Efficient synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose by using new 2-deoxy-d-ribose-5-phosphate aldolase from Streptococcus suis with moderate activity and aldehyde tolerance

The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg⁻¹, K_M of 0.8 mM, and V_max of 32.9 μmol min⁻¹ mg⁻¹ toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.     

Characterization of a newly synthesized carbonyl reductase and construction of a biocatalytic process for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with high space-time yield

A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg^?1. The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K _m and V _max values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min^?1 mg^?1, respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous–organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3 % and e.e. of 99 % was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP⁺ to (S)-CHBE were 26.5 mmol L^?1 h^?1 g^?1 DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.     

Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N backbone and sidechain resonance assignments of a monomeric variant of <Emphasis Type="Italic">E. coli</Emphasis> deoxyribose-5-phosphate aldolase

Deoxyribose-5-phosphate aldolase (DERA) catalyses the reversible conversion of 2-deoxyribose-5-phosphate (dR5P) into glyceraldehyde-3-phosphate (G3P) and acetaldehyde. For industrial applications, this enzyme is used in organic synthesis for aldol reactions between acetaldehyde as a donor and a wide range of aldehydes as acceptors. Here, we present a near complete set of sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments of a 28 kDa monomeric variant of the Escherichia coli DERA. These assignments provide the basis for ongoing structural and dynamic analysis of DERA substrate specificity.     

First Thermostable Endo-β-1,4-Glucanase from Newly Isolated Xanthomonas sp. EC102

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 μmol min^?1 mg^?1, V _max for CMC was 1.44 μmol mg^?1 min^?1, and K _m values was 25.6 mg mL^?1. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²⁺ and Co²⁺. This is the first report about thermostable endoglucanase from Xanthomonas sp.     

In vitro and in vivo analyses of the role of the carboxysomal β-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 μmol mg^?1 protein min^?1) was only 30 % of that observed for the wild-type carboxysomes (0.71 μmol mg^?1 protein min^?1), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO₂ in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO₂ for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO₂ by the cytoplasmically localized Rubisco by counteracting the action of the CO₂ hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO₂ fixation by the cyanobacterium at CO₂ levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.     

Cloning, expression and characterization of a phosphoglucomutase/phosphomannomutase from sphingan-producing Sphingomonas sanxanigenens

Several strains of the genus Sphingomonas produce sphingans, extracellular polysaccharides used as thickeners, emulsifiers and gelling agents. The pgmG gene from Sphingomonas sanxanigenens, which encodes a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, was cloned and sequenced. The predicted amino acid sequence of the PgmG protein possessed 460 amino acids and a calculated molecular mass of 49.8 kDa, and it was 80 % identical to PGM/PMM from S. elodea. We overexpressed pgmG in Escherichia coli, and the purified protein displayed a K _m of 0.2 mM and a V _max of 1.3 μmol min^?1 mg^?1 with glucose 1-phosphate as substrate. The catalytic efficiency (K _cat/K _m) of PgmG was about 15-fold higher for glucose 1-phosphate than for mannose 1-phosphate. Overexpression of pgmG in S. sanxanigenens resulted in a 17 ± 0.3 % increase in sphingan production to ~12.5 g l^?1.     

Heterologous expression of β-xylosidase gene from Paecilomyces thermophila in Pichia pastoris

β-xylosidase from thermophilic fungi Paecilomyces thermophila was functionally expressed in Pichia pastoris with a his tag in the C-terminal under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 0.22 mg l^?1. Its molecular mass was estimated to be 52.3 kDa based on the SDS-PAGE analysis, which is 1.3 times higher than the predicted 39.31 kDa from its amino acid compositions, although no potential N- or O- glycosylation sites were predicted from its amino acid sequence. This is presumed to be caused by some unpredictable posttranslational modifications based on mass spectrum analysis of the recombinant protein. The enzyme was most active at 60 °C and pH 7. It showed not only a β-xylosidase activity with a K_m of 8 mM and a V_max of 54 μmol min^?1 mg^?1 for hydrolysis of p-nitrophenyl β-d-xylopyranoside but also an arabinofuranosidase activity (6.2 U mg^?1) on p-nitrophenyl arabinofuranoside.     

Characterization and expression of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris

Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml^?1 for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein ^?1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The K_m and V_max of the GlcN-6-P synthase towards d-fructose 6-phosphate were 2.8 mM and 6.9 μmol min^?1 mg^?1, respectively.     

