Molecular characterization of the Bambusa oldhamii BoPAL3–encoded phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase (PAL), which is ubiquitous in plants, catalyzes the formation of trans-cinnamic acid via the nonoxidative deamination of l-phenylalanine. Bambusa oldhamii contains four different forms of PAL proteins that differ in substrate specificity. Full-length BoPAL3 cDNA was cloned by a combination bamboo cDNA library screening and PCR-based cloning methods. Sequence alignment showed high homology between the deduced amino acid sequences of the BoPAL2 and BoPAL3 proteins (90%). Obvious PAL and tyrosine ammonia-lyase (TAL) activities were detected in Escherichia coli Top10 expressing recombinant BoPAL3 protein. Size-exclusion chromatography and denatured SDS–PAGE showed that the estimated molecular mass of recombinant BoPAL3 and the subunit form were approximately 330 kDa and 80 kDa, indicating that BoPAL3 presents as a tetrameric protein. The optimum temperature and pH for BoPAL3 activity were 50 °C and 8.5, respectively. The K_m value of BoPAL3 for l-phenylalanine was 200 μM.     

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.     

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.     

Cloning of Intron-Removed Enolase Gene and Expression,Purification, Kinetic Characterization of the Enzyme from Theileria annulata

Tropical theileriosis is a disease caused by infection with an apicomplexan parasite, Theileria annulata, and giving rise to huge economic losses. In recent years, parasite resistance has been reported against the most effective antitheilerial drug used for the treatment of this disease. This emphasizes the need for alternative methods of treatment. Enolase is a key glycolytic enzyme and can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in T. annulata enolase gene was removed by PCR-directed mutagenesis, and the gene was first cloned into pGEM-T Easy vector and then subcloned into pLATE31 vector, and expressed in Escherichia coli cells. The enzyme was purified by affinity chromatography using Ni–NTA agarose column. Steady-state kinetic parameters of the enzyme were determined using GraFit 3.0. High quantities (~65 mg/l of culture) of pure recombinant T. annulata enolase have been obtained in a higly purified form (>95 %). Homodimer form of purified protein was determined from the molecular weights obtained from a single band on SDS-PAGE (48 kDa) and from size exclusion chromatography (93 kDa). Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of ~40 U/mg, K _m: 106 μM, k_cat: 37 s^?1, and k _cat/K _m: 3.5 × 10⁵ M^?1 s^?1. These values have been determined for the first time from this parasite enzyme, and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization toward design of new antitheilerial drugs.     

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.     

Isolation and characterization of novel ginsenoside-hydrolyzing glycosidase from Microbacterium esteraromaticum that transforms ginsenoside Rb2 to rare ginsenoside 20(S)-Rg3

Ginsenoside Rb2 was transformed by recombinant glycosidase (Bgp2) into ginsenosides Rd and 20(S)-Rg3. The bgp2 gene consists of 2,430 bp that encode 809 amino acids, and this gene has homology to the glycosyl hydrolase family 2 protein domain. SDS-PAGE was used to determine that the molecular mass of purified Bgp2 was 87 kDa. Using 0.1 mg ml^?1 of enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg ml^?1 ginsenoside Rb2 was transformed into 0.47 mg ml^?1 ginsenoside 20(S)-Rg3 within 120 min, with a corresponding molar conversion yield of 65 %. Bgp2 hydrolyzed the ginsenoside Rb2 along the following pathway: Rb2 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb2 to ginsenoside 20(S)-Rg3 using the recombinant glycosidase.     

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.     

A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning,heterologous expression and biochemical characterization

A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25 %) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39 % activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93 % activity even at 4 M NaCl. The catalytic efficiency (k _cat/K _m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg^?1 s^?1 (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl₂. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.     

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25–60 °C and pH 3.0–9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL^?1. In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.     

Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

A new gene encoding a superoxide dismutase (SOD) was identified from a thermophile Geobacillus sp. EPT3 isolated from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 437 amino acid residues. It was cloned, overexpressed in Escherichia coli (DE3), and the recombinant protein was purified to homogeneity. Geobacillus sp. EPT3 SOD was of the manganese-containing SOD type, as judged by the insensitivity of the recombinant enzyme to both KCN and H₂O₂, and the activity analysis of Fe or Mn reconstituted SODs by polyacrylamide gel electrophoresis. The recombinant SOD was determined to be a homodimer with monomeric molecular mass of 59.0 kDa. In comparison with other Mn–SODs, the manganese-binding sites are conserved in the sequence (His260, His308, Asp392, His396). The recombinant enzyme had high thermostability at 50 °C. It retained 57 % residual activity after incubation at 90 °C for 1 h, which indicated that this SOD was thermostable. The enzyme also showed striking stability over a wide range of pH 5.0–11.0. At tested conditions, the recombinant SOD from Geobacillus sp. EPT3 showed a relatively good tolerance to some inhibitors, detergents, and denaturants, such as β-mercaptoethanol, dithiothreitol, phenylmethylsulfonyl fluoride, Chaps, Triton X-100, urea, and guanidine hydrochloride.     

Cloning, Large Scale Over-Expression in E. coli and Purification of the Components of the Human LAT 1 (SLC7A5) Amino Acid Transporter

The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni²⁺-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.     

Cloning, Expression and Characterization of a Trehalose Synthase Gene From Rhodococcus opacus

Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 °C and pH optimum around 7.0. Kinetic analysis revealed that the K _m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k _cat/K _m. Both 1 and 10 mM of Hg²⁺, Cu²⁺ and Al³⁺ could inhibit the TreS activity, while only 1 mM of Ca²⁺ and Mn²⁺ could increase its activity. Five amino acid residues, Asp²⁴⁴, Glu²⁸⁶, Asp³⁵⁴, His¹⁴⁷ and His³⁵³, were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.     

Isolation and characterization of a thermostable esterase from a metagenomic library

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₁₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K _m and k _cat of 0.33 mM and 36.21 s^?1, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.     

Recombinant expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli

The goal of this work was to produce high levels of endoglucanase in Escherichia coli for its potential usage in different industrial applications. Endoglucanase gene was amplified from genomic DNA of Bacillus subtilis JS2004 by PCR. The isolated putative endoglucanase gene consisted of an open reading frame of 1,701 nucleotides and encoded a protein of 567 amino acids with a molecular mass of 63-kDa. The gene was cloned into pET-28a(+) and expressed in E. coli BL21 (DE3). Optimum temperature and pH of the recombinant endoglucanase were 50 °C and 9, respectively which makes it very attractive for using in bio-bleaching and pulp industry. It had a K _M of 1.76 μmol and V _max 0.20 μmol/min with carboxymethylcellulose as substrate. The activity of recombinant endoglucanse was enhanced by Mg²⁺, Ca²⁺, isopropanol and Tween 20 and inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ni²⁺ and SDS. The activity of this recombinant endoglucanase was significantly higher than wild type. Therefore, this recombinant enzyme has potential for many industrial applications involving biomass conversions, due to characteristic of broad pH and higher temperature stability.     

Cloning,expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1

Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β-d-galactopyranoside but not p-nitrophenyl-α-d-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K _m and V _max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and ¹³C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.     

Molecular cloning, expression, purification and characterization of thioredoxin from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178

Thioredoxin (Trx) is a highly conserved and multi-functional protein that plays a pivotal role in maintaining the redox state of the cell and in protecting the cell against oxidative stress. Trx gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178 was cloned and expressed as soluble protein in Escherichia coli (designated as PsTrx). Trx gene consisted of an open reading frame of 324-bp nucleotides encoding a protein of 108 amino acids with a calculated molecular mass of 11.88 kDa. The deduced protein included the conserved Cys–Gly–Pro–Cys active-site sequence. After purification by a single step Ni–NTA affinity chromatography, recombinant PsTrx with a high specific activity of 96.67 U/mg was obtained. The purified PsTrx had an optimal temperature and pH of 25 °C and 7.0, respectively, and showed about 55 % of the residual catalytic activity even at 0–10 °C. It had high tolerance to a wide range of NaCl concentrations (0–2 M NaCl) and was stable in the presence of H₂O₂. This research suggested that PsTrx displayed unique catalytic properties.     

