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 characterization of a carboxymethyl cellulase from Artemia salina

Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cellulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase activity was purified and the activity analyzed under different conditions. After initial identification of cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the final purification, a 70-fold increase in specific enzyme activity was observed. SDS–PAGE results revealed that the cellulase enzyme had a molecular mass of 96 kDa. Temperature, pH, and salinity values were found to be optimal at 55 °C, pH 8.0, and 600 mM NaCl, respectively. Specifically, the enzyme showed a fivefold increase in enzyme activity in seawater compared to 600 mM NaCl in phosphate buffer. Further analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indicating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol from marine macroalgae.     

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.     

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 cellulase fromBacillus thermoalcaliphilus isolated from a termite mound

An extracellular carboxymethyl cellulase (CMCase) was purified to homogeneity fromBacillus thermoalcaliphilus sp. nov. The cellulase was composed of a single subunit with molar mass of 46 kDa. The apparentK _m was at 3.5 mg cellulose per mL. Its optimum pH was 8.5, it was most stable at pH 8.5–9.5 but 50% of enzyme activity was present after 30 min at pH 11.0. The activity was highest at 70°C.     

Purification and some properties of a carboxymethyl cellulase from Favolus arcularius

Favolus arcularius, a wood-rotting basidiomycete, produced carboxymethyl cellulose-hydrolyzing enzymes (CMCases) in culture media. Three main peaks of CMCase activity were separated as CMCase I, II and III at pHs from 4.4 to 5.2 by isoelectric focusing. Further, CMCase IIIa was purified from the CMCase III fraction. The molecular weight of CMCase IIIa was determined to be about 28,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was not active on avicel, cellobiose and laminarin, but could randomly hydrolyze cellooligosaccharides to form G₁ and G₂ units as the end products. The apparent K_m value of the enzyme against CMC was 0.28%.     

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.     

芽胞杆菌BSD-8肌氨酸氧化酶的纯化与性质

对一株从土壤中分离到的芽胞杆菌Bacillus sp.BSD-8菌株所产生的热稳定性较高的肌氨酸氧化酶进行纯化,并对该酶的特性进行了研究。通过硫酸铵分级沉淀、DEAE-纤维素离子交换柱、Toyopearl疏水层析柱和Sephadex G-75分子筛层析,使酶提纯25倍,比活力达到5.3U/mg。研究了纯化后的酶的生化特性,确定了该酶的主要特性:该酶为黄素蛋白,与黄素以非共价键的方式结合,由单一亚基组成,其亚基分子量为51kDa。酶的最适反应温度及pH分别为60℃与8.5。该酶在60℃及pH8.0~10.0条件下稳定。以Lineveaver-Burk作图法求得该酶米氏常数Km值为3.1mmol/L。Ag+、Hg2+、SDS及Tween80对该酶有强抑制作用,而Tween20和Triton X-100对酶活性无影响。该肌氨酸氧化酶在耐热性质上比以前所报道的肌氨酸氧化酶有很大的提高,在酶法肌酐测定应用中有明显的优势。     

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.     

Purification and characterization of a propanol-tolerant neutral protease from Bacillus sp. ZG20

Abstract

A propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655?U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29?kDa. The optimal temperature and pH of the protease were 45?°C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg²⁺, Zn²⁺, K⁺, Na⁺ and Tween-80 could improve its activity. The calculated K_m and V_max values of the protease towards α-casein were 12.74?mg/mL and 28.57?µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20.     

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.