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     

Bacillus huizhouensis sp. nov., isolated from a paddy field soil

A Gram-stain positive, facultative aerobic bacterium, designated as strain GSS03^T, was isolated from a paddy field soil. The cells were observed to be endospore forming, rod-shaped and motile with flagella. The organism was found to grow optimally at 35 °C at pH 7.0 and in the presence of 1 % NaCl. The strain was classified as a novel taxon within the genus Bacillus on the basis of phenotypic and phylogenetic analyses. The closest phylogenetic relatives were identified as Bacillus psychrosaccharolyticus DSM 6^T (97.61 %), Bacillus muralis DSM 16288^T (97.55 %), Bacillus asahii JCM 12112^T (97.48 %), Bacillus simplex DSM 1321^T (97.48 %) and “Bacillus frigoritolerans” DSM 8801^T (97.38 %). The menaquinone was identified as MK-7, the major cellular fatty acid was identified as anteiso-C_15:0 and the major cellular polar lipids as phosphatidylethanolamine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and three unknown polar lipids. The DNA G+C content was determined to be 40.2 mol%. The DNA–DNA relatedness with the closest relatives was below 48 %. Therefore, on the basis of all the results, strain GSS03^T is considered to represent a novel species within the genus Bacillus, for which the name Bacillus huizhouensis sp. nov. is proposed. The type strain is GSS03^T (=KCTC 33172^T =CCTCC AB 2013237^T).     

Purification and characterization of an organic-solvent-tolerant cellulase from a halotolerant isolate, Bacillus sp. L1

A halotolerant isolate Bacillus sp. L1 producing extracellular cellulase was isolated from Yuncheng, China. Production of the enzyme started from mid-exponential phase of bacterial growth and reached a maximum level during the post-stationary phase. The cellulase was purified to homogeneity with molecular mass of 45 kDa. Substrate specificity test indicated that it was an endoglucanase for soluble cellulose. Optimal enzyme activity was found to be at 60 °C, pH 8.0, and 7.5 % NaCl. Furthermore, it was highly active and stable over broad ranges of temperature (30-80 °C), pH (7.0-9.0), and NaCl concentration (2.5-15 %), thus showing its excellent thermostable, alkali-stable, and halotolerant nature. The cellulase activity was greatly inhibited by ethylenediaminetetraacetic acid, indicating that it was a metalloenzyme. Significant inhibition by phenylmethylsulfonyl fluoride and phenylarsine oxide revealed that serine and cysteine residues were essential for the enzyme catalysis. Moreover, the cellulase was highly active in the presence of surfactants, and it showed high stability in the presence of water-insoluble organic solvents with log P (ow)at least 0.88. Results from this study indicate that the purified cellulase from isolate L1 may have considerable potential for industrial application owing to its useful properties.     

Purification and biochemical characterization of an acidophilic amylase from a newly isolated Bacillus sp. DR90

An acidophilic and Ca²⁺-independent amylase was purified from a newly isolated Bacillus sp. DR90 by ion-exchange chromatography, and exhibited a molecular weight of 68.9 kDa by SDS-PAGE. The optimum pH and temperature of the enzyme were found to be 4.0 and 45 °C, respectively. The enzyme activity was increased by Ba²⁺, Fe²⁺ and Mg²⁺, and decreased by Hg²⁺ and Zn²⁺, while it was not affected by Na⁺, K⁺, phenylmethylsulfonyl fluoride and β-mercaptoethanol. Ca²⁺ and EDTA did not have significant effect on the enzyme activity and thermal stability. The values of K _m and V _max for starch as substrate were 4.5 ± 0.13 mg/ml and 307 ± 12 μM/min/mg, respectively. N,N-dialkylimidazolium-based ionic liquids such as 1-hexyl-3-methylimidazolium bromide [HMIM][Br] have inhibitory effect on the enzyme activity. Thin layer chromatography analyses displayed that maltose and glucose are the main products of the enzyme reaction on starch. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.     

