Purification and characterization of a novel neutral β-glucanase and an alkaline β-glucanase from an alkaliphilic Bacillus isolate

A relatively high β-glucanase-producing strain Bacillus sp. III-3 was isolated from soda lakes in Neimenggu, China. This alkaliphilic strain was found to produce two thermostable β-glucanases including a novel neutral β-glucanase III-3-A and an alkaline β-glucanase III-3-B. The β-glucanases were purified to homogeneity from the culture supernatant with a two-step column chromatographic procedure. III-3-A and III-3-B had molecular mass of approximately 45 and 85 kDa, respectively. Mass spectrometry analysis indicated that III-3-A was probably different from the β-glucanases reported, whereas III-3-B showed high homology with those of family A5 alkaline β-glucanases from alkaliphilic bacilli. The optimum pH of III-3-A was about 7.0, while that of III-3-B was 8.0–10.0. Both enzymes exhibited maximum activity at 45 °C and were stable up to 50 °C. Ca²⁺ ion stimulated the activity of III-3-A but enhanced the thermostability of III-3-B. The two enzymes were resistant to most metal ions and reagents examined.     

The Na+/H+ antiporter of alkaliphilic Bacillus sp.

The Na⁺/H⁺ antiporter, which appears to predominantly contribute to the alkaliphily of Bacillus halodurans C-125, was studied in an alkali-sensitive mutant of this strain and a transformant with restored alkaliphily. The alkali-sensitive mutant, strain 38154, which has lost the ability to grow above pH 9.5, was found to lack electro-genic Na⁺/H⁺ antiport activity driven by ΔΨ (membrane potential, interior negative), and it showed defective regulation of intracellular pH under alkaline conditions. On the other hand, a transformant carrying a 2.0-kb DNA fragment from the parental genome that complemented this defect was able to maintain an intracellular pH lower than that of the external milieu, and it was found to have recovered the Na⁺/H⁺ antiport activity driven by ΔΨ. Sequence analyses found that a 5.1-kb DNA region contained four open reading frames (ORF-1 to ORF-4). Direct sequencing of the corresponding region in mutant 38154 revealed a G-to-A substitution, which resulted in an amino acid substitution from Gly-393 to Arg in the putative ORF-1 product. It has been recently found that a region homologous to the DNA fragment responsible for the alkaliphily of strain C-125 exists in the genomes of Bacillus subtilis, Sinorhizobium (Rhizobium) meliloti, and Staphylococcus aureus. These homologues are present as a cluster of seven ORFs in each case. The shaA gene product of B. subtilis shows significant similarity to the ORF-1 product of strain C-125. Disruption of the shaA gene resulted in a decrease in Na⁺/H⁺ antiport activity, and growth of the shaA-disrupted strain was impaired when the external Na⁺ concentration was increased. We conclude that the shaA gene encodes a Na⁺/H⁺ antiporter, which plays an important role in extrusion of cytotoxic Na⁺. Received: May 29, 2000 / Accepted: July 18, 2000     

Truncation analysis of an alkaline cellulase from an alkalophilic Bacillus species

A series of truncated gene products from an alkaline cellulase from an alkalophilic Bacillus sp. No. 1139 has been prepared. The variously sized proteins were products of in vitro insertional mutagenesis constructs made by gene inserts containing translational terminators. One product, a 46-kDa protein, which had about half the M_r of the original cellulase, had a similar enzyme activity and pH optimum to the original 92-kDa protein. In contrast, a slightly smaller product protein (43 kDa) did not show cellulase activity.     

