Purification and characterization of a cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3

Purification of a cis-epoxysuccinic acid hydrolase was achieved by ammonium sulfate precipitation, ionic exchange chromatography, hydrophobic interaction chromatography followed by size-exclusion chromatography. The enzyme was purified 177-fold with a yield of 14.4%. The apparent molecular mass of the enzyme was determined to be 33 kDa under denaturing conditions. The optimum pH for enzyme activity was 7.0, and the enzyme exhibited maximum activity at about 45 °C in 50 mM sodium phosphate buffer (pH 7.5). EDTA and o-phenanthrolin inhibited the enzyme activity remarkably, suggesting that the enzyme needs some metal cation to maintain its activity. Results of inductively coupled plasma mass spectrometry analysis indicated that the cis-epoxysuccinic acid hydrolase needs Zn²⁺ as a cofactor. Eight amino acids sequenced from the N-terminal region of the cis-epoxysuccinic acid hydrolase showed the same sequence as the N-terminal region of the beta subunit of the cis-epoxysuccinic acid hydrolase obtained from Alcaligenes sp.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning,expression, and biochemical characterization

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K _m and k _cat values of 0.197 mM and 2,804.32 s^?1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn²⁺ and inhibited by Cu²⁺, Zn²⁺, Ni²⁺, and Fe²⁺. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.     

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.     

Recombinant xylanase from Streptomyces coelicolor Ac-738: characterization and the effect on xylan-containing products

A xylanase gene was isolated from the genomic DNA of Streptomyces coelicolor Ac-738. The 723-bp full-length gene encoded a 241-amino acid peptide consisting of a 49-residue putative TAT signal peptide and a glycoside hydrolase family-11 domain. The mature enzyme called XSC738 was expressed in Escherichia coli M15[pREP4]. The electrophoretically homogeneous protein with a specific activity of 167 U/mg for beechwood xylan was purified. The pH optimum of XSC738 was at pH 6; a high activity was retained within a pH range of 4.5–8.5. The enzyme was thermostable at 50–60 °C and retained an activity at pH 3.0–7.0. Xylanase XSC738 was activated by Mn²⁺, Co²⁺ and largely inhibited by Cd²⁺, SDS and EDTA. The products of xylan hydrolysis were mainly xylobiose, xylotriose, xylopentaose and xylohexose. Xylotetraose appeared as a minor product. Processing of such agricultural xylan-containing products as wheat, oats, soy flour and wheat bran by xylanase resulted in an increased content of sugars.     

A Review of: “HPLC of Biological Macromolecules (Methods and Applications) K.M. Goodring F.E. Regnier,Eds Marcel Dekker,New York, 1990; hardbound, 676 pages, $150”

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0–9.0) and temperature (30–90°C). From the thermal inactivation studies in the range 60–75°C, the half-life (t_1/2) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol^?1. It showed higher specificity with catechol (K_m = 8 mM) as compared to 4-methylcatechol (K_m = 10 mM). Among metal ions and reagents tested, Cu²⁺, Fe²⁺, Hg²⁺, Mn²⁺, Ni²⁺, protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K⁺, Na⁺, Co²⁺, kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Purification and characterization of a class II α-Mannosidase from Moringa oleifera seed kernels

α-Mannosidase (EC. 3.2.1.114) belonging to class II glycosyl hydrolase family 38 was purified from Moringa oleifera seeds to apparent homogeneity by conventional protein purification methods followed by affinity chromatography on Con A Sepharose and size exclusion chromatography. The purified enzyme is a glycoprotein with 9.3 % carbohydrate and exhibited a native molecular mass of 240 kDa, comprising two heterogeneous subunits with molecular masses of 66 kDa (α-larger subunit) and 55 kDa (β-smaller subunit). Among both the subunits only larger subunit stained for carbohydrate with periodic acid Schiff’s staining. The optimum temperature and pH for purified enzyme was 50 °C and pH 5.0, respectively. The enzyme was stable within the pH range of 3.0–7.0. The enzyme was inhibited by EDTA, Hg²⁺, Ag²⁺, and Cu²⁺. The activity lost by EDTA was completely regained by addition of Zn²⁺. The purified enzyme was characterized in terms of the kinetic parameters K _m (1.6 mM) and V _max (2.2 U/mg) using para-nitrophenyl-α-D-mannopyranoside as substrate. The enzyme was very strongly inhibited by swainsonine (SW) at 1 μM concentration a class II α-Mannosidase inhibitor, but not by deoxymannojirimycin (DMNJ). Chemical modification studies revealed involvement of tryptophan at active site. The inhibition by SW and requirement of the Zn²⁺ as a metal ion suggested that the enzyme belongs to class II α-Mannosidase.     

