Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Effect of trace metals on ethanol production from synthesis gas by the ethanologenic acetogen, <Emphasis Type="Italic">Clostridium</Emphasis><Emphasis Type="Italic">ragsdalei</Emphasis>

The effect of trace metal ions (Co²⁺, Cu²⁺, Fe²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²⁺. At optimum concentrations of WO₄ ⁻ and SeO₄ ⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄ ⁻ from the medium. Although increased concentration of Zn²⁺ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²⁺ concentration. Omitting Fe²⁺ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²⁺ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.     

Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis–Menten kinetics with a K _m of 0.1013 mM, V _max of 4.858 μmol min⁻¹, K _cat of 3.36 S⁻¹, and K _cat/K _m is 33,168 M⁻¹ S⁻¹. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol⁻¹ when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg²⁺, Pb²⁺, and Zn²⁺) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K _i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K _i = 0.056 μM.     

Genetic and biochemical characterization of a protease-resistant mesophilic β-mannanase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. S27

A β-mannanase gene, designated as man5S27, was cloned from Streptomyces sp. S27 using the colony polymerase chain reaction (PCR) method and expressed in Escherichia coli BL21 (DE3). The open reading frame consisted of 1,161 bp and encoded a 386-amino-acid polypeptide (Man5S27) with calculated molecular mass of 37.2 kDa. The encoded protein comprised a putative 38-residue signal peptide, a family 5 glycoside hydrolase domain, and a family 10 carbohydrate-binding module. Purified recombinant Man5S27 had high specific activity of 2,107 U mg⁻¹ and showed optimal activity at pH 7.0 and 65°C. The enzyme remained stable at pH 5.0–9.0 and had good thermostability at 50°C. The K _m values for locust bean gum and konjac flour were 0.16 and 0.41 mg ml⁻¹, with V _max values of 3,739 and 1,653 μmol min⁻¹ mg⁻¹, respectively. Divalent metal ions such as Mn²⁺, Zn²⁺, Ca²⁺, Pb²⁺, and Fe²⁺ enhanced the enzyme activity, but Ag⁺ and Hg²⁺ strongly inhibited the activity. Man5S27 also showed resistance to various neutral proteases (retaining >95% activity after proteolytic treatment for 2 h).     

Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.) heterotrophic tissues

Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K _m and V _max values of 82 ± 1.05 μM and 4.20 ± 0.20 nmol min⁻¹ mg⁻¹ protein, respectively. The enzyme’s pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells.     

A cold-active β-glucosidase (Bgl1C) from a sea bacteria <Emphasis Type="Italic">Exiguobacterium oxidotolerans</Emphasis> A011

By constructing a genomic library, a new gene encoding β-glucosidase (Bgl1C) was cloned from Exiguobacterium oxidotolerans A011, which was isolated from deep sea mud. The putative β-glucosidase gene consisted of an open reading frame (ORF) of 1,347 nucleotides, and encoded a protein of 448 amino acids with a predicted molecular weight of 51.6 kDa. Bgl1C belonged to the glycoside hydrolase family 1, and the deduced amino acid sequence displayed the highest identity (68%) to the β-glucosidase from Bacillus coahuilensis m4-4. Optimal conditions for activity were pH 7 and a temperature of 35°C and Bgl1C was stable in buffers ranging from pH 6.6 to 9. The specific activity, K _m, and V _max for the substrate p-nitrophenyl-β-d-glucopyranoside were 41 U mg⁻¹, 1.72 mg ml⁻¹ and 0.45 μg ml⁻¹ s⁻¹, respectively. Na⁺, Ca²⁺, EDTA and β-mercaptoethanol had no effect on the activity, while Hg²⁺, Cu²⁺, Co²⁺ strongly inhibited it. It is noteworthy that Bgl1C is a cold active enzyme that retains about 61% of its maximum activity at 10°C. Structural model of Bgl1C revealed that some amino acids (glycine, alanine, serine, valine) concerned with plasticity and flexibility were located around the active sites, this may contributed to the cold adaption of Bgl1C. These favorable features make Bgl1C a potential candidate for various industrial applications.     

