Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Purification and characterization of an intracellular α-glucosidase with high transglycosylation activity from A. niger M-1

An intracellular α-glucosidase with high transglycosylation activity was purified from a mutant strain of Aspergillus niger M-1 by sequential chromatography using a DEAE-cellulose 52 column, a DEAE-Sepharose CL-6B column, and a Sephadex G-100 column. The molecular mass of the purified enzyme was determined to be 116?kD with no subunits and a pI of 5.23. Maximal α-glucosidase activity occurred at pH 6.0 and 50°C. The N-terminal amino acid sequences were identified as N-SVPGTEYVV-. The presence of Ca(2+) enhanced the enzyme activity by 20%, while the α-glucosidase activity was strongly inhibited by p-chloromercuribenzoate, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, monochloroacetic acid, and 2-mercaptoethanol. In addition, Ag(+), n-bromosuccinimide, and acetylacetone inhibited enzyme activity by 70%, 50%, and 22%, respectively. K(m) values of 4.32?m?mol?L(-1) and V(max) of 3.10?×?10(-2)?mol?L(-1) min(-1) were found for methyl-α-D-glucopyranoside (α-MG). Maltose was identified as the preferred substrate. The high-performance liquid chromatography (HPLC) analysis indicated that the oligosaccharide products contained 10.54% of isomaltose, 8.08% of panose, and 9.29% of isomaltotriose, and the amount of glucose, maltose, maltotriose, and maltotetrose was dropped from 22.21% to 15.80% using the purified enzyme in the solution of 25% maltose and 3% glucose. This intracellular α-glucosidase has potential applications in the synthesis of sugar derivatives and the investigation of associated mechanisms.     

Recombinant α-l-rhamnosidase from Aspergillus terreus in selective trimming of rutin

α-l-Rhamnosidase (EC 3.2.1.40) is a biotechnologically important enzyme used for derhamnosylation of many natural compounds. The extracellular α-l-rhamnosidase was purified from the culture of Aspergillus terreus grown on l-rhamnose-rich medium. This enzyme was found to be thermo- and alkali-tolerant, able to operate at 70 °C and pH 8.0. The α-l-rhamnosidase cDNA was cloned from A. terreus, sequenced, and expressed in the yeast Pichia pastoris as a fully functional protein. The recombinant protein was purified to apparent homogeneity and biochemically characterized. Both the native and the recombinant α-l-rhamnosidases catalyzed the conversion of rutin into quercetin-3-glucopyranoside (isoquercitrin), a pharmacologically significant flavonoid usable in nutraceutics. This procedure has high volumetric productivity (up to 300 g/L) and yields the product void of unwanted quercetin. The significant advantage of our expression system consists in shorter production times, up to fourfold increase in enzyme yields and the absence of unwanted β-d-glucosidase as compared to the native production system. Thanks to its unique properties, this enzyme is applicable in a selective synthesis/hydrolysis of various rhamnose containing structures.     

Purification and characterization of α-l-rhamnosidase from Penicillium corylopholum MTCC-2011

An extracellular α-l-rhamnosidase has been purified to electrophoretic homogeneity from the culture filtrate of Penicillium corylopholum MTCC-2011 using a simple procedure consisting of concentration by ultrafiltration and cation exchange column chromatography on carboxymethyl cellulose. The sodium dodesyl sulphate polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass of 67.0 kDa. The native – polyacrylamide gel electrophoresis analysis also gave a single protein band confirming the purity of the enzyme and also showing that the enzyme is a monomer in the native state. The K_m and k_cat values of the enzyme were 0.42 mM and 35.7 s^?1, respectively, using p-nitrophenyl α-l-rhamnopyranoside as the substrate. The pH and temperature optima of the enzyme were 6.5 and 57.0 °C, respectively. The purified enzyme preparation successfully hydrolyzed naringin and rutin to prunin and quercetin glucoside, respectively. Thus it can be used for the preparation of these pharmaceutically important compounds.     

