Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Purification and properties of a chitinase from Penicillium sp. LYG 0704

The chitinase producing Penicillium sp. LYG 0704 was procured from soil of the Chonnam National University crop field. The chitinase activity was detected after the first day which increased gradually and reached its maximum after 3 days of cultivation. The chitinase was purified from a culture medium by precipitation with isopropanol and column chromatography with Mono Q and Butyl-Sepharose. The molecular mass of chitinase was estimated to be 47 kDa by SDS–PAGE. Optimal pH and temperature were 5.0 and 40 °C, respectively. The N-terminal amino acid sequence of the enzyme was determined to be ¹AGSYRSVAYFVDWAI¹⁵. The fully cloned gene, 1287 bp in size, encoded a single peptide of 429 amino acids. BLAST search of the chitinase gene sequence showed similarity with chitinase of Aspergillus fumigatus Af293 chitinase gene (58%) and A. fumigatus class V chitinase ChiB1 gene (56%).     

Gene cloning,expression, and characterization of a novel trehalose synthase from <Emphasis Type="Italic">Arthrobacter aurescens</Emphasis>

Trehalose synthase (TreS) is an intramolecular transglycosylase. It specially catalyzes the conversion of maltose and trehalose. In this study, a novel treS gene, which had a length of 1,797 bp and encoded 598 amino acids, was cloned from Arthrobacter aurescens CGMCC 1.1892 and expressed in Escherichia coli. Thin layer chromatography results indicated that it could catalyze the conversion between maltose and trehalose in one step. However, the ion chromatography results showed that, as a byproduct, about 13% glucose was also produced. The purified recombinant enzyme had a molecular weight of 68 kDa and showed its optimal activity at 35 °C and pH 6.5. This enzyme was not thermostable, and its activity was increased by 1 mM Mg²⁺, Mn²⁺, and Ca²⁺ while strongly inhibited by 5 mM Cu²⁺ and SDS.     

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 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.     

Identification, characterization and functional analysis of a GH-18 chitinase from Streptomyces roseolus

A 40 kDa chitinase from Streptomyces roseolus DH was purified to homogeneity from culture medium. The N-terminal sequence was TPPPAKAVKLGYFTNWGVYG, which was highly homologous to the glycoside hydrolase (GH) 18 conserved domain of Streptomyces chitinases and included the two crucial Trp and Tyr sites. The purified enzyme showed maximal activity at 60 °C, pH 6.0 and exhibited good thermal and pH stabilities. The enzyme displayed strict substrate specificity on colloidal or glycol chitin, but not on chitosan derivatives. It was activated by Mg²⁺, Ba²⁺ and Ca²⁺, and inhibited by Cu²⁺, Co²⁺, Mn²⁺, whereas Zn²⁺ and ethylenediamine tetraacetic acid showed little inhibitory effects. Morphological changes observed by scanning electron microscopy revealed the occurrence of regular pores on the surface with the progress of enzymatic chitinolysis. Additionally, this GH-18 chitinase had a marked inhibitory effect on fungal hyphal extensions. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.     

Purification and properties of a collagenolytic protease produced by marine bacteriumVibrio vulnificus CYK279H

A collagenolytic enzyme, produced byVibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0 kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Asn. The optimum temperature and pH for the enzyme activity were 35°C and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8∼8.0 and 20∼35°C, respectively. The purified enzyme was strongly activated by Zn²⁺, Li²⁺, and Ca²⁺, but inhibited by Cu²⁺. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.     

Characterization of Antifungal Chitinase from Marine Streptomyces sp. DA11 Associated with South China Sea Sponge Craniella Australiensis

The gene cloning, purification, properties, kinetics, and antifungal activity of chitinase from marine Streptomyces sp. DA11 associated with South China sponge Craniella australiensis were investigated. Alignment analysis of the amino acid sequence deduced from the cloned conserved 451 bp DNA sequence shows the chitinase belongs to ChiC type with 80% similarity to chitinase C precursor from Streptomyces peucetius. Through purification by 80% ammonium sulfate, affinity binding to chitin and diethylaminoethyl-cellulose anion-exchange chromatography, 6.15-fold total purification with a specific activity of 2.95 Umg⁻¹ was achieved. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular weight of approximately 34 kDa and antifungal activities were observed against Aspergillus niger and Candida albicans. The optimal pH, temperature, and salinity for chitinase activity were 8.0, 50°C, and 45 g‰ psu, respectively, which may contribute to special application of this marine microbe-derived chitinase compared with terrestrial chitinases. The chitinase activity was increased by Mn²⁺, Cu²⁺, and Mg²⁺, while strongly inhibited by Fe²⁺ and Ba²⁺. Meanwhile, SDS, ethyleneglycoltetraacetic acid, urea, and ethylenediaminetetraacetic acid were found to have significantly inhibitory effect on chitinase activity. With colloidal chitin as substrates instead of powder chitin, higher V _max (0.82 mg product/min·mg protein) and lower K _m (0.019 mg/ml) values were achieved. The sponge’s microbial symbiont with chitinase activity may contribute to chitin degradation and antifungal defense. To our knowledge, it was the first time to study sponge-associated microbial chitinase.     

