Production and partial purification of naringinase by Penicillium decumbens PTCC 5248

Penicillium decumbens PTCC 5248 produced naringinase when grown in a medium contained naringin as a source of carbon. Rhamnose also induced production of naringinase. Prunin disappeared as the time of enzymatic reaction increased. On fractionation with isopropanol 24-fold purification was achieved. Optimum pH and temperature for naringinase activity were determined to be 4.5 and 55 °C respectively. The K_m value of the enzyme with respect to naringin was found to be 1.7 mM. Citric acid, glucose, Ca²⁺, Mg²⁺, Zn²⁺ all inhibited naringinase activity.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Purification and characterization of chitinase from Alcaligenes xylosoxydans

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecularmass of chitinase was estimated to be 45 kDa and44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 °C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l^–1. Cu²⁺ and Na⁺ at 5 mM inhibited chitinase activity to 25% while Ca²⁺, Mg²⁺ and Ba²⁺ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Partial purification and some properties of an alkaline cellulase from an alkalophilic Bacillus sp.

A newly isolated Bacillus species, which grew optimally at 30°C and pH 10, produced a carboxymethylcellulase in a medium containing 10 g CM-cellulose/l. The enzyme, when partially purified by gel filtration, had a mass of about 29 kDa as determined by both SDS-PAGE and gel filtration chromatography. It was optimally active at pH 9.5 and 40°C, and was stable from pH 7 to 11 at 4°C for 24 h. The enzyme was stimulated by Ca²⁺ (1mm) but was completely inhibited by Hg²⁺ (1mm). Neither EDTA nor EGTA (10mm) affected the activity.The author is with the Department of Biological Sciences, University of Jordan. PO Box 2686, Amman 11181, Jordan     

Thermostable,salt-tolerant amylase fromBacillus sp. 64

A thermostable, salt-tolerant amylase was produced byBacillus sp. 64, with maximum amylase production (8.0 U/ml culture filtrate) after 24-h growth. Partially purified amylase was stable at 60°C for 30 min and 80% of the original activity was retained when incubated in 5m NaCl over 24 h. Starch or dextrin was the best carbon source and peptone the best nitrogen source for the production of the enzyme. Amylase was secreted over a wide pH range (5 to 11) with the maximum activity between pH 7 and 8. Ca²⁺ and Mg²⁺ stimulated growth and enzyme production.NCL Communication No. 5209.     

Isolation and characterization of a d-glucose/xylose isomerase from a new thermophilic strain Streptomyces sp. (PLC)

A thermostable d-xylase isomerase from a newly isolated thermophilic Streptomyces sp. (PLC) strain is described. The enzyme was purified to homogeneity. It is a homotetramer with a native molecular mass of 183 kDa and a subunit molecular mass of 46 kDa. The enzyme has a K _m of 35 mM for d-xylose and also accepts d-glucose as substrate, however, with a tenfold higher K _m (0.4 M) and half the maximum velocity. Both the activity and stability of this d-xylose isomerase depend strongly on divalent metal ions. Two metal ions bind per subunit to non-identical sites. Mg²⁺, Mn²⁺ and Co²⁺ are of comparable efficiency for the d-xylose isomerase reaction. Con²⁺ is the most efficient cofactor for d-glucose isomerization. The enzyme remains fully active up to 95°C. The activity decreases at 53°C in the presence of Co²⁺ and Mg²⁺ with a half-life of 7 and 9 days respectively. In the presence of Mn²⁺ the enzyme activity remains constant for at least 10 days and at 70°C 50% of the activity is lost after 5 days.     

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete <Emphasis Type="Italic">Pseudonocardia thermophila</Emphasis>

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg^–1. The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4–9) and temperature range (40–80°C) and has a half-life of 1.2 h at 70°C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K_m values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the d,l-tryptophanamide and d,l-methioninamide were converted to the corresponding d,l-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.     

