Lysosomal arylsulfatases A and B from horse blood leukocytes: purification and physico-chemical properties

Lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolases, EC 3.1.6.1) from horse leukocytes were purified about 680-fold and 70-fold, respectively, starting from a crude extract of the azurophil and specific granules of leukocytes, by affinity, ion exchange, and gel filtration chromatography. Purified arylsulfatase A displayed anomalous kinetics, a pH optimum at 5.2, an isoelectric point at 4.3, and a Km value for p-nitrocatechol sulfate (pNCS) of 0.37 mM. This enzyme was found to exist in two association states depending on pH: a high molecular weight form at pH 5.0 and a low molecular weight form at pH 7.5. Arylsulfatase B displayed normal kinetics, a pH optimum at 5.8, two isoelectric points at pH 8.6 and 8.9, and a Km value for pNCS of 3.38 mM. The thermostability of the two enzymes was different: arylsulfatase B was found to be more stable than arylsulfatase A. Arylsulfatase A was inhibited by sulfate, sulfite, silver, magnesium, manganese and calcium ions and arylsulfatase B by chloride, sulfate, sulfite and silver ions.     

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.     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.     

Purification and properties of arylsulfatase B from high- and low-activity mouse strains

Arylsulfatase B (arylsulfate sulfohydrolase; EC 3.1.6.1) activities in C57BL/6J, SWR/J, and A/J mouse liver approximate a 5:3:1 ratio. Each enzyme was purified to apparent homogeneity, and the properties of the three purified enzymes were compared. The purified enzyme behaved as a monomer with an apparent molecular weight of 50,000. The purified enzyme catalyzed the hydrolysis of p-nitrocatechol sulfate (pNCS), 4-methylumbelliferyl sulfate (4MUS), and chondroitin-4-sulfate (C4S) heptasaccharide. Purified SWR/J arylsulfatase B possessed a higher relative electrophoretic mobility at pH 4.0 than the A/J and C57BL/6J isozymes, and the SWR/J enzyme was more thermostable than either the C57BL/6J or the A/J enzyme. No differences were observed among the three enzymes with respect to their Michaelis constants for 4MUS and pNCS, isoelectric points, responses to inhibitors, pH optima, or electrophoretic mobilities at pH 8.3. The relative in vivo rates of synthesis of C57BL/6J, A/J, and SWR/J arylsulfatase B were comparable.     

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.     

A novel thermostable α-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: Purification and characterization

High levels of an extracellular α-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH₄)₂SO₄ fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric α-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5–5.5 and 65 °C, respectively. The purified enzyme retains more than 90% of its activity at 45 °C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-α-galactopyranoside, raffinose and stachyose and very similar K_m values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn⁺⁺ ions activates enzyme activity, whereas inhibitory effects can be observed with Ca⁺⁺, Zn⁺⁺ and Hg⁺⁺. Five min incubation at 65° with 10 mM Ag⁺ results in complete inactivation of the purified α-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the α-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.     

Purification and characterization of a thiol amylase over produced by a non-cereal non-leguminous plant, Tinospora cordifolia

A 43 kDa α-amylase was purified from Tinospora cordifolia by glycogen precipitation, ammonium sulfate precipitation, gel filtration chromatography, and HPGPLC. The enzyme was optimally active in pH 6.0 at 60 °C and had specific activity of 546.2 U/mg of protein. Activity was stable in the pH range of 4-7 and at temperatures up to 60 °C. PCMB, iodoacetic acid, iodoacetamide, DTNB, and heavy metal ions Hg²⁺ > Ag⁺ > Cd²⁺ inhibited enzyme activity while Ca²⁺ improved both activity and thermostability. The enzyme was a thiol amylase (3 SH group/mole) and DTNB inhibition of activity was released by cysteine. N-terminal sequence of the enzyme had poor similarity (12-24%) with those of plant and microbial amylases. The enzyme was equally active on soluble starch and amylopectin and released maltose as the major end product.     

Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336

A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0–8.0) and temperatures below 50 °C. Metal ions including Na⁺, Mg²⁺, Mn²⁺, and Ca²⁺ notably increased the activity of the enzyme. With sodium alginate as the substrate, the K_m and V_max were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees.     