Induction,purification, and characterization of a thermo and pH stable laccase from Abortiporus biennis J2 and its application on the clarification of litchi juice

A fungus J2 producing laccase with high yield was screened in soils and identified as Abortiporus biennis. The production of laccase was induced by 0.1 mM Cu²⁺, 0.1 mM tannic acid, and 0.5 M ethanol. The laccase from Abortiporus biennis J2 was purified to electrophoretic homogeneity by a couple of steps. The N-terminal amino acid sequence of the enzyme was AIGPTADLNISNADI. The properties of the purified laccase were investigated. The result showed the laccase from Abortiporus biennis J2 is a thermo and pH stable enzyme. The laccase activity was inhibited by Hg²⁺, Cd²⁺, Fe²⁺, Ag⁺, Cu²⁺, and Zn²⁺, while promoted by Mg²⁺, Mn²⁺ at 10 mM level. Purified laccase was used to the clarification of litchi juice. After treatment with this laccase, the phenolic content of litchi juice had been found to be greatly reduced along with an increase in the clarity of the juice. The result indicated the potential of this laccase for application in juice procession.     

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.     

Functional expression and characterization of a chitinase from the marine archaeon Halobacterium salinarum CECT 395 in Escherichia coli

The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6–8.5) and temperature (25–45 °C) ranges. Protein activity was stimulated by the metal ions Mg⁺², K⁺, and Ca⁺² and strongly inhibited by Mn⁺². HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20 % of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc)₃, p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.     

Influence of temperature,pH and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis

The bark beetle Dendroctonus armandi is able to kill living Pinus armandi and has caused serious damage to pine forest in Northern China. As the most important symbiotic fungus of D. armandi, Leptographium qinlingensis plays an important role in the invasion process of the bark beetle. The laccase secreted by it are involved in lignin degradation to provide utilizable nutrition for D. armandi, and catalyze some biochemical reactions, causing the damages of tree tissue. In present study, the extracellular laccase of L. qinlingensis was purified by using the ammonium sulfate precipitation and DEAE-cellulose (DE-52) column chromatography. Furthermore, the effects of temperature, pH value and metal ions on it were investigated and characterized. The purified enzyme exerted its optimal activity with guaiacol. The catalytic efficiencies K_m and V_max determined for substrate guaiacol were 15.4 μM and 372.9 IU mg^?1, respectively. The optimum pH and temperature for the purified enzyme was 4.4 and 45 °C, respectively, with the highest enzyme specific activity of 7,000 IU mg^?1. Moreover, the metal ions, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺, Fe²⁺ and Cd²⁺, especially Hg²⁺, showed significantly inhibition effects on its activity. To understand the characteristics of this laccase might provide an opportunity and theoretical basis to promote integrated pest management of D. armandi.     

β-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties

The gene encoding the cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) of Paenibacillus illinoisensis was isolated, cloned, sequenced and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34-residues. The deduced amino acid sequence of the CGTase from P. illinoisensis ZY-08 exhibited highest identity (99 %) to the CGTase sequence from Bacillus licheniformis (P14014). The four consensus regions of carbohydrate converting domain and Ca²⁺ binding domain could be identified in the sequence. The CGTase was purified by using cold expression vector, pCold I, and His-tag affinity chromatography. The molecular weight of the purified enzyme was about 74 kDa. The optimum temperature and pH of the enzyme were 40 °C and pH 7.4, respectively. The enzyme activity was increased by the addition of Ca²⁺ and inhibited by Ba²⁺, Cu²⁺, and Hg²⁺. The K _m and V _max values calculated were 0.48 mg/ml and 51.38 mg of β-cyclodextrin/ml/min. The ZY-08 and recombinant readily converted soluble starch to β-cyclodextrin but ZY-08 did not convert king oyster mushroom powder and enoki mushroom powder. However the recombinant CGTase converted king oyster mushroom powder and enoki mushroom powder to β-cyclodextrin.     