Characterization of β-N-acetylglucosaminidase from a marine Pseudoalteromonas sp. for application in N-acetyl-glucosamine production

The psychrotolerant Pseudoalteromonas issachenkonii PAMC 22718 was isolated for its high exo-acting chitinase activity in the Kara Sea, Arctic. An exo-acting chitinase (W-Chi22718) was homogeneously purified from the culture supernatant of PAMC 22718, the molecular weight of which was estimated to be approximately 112?kDa. Due to its β-N-acetylglucosaminidase activity, W-Chi22718 was able to produce N-acetyl-D-glucosamine (GlcNAc) monomers from chitin oligosaccharide substrates. W-Chi22718 displayed chitinase activity from 0 to 37°C (optimal temperature of 30°C) and maintained activity from pH 6.0 to 9.0 (optimal pH of 7.6). W-Chi22718 exhibited a relative activity of 13 and 35% of maximal activity at 0 and 10°C, respectively, which is comparable to the activities of previously characterized, cold-adapted bacterial chitinases. W-Chi22718 activity was enhanced by K⁺, Ca²⁺, and Fe²⁺, but completely inhibited by Cu²⁺ and SDS. We found that W-Chi22718 can produce much more (GlcNAcs) from colloidal chitin, working together with previously characterized cold-active endochitinase W-Chi21702. Genome sequencing revealed that the corresponding gene (chi22718_IV) was 2,856?bp encoding a 951?amino acid protein with a calculated molecular weight of approximately 102?kDa.     

Purification and Characterization of α-Hydroxyglutarate Dehydrogenase from Alcaligenes sp.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD⁺ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.     

Molecular Characterization and Therapeutic Potential of a Marine Bacterium Pseudoalteromonas sp. KMM 701 α-Galactosidase

An α-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic and requires Na⁺ ions and temperature up to 34°C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes. Its single intracellular α-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The α-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric α-galactosidase indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This α-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme are likely to be highly beneficial for medicinal purposes.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria

The α-galactosidase gene, galA17, was cloned from Flavobacterium sp. TN17, a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae. The 2,205-bp full-length gene encodes a 734-residue polypeptide (GalA17) containing a putative 28-residue signal peptide and a catalytic domain belonging to glycosyl hydrolase family 36 (GH 36). The deduced amino acid sequence of galA17 was most similar to a putative α-galactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 α-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for α-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45°C; exhibited strong resistance to digestion by trypsin, α-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-α-d-galactopyranoside, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum).     

Raffinose production using α-galactosidase from Paraphaeosphaeria sp.

An α-galactosidase for raffinose synthesis by a reverse hydrolysis reaction was searched from intracellular of endophytic fungi. About 500 strains were screened, one strain was selected and identified as Paraphaeosphaeria sp. Partially purified α-galactosidase (1 U/ml in the reaction mixture), 100 g/L galactose, and 500 g/L sucrose at 60 °C for 48 h resulted in synthesis of 13.3 g/L raffinose and 4.6 g/L planteose at a ratio of about 3:1. The data suggest that α-galactosidase was able to synthesize raffinose at a ratio higher than that of α-galactosidases derived from other fungi.     

Production, purification and characterization of a constitutive intracellular α-galactosidase from the thermophilic fungus Humicola sp

The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg⁻¹ protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K _m S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag⁺ and Hg²⁺. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K _i) for galactose was 11 mM. Received 28 January 1999/ Accepted in revised form 07 April 1999     

Characterization of a α-l-rhamnosidase from Bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C

In this study, a α-l-rhamnosidase gene from Bacteroides thetaiotaomicron VPI-5482 was cloned and expressed in Escherichia coli. The specific activity of rhamnosidase was 0.57 U/mg in LB medium with 0.1 mM Isopropyl β-d-Thiogalactoside (IPTG) induction at 28 °C for 8 h. The protein was purified by Ni-NTA affinity, which molecular weight approximately 83.3 kDa. The characterization of BtRha was determined. The optimal activity was at 55 °C and pH 6.5. The enzyme was stable in the pH range 5.0–8.0 for 4 h over 60%, and had a 1-h half-life at 50 °C. The Kcat and Km for p-nitrophenyl-α-l-rhamnopyranoside (pNPR) were 1743.29 s⁻¹ and 2.87 mM, respectively. The α-l-rhamnosidase exhibited high selectivity to cleave the α-1,2 and α-1,6 glycosidic bond between rhamnoside and rhamnoside, rhamnoside and glycoside, respectively, which could hydrolyze rutin, hesperidin, epimedin C and 2″-O-rhamnosyl icariside II. Under the optimal conditions, BtRha transformed epimedin C (1 g/L) to icariin by 90.5% in 4 h. This study provides the first demonstration that the α-l-rhamnosidase could hydrolyze α-1,2 glycosidic bond between rhamnoside and rhamnoside.     

Purification and Characterization of a Novel α-Agarase from a Thalassomonas sp.

