Octyl-Sepharose CL-4B Research Articles

The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability

Thermal stability is one of the essential parameters characterizing biocatalysts with potential applications in the chemical and pharmaceutical industries. Therefore, it is extremely important to develop standardized procedures for enzyme stability studies. The paper attempts to assess the thermal stability of immobilized lipases in aqueous buffers: lipase B from Candida antarctica (CALB) and lipase from Candida rugosa (CRL-OF) immobilized on the Octyl-Sepharose CL-4B carrier. As part of the optimization conditions of the immobilization, the influence of time on the catalytic activity and lipase loading, as well as the effect of temperature on lipase activity (optimal incubation—14 h at 4 °C), was determined. The thermal stability test procedure was carried out for 7 days using a climatic chamber (65 °C) and a refrigerator (4 °C). The studies of immobilized lipases included the assessment of the impact of various solvents (water, citrate buffer, 1,2-dichloropropane—DCP), temperature, light in the visible spectral range (400–800 nm), and additions of calcium ions. The highest value of residual activity (564.5 ± 21.6%) was received by storing the immobilized CALB in citrate buffer (pH 4.0, 500 mM) with the addition of calcium ions (Ca2+). On the other hand, residual activity values for immobilized CRL-OF after storage in the climatic chamber were lower than 5%. A combining of techniques: immobilization onto the support in high ionic strength and low pH, with a technique of extremally high-temperature applied in a climatic chamber, with the addition of Ca2+ allowed to achieve of excellent thermal stability of the immobilized CALB, with increasing of catalytic activity more than five-fold. Additionally, performing studies on the thermal stability of the tested lipases using a climatic chamber seems to be particularly promising in the context of unifying and standardizing storage guidelines, enabling the comparison of results between different laboratories, as well as enhancing catalytic activity.

Climatic Chamber Stability Tests of Lipase-Catalytic Octyl-Sepharose Systems

The application of the climatic chamber presented in this paper to assess the storage stability of immobilized lipases is a new approach characterized by the potential of unifying the study conditions of biocatalysts created in various laboratories. The data achieved from storing lipases in the climatic chambers may be crucial for the chemical and pharmaceutical industry. Our paper describes the developed protocols for immobilization via interfacial activation of lipase B from Candida antarctica (CALB) and lipase OF from Candida rugosa (CRL-OF) on the Octyl-Sepharose CL-4B support. Optimization included buffers with different pH values of 4–9 and a wide range of ionic strength from 5 mM to 700 mM. It has been shown that the optimal medium for the CALB immobilization process on the tested support is a citrate buffer at pH 4 and high ionic strength of 500 mM. Implementing new optimal procedures enabled the hyperactivation of immobilized CALB (recovery activity 116.10 ± 1.70%) under the applicable reaction conditions using olive oil as a substrate. Importantly, CALB storage stability tests performed in a climatic chamber under drastic temperature and humidity conditions proved good stability of the developed biocatalyst (residual activity 218 ± 7.3% of dry form, after 7 days). At the same time, the low storage stability of CRL OF in a climatic chamber was demonstrated. It should be emphasized that the use of a climatic chamber to test the storage stability of a dry form of the studied lipases immobilized on Octyl-Sepharose CL-4B is, to our knowledge, described for the first time in the literature.

Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7.

There are numerous reports on poly-β-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30°C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg-1mL-1). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the natural soil condition is the result of the activity of soil microbes that complemented the biodegradation of PHB by Stenotrophomonas sp. RZS7.

Optimal immobilization of trypsin from the spleen of albacore tuna (Thunnus alalunga) and its characterization

Trypsin purified from the spleen of albacore tuna was immobilized onto Octyl Sepharose CL-4B, glutaraldehyde activated silica and 5′-4,4′-dimethyltryptamine-thymidine-succinyl controlled pore glass. Trypsin was highly and efficiently immobilized onto Octyl Sepharose CL-4B, with the highest activity (6.26 U/g support) and specific activity (1.45 U/mg bound protein). The optimum conditions for trypsin immobilization onto Octyl Sepharose CL-4B were 40 mg/mL trypsin solution, pH 7 at 4 °C for 6 h of incubation time. The optimal temperature and pH for the hydrolysis of N-α-benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA) by the immobilized trypsin were 55 °C and 8.5, both of which were higher than that of the free form. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and organic solvents. The immobilized enzyme was less sensitive to inhibition by the soybean trypsin inhibitor compared with the free soluble form of the enzyme. According to the results, the immobilized trypsin and free enzyme retained 83% and 47% of their activity, respectively, when they were incubated with 1 μM of the soybean trypsin inhibitor. For the reusability study, the immobilized trypsin maintained 60% of its activity after 4 periods of activity, indicating that the immobilized trypsin had appropriate stability and could be reused.

