Identification of the first archaeal arylsulfatase from Pyrococcus furiosus and its application to desulfatation of agar

A gene encoding a putative arylsulfatase from the hyperthermophilic archaeon Pyrococcus furiosus was identified, cloned, and expressed as a fusion protein with a Sce VMA intein and chitin binding domain (CBD) residue. The gene (PF1345) from P. furiosus encoding a 35 kDa protein showed some similarity (17 ～ 19%) with other arylsulfatases from the bacteria. The recombinant fusion arylsulfatase was overexpressed in E. coli and partially purified. Its molecular mass was estimated to be 90 kDa by SDS-PAGE. The optimal temperature and pH for arylsulfatase activity were found to be 45°C and 9.5, respectively. Various divalent cations (Ca²⁺, Mg²⁺, Co²⁺, Cu²⁺, Zn²⁺, and Mn²⁺) slightly activated the arylsulfatase activity in a narrow range of concentrations (below 0.5 mM), whereas Zn²⁺ concentrations above 2.0 mM significantly inhibited the activity. After the reaction of agar with recombinant fusion arylsulfatase for 12 h at 50°C, 75% of the sulfate in the agar was removed, and the DNA migration was greatly enhanced. Therefore, the arylsulfatase in this study could be applicable for the production of electrophoretic grade agarose by removing sulfate groups in agar.     

Purification and characterization of arylsulfatase from<Emphasis Type="Italic"> Sphingomonas</Emphasis> sp. AS6330

Arylsulfatase was purified from Sphingomonas sp. AS6330 through ionic exchange, hydrophobic- and gel-chromatographies. The purity increased 12,800-fold with approximately 19.1% yield against cell homogenate. The enzyme was a monomeric protein with apparent molecular weight of 62 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 41 kDa as determined by gel filtration. The enzyme had optimum reaction conditions for hydrolysis of sulfate ester bonds in agar and p-nitrophenyl sulfate (NPS) at pH 7.0 and 45°C, with a specific activity of 3.93 and 97.2 U, respectively. The enzyme showed higher activity towards agar than other sulfated marine polysaccharides such as porphyran, fucoidan and carrageenan. The K _m and V _max of the enzyme for hydrolysis of NPS were 54.9 M and 113 mM/min, respectively. With reaction of 200 g agar with 100 U arylsulfatase for 8 h at 45°C, gel strength increased 2.44-fold, and 97.7% of the sulfate in the agar was hydrolyzed.     

Heterologous expression of a newly screened β-agarase from Alteromonas sp. GNUM1 in Escherichia coli and its application for agarose degradation

The gene of agaG1 from Alteromonas sp. GNUM1 encoding a β-agarase (AgaG1) was heterologously expressed in E. coli BL21 (DE3). The recombinant strain was cultured at 37 °C and then AgaG1 was expressed at 25 °C and 0.5 mM IPTG. The optimum conditions for AgaG1 to hydrolyze agarose were pH 7.0 and 40 °C. The main products of agarose hydrolysis by AgaG1 were confirmed to be neoagarobiose and neoagarotetraose. A new agarose hydrolysis process using AgaG1 was developed, in which the reaction temperature was adjusted stepwise to avoid gelation problem with no chemical pretreatment step. The enzyme AgaG1 was found to be very effective and highly selective. When 10.0 g/L agarose was hydrolyzed, 98% of the agarose added was converted to 3.8 and 6.4 g/L of neoagarobiose and neoagarotetraose, respectively.     

Gene cloning,expression and characterisation of a new β-agarase,AgWH50C,producing neoagarobiose from Agarivorans gilvus WH0801

agWH50C, a novel β-agarase gene, was cloned from Agarivorans gilvus WH0801 by degenerate PCR and nested PCR. The gene agWH50C comprized a 2,223-bp, encoding a protein of 740 amino acids. Sequencing results demonstrated that AgWH50C shared 45 % sequence identity with a well characterized β-agarase, Aga50D, from Saccharophagus degradans 2–40. The mature agarase was expressed in Escherichia coli and purified by affinity chromatography. The optimum pH and temperature for AgWH50C activity were 6.0 and 30 °C. The K _m and V _max values for agarose were 12.55 mg/ml and 1.17 U/mg. Analysis of the hydrolysis products using linear ion trap mass spectrometry, Fourier transform-nuclear magnetic resonance spectrometry and thin-layer chromatography confirmed that the reaction product of AgWH50c was α-neoagarobiose alone. Therefore, our novel agarase has the potential for industrial applications to produce neoagarobiose as well as provides a key β-agarase for fermentation of agar biomass.     