Nucleotide pyrophosphatase/phosphodiesterase from Euphorbia characias latex: Purification and characterization

An authentic soluble metallo-protein nucleotide pyrophosphatase/phosphodiesterase (ELNPP) was purified to homogeneity from Euphorbia characias latex. The native protein had a molecular mass of 80 ± 5 kDa and was shown to be formed by two apparently identical subunits, each containing 1 Ca²⁺ and 1 Mg²⁺ ion. Whereas Mg²⁺ was shown to be strongly bound to the enzyme, Ca²⁺ was easily removed by treatment with EDTA. Ca²⁺-demetalated enzyme was shown to be almost totally inactive and the activity was fully restored incubating the demetalated ELNPP with Ca²⁺ ions. ELNPP exhibited hydrolytic activities toward pyrophosphate/phosphodiester bonds of a broad range of substrates and very efficiently hydrolyzed the artificial substrate thymidine 5′-monophosphate 4-nitrophenyl ester generating 4-nitrophenolate as a final product, and it has been used for enzyme kinetic experiments. ELNPP represents the first example of a nucleotide pyrophosphatase/phosphodiesterase enzyme purified from the latex of a plant belonging to the large genus Euphorbia.     

Molecular cloning and characterization of 58 kDa chitinase gene fromSerratia marcescens KCTC 2172

A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from theSerratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids.Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using a chitin affinity column and mono-S column. A nucleotide andN-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a 98% similarity to that ofS. marcescens QMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N′-diacetyl chitobiose (NAG2) as the major hydrolysis end-product with a trace amount ofN-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG monomer (analogue of NAG₂), thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the enzyme were 50°C and 5.0, respectively.     

Partial purification and properties of extracellular chitinase produced by Acremonium obclavatum,an antagonist to the groundnut rust,Puccinia arachidis

An extracellular chitinase of Acremonium obclavatum was partially purified. It had an M _r of 45 kDa on SDS-PAGE, and was optimally active at pH 3 to 4 and 50°C. Hg and Mn (10 mm) inhibited activity. The chitinase hydrolysed colloidal chitin more rapidly than crude chitin or isolated A. obclavatum cell walls. The partially-purified enzyme inhibited uredospore germination and germ-tube growth of Puccinia arachidis.The authors are with the Centre for Advanced Study in Botany, University of Madras, Guindy campus, Madras 600 025, India     

An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product

An extracellular chitinase of Bacillus sp. WY22 was purified by 9.6-fold. It had a Mr of 35 kDa, an apparent K _m value for colloidal chitin of 3 mg ml^–1 and was optimally active at 37 °C and pH 5.5 over 1 h. The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin. It formed chitotriose as a major product from colloidal chitin and glycol chitin.     

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.     

A novel fibrinolytic enzyme from <Emphasis Type="Italic">Cordyceps militaris</Emphasis>, a Chinese traditional medicinal mushroom

A novel fibrinolytic enzyme from Cordyceps militaris was purified and partially characterized for the first time, which was designated C. militaris fibrinolytic enzyme (CMase). This extracellular enzyme from C. militaris was isolated by ammonium sulphate fraction, and purified to electrophoretic homogeneity using gel filtration chromatography. The apparent molecular mass of the purified enzyme was estimated to be 27.3 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 6.0 and 25 °C, respectively. In the presence of metal ions such as Mg²⁺ and Fe²⁺ ions the activity of the enzyme increased, whereas EDTA and Cu²⁺ ion inhibited the enzyme activity. Interestingly the N-terminal amino acid sequences of the enzyme is extremely similar to those of the trypsin proteinases from insects, and has no significant homology with those of the fibrinolytic enzyme from other medicinal mushroom. In conclusion, C. militaris produces a strong fibrinolytic enzyme CMase and may be considered as a new source for thrombolytic agents.     

Purification and some properties of a carboxypeptidase B from dogfish Scyliorhinus canicula

A carboxypeptidase B (CPB) has been purified from dogfish (Scyliorhinus canicula) pancreas and partially characterized. The purification procedure included acetone precipitation, ion-exchange chromatography on a CM-cellulose column and gel filtration on Sephadex G-75. The purified enzyme migrates as a single band both on PAGE and SDS-PAGE. Its molecular mass is estimated to be about 32 kDa. The optimum of activity is obtained at pH 7.5–8.2. The enzyme is inhibited by typical metal-chelating agents (EDTA and o-phenanthroline) and by Hg²⁺. It is activated by Co²⁺, l-cysteine and by heat treatment at 40° and 50°C. Kinetic parameters, K_m and k_cat, of native enzyme, Co²⁺-activated CPB and heat-treated CPB have been determined     

Hyaluronic acid production by recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at 25°C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml⁻¹. The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 40°C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The K _m and V _max values for birchwood xylan are 2.06 mg ml⁻¹ and 0.49 mmol min⁻¹mg⁻¹, respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu²⁺ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity is unaffected by EDTA even at a concentration of 5 mM.     

Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK _m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg²⁺ but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I₂, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.     

Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum

Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent K_mS for -ketoglutarate, NADPH and NH _inf4 ^sup+ are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn²⁺ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - Tris Tris(hydroxymethyl)aminomethane - Bis-tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - TRITON X-100 iso-octylphenoxypoly-ethoxyethanol - pHMB p-Hydroxymercuribenzoic acid