Purification and characterization of the recombinant arylsulfatase cloned from Pseudoalteromonas carrageenovora

Arylsulfatase cloned from a marine aerobic Gram-negative bacterium, Pseudoalteromonas carrageenovora, was overexpressed in Escherichia coli with 10 microM IPTG induction. The expressed recombinant arylsulfatase was purified to homogeneity from the harvested cells through osmotic disruption and column chromatography methods, such as DEAE-cellulose anion exchange chromatography and Heparin-Sepharose affinity chromatography. The purified arylsulfatase was kinetically characterized using the synthetic substrate of phenolic ester, p-nitrophenyl sulfate (pNPS). One unit of arylsulfatase catalyzes the liberation of 1.0 micromol p-nitrophenol from pNPS per minute. The purified enzyme has a specific activity of 468 U/mg with a purification yield of 27% from the cell lysate, and exhibited an estimated molecular mass of 33 kDa in SDS-PAGE analysis. The precursor polypeptide of 36 kDa was processed by releasing a putative signal peptide, and the mature arylsulfatase of 33.1 kDa with a N-terminal sequence of S-E-T-K-N was trafficked to periplasmic space. The enzyme had optimum reaction conditions for activity at pH 7.0 and at a temperature range of 40-45 degrees C. The apparent K(M) and k(cat) of the enzyme for hydrolysis of pNPS at pH 7.0 and at 45 degrees C were determined to be 1.15 mM and 1000 s-1, respectively. Based on inhibitor studies along with optimal pH values and preferential periplasmic location of the enzyme, we suggest that the recombinant arylsulfatase from P. carrageenovora is probably similar to the Klebsiella sulfatase with serine residue in the active site.     

Properties of laccase purified from nitrogen limited culture of white-rot fungus Coriolus hirsutus

Laccase produced by nitrogen-limited culture of Coriolus hirsutus was purified to electrophoretic homogeneity (133-fold) with an overall yield of 40%. The molecular mass of the enzyme was determined as 82 kDa by SDS-PAGE and 80 kDa using gel filtration. It had a pI of 3.50. With ferulic acid and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as the substrate, the enzyme had optimal activity at pH 4.0 and 2.5, respectively. The enzyme was stable in the range pH 5.5 to 7.0 at 30 °C for 1 h. The enzyme was optimally active at 70 °C and it lost all activity within 15 min at 80 °C. The apparent Km value of enzyme toward ABTS was 67 °M and had highest affinity toward sinapinic acid. The enzyme was totally inhibited by 0.01 mM cysteine.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura

An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81 kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35 °C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35 °C for 1 h. Higher activity was observed in the presence of surfactants, Na⁺, NH₄⁺ ions, NaN₃ and ethylenediaminetetraacetic acid (EDTA), while Co²⁺ and Cu²⁺ ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10 days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone.     

Purification and characterization of a carboxymethyl cellulase from Artemia salina

Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cellulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase activity was purified and the activity analyzed under different conditions. After initial identification of cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the final purification, a 70-fold increase in specific enzyme activity was observed. SDS–PAGE results revealed that the cellulase enzyme had a molecular mass of 96 kDa. Temperature, pH, and salinity values were found to be optimal at 55 °C, pH 8.0, and 600 mM NaCl, respectively. Specifically, the enzyme showed a fivefold increase in enzyme activity in seawater compared to 600 mM NaCl in phosphate buffer. Further analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indicating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol from marine macroalgae.     

Purification and partial biochemical characterization of polyphenol oxidase from mamey (Pouteria sapota)

While a long shelf life for fruit products is highly desired, enzymatic browning is the main cause of quality loss in fruits and is therefore a main problem for the food industry. In this study polyphenol oxidase (PPO), the main enzyme responsible for browning was isolated from mamey fruit (Pouteria sapota) and characterized biochemically. Two isoenzymes (PPO 1 and PPO 2) were obtained upon ammonium sulfate precipitation and hydrophobic and ion exchange chromatography; PPO 1 was purified up to 6.6-fold with 0.28% yield, while PPO 2 could not be characterized as enzyme activity was completely lost after 24 h of storage. PPO 1 molecular weight was estimated to be 16.1 and 18 kDa by gel filtration and SDS-PAGE, respectively, indicating that the native state of the PPO 1 is a monomer. The optimum pH for PPO 1 activity was 7. The PPO 1 was determined to be maximum thermally stable up to 35 °C. Kinetic constants for PPO 1 were K_m = 44 mM and K_m = 1.3 mM using catechol and pyrogallol as substrate, respectively. The best substrates for PPO 1 were pyrogallol, 4-methylcatechol and catechol, while ascorbic acid and sodium metabisulfite were the most effective inhibitors.     