Isolation and characteristics of new thermostable DNA ligase from archaea of the genus Thermococcus

The DNA ligase gene from thermophilic archaea of the genus Thermococcus (strain 1519) was identified and sequenced in the polymerase chain reaction. The recombinant enzyme LigTh1519 was expressed in Escherichia coli, purified, and characterized. LigTh1519 was capable of ligating the cohesive ends and single-strand breaks in double-stranded DNA (ATP as a cofactor). The optimum conditions for the ligase reaction appeared as follows: 100 mM NaCl, 50 mM MgCl₂, pH 7.0–10.5, and temperature 70°C. More than 50% Lig1519 activity were preserved after incubation of the enzyme at 80°C for 30 min. New thermostable DNA ligase LihTh1519 may be used for basic and applied researches in molecular biology and genetic engineering.     

Cloning, expression, and biochemical characterization of a thermostable lipase from Geobacillus stearothermophilus JC

A thermophilic lipase gene of Geobacillus stearothermophilus JC was cloned and expressed in a pET 28-a (+) expression vector. The biochemical properties of the recombinant enzyme and its enantioselective hydrolysis of (RS)-1-phenylethyl acetate were studied. Removal of the signal peptide greatly increased the enzyme’s expression level by 4.3 times. The purified JC lipase had an optimum temperature of 55°C and optimum pH of 9. Furthermore, comparisons with other enzymes suggest that a few amino acid alterations may significantly change the thermostability of this enzyme. The hydrolysis of (RS)-1-phenylethyl acetate with the crude recombinant JC lipase at 25°C produce (R)-1-phenylethanol in 97.7% e.e. and 46.1% yield after 24 h, corresponding to an E value of 237.     

Production and characteristics of some new β-agarases from a marine bacterium,Vibrio sp. strain JT0107

Vibrio sp. strain JT0107 is one of the marine bacteria that secrete β-agarases which catalyze the hydrolysis of agarose. The optimum culture conditions for the production of some β-agarases have been determined. To increase agarase activity, aeration and a sufficient concentration of agarose are needed. One of the enzymes that the bacteria secreted into the culture medium was isolated and purified 39-fold using a combination of ultrafiltration and subsequent anion exchange column chromatography. The purified protein migrated as a single band (72 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and its isoelectric point was 4.7. Amino acid sequence analysis revealed a single N-terminal sequence that had no sequence identity to other marine bacterial agarases. This novel enzyme was found to be an endo-type β-agarase (EC 3.2.1.81) that catalyzes the hydrolysis of the β-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-O-β-d-galactopyranosyl(1→4)-O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d -galactose] and neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d-galactose]. The optimum pH and temperature for obtaining high activity of the enzyme were at around 8 and 30°C, respectively. The enzyme did not degrade sodium alginate, λ-carrageenan, ι-carrageenan or κ-carrageenan.     

Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3

An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel β-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0?~?9.0 and at temperatures below 42 °C. The K _m and V _max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.     

Cloning, expression and characterization of xylose isomerase, XylA, from Caldanaerobacter subterraneus subsp. yonseiensis

The xylA gene, coding for xylose isomerase, from the extreme thermophile, Caldanaerobacter subterraneus subsp. yonseiensis was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the xylA gene encoded a polypeptide of 438 residues with a calculated molecular weight of 50,170 Da. The purified XylA showed high sequence homology (92% identity) with that of Thermoanaerobacter thermohydrosulfuricus. The recombinant enzyme expressed in Escherichia coli was purified by heat treatment and gel chromatography. The purified enzyme was thermostable with optimal activity at 95°C. The enzyme required divalent cations including Zn²⁺ for its maximal activity and thermostability.     

Heterologous expression and characterization of a malathion-hydrolyzing carboxylesterase from a thermophilic bacterium, Alicyclobacillus tengchongensis

A carboxylesterase gene from thermophilic bacterium, Alicyclobacillus tengchongensis, was cloned and expressed in Escherichia coli BL21 (DE3). The gene coded for a 513 amino acid protein with a calculated molecular mass of 57.82 kDa. The deduced amino acid sequence had structural features highly conserved among serine hydrolases, including Ser204, Glu325, and His415 as a catalytic triad, as well as type-B carboxylesterase serine active site (FGGDPENITIGGQSAG) and type-B carboxylesterase signature 2 (EDCLYLNIWTP). The purified enzyme exhibited optimum activity with β-naphthyl acetate at 60 °C and pH 7 as well as stability at 25 °C and pH 7. One unit of the enzyme hydrolyzed 5 mg malathion l^?1 by 50 % within 25 min and 89 % within 100 min. The enzyme strongly degraded malathion and has a potential use for the detoxification of malathion residues.     