SeGSTo,a novel glutathione <Emphasis Type="Italic">S</Emphasis>-transferase from the beet armyworm (<Emphasis Type="Italic">Spodoptera exigua</Emphasis>), involved in detoxification and oxidative stress

Members of the glutathione S-transferase superfamily can protect organisms against oxidative stress. In this study, we characterized an omega glutathione S-transferase from Spodoptera exigua (SeGSTo). The SeGSTo gene contains an open reading frame (ORF) of 744 nucleotides encoding a 248-amino acid polypeptide. The predicted molecular mass and isoelectric point of SeGSTo are 29007 Da and 7.74, respectively. Multiple amino acid sequence alignment analysis shows that the SeGSTo sequence is closely related to the class 4 GSTo of Bombyx mori BmGSTo4 (77 % protein sequence similarity). Homologous modeling and molecular docking reveal that Cys35 may play an essential role in the catalytic process. Additionally, the phylogenetic tree indicates that SeGSTo belongs to the omega group of the GST superfamily. During S. exigua development, SeGSTo is expressed in the midgut of the fifth instar larval stage, but not in the epidermis or fat body. Identification of recombinant SeGSTo via SDS-PAGE and Western blot shows that its molecular mass is 30 kDa. The recombinant SeGSTo was able to protect super-coiled DNA from damage in a metal-catalyzed oxidation (MCO) system and catalyze the 1-chloro-2,4-dinitrobenzene (CDNB), but not 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrophenethyl bromide (4-NPB), or 4-nitrobenzyl chloride (4-NBC). The optimal reaction pH and temperature were 8 and 50 °C, respectively, in the catalysis of CDNB by recombinant SeGSTo. The mRNA expression of SeGSTo was up-regulated by various oxidative stresses, such as CdCl_2, CuSO₄, and isoprocarb, and the catalytic activity of recombinant SeGSTo was noticeably inhibited by heavy metals (Cu²⁺ and Cd²⁺) and various pesticides. Taken together, these results indicate that SeGSTo plays an important role in the antioxidation and detoxification of pesticides.     

A low molecular mass cutinase of Thielavia terrestris efficiently hydrolyzes poly(esters)

A low molecular mass cutinase (designated TtcutA) from Thielavia terrestris was purified and biochemically characterized. The thermophilic fungus T. terrestris CAU709 secreted a highly active cutinase (90.4 U ml^?1) in fermentation broth containing wheat bran as the carbon source. The cutinase was purified 19-fold with a recovery yield of 4.8 %. The molecular mass of the purified TtcutA was determined as 25.3 and 22.8 kDa using SDS-PAGE and gel filtration, respectively. TtcutA displayed optimal activity at pH 4.0 and 50 °C. It was highly stable up to 65 °C and in the broad pH range 2.5–10.5. Extreme stability in high concentrations (80 %, v/v) of solvents such as methanol, ethanol, acetone, acetonitrile, isopropanol, and dimethyl sulfoxide was observed for the enzyme. The K _m values for this enzyme towards p-nitrophenyl (pNP) acetate, pNP butyrate, and pNP caproate were 7.7, 1.0, and 0.52 mM, respectively. TtcutA was able to efficiently degrade various ester polymers, including cutin, polyethylene terephthalate (PET), polycaprolactone (PCL), and poly(butylene succinate) (PBS) at hydrolytic rates of 3 μmol h^?1 mg^?1 protein, 1.1 mg h^?1 mg^?1 protein, 203.6 mg h^?1 mg^?1 protein, and 56.4 mg h^?1 mg^?1 protein, respectively. Because of these unique biochemical properties, TtcutA of T. terrestris may be useful in various industrial applications in the future.     

Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense

An extracellular haloalkaliphilic thermostable α-amylase producing archaeon was isolated from the saltwater Lake Urmia and identified as Halorubrum xinjiangense on the basis of morphological, biochemical, and molecular properties. The enzyme was purified to an electrophoretically homogenous state by 80 % cold ethanol precipitation, followed by affinity chromatography. The concentrated pure amylase was eluted as a single peak on fast protein liquid chromatography. The molecular mass of the purified enzyme was about 60 kDa, with a pI value of 4.5. Maximum amylase activity was at 4 M NaCl or 4.5 M KCl, 70 °C, and pH 8.5. The K _m and V _max of the enzyme were determined as 3.8 mg ml^?1 and 12.4 U mg^?1, respectively. The pure amylase was stable in the presence of SDS, detergents, and organic solvents. In addition, the enzyme (20 U) hydrolyzed 69 % of the wheat starch after a 2-h incubation at 70 °C in an aqueous/hexadecane two-phase system.