Identification, expression, and characterization of a novel bacterial RGI lyase enzyme for the production of bio-functional fibers

A gene encoding a putative rhamnogalacturonan I (RGI) Lyase (EC 4.2.2.-) from Bacillus licheniformis (DSM13) was selected after a homology search and phylogenetic analysis and optimized with respect to codon usage. The designed gene was transformed into Pichia pastoris and the enzyme was produced in the eukaryotic host with a high titer in a 5 l bioreactor. The RGI Lyase was purified by Cu²⁺ affinity chromatography and 1.1 g pure enzyme was achieved pr. L. When the denatured protein was deglycosylated with EndoH, the molecular weight of the protein decreased to 65 kDa, which correlated with the predicted molecular weight of the mature RGI Lyase of 596 amino acids. By use of a statistical design approach, with potato rhamnogalacturonan as the substrate, the optimal reaction conditions for the RGI Lyase were established to be: 61 °C, pH 8.1, and 2 mM of both Ca²⁺ and Mn²⁺ (specific activity 18.4 U/mg; K_M 1.2 mg/ml). The addition of both Ca²⁺ and Mn²⁺ was essential for enzyme activity. The enzyme retained its catalytic activity at higher temperatures and the enzyme has a half life at 61 °C of 15 min. The work thus demonstrated the workability of in silico based screening coupled with a synthetic biology approach for gene synthesis for identification and production of a thermostable enzyme.     

Heterologous expression and characterization of a recombinant thermophilic arylsulfatase from Thermotoga maritima

Production of low sulfated agar or agarose from agar or agaropectins by enzymatic hydrolysis has advantages but a high melting temperature is needed. The arylsulfatase gene from thermophilic Thermotoga maritima was cloned and expressed in Escherichia coli W3110 with pCol-MICT as the vector. The gene was comprised of 1,782 bp and encoded a protein of 593 amino acids with a molecular weight of 65 kDa. The recombinant arylsulfatase was partially purified by heat treatment (70°C, 30 min) and characterized. The enzyme was prepared with a total protein content of 2.4 mg and a specific activity of 20.63 U/mg. Optimal temperature and pH of the enzyme were 80°C and 7.0, respectively, for hydrolysis of p-nitrophenyl sulfate and sulfate content of agar was diminished to 40% after a 12 h treatment at that condition. Enhanced electrophoretic movement of DNA was observed in enzymetreated agar gel compared to that in a non-treated agar gel. These results suggest that thermophilic arylsulfatase expressed in E. coli could be useful for producing a low sulfated agar and electrophoretic grade agarose.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

A method of purification, identification and characterization of β-glucosidase from Trichoderma koningii AS3.2774

In this study, we used native gradient-polyacrylamide gel electrophoresis and electroelution (NGGEE) to purify enzymatic proteins from Trichoderma koningii AS3.2774. With this method, we purified eight enzymatic proteins and classified them to the cellulase system by comparing secretions of T. koningii in inductive medium and in repressive medium. It resulted in 24-fold β-glucosidase (BG) purification with a recovery rate of 5.5%, and a specific activity of 994.6 IU mg^− 1 protein. The final yield of BG reached 8 μg under purifying procedure of NGGEE. We also identified BG using the enzyme assay with thin-layer chromatography and MALDI-TOFMS. This BG had one subunit with a molecular mass of 69.1 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The hydrolytic activity of the BG had an optimal pH of 5.0, an optimal temperature of 50 °C, an isoelectric point of 5.68 and a K_m for p-nitrophenyl-β-d-glucopyranoside of 2.67 mM. Taken together, we show that NGGEE is a reliable method through which μg grade of active proteins can be purified.     

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum)

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex® G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH₂-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1′. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 °C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 °C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3 h at 60 °C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.     

Chitobiose production by using a novel thermostable chitinase from Bacillus licheniformis strain JS isolated from a mushroom bed

The thermophilic Bacillus licheniformis strain JS was isolated from a bed of mushrooms, Pleurotus sajor-caju. The organism could produce a novel, single-component, thermostable chitinase that was purified by ion-exchange chromatography using DEAE-cellulose in 7.64% yield and in an 8.1-fold enhancement in purity. Its molecular weight is 22 kDa. The enzyme is a chitobiosidase, since the chitin hydrolysate is N^I,N^II-diacetylchitobiose. The optimum temperature for enzyme activity is 55 °C, and the optimum pH is 8.0. It was completely inhibited by Hg²⁺ ions whereas Co²⁺ ions served as an activator. The thermostability of this enzyme is important in the bioconversion of chitinous waste and for the production of chitooligosaccharides.