Formation and Purification of Serratia marcescens Arylsulfatase

The effects of culture conditions on arylsulfatase production by six strains of the genus Serratia were studied. Synthesis of arylsulfatases in all six strains was repressed in media with inorganic sulfate or methionine as the sole source of sulfur and derepressed by the addition of tyramine. Serratia marcescens IFO 3046 grew most rapidly and produced a high level of arylsulfatase when cultured on mannitol with inorganic sulfate and tyramine. The derepressed synthesis of arylsulfatase in S. marcescens was not subject to strong catabolite repression. The molecular weight of purified arylsulfatase was determined to be between 46,000 and 49,000. Arylsulfatase from S. marcescens differed in K_m and V_max values, substrate specificities, fluoride inhibition, and electrophoretic mobility from the enzyme from K. aerogenes, but had the same molecular weight as the latter.     

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.     

Cloning,purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1

Thermostable amylopullulanases can catalyse the hydrolysis of both α-1,4 and α-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 °C. Apu-Tk was also active at a broad range of pH (4–7), with an optimum pH of ~5.0–5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% β-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications.     

Biochemical and conformational characterization of a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2

In order to search for valuable and extremely thermo-stable enzymes that could be used in the protein hydrolysis industry, the gene corresponding to a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2 (GtLAP) was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined. Meanwhile, potential applications of GtLAP in the hydrolysis of anchovy proteins were also investigated. GtLAP was overexpressed in IPTG-induced E. coli BL21 (pET28a-LAP) as a soluble protein, and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 125?±?8.75 U/mg proteins. The molecular mass of GtLAP was estimated to be 55?kDa by SDS-PAGE analysis. The optimal reaction temperature and pH of GtLAP were 70?°C and 8.0, respectively. Under optimal conditions, GtLAP showed a marked preference for Leu-p-nitroanilide, followed by Met- and Phe-derivatives. Activity of GtLAP was strongly stimulated by Ni²⁺ ions, but was strongly inhibited by Hg²⁺. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of GtLAP to various extents and further induce changes in enzymatic activity. Results of hydrolytic experiment showed that combining GtLAP with endogenous enzymes could significantly increase the degree of hydrolysis to anchovy proteins and concentrations of free amino acids in hydrolysates. In this regard, GtLAP could potentially be used in the protein hydrolysis industry.     

Electroendosmosis correction for electrophoretic mobility determined in cells

Electroendosmosis is a complicating factor in gel electrophoresis. Determination of electroendosmotic mobility by the use of vitamin B₁₂ as a marker in agar and agarose gels at different concentrations revealed that electroendosmosis was not reduced to zero by extrapolation of observed mobility values to zero gel concentration. It is shown that a Ferguson plot of the observed values of electrophoretic mobilities yields the correct values for K_R; however the extrapolated values of electrophoretic mobility must still be corrected for electroendosmosis to obtain the true electrophoretic mobilities.     

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5–30 °C) and weakly basic conditions (pH 8.5–10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.     

Characterization of human arylsulfatase a glycans

Despite numerous studies on arylsulfatase A, the structure of the glycans present in each of its two subunits has not been determined. This is important because the carbohydrate component of human arylsulfatase A synthesized in tumor tissues and transformed cells has been shown to undergo apparent changes. This study elucidates some of their major features.Glycan chain analysis of native and deglycosylated arylsulfatase A as well as its subunits was performed with the use of a Glycan Differentiation Kit and lectin affinity chromatography. Each of the two subunits of arylsulfatase A from placenta, separated electrophoretically on polyacrylamide gel in reducing conditions, reacted with digoxigenin-labelled Galantus nivalis agglutinin and Aleuria awantia agglutinin, while those from liver enzyme reacted with the former only. The subunits of both enzymes did not react with Sambucus nigra, Maakia amuriensis, Datura stramonium or Peanut agglutinin. Deglycosylation of arylsulfatase A with peptide N-glycosidase F and endo-β-N-acetylglucosaminidase F resulted in complete cleavage of its carbohydrate component from each subunit. Their molecular weights decreased by 3 kDa. Neuraminidase treatment of the enzyme from liver and placenta followed by isoelectrofocusing separation showed the presence of sialylated forms which constituted a small percentage of total enzyme activity. Placental arylsulfatase A became bound to Lens culinaris agglutinin agarose, while no interaction with Ricinus communis or Griffonia simplicifolia agglutinin agarose was observed.The study shows that both subunits of arylsulfatase A from human placenta possess two high mannose/hybrid type glycans as major structures, with at least one 6-O-l-fucose bound to the innermost N-acetylglucosamine on each. The enzyme from liver does not possess fucose. Complex type glycans containing sialic acid constitute a small percentage of the total carbohydrate component.     