Production and characterization of laccase from Pleurotus ferulae in submerged fermentation

Pleurotus ferulae is a mushroom typically found in arid steppe that is distributed widely in the Junggar Basin of Xinjiang, China. In this work, laccase production by P. ferulae JM30X was optimized in terms of medium composition and culture conditions. After optimization, the highest laccase activity obtained was 6,832.86 U/L. A single isozyme with a molecular weight of 66 kDa was observed by SDS-PAGE and native-PAGE. Optimum pH and temperature were 3.0 and 50–70 °C, respectively. The best laccase substrate was ABTS, for which the Michaelis-Menten constant (K _m) and catalytic efficiency (K _cat/K _m) value for P. ferulae laccase were 0.193 mM and 2.73?×?10⁶ (mM s)^?1, respectively. The activity of purified laccase was increased by more than four-fold by Cu²⁺, Mn²⁺ and Mg²⁺, while it was completely inhibited by Fe²⁺ and Fe³⁺. The production of laccase was influenced by the initial pH and K⁺ concentration, and the activity of purified laccase was enhanced by Cu²⁺, Mn²⁺ and Mg²⁺. This Pleurotus genus laccase from P. ferulae JM30X was analyzed by MS spectrum and the results are conducive to furthering our understanding of Pleurotus genus laccases.     

The plant Selaginella moellendorffii possesses enzymes for synthesis and hydrolysis of the compatible solutes mannosylglycerate and glucosylglycerate

A mannosylglycerate synthase (MgS) gene detected in the genome of Selaginella moellendorffii was expressed in E. coli and the recombinant enzyme was purified and characterized. A remarkable and unprecedented feature of this enzyme was the ability to efficiently synthesize mannosylglycerate (MG) and glucosylglycerate (GG) alike, with maximal activity at 50 °C, pH 8.0 and with Mg²⁺ as reaction enhancer. We have also identified a novel glycoside hydrolase gene in this plant’s genome, which was functionally confirmed to be highly specific for the hydrolysis of MG and GG and named MG hydrolase (MgH), due to its homology with bacterial MgHs. The recombinant enzyme was maximally active at 40 °C and at pH 6.0–6.5. The activity was independent of cations, but Mn²⁺ was a strong stimulator. Regardless of these efficient enzymatic resources we could not detect MG or GG in S. moellendorffii or in the extracts of five additional Selaginella species. Herein, we describe the properties of the first eukaryotic enzymes for the synthesis and hydrolysis of the compatible solutes, MG and GG.     

The acid tolerant and cold-active β-galactosidase from Lactococcus lactis strain is an attractive biocatalyst for lactose hydrolysis

The gene encoding the β-galactosidase from the dairy Lactococcus lactis IL1403 strain was cloned, sequenced and overexpressed in Escherichia coli. The purified enzyme has a tetrameric arrangement composed of four identical 120 kDa subunits. Biochemical characterization showed that it is optimally active within a wide range of temperatures from 15 to 55 °C and of pH from 6.0 to 7.5. For its maximal activity this enzyme requires only 0.8 mM Fe²⁺ and 1.6 mM Mg²⁺. Purified protein displayed a high catalytic efficiency of 102 s^?1 mM^?1 for lactose. The enzyme stability was increased by immobilization mainly at low pH (from 4.0 to 5.5) and high temperatures (55 and 60 °C). The bioconversion of lactose using the L. lactis β-galactosidase allows the production of lactose with a high bioconversion rate (98 %) within a wide range of pH and temperature.     