Purification and characterization of a new bacteriocin isolated from a Carnobacterium sp.

A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.     

Purification and characterization of a novel thermostable lipase from Bacillus sp

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.     

Purification and characterization of alkaline soda-bleach stable protease from Bacillus sp. APP-07 isolated from Laundromat soil

The detergent-compatible alkaline protease was produced from the bacterial strain Bacillus sp. APP-07 isolated from Laundromat soil of Solapur, Maharashtra, India. The culture was grown in 1000?ml capacity baffled flask with a working volume of 100?ml and incubated at 55?°C for 33?h on a rotary shaker. After incubation, alkaline protease was partially purified by the sequential method of acetone precipitation followed by nominal molecular weight limit (NMWL) cut-off ultrafiltration using 50?K and 10?K filters. Finally, Sephadex G-100 gel filtration chromatographic purification was performed to obtain 3.12 fold purified alkaline protease enzyme with a 66.67% final yield. The purified enzyme showed 31907.269 units (U) of enzyme activity containing 8741.718?U/mg of specific enzyme activity. The molecular weight of the enzyme was confirmed about 33.0?kDa (kDa) by the SDS-PAGE analysis. The purified enzyme was stable at higher pH and temperature range, with an optimum pH 10.5 and temperature 55?°C. The enzyme showed excellent stability and compatibility in various detergents, surfactants, bleach, and oxidizing agents. The enzyme activity enhanced in the presence of Ca²⁺, Cu²⁺, and surfactants, whereas; the phenylmethylsulphonyl fluoride (PMSF) and Diisopropyl fluorophosphate (DFP) completely inhibit the enzymatic activity, which pointed out that the enzyme affiliated to serine-centered metalloproteases family.In conclusion, the remarkable tolerance and stability of the enzyme explored the promising candidature for the several potential applications in the laundry detergents. The sustainability of the enzyme might serve several possible applications in the laundry detergents, leather industries, and other harsh industrial processes.     

Bacillus haikouensis sp. nov., a facultatively anaerobic halotolerant bacterium isolated from a paddy soil

A Gram-stain positive, rod-shaped, endospore-forming and facultatively anaerobic halotolerant bacterium, designated as C-89^T, was isolated from a paddy field soil in Haikou, Hainan Province, People’s Republic of China. Optimal growth was observed at 37 °C and pH 7.0 in the presence of 4 % NaCl (w/v). The predominant menaquinone was identified as MK-7, the major cellular fatty acids were identified as anteiso-C_15:0 and iso-C_15:0, and the major cellular polar lipids were identified as phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and two unknown phospholipids. The peptidoglycan type was determined to be based on meso-DAP. Based on 16S rRNA gene sequence similarity, the closest phylogenetic relatives were identified as Bacillus vietnamensis JCM 11124^T (98.8 % sequence similarity), Bacillus aquimaris JCM 11545^T (98.6 %) and Bacillus marisflavi JCM 11544^T (98.5 %). The DNA G+C content of strain C-89^T was determined to be 45.4 mol%. The DNA–DNA relatedness values of strain C-89^T with its closest relatives were below 18 %. Therefore, on the basis of phylogenetic, chemotaxonomic, and phenotypic results, strain C-89^T can be considered to represent a novel species within the genus Bacillus, for which the name Bacillus haikouensis sp. nov., is proposed. The type strain is C-89^T (=KCTC 33545^T = CCTCC AB 2014076^T).     