The effects of copper on the photosynthetic response of <Emphasis Type="Italic">Phaeocystis cordata</Emphasis>

We investigated the effects of limiting (1.96 × 10⁻⁹ mol l⁻¹ total Cu, corresponding to pCu 14.8; where pCu = −log [Cu²⁺]) and toxic Cu concentrations up to 8.0 × 10⁻⁵ mol l⁻¹ total Cu (equivalent to pCu 9.5) on growth rates and photosynthetic activity of exponentially grown Phaeocystis cordata, using batch and semi-continuous cultures. With pulse amplitude modulated (PAM) fluorometry, we determined the photochemical response of P. cordata to the various Cu levels, and showed contrasting results for the batch and semi-continuous cultures. Although maximum photosystem II (PSII) quantum yield (Φ_M) was optimal and constant in the semi-continuous P. cordata, the batch cultures showed a significant decrease in Φ_M with culture age (0–72 h). The EC50 for the batch cultures was higher (2.0 × 10⁻¹⁰ mol l⁻¹, pCu9.7), than that for the semi-continuous cultures (6.3 × 10⁻¹¹ mol l⁻¹, pCu10.2). The semi-continuous cultures exhibited a systematic and linear decrease in Φ_M as Cu levels increased (for [Cu²⁺] < 1.0 × 10⁻¹² mol l⁻¹, pCu12.0), however, no effect of high Cu was observed on their operational PSII quantum yield (Φ′_M). Similarly, semi-continuous cultures exhibited a significant decrease in Φ_M, but not in Φ′_M, because of low-Cu levels. Thus, Cu toxicity and Cu limitation damage the PSII reaction centers, but not the processes downstream of PSII. Quenching mechanisms (NPQ and Q _n) were lower under high Cu relative to the controls, suggesting that toxic Cu impairs photo-protective mechanisms. PAM fluorometry is a sensitive tool for detecting minor physiological variations. However, culturing techniques (batch vs. semi-continuous) and sampling time might account for literature discrepancies on the effects of Cu on PSII. Semi-continuous culturing might be the most adequate technique to investigate Cu effects on PSII photochemistry.     

Characterization of trehalose synthase from <Emphasis Type="Italic">Corynebacterium nitrilophilus</Emphasis> NRC

Trehalose synthase (TSII) from Corynebacterium nitrilophilus NRC was successively purified by ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-100 columns. The specific activity of the trehalose synthase was increased ~200-fold, from 0.14 U mg⁻¹ protein to 28.3 U mg⁻¹ protein. TSII was found to be a monomeric protein with a molecular weight of 67–69 kDa. Characterization of the enzyme exhibited optimum pH and temperature were 7.5 and 35°C, respectively. The purified enzyme was stable from pH 6.6 to 7.8 and able to prolong its thermal stability up to 35°C. The enzyme activity was inhibited strongly by Zn²⁺, Hg²⁺ and Cu²⁺ and moderately by Ba²⁺, Fe²⁺, Pb²⁺ and Ni²⁺. Other metal ions Ca²⁺, Mg²⁺, Co²⁺, Mn²⁺ and EDTA had almost no effect.     

A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris

Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid β-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-β-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml⁻¹. The enzyme was acidophilic, with highest activity at pH 1.0–1.5, lower than any known mannanases, and optimal temperature for activity was 65°C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K _m, and V _max for locust bean gum substrate was 3,373 U mg⁻¹, 1.56 mg ml⁻¹, and 6,587.6 μmol min⁻¹ mg⁻¹, respectively. The enzymatic activity was not significantly affected by ions such as Ca²⁺, Cr³⁺, Co²⁺, Zn²⁺, Na⁺, K⁺, and Mg²⁺ and enhanced by Ni²⁺, Fe³⁺, Mn²⁺ and Ag⁺. These favorable properties make MAN5A a potential candidate for use in various industrial applications.     

Identification and characterization of the propanediol utilization protein PduP of Lactobacillus reuteri for 3-hydroxypropionic acid production from glycerol

Although the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol-fermenting microorganisms is still unclear, the propanediol utilization protein (PduP) of Lactobacillus species has been suggested to be a key enzyme in this regard. To verify this hypothesis, a pduP gene from Lactobacillus reuteri was cloned and expressed, and the encoded protein was characterized. Recombinant L. reuteri PduP exhibited broad substrate specificity including 3-hydroxypropionaldehyde and utilized both NAD⁺ and NADP⁺ as a cofactor. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37 °C. The K _m and V _max values for propionaldehyde in the presence of NAD⁺ were 1.18 mM and 0.35 U mg⁻¹, respectively. When L. reuteri pduP was overexpressed in Klebsiella pneumoniae, 3-HP production remarkably increased as compared to the wild-type strain (from 0.18 g L⁻¹ to 0.72 g L⁻¹) under shake-flask culture conditions, and the highest titer (1.38 g L⁻¹ 3-HP) was produced by the recombinant strain under batch fermentation conditions in a bioreactor. This is the first report stating the enzymatic properties of PduP protein and the probable role in biosynthesis of 3-HP in glycerol fermentation.     