An agar-degrading Thalassomonas bacterium, strain JAMB-A33, was isolated from the sediment off Noma Point, Japan, at a depth of 230 m. A novel -agarase from the isolate was purified to homogeneity from cultures containing agar as a carbon source. The molecular mass of the purified enzyme, designated as agaraseA33, was 85 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that it is a monomer. The optimal pH and temperature for activity were about 8.5 and 45°C, respectively. The enzyme had a specific activity of 40.7 U/mg protein. The pattern of agarose hydrolysis showed that the enzyme is an endo-type -agarase, and the final main product was agarotetraose. The enzyme degraded not only agarose but also agarohexaose, neoagarohexaose, and porphyran.     

Properties of an α-1,3-Glucanase from Streptomyces sp. KI-8

Srome properties were examined of purified α-l,3-glucanase isolated from the culture supernatant of the soil microorganism Streptomyces KI-8.

The optimum pH and temperature were pH 5.4 and 60°C, respectively. The α-1,3-glucanase was stable up to 50°C on heating for 10 min. This enzyme hydrolyzed the substrate α-l,3-glucan into glucose and nigerose by an endo-type of action. Nigerotriose, nigerotetraose and nigeropentaose were hydrolyzed into glucose and nigerose, whereas nigerose was not attacked. The degree of hydrolysis of pseudonigeran, Lentinus α-1,3-glucan, mutan IG-1 (less soluble fraction) and IG-2 (more soluble fraction) by the α-1,3-glucanase were 28.5%, 14.3%, 8.8% and 10.0%, respectively. Km values (mg/ml) for pseudonigeran, Lentinus α-l,3-glucan, mutan IG-1 and IG-2 were 1.12, 1.98, 8.00 and 5.00. The enzyme solubilized 50 to 80% of mutan by concerted action with dextranase.     

Studies of A.A. Neĭfakh on periodicity of morphogenetic nuclear activity and their significance

The studies of A.A. Neyfakh on morphogenetic nuclear activity are considered in the light of experimental embryology data.     

Structure and activity of exo-1,3/1,4-β-glucanase from marine bacterium Pseudoalteromonas sp. BB1 showing a novel C-terminal domain

Following the discovery of an exo-1,3/1,4-β-glucanase (glycoside hydrolase family 3) from a seaweed-associated bacterium Pseudoalteromonas sp. BB1, the recombinant three-domain protein (ExoP) was crystallized and its structure solved to 2.3 ? resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two-domain barley homologue β-d-glucan exohydrolase (ExoI) with a distinctive Trp-Trp clamp at the +1 subsite, although ExoI displays broader specificity towards β-glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a β-sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A-ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small-angle X-ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate-binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 3UT0, 3USZ, 3F95 and 3RRX.     

GC–MS volatolomic approach to study the antimicrobial activity of the antarctic bacterium Pseudoalteromonas sp. TB41

Many bacteria produce a wide range of volatile info-chemicals compounds (mVOCs) that constitute an important regulatory factor in the interrelationships among different organisms in microbial ecosystems. It has been shown that Antarctic bacteria isolated from three different sponge species, by producing mVOCs, are able to inhibit specifically the growth of Burkholderia cepacia complex (Bcc) strains (i.e. opportunistic pathogens of cystic fibrosis patients) as demonstrated by cross-streaking inhibition assays. This study reports a metabolomics approach to investigate the volatile profile of both the Antarctic sponge-associated Pseudoalteromonas sp. TB41 (P-sp-TB41) and Burkholderia cenocepacia strain LMG16654 (Bc-LMG16654) under aerobic conditions. Solid phase micro extraction (SPME) in head space of biological samples allowed an in vivo sampling of mVOCs with minimal specimen disturbance. The SPME fiber was termically desorbed in the injection port of gas chromatography–mass spectrometer (GC–MS) system setted in EI scan mode. The raw data were processed using both an automated mass spectra deconvolution and identification system and a metabolomic approach, which allowed a selection of 30 compounds presumably responsible for the inhibition of Bc-LMG16654 growth. The results obtained from samples prepared under cross-streaking conditions also suggest that the presence of Bc-LMG16654 cells neither interferes with the production of mVOCs nor induces the synthesis of different mVOCs. The employing of mass spectrometry played a key role in tuning the experimental system and in the evaluation of results. The use of this approach to study the interaction, in aerobic condition, among other Antarctic bacteria and Bcc and the possibility to extend this approach to other pathogen-antagonist relationship, is currently in progress.