Overexpression of acid protease of <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and characterization of the recombinant acid protease for skimmed milk clotting

The gene encoding an acid protease natively produced by Saccharomycopsis fibuligera was cloned and overexpressed in Yarrowia lipolytica and the resultant recombinant acid protease was purified and characterized. The molecular mass of the purified enzyme was estimated as 94.8 kDa by gel filtration chromatography. The optimal pH and temperature of the purified acid protease were 3.5 and 33°C, respectively, and the enzyme was very stable over a pH range of 1.0 ∼ 3.0. The recombinant acid protease was activated by Zn²⁺, but was inhibited by Hg²⁺, Fe²⁺, Fe³⁺, and Mg²⁺, EDTA, EGTA, iodoacetic acid, and pepstatin. The purified recombinant acid protease from the positive transformant 71 had high milk clotting activity, suggesting that it may be used as a rennet substitute in the cheese industry.     

Purification and characterization of a thermostable aliphatic amidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1

Amidases catalyze the hydrolysis of amides to free carboxylic acids and ammonia. Hyperthermophilic archaea are a natural reservoir of various types of thermostable enzymes. Here, we report the purification and characterization of an amidase from Pyrococcus yayanosii CH1, the first representative of a strict-piezophilic hyperthermophilic archaeon that originated from a deep-sea hydrothermal vent. An open reading frame that encoded a putative member of the nitrilase protein superfamily was identified. We cloned and overexpressed amiE in Escherichia coli C41 (DE3). The purified AmiE enzyme displayed maximal activity at 85 °C and pH 6.0 (NaH₂PO₄–Na₂HPO₄) with acetamide as the substrate and showed activity over the pH range of 4–8 and the temperature range of 4–95 °C. AmiE is a dimer and active on many aliphatic amide substrates, such as formamide, acetamide, hexanamide, acrylamide, and l-glutamine. Enzyme activity was induced by 1 mM Ca²⁺, 1 mM Al³⁺, and 1–10 mM Mg²⁺, but strongly inhibited by Zn²⁺, Cu²⁺, Ni²⁺, and Fe³⁺. The presence of acetone and ethanol significantly decreased the enzymatic activity. Neither 5 % methanol nor 5 % isopropanol had any significant effect on AmiE activity (99 and 96 % retained, respectively). AmiE displayed amidase activity although it showed high sequence homology (78 % identity) with the known nitrilase from Pyrococcus abyssi. AmiE is the most characterized archaeal thermostable amidase in the nitrilase superfamily. The thermostability and pH-stability of AmiE will attract further studies on its potential industrial applications.     

Purification,characterization, and overexpression of an endo-1,4-β-mannanase from thermotolerant <Emphasis Type="Italic">Bacillus</Emphasis> sp. SWU60

Endo-β-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the β-1,4-linkage in the mannan backbone and release short β-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300 ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38 kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60?°C, respectively. The enzyme was stable up to 60?°C for 1 h and at pH 5–9 at 4?°C for 16 h. Its enzyme activity was inhibited by Hg²⁺. The full-length mans2 gene was 1,008 bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.     

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.     

Purification and partial characterization of NAD aminohydrolase from Aspergillus oryzae NRRL447

Aspergillus oryzae aminohydrolase free acid phosphodiesterase catalyzes nicotinamide adenine dinucleotide to deamino-NAD and ammonia. The enzyme was purified to homogeneity by a combination of acetone precipitation, anion exchange chromatography and gel filtration chromatography. The enzyme was purified 230.5 fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of MW 94 kDa. The enzyme displayed maximum activity at pH 5 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable up to 40 °C. The enzyme activity was enhanced slightly by addition of Na⁺ and K⁺, whereas inhibited strongly by addition of Ag⁺, Mn²⁺, Hg²⁺ and Cu²⁺ to the reaction mixtures. The enzyme hydrolyzes several substrates, suggesting a probable non-specific nature. The enzyme catalyzes the hydrolytic cleavage of amino group of NAD, adenosine, AMP, CMP, GMP, adenosine, cytidine and cytosine to the corresponding nucleotides, nucleosides or bases and ammonia. The substrate concentration–activity relationship is the hyperbolic type and the apparent K_m and K_cat for the tested substrates were calculated.