Purification and some properties of a starch debranching enzyme ofHendersonula toruloidea

An extracellular, debranching isoamylase fromHendersonula toruloidea ATCC 64930, grown on starch, was purified 12-fold to an electrophoretically homogeneous state. The purified enzyme (estimated mol wt 83000) was optimally active at pH 6.0 and 50°C and remained active when held at 70°C (30 min) and at pH 6 to 8 for 24 h. Na⁺, Fe²⁺ and Ba²⁺ (at 5mm) enhanced enzyme activity while Hg²⁺, Zn²⁺ and Cu²⁺ (at 5mm) were inhibitory. The enzyme hydrolysed amylopectin (Km, 0.25 mg/ml), forming maltose, maltotriose and maltotetraose and hydrolyzed glycogen (Km, 0.29 mg/ml) and soluble starch (Km, 0.42 mg/ml) forming maltotriose and maltotetraose. Pullulan was not hydrolyzed.     

Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.     

Production,partial purification and characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase from <Emphasis Type="Italic">Penicillium ulaiense</Emphasis>

Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism as an α-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. α-l-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification, including (NH₄)₂SO₄ precipitation, incubation at pH 12 and DEAE-sepharose chromatography yielded an enzyme with very low β-glucosidase activity. The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis–Menten constants for the hydrolysis of p-nitrophenyl-α-l-rhamnoside were V _max = 26 ± 4 IU ml⁻¹ and K _m  = 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co²⁺ affected positively the activity; EDTA, Mn²⁺, Mg²⁺, dithiotreitol and Cu²⁺ reduced the activity by different amounts, and Hg²⁺ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-α-l-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries since P. ulaiense does not produce mycotoxins.     

Short communication: Purification and properties of a glucose-forming amylase of Lactobacillus brevis

An extracellular glucose-forming amylase was produced by Lactobacillus brevis isolated from Kagasok tea. The enzyme was purified 70-fold and had optimal activity at 55°C and pH 6.5. Its K _m value for starch was 0.27 mg ml^-1 and its M _r was approx. 75,900 Da. The activity of the enzyme was enhanced by Ca²⁺, Mg²⁺, Na⁺ or K⁺ and inhibited by EDTA, KCN, citric acid and l-cysteine.     

A Novel Ferric Reductase Purified from <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> MSR-1

A ferric reductase was purified into an electrophoretically homologous state from Magnetospirillum gryphiswaldense MSR-1 strain. The enzyme was found within the cytoplasm and associated with the cytoplasmic membrane. The molecular weight of the purified enzyme was calculated as 16.1 kDa using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and was almost identical to that calibrated using agarose gel filtration chromatography. It was NADH-dependent and required flavin mononucleotide as a cofactor. The optimal reaction temperature and pH values were 30°C and 6.5, respectively. The K _m and Vmax values for ferric citrate were 45.1 μM and 1.216 μM min⁻¹, respectively. Though ferric reductase activity could be inhibited by Co²⁺, Cu²⁺, Mn²⁺, and Zn²⁺, even high concentrations of Mg²⁺ ions have failed to accomplish such enzyme inhibition. Furthermore, the molecular weight, the N-terminal sequence, and the activity of ferric reductase from MSR-1 are not matching with the enzyme preparation obtained from an analogous strain M. magnetotacticum (MS-1). Therefore, it is concluded that the ferric reductase of M. grysphiwaldense and M. magnetotacticum strains are two different enzymes.     

Purification and characterization of an alkaline cellulase from a newly isolated alkalophilic Bacillus sp. HSH-810

An alkaline cellulase from Bacillus sp. HSH-810 was purified 8.7-fold with a 30% yield and a specific activity of 71 U mg^–1 protein. It was optimally active at pH 10 and 50 °C and was stable from pH 6 to 10 with more than 60% activity remaining after heating at 60 °C for 60 min. The molecular mass of cellulase was 80 kDa. It was inhibited by 50% by Fe³⁺ (1 mM) and Mn²⁺ (0.1 mM) but was relatively insensitive to Hg²⁺ and Pb²⁺ at 1 mM.Revisions requested: 8 October 2004/1 December 2004; Revisions received 29 November 2004/5 January 2005     

Purification and characterization of a novel alkaline α-<Emphasis Type="SmallCaps">L</Emphasis>-rhamnosidase produced by <Emphasis Type="Italic">Acrostalagmus luteo albus</Emphasis>