Purification and characterization of a small size protease from Bacillus sp. APR-4

A thermostable extracellular protease of Bacillus sp. APR-4 was purified by size-exclusion and ion-exchange chromatographic methods and its properties were studied. The purified enzyme had a specific activity of 21,000 U/mg of protein and gave single band on SDS/PAGE with a molecular mass of 16.9 KDa. This protease had an optimal pH of 9 and exhibited its highest activity at 60 degrees C. The enzyme activity was inhibited by EDTA, suggesting the presence of metal residue at the active site. Ca2+ (5 mM) had stabilising effect on the activity of protease, but Cu2+ (5 mM) had inhibitory effect. The enzyme exhibited highest specificity towards casein (1%) and had a Km of 26.3 mg/ml and a Vmax of 47.6 U/mg with casein as a substrate. The stability of this enzyme was evaluated in the presence of some organic solvents and the enzyme was stable in methanol, petroleum ether and ethanol. Detergents (Wheel, Farishta) had stimulatory effect on the activity of this enzyme.     

Purification and characterization of maltose phosphorylase from Bacillus sp. RK-1

Bacillus sp. RK-1 was isolated as a bacterium that produced maltose phosphorylase (MPase) in the culture supernatant. Screening was done from among about 400 isolates that could grow at 55 degrees C in a medium containing maltose as the sole carbon source. The enzyme was purified to an electrophoretically homogeneous state and some properties were investigated. The Mr of the enzyme was estimated to be 170 kDa by gel filtration and 88.5 kDa by SDS-PAGE, suggesting that it consisted of two identical subunits. The enzyme showed optimum activity around pH 6.0-7.0 and the optimum temperature was about 65 degrees C. The enzyme was stable in the range of pH 5.5-8.0 after keeping it at 4 degrees C for 24 h and retained the activity up to about 55 degrees C after keeping it for 15 min. This is the first report about an MPase that could be produced in the culture supernatant. Furthermore, these investigations showed that this MPase is one of the most thermostable ones reported so far.     

A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: Purification and characterization

A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60–65 ^∘C and retained 100% activity after incubation at 60 ^∘C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ^∘C and 23 min incubation at 80 ^∘C. Catalytic function of lipase was activated by Mg⁺⁺ (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, βME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m and V _max of 0.5 mM and 0.139 μM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap.     

Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05

A halostable cellulase with a molecular mass of 29 kDa was purified from culture supernatants of the halophilic bacterium Salinivibrio sp. NTU-05 by way of the Fast Protein Liquid Chromatography method and the biochemical properties of the halostable cellulase was studied. The enzyme was active over a range of 0–25% sodium chloride examined in culture broth. The optimum cellulase activity was observed at 5% sodium chloride. Results from the salinity stability test indicated 24% of enzyme activity was retained at 25% sodium chloride for 4 h. The enzyme was also shown to be slightly thermostable with 40% residual activity under 60 °C for 4 h. The enzyme has a K_m of 3.03 mg/ml and a V_max of 142.86 mol/min/mg when tested using carboxymethyl-cellulose (CMC). The enzyme activity increased in the presence of K⁺, Mg²⁺, Na⁺ ions and decreased when Hg²⁺ ions were present. The deduced internal amino acid sequence of the Salinivibrio sp. NTU-05 cellulase showed similarity to the sequence of the glycoside hydrolase family protein. These are some of the novel characteristics that make this enzyme have potential applications in cellulose biodegradation.     

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn²⁺, Hg²⁺, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K _m and V _max of the enzyme for CMC were 3.4 mg ml^–1 and 44.7 mol min^–1 (mg protein)^–1, respectively.     

Purification,characterization and thermostability of lipase from a thermophilic Bacillus sp. J33

A thermostable lipase produced by a thermophilic Bacillus sp. J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography. The enzyme is a monomeric protein having molecular weight of 45 kDa. It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C12 and C4. The K_m and V_max for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 M min^-1 ml^-1 respectively. The immobilized enzyme was stable for 12 h at 60°C. Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers. Lipase acquired a remarkable stability, since no deactivation occurred at 70°C for 150 min in the presence of additives.     

Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp.

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.     

Purification and some properties of a Haim-sensitive alpha-amylase from newly isolated Bacillus sp. No. 195.

