe7。利用基因克隆、质粒构建以及在大肠杆菌中表达目标蛋白并纯化等实验方法,获得了该酶的重组蛋白。【结果】以4-甲基乙酸伞形酯(4-Methylumbelliferyl-acetate)作为底物时,该酶的最适反应p H在6.5-7.0之间,最适反应温度为85°C,在最适的温度和p H条件下,Axe7活性半衰期(Half-life)超过4 h。在不同金属离子(1.5 mmol/L)存在下,Axe7活性可保持为最适反应酶活的(66.3±4.6)%-(95.7±2.3)%之间,说明金属离子对其酶活有一定的影响。通过测定酶动力学发现Axe7的Km和kcat值分别为0.39 mmol/L和66.95 s-1。【结论】从高温厌氧微生物中发现并表征一个热稳定性良好的乙酰木聚糖脂酶,为木质纤维素的高温糖化和生物炼制提供了一个可工业化的潜在选择。     

Isolation and properties of an extracellular beta-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2.

An extracellular beta-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a M(r) of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pI of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40 degrees C are 0.25 mM and 27.1 mumol.min-1 x mg-1, respectively; with PNPG as the substrate, the corresponding values are of 0.35 mM and 27.7 mumol.min-1 x mg-1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 1.68 x 10(-2) and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50 degrees C. The enzyme has high activity against sophorose (beta-1,2-glucobiose) and laminaribiose (beta-1,3-glucobiose) but has no activity against gentiobiose (beta-1,6-glucobiose). The activity of the beta-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.     

Characterization of an Acetyl Xylan Esterase from the Anaerobic Fungus Orpinomyces sp. Strain PC-2

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K_m of 0.9 mM and a V_max of 785 μmol min⁻¹ mg⁻¹. It had temperature and pH optima of 30°C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.     

A mannanase, ManA, of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has carbohydrate binding and docking modules

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or beta-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 degrees C, but was rapidly inactivated at 60 degrees C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.     

Two cellulases, CelA and CelC, from the polycentric anaerobic fungus Orpinomyces strain PC-2 contain N-terminal docking domains for a cellulase-hemicellulase complex.

Two cDNAs encoding two cellulases, CelA and CelC, were isolated from a cDNA library of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 constructed in Escherichia coli. Nucleotide sequencing revealed that the celA cDNA (1,558 bp) and celC cDNA (1,628 bp) had open reading frames encoding polypeptides of 459 (CelA) and 449 (CelC) amino acids, respectively. The two cDNAs were 76.9 and 67.7% identical at the nucleotide and amino acid levels, respectively. Analysis of the deduced amino acid sequences showed that starting from the N termini, both CelA and CelC had signal peptides, which were followed by noncatalytic repeated peptide domains (NCRPD) containing two repeated sequences of 33 to 40 amino acid residues functioning as docking domains. The NCRPDs and the catalytic domains were separated by linker sequences. The NCRPDs were homologous to those found in several hydrolases of anaerobic fungi, whereas the catalytic domains were homologous to the catalytic domains of fungal cellobiohydrolases and bacterial endoglucanases. The linker sequence of CelA contained predominantly glutamine and proline residues, while that of CelC contained mainly threonine residues. CelA and CelC did not have a typical cellulose binding domain (CBD). CelA and CelC expressed in E. coli rapidly decreased the viscosity of carboxymethyl cellulose (CMC), indicating that there was endoglucanase activity. In addition, they produced cellobiose from CMC, acid-swollen cellulose, and cellotetraose, suggesting that they had cellobiohydrolase activity. The optimal activity conditions with CMC as the substrate were pH 4.3 to 6.8 and 50 degrees C for CelA and pH 4.6 to 7.0 and 40 degrees C for CelC. Despite the lack of a CBD, CelC displayed a high affinity for microcrystalline cellulose, whereas CelA did not.     

Purification and characterization of an acetyl xylan esterase from Bacillus pumilus

Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55 degrees C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis contant (Km) and Vmax for alpha-naphthyl acetate were 1.54 mM and 360 micromol min-1 mg of protein-1, respectively.     

Characterization of crystals of Penicillium purpurogenum acetyl xylan esterase from high-resolution X-ray diffraction

Acetyl xylan esterase from Penicillium purpurogenum, a single-chain 23 kDa member of a newly characterized family of esterases that cleaves side chain ester linkages in xylan, has been crystallized. The crystals diffract to better than 1 Å resolution at the Cornell High Energy Synchrotron Source (CHESS) and are highly stable in the synchrotron radiation. The space group is P2₁2₁2₁ and cell dimensions are a = 34.9 Å, b = 61.0 Å, c = 72.5 Å.     

Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2.

An extracellular p-coumaroyl esterase produced by the anaerobic fungus Neocallimastix strain MC-2 released p-coumaroyl groups from 0-[5-0-((E)-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-0-beta -D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX). The esterase was purified 121-fold from culture medium in successive steps involving ultrafiltration column chromatography on S-sepharose and hydroxylapatite, isoelectric focusing, and gel filtration. The native enzyme had an apparent mass of 11 kDa under nondenaturing conditions and a mass of 5.8 kDa under denaturing conditions, suggesting that the enzyme may exist as a dimer. The isoelectric point was 4.7, and the pH optimum was 7.2. The purified esterase had 100 times more activity towards PAXX than towards the analogous feruloyl ester (FAXX). The apparent Km and Vmax of the purified p-coumaroyl esterase for PAXX at pH 7.2 and 40 degrees C were 19.4 microM and 5.1 microM min(-1), respectively. p-Coumaroyl tetrasaccharides isolated from plant cell walls were hydrolyzed at rates similar to that for PAXX, whereas a dimer of PAXX was hydrolyzed at a rate 20-fold lower, yielding 4,4'-dihydroxy-alpha-truxillic acid as an end product. Ethyl and methyl p-coumarates were hydrolyzed at very slow rates, if at all. The purified esterase released p-coumaroyl groups from finely, but not coarsely, ground plant cell walls, and this activity was enhanced by the addition of xylanase and other cell wall-degrading enzymes.     

Characterization of an anaerobic fungus from llama faeces.

An anaerobic fungus was isolated from llama faeces. Based on its morphological characteristics, polyflagellated zoospores, extensive rhizoid system and the formation of monocentric colonies, the fungus is assigned to the genus Neocallimastix. Neocallimastix sp. L2 is able to grow on several poly-, oligo- and monosaccharides. It differs from other Neocallimastix isolates in its inability to ferment inulin. Neocallimastix sp. L2 requires CO2 for growth. In the presence of 100% CO2 in the gas phase glucose is fermented to H2, CO2, formate, acetate, lactate, succinate and ethanol (33.8, 15.4, 74.3, 69.2, 26.7, 8.2, and 28.7 mmol per 100 mmol glucose, respectively). Reduced sulphur compounds can be used as sulphur source and ammonium or amino acids as nitrogen source. The temperature range for glucose fermentation is from 37 to 42 degrees C with an optimum of around 38 degrees C. The pH range for glucose fermentation is from pH 6 to pH 8 with a broad optimum between pH 6.5 and pH 7.5. The zoospores of Neocallimastix sp. L2 contain ribosomal 'globules' and hydrogenosomes. In the kinetosomes of the zoospores spurs, scoops and skirts are visible. In both the rhizoids and the sporangia 'crystal bodies' and hydrogenosomes are present. Mitochondria were not detected in either of these life stages.     

Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus

Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan‐degrading organism whose genome encodes a large number of open reading frames annotated as fiber‐degrading enzymes. We have determined the three‐dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains—a C‐terminal SGNH domain and an N‐terminal jelly‐roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para‐nitrophenyl acetate and para‐nitrophenyl butyrate. The suite of fiber‐degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

SGNH hydrolase-type esterase domain containing Cbes-AcXE2: a novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii

Extremophiles - Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium, is rich in hydrolytic and accessory enzymes that can degrade untreated biomass, but the precise role of...     

Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp. strain JW/SL-YS485.

Two acetyl esterases (EC 3.1.1.6) were purified to gel electrophoretic homogeneity from Thermoanaerobacterium sp. strain JW/SL-YS485, an anaerobic, thermophilic endospore former which is able to utilize various substituted xylans for growth. Both enzymes released acetic acid from chemically acetylated larch xylan. Acetyl xylan esterases I and II had molecular masses of 195 and 106 kDa, respectively, with subunits of 32 kDa (esterase I) and 26 kDa (esterase II). The isoelectric points were 4.2 and 4.3, respectively. As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases I and II occurred at pH 7.0 and 80 degrees C and at pH 7.5 and 84 degrees C, respectively. Km values of 0.45 and 0.52 mM 4-methylumbelliferyl acetate were observed for acetyl xylan esterases I and II, respectively. At pH 7.0, the temperatures for the 1-h half-lives for acetyl xylan esterases I and II were 75 degrees and slightly above 100 degrees C, respectively.     