Expression of a functional 78,000 dalton mammalian flavoprotein,NADPH-cytochrome P-450 oxidoreductase,in Escherichia coli

A cDNA containing the complete coding nucleotide sequence for rat liver NADPH-cytochrome P-450 oxidoreductase was constructed from two overlapping cDNA clones. This full-length cDNA was inserted into the plasmid expression vector pCQV2, transfected into Escherichia coli, and expressed reductase was identified in cell lysates by electrophoresis followed by electrophoretic transfer to nitrocellulose and immunodetection. Various strains were screened for maximal expression and minimal intracellular degradation of the expressed protein, and strain C-1A was selected for preparation of the expressed enzyme. Induced cells from 12-liter cultures were pelleted, lysed in a French press, and the 50,000g supernate was fractionated by DEAE-cellulose and 2′5′-ADP agarose chromatography. Thirty-five grams of packed cells yielded approximately 2 mg of affinity-purified protein that was essentially free of E. coli proteins. The final preparation exhibited considerable proteolytic degradation and only an estimated 5–10% of the immunoreactive protein was undegraded. Four principal forms could be distinguished upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular weights of 65,000, 66,000, 74,000, and 78,000, the latter being equivalent to that of intact reductase. High-performance liquid chromatography with a Spherogel-DEAE column resolved these forms but resulted in the loss of the 78-kDa form; three peaks eluted with molecular weights of 65,000. Several of the HPLC fractions exhibited cytochrome c reductase activity, indicating correct incorporation of both flavin prosthetic groups, with the 66-kDa form showing the highest specific activity (44 μmol of cytochrome c reduced/mg reductase/min at 22 °C). HPLC assay of flavin content demonstrated equimolar FMN and FAD concentrations, and spectrophotometric analysis of the 66-kDa form revealed a spectrum essentially identical to that of reductase purified from rat liver. When the affinity-purified preparation was reconstituted with cytochrome P-450c, rates of benzo[a]pyrene metabolism approaching rates observed with liver reductase were obtained, indicating that the undegraded component in the affinity-purified preparation was able to interact with cytochrome P-450 and catalyze electron transfer from NADPH.     

Solubilization and partial purification of steroid sulfatase from rat liver: characterization of estrone sulfatase.

Steroid sulfatase, a membrane-bound enzyme present in many mammalian tissues, was extracted from rat liver microsomes by treatment with Miranol H2M, a zwitterion detergent, and sonication. It has been purified approximately 33-fold. All steps of the purification, which included salt and solvent fractionation, hydroxylapatite treatment, ion-exchange chromatography, and gel filtration were performed in the presence of Miranol H2M, most of which was removed from the final preparation by gel filtration. The final preparation did not contain any detectable NADPH-cytochrome c reductase or glucose-6-phosphate phophatase activities. According to the elution volume on a Sephadex G-200 column, steroid sulfatase has a molecular weight of approximately 130,000. Polyacrylamide-gel electrophoresis in the presence of Miranol H2M revealed one major protein band which was enzymatically active. Purified steroid sulfatase hydrolyzes all the sulfate esters of estrone, dehydroepiandrosterone, pregnenolone, testosterone, and cholesterol as well as p-nitrophenyl sulfate, the substrate for arylsulfatase C, during the purification. However, estrone sulfatase and arylsulfatase C activities were enriched more than the others. Analysis of kinetic data and the effects of different buffers and of Miranol H2M also suggested that estrone sulfatase and arylsulfatase C are identical but that they are distinct from the other sulfatases. Competitive inhibition studies suggest that estrone sulfatase also catalyzes the hydrolysis of the sulfate esters of other estrogens.     

Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus

An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.