Newly isolated Bacillus sp. No. 195 produced an extracellular alpha-amylase sensitive to Haim which was found to inhibit specifically animal alpha-amylases. The enzyme was purified easily by two steps of starch adsorption and gel filtration using Sephacryl S-200. The purified enzyme, which showed a single band on native-PAGE or SDS-PAGE, had a molecular weight of 60,000 as judged on SDS-PAGE. The optimum pH value for activity and the isoelectric point were around 7.0 and 4.5, respectively. The sensitivity of the amylase to Haim was similar to that of animal amylase rather than bacterial amylase. It was suggested that a Haim-amylase complex might be formed at the molar ratio of 1:1. The amino acid sequence F-S-W similar to the triplet F-E-W highly conserved among alpha-amylases sensitive to proteinaceous inhibitors, such as Hoe 467-A or Haim, was found in the amino-terminal part of the No. 195 amylase.     

Purification and characterization of alanine carrier isolated from H-proteins of Bacillus subtilis.

Alanine transport carrier was isolated and purified from H-proteins of Bacillus subtilis. The purified carrier preparation was homogeneous in migration on polyacrylamide gels containing urea or sodium dodecyl sulfate. Electrophoresis on polyacrylamide gels containing dodecyl sulfate showed a single band of molecular weight of about 7500. 1 mol alanine was bound/mol carrier protein with a dissociation constant of 0.2 micron. The binding was inhibited by p-chloromercuribenzoate and the inhibition was reversed by dithiothreitol.     

Purification and characterization of a monoacylglycerol lipase from the moderately thermophilic Bacillus sp. H-257

A thermostable monoacylglycerol lipase [MGLP, EC 3.1.1.23] was purified for the first time from a cell-free extract of the moderately thermophilic Bacillus sp. H-257. The enzyme was purified 3,028-fold to homogeneity by chromatography using Octyl-Sepharose CL-4B, Q-Sepharose FF, and Superose 12 columns. The molecular mass of the MGLP was estimated to be 25 kDa by gel filtration and 24 kDa by SDS-PAGE, suggesting a monomeric protein. The isoelectric point was determined to be 4.66 by isoelectric focusing. The MGLP retained its full activity upon incubation at 60 degrees C for 10 min (pH 7. 3), and was stable at pH 7-10. The optimal temperature for activity at pH 7.5 was 75 degrees C, and the maximum activity was observed from pH 6-8. This enzyme hydrolyzes monoacylglycerols, with the highest activity occurring with 1-monolauroylglycerol. Di- and triacylglycerols, on the other hand, are essentially inert as substrates for the enzyme. The K(m) values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-monooleoylglycerol were determined to be 140, 83 and 59 mM, respectively. The enzyme was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycholate. The amino acid sequence of the N-terminal region of the enzyme (16 residues) was also determined.     

Purification, characterization and cloning of a thermotolerant isoamylase produced from Bacillus sp. CICIM 304

A novel thermostable isoamylase, IAM, was purified to homogeneity from the newly isolated thermophilic bacterium Bacillus sp. CICIM 304. The purified monomeric protein with an estimated molecular mass of 100 kDa displayed its optimal temperature and pH at 70 °C and 6.0, respectively, with excellent thermostability between 30 and 70 °C and pH values from 5.5 to 9.0. Under the conditions of temperature 50 °C and pH 6.0, the K _m and V _max on glycogen were 0.403 ± 0.018 mg/mg and 0.018 ± 0.001 mg/(min mg), respectively. Gene encoding IAM, BsIam was identified from genomic DNA sequence with inverse PCRs. The open reading frame of the BsIam gene was 2,655 base pairs long and encoded a polypeptide of 885 amino acids with a calculated molecular mass of 101,155 Da. The deduced amino acid sequence of IAM shared less than 40 % homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. This enzyme showed its obvious superiority in the industrial starch conversion process.