Heterologous expression and biochemical characterization of acetyl xylan esterase from Coprinopsis cinerea

Acetyl xylan esterase (AXE) from basidiomycete Coprinopsis cinerea Okayama 7 (#130) was functionally expressed in Pichia pastoris with a C-terminal tag under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 1.5 mg l^?1. Its molecular mass was estimated to be 65.5 kDa based on the SDS-PAGE analysis, which is higher than the calculated molecular mass of 40 kDa based on amino acid composition. In-silico analysis of the amino acid sequence predicted two potential N-glycosylation sites. Results from PNGase F deglycosylation and mass spectrum confirmed the presence of N-glycosylation on the recombinant AXE with predominant N-glycans HexNAc₂Hex_9–16. The recombinant AXE showed best activity at 40 °C and pH 8. It showed not only acetyl esterase activity with a K_m of 4.3 mM and a V_max of 2.15 U mg^?1 for hydrolysis of 4-nitrophenyl acetate but also a butyl esterase activity for hydrolysis of 4-nitrophenyl butyrate with a K_m of 0.11 mM and V_max of 0.78 U mg^?1. The presence of two additional amino acid residues at its native N-terminus was found to help stabilize the enzyme against the protease cleavages without affecting its activity.     

Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation

Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.     

Adsorption characteristics of cellulolytic enzymes from the anaerobic fungus Piromyces sp. strain E2 on microcrystalline cellulose.

Characteristics of the cellulolytic system of the anaerobic fungus Piromyces sp. strain E2 with respect to adsorption onto microcrystalline cellulose were examined. Cellulolytic enzymes were separated by gel filtration chromatography into a high-molecular-mass complex with an apparent mass of approximately 1,200 to 1,400 kDa and proteins of lower molecular weights. Adsorption of cellulolytic enzymes was not only very fast (within 2 min, equilibrium was attained) but also very effective: Avicelase, endoglucanase, and beta-glucosidase activities from the high-molecular-mass complex were almost completely removed by Avicel. Adsorption of these enzyme activities was proportional and appeared to obey the Langmuir isotherm. For Avicelase, endoglucanase, and beta-glucosidase activities, the maximum amounts adsorbed (Amax) and apparent adsorption constants (Kad) were 16.8, 600, and 33.5 IU/g and 284, 6.93 and 126 ml/IU, respectively. The results of this study strongly support the existence of a multiprotein enzyme complex. This complex was found not to be specifically associated with cell wall fragments as judged by chitin determination.     

Active site architecture of an acetyl xylan esterase indicates a novel cold adaptation strategy

SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504^T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a T_m value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs.     

Isolation of Extremely AT-Rich Genomic DNA and Analysis of Genes Encoding Carbohydrate-Degrading Enzymes from Orpinomyces sp. Strain PC-2

An effective method for extraction of intact genomic DNA from the extremely AT-rich polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogen-like storage polysaccharides using hexadecyltrimethylammonium bromide (CTAB) and high salt washes. The DNA was digested with various restriction enzymes and was suitable for use as a PCR template, for Southern blotting, and for genomic library construction. Genomic DNA analysis of three representative genes (celE, bgl1, and xynA) encoding (hemi-) cellulolytic enzymes of the fungus revealed multiplicity of family 5 endocellulase genes (celE-like), and family 1 β-glucosidase genes (bgl1-like), but only a single copy of family 11 xylanase gene (xynA).     

A serpin in the cellulosome of the anaerobic fungus Piromyces sp. strain E2

A gene encoding a novel component of the cellulolytic complex (cellulosome) of the anaerobic fungus Piromyces sp. strain E2 was identified. The encoded 538 amino acid protein, named celpin, consists of a signal peptide, a positively charged domain of unknown function followed by two fungal dockerins, typical for components of the extracellular fungal cellulosome. The C-terminal end consists of a 380 amino acid serine proteinase inhibitor (or serpin) domain homologue, sharing 30 % identity and 50 % similarity to vertebrate and bacterial serpins. Detailed protein sequence analysis of the serpin domain revealed that it contained all features of a functional serpin. It possesses the conserved amino acids present in more than 70 % of known serpins, and it contained the consensus of inhibiting serpins. Because of the confined space of the fungal cellulosome inside plant tissue and the auto-proteolysis of plant material in the rumen, the fungal serpin is presumably involved in protection of the cellulosome against plant proteinases. The celpin protein of Piromyces sp. strain E2 is the first non-structural, non-hydrolytic fungal cellulosome component. Furthermore, the celpin protein of Piromyces sp. strain E2 is the first representative of a serine proteinase inhibitor of the fungal kingdom.