Purification and kinetics of the PHB depolymerase of Microbacterium paraoxydans RZS6 isolated from a dumping yard.

Poly-β-hydroxybutyrate (PHB) depolymerase is known to decompose PHB, biodegradable polymers and therefore has great commercial significance in the bioplastic sector. However, reports on PHB depolymerases from isolates obtained from plastic-contaminated sites that reflect the potential of the source organism is scarce. In this study, we evaluated the production of extracellular PHB depolymerase from Microbacterium paraoxydans RZS6 isolated from the plastic-contaminated site in the municipal area of Shahada, Maharashtra, India, for the first time. The isolate was identified using 16S rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters (GC-FAME), and BIOLOG method. Ithydrolyzed PHB on minimal salt medium (MSM) containing PHB as the only source of carbon. The isolate produced PHB depolymerase at 45°C during 48 h of incubation. The enzyme was purified most efficiently using octyl-sepharose CL-4B column, with the highest purification yield of 6.675 Umg-1mL-1. The activity of the enzyme was enhanced in the presence of Ca2+ and Mg2+ ions but inhibited by Fe2+ (1 mM) ions and mercaptoethanol (1000 rpm). the nzyme kinetic analysis revealed that the enzyme was a metalloenzyme; requiring Mg2+ ions, that showed optimum enzyme activity at 30°C (mesophilic) and under neutrophilic (pH 7) conditions. Scale-up from the shake-flask level to a laboratory-scale bioreactor further enhanced the enzyme yield by 0.809 UmL-1. The molecular weight of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, closely resembled the PHB depolymerase of Aureobacterium saperdae. Our findings highlighted the applicability of M. paraoxydans as a producer of extracellular PHB depolymerase having potential of degrading PHB under diverse conditions.

Screening and Immobilization of Interfacial Esterases from Marine Invertebrates as Promising Biocatalyst Derivatives.

Interfacial esterases are useful enzymes in bioconversion and racemic mixture resolution processes. Marine invertebrates are few explored potential sources of these proteins. In this work, aqueous extracts of 41 species of marine invertebrates were screened for esterase, lipase, and phospholipase A activities, being all positive. Five extracts (Stichodactyla helianthus, Condylactis gigantea, Stylocheilus longicauda, Zoanthus pulchellus, and Plexaura homomalla) were selected for their activity values and immobilized on Octyl-Sepharose CL 4B support by interfacial adsorption. The selectivity of this immobilization method for interfacial esterases was evidenced by immobilization percentages ≥ 94% in almost all cases for lipase and phospholipase A activities. Six pharmaceutical-relevant esters (phenylethyl butyrate, ethyl-2-hydroxy-4-phenyl-butanoate, 2-oxyranylmethyl acetate (glycidol acetate), 7-aminocephalosporanic acid, methyl-prostaglandin F2α, and methyl-6-metoxy-α-methyl-2-naphtalen-acetate -naproxen methyl ester-) were bioconverted by at least three of these biocatalysts, with the lowest conversion percentage of 24%. In addition, three biocatalysts were used in the racemic mixture resolution of three previous compounds. The S. helianthus-derived biocatalyst showed the highest enantiomeric ratios for glycidol acetate (2.67, (S)-selective) and naproxen methyl ester (8.32, (R)-selective), and the immobilized extract of S. longicauda was the most resolutive toward the ethyl-2-hydroxy-4-phenyl-butanoate (8.13, (S)-selective). These results indicate the relevance of such marine interfacial esterases as immobilized biocatalysts for the pharmaceutical industry.

Immobilization of Lipase from Grey Mullet

Grey mullet (Mugil cephalus) lipase was isolated using para-aminobenzamidine agarose and immobilized on octyl Sepharose CL-4B (o-Sep). Immobilized grey mullet lipase (GMLi) had a 10°C higher optimum temperature compared to the free enzyme and showed remarkable thermal stability. GMLi was most active within the pH range of 8.0-9.5 with an optimum at 8.5. Immobilization also enhanced the storage stability and reusability of the enzyme with minimal changes in efficiency during repeated batches. GMLi showed variable stabilities in various organic solvents. A signal in the amide I absorption region of the FTIR spectrum of GMLi was attributed to the protein layer on o-Sep. The surface morphology of o-Sep was visualized on a Zeiss stereomicroscope as globular-shaped beads.