A novel β-mannanase from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> A021: gene cloning,expression, purification and characterization

This paper describes the construction of a genomic library from the bacterium Pantoea agglomerans A021 and the subsequent cloning and expression of a novel mannanase gene (man26P). The gene consists of 1,047 bp and encodes a peptide (Man26P) of 348 amino acids with a calculated molecular mass of 38.5 kDa. Man26P is 63% identical with mannanase from Pectobacterium carotovorum at protein level and considered to be a member of the glycoside hydrolase family 26 (GH26). Man26P was expressed efficiently in E.coli BL21 (DE3) after induction with isopropylthiogalactoside (IPTG) and purified with a GST Bind Purification Kit. Maximum activity of purified Man26P was 514 U mg⁻¹, which was seen at pH 6.0 and a temperature of 55°C. Man26P was stable on exposure to buffers ranging from pH 4.0–10.0, and tolerant of temperature below 60°C. Zn²⁺, Mg²⁺ and Co²⁺ enhanced the activity, while Mn²⁺, Cu²⁺ and Hg⁺ had a negative effect. β-mercaptoethanol (1%) increased the activity twofold, while SDS (1%) inhibited it significantly. The enzyme showed optimal activity in a NaCl solution. The properties make it a candidate for various industrial applications.     

Callus formation,plant regeneration,and transient expression in the halophyte sea aster (<Emphasis Type="Italic">Aster tripolium</Emphasis> L.)

An in vitro regeneration and transient expression systems were developed for the halophyte sea aster (Aster tripolium L.), an important genetic resource for salt tolerance. Adventitious shoots were formed from both leaf explants and suspension-cultured cells in a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) basal salts containing 500 mg l⁻¹ casamino acids, and supplemented with 5.4 μM a-naphthaleneacetic acid (NAA) and 4.7 μM kinetin to the culture medium. Hyperhydricity of shoots was avoided by increasing the ventilation of the culture vessel. Root formation from shoots was promoted in the presence of 26.9 μM NAA. A high yield of protoplasts was isolated using 1% cellulase and 0.25% pectinase from both leaf mesophyll and suspension-cultured cells, and these were used for transient expression. The highest level of transient expression of the green fluorescent protein was obtained with 1 × 10⁵ protoplasts ml⁻¹, 25 μg batch⁻¹ of plasmid vector, and 30% polyethylene glycol 4,000.     

Identification and hexavalent chromium reduction characteristics of <Emphasis Type="Italic">Pannonibacter phragmitetus</Emphasis>

A hexavalent chromium [Cr(VI)] reducing bacterial strain was isolated from chromium-containing slag. It was identified as Pannonibacter phragmitetus based on physiological, biochemical characteristics and 16S rRNA gene sequence analysis. This bacterium displayed great Cr(VI) reduction capability. The Cr(VI) could be completely removed in 24 h under anaerobic condition when the initial concentration was 1,917 mg L⁻¹, with the maximum reduction rate of 562.8 mg L⁻¹ h⁻¹. The Cr(VI) reduction rate increased with the increase of Cr(VI) concentration. P. phragmitetus was able to use many carbon sources such as lactose, fructose, glucose, pyruvate, citrate, formate, lactate, NADPH and NADH as electron donors, among which the lactate had the greatest power to promote the reduction process. Zn²⁺, Cd²⁺ and Ni²⁺ inhibited, while Cu²⁺, Pb²⁺, Mn²⁺ and Co²⁺ stimulated the reduction. The optimum pH and temperature for reduction were 9.0 and 30 °C, respectively. The results indicated that this strain had great potential for application in the bioremediation of chromate-polluted soil and water systems.     

Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases

We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-β-d-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 °C, maintained more than 60% of maximal activity when assayed at pH 1.5–4.0, and had good thermal stability at 60 °C and remained stable at pH 1.0–6.0. The enzyme activity was enhanced in the presence of Ni²⁺ and β-mercaptoethanol and inhibited by some metal irons (Hg²⁺, Cu²⁺, Pb²⁺, Mn²⁺, Li⁺, and Fe³⁺) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-d-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg⁻¹, respectively. The apparent K _m and V _max values for beechwood xylan were 5.6 mg ml⁻¹ and 3,622 μmol min⁻¹ mg⁻¹, respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.     

Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata

The effect of polysaccharidases (κ-carrageenase, β-agarase, xylanase, cellulase) on the protein extraction from three rhodophytes has been studied. The kinetic parameters (apparent V _m, apparent K _m) and the optimum activity conditions (pH, temperature) of each enzyme were determined by using pure substrates. All the tested enzymes possess Michaelis Menten mechanism with estimated substrate saturating concentrations of 8 000 mg l⁻¹(carrageenan) for κ-carrageenase, 8 000 mg l⁻¹ (agar) for β-agarase, 5000 mg l⁻¹ (xylane) for β-xylanase and 6 000 mg l⁻¹ (carboxymethylcellulose) for cellulase. The optimum activity conditions are pH 6.5–6.8 at 45°C for carrageenase, pH 6–6.5 at 55°C for agarase, pH 5 at 55°C for xylanase and pH 3.8 at 50°C for cellulose. Different alga/enzymes couples (κ-carrageenase/Chondrus crispus, β-agarase/Gracilaria verrucosa, β-xylanase/Palmaria palmata) were tested under the optimum activity conditions. Alga/cellulase + specific enzyme (e.g. Chondrus crispus/carrageenase + cellulase) systems were also studied at the optimum activity conditions of a specific enzyme (e.g. carageenase). The use of the only cellulose was also tested on each alga. Except for Palmaria palmata, the highest protein yields were observed with the procedures using cellulase coupled with carrageenase or agarase for an incubation period limited to 2 h. The Chondrus crispus/carrageenase + cellulose and Gracilaria verrucosa/agarase + cellulase systems gave ten-fold and three-fold improvements, respectively, in protein extraction yield as compared to the enzyme-free blank procedure. The combined action of xylanase and cellulose on protein extraction from Palmaria palmata does not significantly improve protein yield. The best overall protein yield for P. palmata is for P. palmata/xylanase with a 14-h incubation time. This study shows the interest in the use of a polysaccharidase mixture for improving protein extractibility from certain rhodophytes. This biotechnology approach, adapted from procedures for protoplast production or enzymatic liquefaction of higher plants, could be tested as an alternative method to obtain proteins from seaweeds of nutritional interest.     

Heterologous expression and characterization of a recombinant thermostable alkylsulfatase (<Emphasis Type="Italic">sdsAP</Emphasis>)

A novel alkylsulfatase gene, sdsAP, was cloned from a newly isolated bacterium Pseudomonas sp. S9. It encoded a protein of 675 amino acids with a calculated molecular mass of 74.9 kDa. The protein contained a typical N-terminal signal peptide of 41 amino acid residues, followed by a metallo-β-lactamase like domain at the N-terminus and a SCP-2-like domain at the C-terminus. This domain organization mode suggested that it belonged to the type III sulfatase. The mature alkylsulfatase was overexpressed in Escherichia coli. The optimal temperature and pH of the recombinant SdsAP were 70°C and 9.0, respectively. Notably, at optimal conditions, the purified recombinant SdsAP had a high specific activity of 23.25 μmol min⁻¹ mg⁻¹, a K _{m (app)} of 264.3 μmol, and a V _{max (app)} of 33.8 μmol min⁻¹ mg⁻¹ for SDS. Additionally, it still retained more than 90% activity after incubation at 65°C for 1 h, which was much different from other alkylsulfatases reported. The recombinant enzyme hydrolyzed the primary alkyl sulfate such as sodium octyl sulfate and sodium dodecyl sulfate (SDS). It was a Zn²⁺-containing and Ca²⁺ activated alkylsulfatase. This is the first report to explore the various characteristics of the heterologous recombinant alkylsulfatase in details. These favorable properties could make SdsAP attractive to be useful in the degradation of SDS-containing waste.     

Purification and Characterization of Thermophilic Xylanase Isolated from the Xerophytic-<Emphasis Type="Italic">Cereus pterogonus</Emphasis> sp.

A thermo stable xylanase was purified and characterized from the cladodes of Cereus pterogonus plant species. The enzyme was purified to homogeneity by ammonium sulfate (80%) fractionation, ion exchange and size exclusion chromatography. The enzyme showed a final specific activity of 216.2 U/mg and the molecular mass of the protein was 80 KDa. The optimum pH and temperature for xylanase activity were 5.0 and 80 °C, respectively,. With oat spelt xylan as a substrate the enzyme yielded a Km value of 2.24 mg/mL and a Vmax of 5.8 μmol min⁻¹ mg⁻¹. In the presence of metal ions (1 mM) such as Co²⁺,Mn²⁺, Ni²⁺, Ca²⁺ and Fe³⁺ the activity of the enzyme increased, where as strong inhibition of the enzyme activity was observed with the use of Hg²⁺, Cd²⁺, Cu²⁺, while partial inhibition was noted with Zn²⁺ and Mg²⁺. The substrate specificity of the xylanase yielded maximum activity with oat spelt xylan.     