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal nonreducing L-rhamnose for the bioconversion of natural or synthetic rhamnosides. They are of great significance in the current biotechnological area, with applications in food and pharmaceutical industrial processes. In this study we isolated and characterized a novel alkaline rhamnosidase from Acrostalagmus luteo albus, an alkali-tolerant soil fungus from Argentina. We also present an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In the presence of rhamnose as the sole carbon source, this fungus produces a rhamnosidase with a molecular weight of 109 kDa and a pI value of 4.6, as determined by SDS–PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electrophoretic techniques. Using p-nitrofenil-α-L-rhamnopiranoside as substrate, the enzyme activity showed pH and temperature optima of 8.0 and 55°C, respectively. The enzyme exhibited Michaelis–Menten kinetics, with K _M and V _max values of 3.38 mmol l⁻¹ and 68.5 mmol l⁻¹ min⁻¹, respectively. Neither divalent cations such as Ca²⁺, Mg²⁺, Mn²⁺, and Co²⁺ nor reducing agents such as β-mercaptoethanol and dithiothreitol showed any effect on enzyme activity, whereas this activity was completely inhibited by Zn²⁺ at a concentration of 0.2 mM. This enzyme showed the capacity to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. Based on these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered a potential new biocatalyst for industrial applications.     

Extracellular calcium-inhibited alpha-amylase of Bacillus coagulans B 49

Extracellular α-amylase (EC 3.2.1.1) from Bacillus coagulans B 49 was purified to homogeneity by ion-exchange chromatography and gel filtration. The optimum pH and temperature for dextrinizing activity were 6–7 and 70°C and for saccharolytic activity were 7 and 60°C, respectively. Calcium inhibited α-amylase activity even at low concentrations (10 m ), and most of its activity could be restored by dialysis against EDTA. Other cations such as Mg²⁺, Fe²⁺, and Hg²⁺ also inhibited amylase activity, while Mn²⁺ exhibited a slight stimulatory effect. The activity of the enzyme was not affected by ethylenediaminetetraacetic acid (EDTA).     

ADP-dependent glucokinase from the hyperthermophilic sulfate-reducing archaeon<Emphasis Type="Italic"> Archaeoglobus fulgidus</Emphasis> strain 7324

The hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 has been shown to degrade starch via glucose using a modified Embden-Meyerhof pathway. The first enzyme of this pathway, ADP-dependent glucokinase, was purified 600-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of 50 kDa. It had a temperature optimum at 83 °C and showed a significant thermostability up to 100 °C. The enzyme was highly specific for ADP and glucose as substrates; it did not use ATP, CDP, UDP, or GDP as phosphoryl donors, or mannose, fructose and fructose 6-phosphate as phosphoryl acceptors (at 80 °C). Only glucosamine was phosphorylated at significant rates. The apparent K_m values for ADP and glucose (at 50 °C) were 0.07 mM and 0.78 mM, respectively; the apparent V_max value was about 50 U/mg at 50 °C and 350 U/mg at 80 °C. Divalent cations were required for maximal activity; Mn²⁺, Mg²⁺ and Ca²⁺, which were most effective, could be replaced partially by Cu²⁺, Ni²⁺, Co²⁺ and Zn²⁺. The N-terminal amino acid sequence (42 amino acids) of ADP-dependent glucokinase was almost identical to that of ADP-dependent glucokinase from Thermococcus litoralis. In the genome of the closely related Archaeoglobus fulgidus strain VC16 a homologous gene for ADP-dependent glucokinase could not be identified.     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Tetrahydromethanopterin-dependent serine transhydroxymethylase from Methanobacterium thermoautotrophicum

Serine transhydroxymethylase of Methanobacterium thermoautotrophicum has been purified to within 95% of homogeneity. Activity was strictly dependent on tetrahydromethanopterin, tetrahydrofolate being unable to serve as the acceptor C₁ units from l-serine. The native protein has a molecular weight of about 102,000 daltons. The enzyme shows maximal activity at 60°C, has a pH optimum of 8.1, and required pyridoxal-5-phosphate and Mg²⁺ for optimal activity.