Immobilization and characterization of carbonic anhydrase purified from E. coli MO1 and its influence on CO2 sequestration

The present investigation entails the immobilisation and characterisation of Escherichia coli MO1-derived carbonic anhydrase (CA) and its influence on the transformation of CO₂ to CaCO₃. CA was purified from MO1 using a combination of Sephadex G-75 and DEAE cellulose column chromatography, resulting in 4.64-fold purification. The purified CA was immobilised in chitosan-alginate polyelectrolyte complex (C-A PEC) with an immobilisation potential of 94.5 %. Both the immobilised and free forms of the enzyme were most active and stable at pH 8.2 and at 37 °C. The K _m and V _max of the immobilised enzyme were found to be 19.12 mM and 416.66 μmol min^?1 mg^?1, respectively; whereas, the K _m and V _max of free enzyme were 18.26 mM and 434.78 μmol min^?1 mg^?1, respectively. The presence of metal ions such as Cu²⁺, Fe²⁺, and Mg²⁺ stimulated the enzyme activity. Immobilised CA showed higher storage stability and maintained its catalytic efficiency after repeated operational cycles. Furthermore, both forms of the enzyme were tested for targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The amounts of CaCO₃ precipitated over free and immobilised CA were 267 and 253 mg/mg of enzyme, respectively. The results of this study show that immobilised CA in chitosan-alginate beads can be useful for CO₂ sequestration by the biomimetic route.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Characterization and constitutive expression of an acidic mesophilic endo-1,4-β-d-xylanohydrolase with high thermotolerance and catalytic efficiency in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl11 from Aspergillus niger, encoding a 188-residue xylanase of glycosyl hydrolase family 11, was constitutively expressed in Pichia pastoris. The recombinant Xyl11 exhibited optimal activity at pH 5.0 and 50 °C, and displayed more than 68 % of the maximum activity over the temperature range 35–65 °C and 33 % over the pH range 2.2–7.0. It maintained more than 40 % of the original activity after incubation at 90 °C (pH 5.0) for 10 min and more than 75 % of the original activity after incubation at pH 2.2–11.0 (room temperature) for 2 h. The specific activity, K _m and V _max of purified Xyl11 were 22,253 U mg^?1, 6.57 mg ml^?1 and 51,546.4 μmol min^?1 mg^?1. It could degrade xylan to a series of xylooligosaccharides and no xylose was detected. The recombinant enzyme with high stability and catalytic efficiency could work over wide ranges of pH and temperature and thus has the potential for various industrial applications.     

Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K _m and V _max values of 0.05 mM and 212.73 μmoL min^?1 mg^?1, respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe²⁺, Zn²⁺, Cd²⁺ and Mn²⁺, while Cu²⁺ acted as inducer. EDTA (10 mM) and NaN₃ (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL^?1 of laccase and 2 mM HBT.     

Characterization and constitutive expression of a novel endo-1,4-β-d-xylanohydrolase from Aspergillus niger in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl10 from Aspergillus niger, encoding a 308-residue mature xylanase belonging to glycosyl hydrolase family 10, was constitutively expressed in Pichia pastoris. The recombinant Xyl10 exhibited optimal activity at pH 5.0 and 60 °C with more than 50 % of the maximum activity from 40 to 70 °C. It retained more than 90 % of the original activity after incubation at 60 °C (pH 5.0) for 30 min and more than 74 % after incubation at pH 3.0–13.0 for 2 h (25 °C). The specific activity, K _m and V _max values for purified Xyl10 were, respectively, 3.2 × 10³ U mg^?1, 3.6 mg ml^?1 and 5.4 × 10³ μmol min^?1 mg^?1 towards beechwood xylan. The enzyme degraded xylan to a series of xylooligosaccharides and xylose. The recombinant enzyme with these properties has the potential for various industrial applications.     

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg^?1. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5–11.5) and temperature (40–90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K _m and V _max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min^?1 mg^?1 protein by analyzing Michaelis–Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.     

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min^?1 mg protein^?1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min^?1 mg protein^?1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (?arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.     

Characterization of the ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides

This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum KACC 20028^T in order to biotransform ginsenosides efficiently. The gene, termed as bglAm, encoding a β-glucosidase (BglAm) belonging to the glycoside hydrolase family 3 was cloned. bglAm consisted of 1,830 bp (609 amino acid residues) with a predicted molecular mass of 65,277 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The recombinant BglAm was purified with a GST·bind agarose resin and characterized. The optimum conditions of the recombinant BglAm were pH 7.0 and 37 °C. BglAm could hydrolyze the outer and inner glucose moieties at the C3 and C20 of the protopanaxadiol-type ginsenosides (i.e., Rb₁ and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F₂, and Rh₂(S) with various pathways. BglAm can effectively transform the ginsenoside Rb₁ to gypenoside XVII and Rd to F₂; the K _m values of Rb₁ and Rd were 0.69?±?0.06 and 0.45?±?0.02 mM, respectively, and the V _max values were 16.13?±?0.29 and 51.56?±?1.35 μmol min^?1 mg^?1 of protein, respectively. Furthermore, BglAm could convert the protopanaxatriol-type ginsenoside Re and Rg₁ into Rg₂(S) and Rh₁(S) hydrolyzing the attached glucose moiety at the C6 and C20 positions, respectively. These various ginsenoside-hydrolyzing pathways of BglAm may assist in producing the minor ginsenosides from abundant major ginsenosides.     