Involvement of Acinetobacter sp. in the floc-formation in activated sludge process

The coaggregation behavior of Acinetobacter johnsonii S35 isolate with sewage bacteria was assessed by a spectrophotometric assay using different samples from a municipal wastewater treatment plant and a community plant. A. johnsonii S35 coaggregated well with other free bacteria and microflocs at the mixing ratios of 0.2:1-0.6:1 of A. johnsonii S35 and sewage samples. In addition, the size of coaggregates became larger (100 μm or more) under the same conditions. A. johnsonii S35 cells were highly adsorbed (adsorption=93-99%) onto sludge samples. Microbial adhesion to hydrocarbon (MATH) test and adsorption to octyl-Sepharose CL-4B showed that A. johnsonii S35 cells and sludge samples had a hydrophobic character. The population of Acinetobacter spp. in sewage treatment plants was 2-7% and its role in bioflocculation was discussed. The present study revealed that A. johnsonii S35 isolate can play as a bridging organism and contribute in floc-formation in activated sludge process.

Purification and characterization of an extracellular medium-chain length polyhydroxyalkanoate depolymerase from Thermus thermophilus HB8

During growth on medium-chain length (mcl) polyhydroxyalkanoates (PHAs), or on sodium octanoate Thermus thermophilus HB8 produces an extracellular mcl-PHA depolymerase. This enzyme was purified from the culture medium of sodium octanoate-grown cells to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the purified enzyme was approximately 28 kDa. A part of the gene TTHA1605 encoding a 24.17 kDa protein was demonstrated to encode the mcl-PHA depolymerase of T. thermophilus. The primary amino-acid sequence of purified enzyme reveals similarity to all reported so far extracellular mcl-PHA depolymerases. The purified enzyme could hydrolyze mcl – PHAs and p-nitrophenyl (pNP) esters but not short chain length (scl) – PHAs. The optimum pH range was 7.5–9 and the optimum temperature was 70 °C for pNP-octanoate (pNPO) hydrolysis. The Km value for pNPO was 53.2 μM. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and non-ionic detergents (Tween 20, Tween 80 and Triton X-100). The results demonstrated in this study revealed that the mcl-PHA depolymerase from T. thermophilus is a distinct enzyme, which is different from those of other mcl-PHA-degrading bacteria.

Role of Acinetobacter johnsonii S35 isolate in floc-formation in activated sludge process

Activated sludge process is widely used for removing organic and inorganic substances from municipal as well as industrial wastewaters. The process depends on the settleability of flocs formed by microorganisms (mainly bacteria). Therefore, the bioflocculation, mentioned mainly to the isolation and flocculation mechanism of floc-forming bacteria, have been investigated by many workers. Meanwhile, many nonflocculating bacteria are known tobepresent in any sludge samples.Wepreviously reported that nonflocculating bacterium, Acinetobacter johnsonii S35 isolate, coaggregated with several kinds of Gram-positive and -negative bacteria, obtained from the same sewage activated sludge. In the present study, the coaggregation behaviour of A. johnsonii S35 isolate with sewage bacteria was investigated using different samples from a municipal wastewater treatment plant and a community plant. Thecoaggregationexperimentwasassessedbya spectrophotometric assay and the mixing ratios of A. johnsonii S35 and settled sewage sample were 0.2:1–1.5:1 on the basis of their turbidity. The relationship between coaggregation behaviour and cell surface characteristics was also studied using the microbial adhesion to hydrocarbon (MATH) test and adsorption to octyl-Sepharose CL4B. Population and role of Acinetobacter spp. were also examined using fluorescence in situ hybridization (FISH) technique. The results showedthatA. johnsoniiS35coaggregatedwithother free-floating bacteria or microflocs at the mixing ratios of 0.2:10.6:1 of A. johnsonii S35 and sewage samples. In addition, the size of coaggregates became larger under the same conditions. These indicated that A. johnsonii S35 played a role as a bridging bacterium in the coaggregation. A. johnsonii S35 cells were highly adsorbed onto sludge samples. MATH test and adsorption to octyl-Sepharose CL4B showed that A. johnsonii S35 cells and sludge samples had a hydrophobic character. According to the result of FISH, the population of Acinetobacter spp. presented in settled sludge and settled sewage in a primary settling tank as well as in mixed liquor in an aeration tank of the municipal wastewater treatment plant were 6.7%, 4.0%, and2.7%, respectively. On the other hand, the population ofAcinetobacter spp. in influent andmixed liquor in anaeration tank of the community plantwere 6.2% 2.2%, respectively. These results suggest that Acinetobacter spp. were derived from the influent and play an important role in the formation of sludge flocs. The overall results demonstrated that A. johnsonii S35 isolate had a high affinity for many of settled sewage bacteria and activatedsludgeflocs.A. johnsoniiS35cells andsewageactivatedsludge used showed a hydrophobic character. The population of Acinetobacter spp. were higher in primary settling tank, suggesting that Acinetobacter spp. were derived from the influent. These suggest that bacteriawith a hydrophobic cell surface like Acinetobacter spp. isolates may play similar function as bridging organisms and contribute in solid–liquid separation in activated sludge process.

Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced byBacillus thuringiensisSF361

Thurincin H, a bacteriocin produced by Bacillus thuringiensis SF361 isolated from honey, strongly inhibited the growth of Bacillus cereus F4552. The bacteriocin was purified by 65% ammonium sulfate precipitation of the culture supernatant, followed by octyl-sepharose CL-4B and reverse-phase HPLC. The molecular mass of the bacteriocin was determined to be 3139.51 Da and the 14 amino acids of the bacteriocin at the N-terminus were identified. The complete amino acid sequence of mature thurincin H was deduced from three structural genes, thnA1, thnA2, and thnA3 found in tandem repeats on the chromosome, all of which encode for the same bacteriocin, thurincin H. The genetic determinants for thurincin H biosynthesis consist of 10 ORFs, including three thurincin H structural genes. Northern hybridization elucidated that the transcription of all three bacteriocin structural genes was regulated by a putative promoter located upstream of thnA1.

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active againstByssochlamys fulvaH25

Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages. A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16. The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F. The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100 degrees C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.

Two Step Purification of Acinetobacter sp. Lipase and Its Evaluation as a Detergent Additive at Low Temperatures

Acinetobacter sp. lipase was purified to homogeneity by a two-step process. The crude enzyme (along with biomass) was subjected to partial purification by aqueous two phase system (ATPS), avoiding centrifugation and filtration steps. Conditions for lipase partitioning by ATPS were optimized by response surface methodology (RSM) and a combination of 29.45% polyethylene glycol 8000, 15.5% phosphate, and a pH of 7.0 resulted in an optimal partition coefficient. Partially pure lipase was further purified by a modified batch process using Octyl Sepharose CL-4B in a vacuum filtration apparatus. This two-step process resulted in a purified lipase with a yield of 74.6% having a specific activity of 88.8 U/mg of protein and a purification fold of 14.92. The homogeneity of the lipase preparation obtained by the purification process was confirmed by reversed phase high performance liquid chromatography profile. The molecular weight of the purified lipase was found to be around 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified lipase exhibited pH and temperature optima of 8.5 and 37 degrees C, respectively. The lipase was active at low temperatures and it retained 86.8% activity at 10 degrees C. It also displayed other features such as stability over a broad range of pH (3.0-9.0) as well as stability in the presence of hydrogen peroxide and commercial detergents. Based on these characteristics, the potential of this lipase as an additive in laundry detergent formulation was evaluated under low temperature wash conditions. The results indicated that Acinetobacter sp. lipase increased the washing efficiency of the detergent Nirma by 21-24% at 15 degrees C-20 degrees C, respectively.

Screening and Immobilization Burkholderia sp. GXU56 Lipase for Enantioselective Resolution of (R,S)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 degrees C (free lipase) to 50 degrees C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0-8.0, at the temperatures below 50 degrees C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.

CONSTITUENT PROTEOGLYCANS OF THE HUMAN YELLOW LIGAMENT EXTRACELLULAR MATRIX

Three types of proteoglycans (PG-I, II and III) present in the human yellow ligament extracellular matrix were separated using a combination of CsCl isopycnic density gradient centrifugation, DEAE-Sephacel ion-exchange chromatography, gel-filtration on Sepharose CL-4B and CL-2B, and Octyl-Sepharose CL-4B chromatography. Proteoglycan-I was a high-molecular-weight proteoglycan, which was excluded by Sepharose CL-4B, and its molecular weight, estimated using sodium dedecvl sulfate-polyacrylamide gel electrophoresis, was over 350,000. The glycosaminoglycan chain of PG-I was composed of chondroitin 6-sulfate, and PG-I appeared to be an aggrecan-type proteoglycan. Proteoglycan-Il and III were low-molecular-weight proteoglycans with molecular weights of 190,000 and 105,000, respectively. The core protein sizes of PG-II and III were similar, 44,000, but anti-human decorin antibody reacted with PG--III, not with PG-II. The glycosaminoglycan chain of PG-II contained dermatan sulfate and chondroitin 6-sulfate, whereas that of PG-III contained the former only. In the light of these results, PG-II and III were suggested to be bigIycan- and decorin-type proteoglycans, respectively. The major proteoglvcan in the human yellow ligament extracellular matrix was PG-IIL Hirosaki Med. 1. 57: 49-58, 2006