Effects of warm acclimation on serum osmolality,cortisol and hematocrit levels in the Antarctic fish, <Emphasis Type="Italic">Trematomus bernacchii</Emphasis>

Antarctic fish, such as the Trematomus bernacchii, living at −1.9°C maintain a serum osmolality of around 600 mOsm kg⁻¹, nearly twice that of temperate fish. Upon warm acclimation, Antarctic fish significantly lower their serum osmolality. It has been suggested that this response to warm acclimation is due to stress. The purpose of this study was to determine, whether upon warm acclimation there was a change in the levels of the stress hormone cortisol and hematocrit associated with the decrease in serum osmolality. T. bernacchii were warm acclimated up to 4 weeks and serum osmolality, cortisol and hematocrit were measured. Upon warm acclimation to +1.6 and +3.8°C over the course of 4 weeks, T. bernacchii significantly lowered their serum osmolality (from 547 ± 4 mOsm kg⁻¹ to 494 ± 6 and 489 ± 4 mOsm kg⁻¹, respectively), yet did not alter their serum cortisol (29 ± 6 nl ml⁻¹) or hematocrit (22 ± 1%) levels. These results suggest that warm acclimation does not induce a stress response in T. bernacchii.     

Conversion of glycerol to pyruvate by Escherichia coli using acetate- and acetate/glucose-limited fed-batch processes

We report the conversion of glycerol to pyruvate by E. coli ALS929 containing knockouts in the genes encoding for phosphoenolpyruvate synthase, lactate dehydrogenase, pyruvate formate lyase, the pyruvate dehydrogenase complex, and pyruvate oxidase. As a result of these knockouts, ALS929 has a growth requirement of acetate for the generation of acetyl CoA. In steady-state chemostat experiments using excess glycerol and limited by acetate, lower growth rates favored the formation of pyruvate from glycerol (0.60 g/g at 0.10 h⁻¹ versus 0.44 g/g at 0.25 h⁻¹), while higher growth rates resulted in the maximum specific glycerol consumption rate (0.85 g/g h at 0.25 h⁻¹ versus 0.59 g/g h at 0.10 h⁻¹). The presence of glucose significantly improved pyruvate productivity and yield from glycerol (0.72 g/g at 0.10 h⁻¹). In fed-batch studies using exponential acetate/glucose-limited feeding at a constant growth rate of 0.10 h⁻¹, the final pyruvate concentration achieved was about 40 g/L in 36 h. A derivative of ALS929 which additionally knocked out methylglyoxal synthase did not further increase pyruvate productivity or yield, indicating that pyruvate formation was not limited by accumulation of methylglyoxal.     

Purification and characterization of an endoglucanase from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> highly active against barley β-glucan and xyloglucan

An endoglucanase (1, 4-β-d glucan glucanohydrolase, EC 3.2.1.4) which was catalytically more active and exhibited higher affinity towards barley β-glucan, xyloglucan and lichenin as compared to carboxymethylcellulose (CMC) was purified from Aspergillus terreus strain AN₁ following ion-exchange and hydrophobic interaction chromatography and gel filtration. The purified enzyme (40-fold) that apparently lacked a cellulose-binding domain showed a specific activity of 60 μmol mg⁻¹ protein⁻¹ against CMC. The purified enzyme had a molecular weight of 78 and 80 KDa as indicated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and gel filtration, respectively, and a pI of 3.5. The enzyme was optimally active at temperature 60°C and pH 4.0, and was stable over a broad range of pH (3.0–5.0) at 50°C. The endoglucanase activity was positively modulated in the presence of Cu²⁺, Mg²⁺, Ca²⁺, Na⁺, DTT and mercaptoethanol. Endoglucanase exhibited maximal turn over number (K _cat) and catalytic efficiency (K _cat/km) of 19.11 × 10⁵ min⁻¹ and 29.7 × 10⁵ mM⁻¹ min⁻¹ against barley β-glucan as substrate, respectively. Hydrolysis of CMC and barley β-glucan liberated cellobiose, cellotriose, cellotetraose and detectable amount of glucose. The hydrolysis of xyloglucan, however, apparently yielded positional isomers of cellobiose, cellotriose and cellotetraose as well as larger oligosaccharides.