Purification and characterization of two distinct acidic phytases with broad pH stability from Aspergillus niger NCIM 563

Aspergillus niger NCIM 563 produced two different extracellular phytases (Phy I and Phy II) under submerged fermentation conditions at 30°C in medium containing dextrin-glucose-sodium nitrate-salts. Both the enzymes were purified to homogeneity using Rotavapor concentration, Phenyl-Sepharose column chromatography and Sephacryl S-200 gel filtration. The molecular mass of Phy I and II as determined by SDS–PAGE and gel filtration were 66, 264, 150 and 148 kDa respectively, indicating that Phy I consists of four identical subunits and Phy II is a monomer. The pI values of Phy I and II were 3.55 and 3.91, respectively. Phy I was highly acidic with optimum pH of 2.5 and was stable over a broad pH range (1.5–9.0) while Phy II showed a pH optimum of 5.0 with stability in the range of pH 3.5–9.0. Phy I exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. Similarly Phy II was strongly inhibited by Ag⁺, Hg²⁺ (1 mM) metal ions and Phy I was partially inhibited. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF also indicated that both the proteins were totally different. The K _m for Phy I and II for sodium phytate was 2.01 and 0.145 mM while V _max was 5,018 and 1,671 μmol min^?1 mg^?1, respectively. The N-terminal amino acid sequences of Phy I and Phy II were FSYGAAIPQQ and GVDERFPYTG, respectively. Phy II showed no homology with Phy I and any other known phytases from the literature suggesting its unique nature. This, according to us, is the first report of two distinct novel phytases from Aspergillus niger.     

Arginine Metabolising Enzymes as Therapeutic Tools for Alzheimer’s Disease: Peptidyl Arginine Deiminase Catalyses Fibrillogenesis of β-amyloid Peptides

The accumulation of arginine in the cerebrospinal fluid and brains of patients suffering from acute neurodegenerative diseases like Alzheimer’s disease, point to defects in the metabolic pathways involving this amino acids. The deposits of neurofibrillary tangles and senile plaques perhaps as a consequence of fibrillogenesis of β-amyloid peptides has also been shown to be a hallmark in the aetiology of certain neurodegenerative diseases. Peptidylarginine deiminase (PAD II) is an enzyme that uses arginine as a substrate and we now show that PAD II not only binds with the peptides Aβ_1-40, Aβ_22-35, Aβ_17-28, Aβ_25-35 and Aβ_32-35 but assists in the proteolytic degradation of these peptides with the concomitant formation of insoluble fibrils. PAD was purified in 12.5% yield and 137 fold with a specific activity of 59 μmol min^?1?mg^?1 from bovine brain by chromatography on diethylaminoethyl (DEAE)-Sephacel. Characterisation of the enzyme gave a pH and temperature optima of 7.5°C and 68°C, respectively, and the enzyme lost 50% activity within 38 min at this temperature. Michaelis-Menten kinetics established a V _max and K _m of 1.57 μmol min^?1?ml^?1 and 1.35 mM, respectively, with N-benzoyl arginine ethyl ester as substrate. Kinetic analysis was used to measure the affinity (K _i) of the amyloid peptides to PAD with values between 1.4 and 4.6 μM. The formation of Aβ fibrils was rate limiting involving an initial lag time of about 24 h that was dependent on the concentration of the amyloid peptides. Turbidity measurements at 400 nm, Congo Red assay and Thioflavin-T staining fluorescence were used to establish the aggregation kinetics of PAD-induced fibril formation.     

Molecular cloning and characterization of a GH11 endoxylanase from Chaetomium globosum, and its use in enzymatic pretreatment of biomass

An endo-1,4-β-xylanase gene, xylcg, was cloned from Chaetomium globosum and successfully expressed in Escherichia coli. The complete gene of 675 bp was amplified, cloned into the pET 28(a) vector, and expressed. The optimal conditions for the highest activity of the purified recombinant XylCg were observed at a temperature of 40 °C and pH of 5.5. Using oat-spelt xylan, the determined K _m, V _max, and k _cat/K _m values were 0.243 mg?ml^?1, 4,530 U?mg^?1 protein, and 7,640 ml?s^?1?mg^?1, respectively. A homology model and sequence analysis of XylCg, along with the biochemical properties, confirmed that XylCg belongs to the GH11 family. Rice straw pretreated with XylCg showed 30 % higher conversion yield than the rice straw pretreated with a commercial xylanase. Although xylanases have been characterized from fungal and bacterial sources, C. globosum XylCg is distinguished from other xylanases by its high catalytic efficiency and its effectiveness in the pretreatment of lignocellulosic biomass.