CHARACTERIZATION OF DECORIN OF THE HUMAN YELLOW LIGAMENT

Decorin, a component of the low-molecular-weight proteoglycans (PG) of yellow Iigaments was purified, and the structures of the core protein and the glycosaminoglycan (GAG) chains were analyzed. The crude PG fraction extracted under dissociative conditions was subjected to DEAE-Sephacel ion-exchange chromatography, gel-filtration on Sepharose CL-4B, and Octyl-Sepharose CL-4B chromatography, and decorin was obtained. The amino-terminal amino acid sequence of decorin was found to be identical to those of human bone PG-II and human fetal membrane PG-II. The analyses of the GAG chains using two-dimensional electrophoresis on cellulose acetate membranes, affinity high-performance liquid chromatography (HPLC) with a hydroxyapatite column and gel-filtration HPLC after chondroitinase digestion, showed the presence of dermatan sulfate (DS) chains and chondroitin sulfate (CS). Further, it was suggested that CS located in the reducing termini and DS in the non-reducing termini in the hybrid chain. Hirosaki Med. J. 56: 45-54, 2005 ~~ g~~~0~~~0h~~m~~~ ~~0~~.~.*~GM~G, ~.~0~~~57~-~M~G~ ~, ~0~ 7 -3l J\~:BetU!f I) -!t -2 J ~ 0 fJ (GAG) mt~ji0t~m~~~fJT Gt.::. 7 -3l~)\~ 0N*jJiM7 -2 J ~~2:71J~J: e: l-~PG-II:B etUe: ~ij~ij~PG-II0~hC :-3G)t.::. GAGM~ji0-t)vD-;Z 7-t~-~ij~=jzjI;~%r*J} ), /\-1 ~0:::f:~7/\-3l-1 ~fJ ~~fJT~ ~J:, GAGM~ji~J:JijI;*j/iM{WH::~ 0 -1T ~1Jj1t~ (CS), ~FJijI;*j/iM{J!lH::~)v~ -3l ~~1rE~ (DS) i6~~tt9~/\-1 :1' I) 'Y ~~M 1) ~b*tk~~~ff~~*H1. *4~~fiJ~ 2) %iW*~~~ff~~-j::1 t;~~fiJ~

Purification and characterization of NADH dehydrogenase from Bacillus subtilis.

NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.

Roles of Conserved Proline and Glycosyltransferase Motifs of EmbC in Biosynthesis of Lipoarabinomannan

D-Arabinans, composed of D-arabinofuranose (D-Araf), dominate the structure of mycobacterial cell walls in two settings, as part of lipoarabinomannan (LAM) and arabinogalactan, each with markedly different structures and functions. Little is known of the complexity of their biosynthesis. beta-D-Arabinofuranosyl-1-monophosphoryldecaprenol is the only known sugar donor. EmbA, EmbB, and EmbC, products of the paralogous genes embA, embB, and embC, the sites of resistance to the anti-tuberculosis drug ethambutol (EMB), are the only known implicated enzymes. EmbA and -B apparently contribute to the synthesis of arabinogalactan, whereas EmbC is reserved for the synthesis of LAM. The Emb proteins show no overall similarity to any known proteins beyond Mycobacterium and related genera. However, functional motifs, equivalent to a proline-rich motif of several bacterial polysaccharide co-polymerases and a superfamily of glycosyltransferases, were found. Site-directed mutagenesis in glycosyltransferase superfamily C resulted in complete ablation of LAM synthesis. Point mutations in three amino acids of the proline motif of EmbC resulted in marked reduction of LAM-arabinan synthesis and accumulation of an unknown intermediate and of the known precursor lipomannan. Yet the pattern of the differently linked d-Araf units observed in wild type LAM-arabinan was largely retained in the proline motif mutants. The results allow for the presentation of a unique model of arabinan synthesis.

Purification and localization of a 10 kDa calcareous corpuscle binding protein of Spirometra mansoni plerocercoid.

In cestode parasites, calcareous corpuscles are thought to be associated with a number of intracellular physiologies by regulating the trafficking of mineral components. We previously separated these particular components by Ficoll, and their binding proteins of 10 kDa and 35 kDa in the Spirometra mansoni plerocercoid (sparganum). In the present study, we purified a 10 kDa protein employing octyl-Sepharose CL-4B affinity chromatography. Anti-serum raised against the purified protein showed specific reactions to the calcareous corpuscles of the worm section by immunohistochemistry, and recognized the 10 kDa protein by immunoblotting.

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.