青海可可西里嗜碱芽胞杆菌资源调查

【目的】了解可可西里嗜碱芽胞杆菌资源多样性及产酶多样性,为芽胞杆菌功能资源挖掘和菌剂开发提供基础。【方法】采用Horikoshi I培养基,通过可培养法分离青海可可西里土壤中的嗜碱芽胞杆菌,利用16S r RNA基因序列初步鉴定分离获得的芽胞杆菌。采用透明圈法分析分离菌株的产蛋白酶、纤维素酶及木聚糖酶活性。【结果】从青海可可西里土壤中共分离获得66株嗜碱芽胞杆菌,根据16S r RNA基因序列相似性分析,发现它们隶属于6个属22个种,分别为芽胞杆菌属(Bacillus)、纤细芽胞杆菌属(Gracilibacillus)、喜盐芽胞杆菌属(Halobacillus)、咸海鲜芽胞杆菌属(Jeotgalibacillus)、类芽胞杆菌属(Paenibacillus)和嗜冷芽胞杆菌属(Psychrobacillus),其中以芽胞杆菌属(Bacillus)为优势属。2株嗜碱芽胞杆菌与它们最近匹配模式菌株的16S r RNA基因序列相似性为97.00%和98.65%,为潜在新种。三种酶活检测结果表明产酶菌株约占总分离菌株的95.00%,其中55株具有产蛋白酶活性,27株具有产纤维素酶活性,8株能够产木聚糖酶。【结论】青海可可西里蕴藏着较丰富的嗜碱芽胞杆菌资源及丰富的产酶资源,为后续嗜碱芽胞杆菌的挖掘提供理论基础。     

Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

Partial purification and some properties of an alkaline cellulase from an alkalophilic Bacillus sp.

A newly isolated Bacillus species, which grew optimally at 30°C and pH 10, produced a carboxymethylcellulase in a medium containing 10 g CM-cellulose/l. The enzyme, when partially purified by gel filtration, had a mass of about 29 kDa as determined by both SDS-PAGE and gel filtration chromatography. It was optimally active at pH 9.5 and 40°C, and was stable from pH 7 to 11 at 4°C for 24 h. The enzyme was stimulated by Ca²⁺ (1mm) but was completely inhibited by Hg²⁺ (1mm). Neither EDTA nor EGTA (10mm) affected the activity.The author is with the Department of Biological Sciences, University of Jordan. PO Box 2686, Amman 11181, Jordan     

嗜碱菌(Bacillus sp.)ZBAW6的木聚糖酶的分离纯化及其性质

通过硫酸铵分级沉淀 ,阴离子交换层析 ,凝胶过滤 3步从嗜碱菌Bacillussp .ZBAW6纯化了木聚糖酶。结果表明该酶分子量为 4 5kD。N末端序列为DPFAAAVAPL。在pH5 5～ 1 0 5范围内均具有较高酶活性和稳定性 ;最适反应温度为 6 5℃ ,酶活力基本不变。该酶作用于Beech xylan的Km 为 0 1 1mg mL ,Vmax 为 2 3 89μmol (min·mg)。Hg2 + 对该酶有强的抑制作用     

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.     

Bleach-stable, alkaline protease from Bacillus sp.

A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolated Bacillus SB5 is reported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at 40°C, after 1 h. The enzyme was optimal at pH 10 and 60°C to 70°C. Enzyme activity was enhanced 30 to 80% in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.     

K+/H+ antiporter in alkaliphilic Bacillus sp. no. 66 (JCM 9763)

A K⁺/H⁺ antiport system was detected for the first time in right-side-out membrane vesicles prepared from alkaliphilic Bacillus sp. no. 66 (JCM 9763). An outwardly directed K⁺ gradient (intravesicular K⁺ concentration, K_in, 100 mM; extravesicular K⁺ concentration, K_out, 0.25 mM) stimulated uphill H⁺ influx into right-side-out vesicles and created the inside-acidic pH gradient (ΔpH). This H⁺ influx was pH-dependent and increased as the pH increased from 6.8 to 8.4. Addition of 100 μM quinine inhibited the H⁺ influx by 75%. This exchange process was electroneutral, and the H⁺ influx was not stimulated by the imposition of the membrane potential (interior negative). Addition of K⁺ at the point of maximum ΔpH caused a rapid K⁺-dependent H⁺ eflux consistent with the inward exchange of external K⁺ for internal H⁺ by a K⁺/H⁺ antiporter. Rb⁺ and Cs⁺ could replace K⁺ but Na⁺ and Li⁺ could not. The H⁺ efflux rate was a hyperbolic function of K⁺ and increased with increasing extravesicular pH (pH_out) from 7.5 to 8.5. These findings were consistent with the presence of K⁺/H⁺ antiport activity in these membrane vesicles. Received: March 20, 1997 / Accepted: May 22, 1997     

Tandem location of the cellulase genes on the chromosome of Bacillus sp. strain N-4

Abstract A 5.7-kb Eco RI DNA fragment has been isolated from Bacillus sp. strain N-4 chromosome DNA. This fragment contained both the pNK1-encoded cellulase ( celB ) gene and the pNK2-encoded cellulase ( celA ) gene which were highly homologous [13]. These results demonstrate the tandem location of these genes on the chromosomal DNA. The homologous sequence, which may play an important role for the gene duplication, were observed 5' upstream of the celA gene, between the celA and celB genes, and 3' downstream from the celB gene.     

Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkalinesoda lake

An alkaline xylanase-producing alkaliphilic Bacillus sp. AR-009 was isolated from analkaline soda lake in Ethiopia. The enzyme was optimally active at pH 9 and was stable over abroad pH range. The optimum temperature for xylanase activity, assayed at pH 9, was60°–65°C. Measured at pH 8 and 9, the enzyme had good stability at 55° and60°C. At both pH values, over 80% of its original activity was retained after heating for2·5 h at 55°C. At 60°C, the enzyme maintained 63% of its original activity after2·5 h incubation while at pH 9 it retained 54% of its original activity after 1 h heating. Theseproperties qualify the enzyme to be novel and potentially important for application in someindustrial processes.     

Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability

Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca²⁺ on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca²⁺ with Ba²⁺, Mn²⁺, Mg²⁺ and Sr²⁺ in the phytase resulted in the expression of > 90% of the maximal activity with calcium-phytate as the substrate, while Fe²⁺ and Zn²⁺ rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg²⁺, Sn²⁺ and Sr²⁺, respectively. On replacing Ca²⁺ on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg²⁺ and Sr²⁺, respectively. Only Ca²⁺ resulted in a marked increase in the melting temperature (T_m) of the enzyme by 12-21 °C, while Ba²⁺, Mn²⁺, Sr²⁺ or Cu²⁺ resulted in a modest (2-3.5 °C) increase in T_m. In the presence of 1-5 mM Ca²⁺, the optimum temperature of the phytase activity was increased from 40 °C to 70 °C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region.     

Cation-induced thermal stability of an alkaline protease from a Bacillus sp.

An alkaline protease produced from a Bacillus sp. was stimulated by the metal ions Ca²⁺, Mg²⁺ and Mn²⁺; with Ca²⁺ having the maximum effect. The thermal stability of the enzyme was also enhanced to varying degrees in the presence of these ions.     

Production of alkaline xylanase by a newly isolated alkaliphilic Bacillus sp. strain 41M-1

Alkaliphilic Bacillus sp. strain 41M-1, isolated from soil, produced xylan-degrading enzymes extracellularly. Optimum pH for the crude xylanase preparation was about pH 9, confirming the production of novel alkaline xylanase(s) by the isolate. Xylanases were induced by xylan, but were not produced in the presence of xylose, arabinose or glucose. Xylanase productivity was influenced by culture pH, and production at pH 10.5 was higher than that at pH 8.0. Zymogram analysis of the culture supernatant showed the alkaline xylanase with a molecular mass of 36 kDa.     

Purification and properties of mangano-superoxide dismutase from a strain of alkaliphilic Bacillus

A mangano-superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a strain of alkaliphilic Bacillus for the first time. The purified protein, with an isoelectric point of pH 4.5, had a molecular mass of approximately 50 kDa and consisted of two identical subunits (25 kDa). The N-terminal amino acid sequence was Ala-Tyr-Lys-Leu-Pro-Glu-Leu-Pro-Tyr-Ala-Ala-Asn-Ala-Leu-Glu-Pro-His-Ile-Asp-Glu-Ala. The optimum pH and temperature for the reaction were 7.5 and 35°C, respectively. The properties of the superoxide dismutase were compared with those of the enzyme from thermophilic Bacillus stearothermophilus. Received: September 3, 1996 / Accepted: October 4, 1996     

Alkaline cellulases from alkaliphilic Bacillus: Enzymatic properties, genetics, and application to detergents

We have isolated a number of alkaliphilic Bacillus that produce alkaline exoenzymes and found a possible use for alkaline cellulase (carboxymethylcellulase) as an additive for improving the cleaning effect of detergents. Enzymatic properties of some candidate cellulases fulfilled the essential requirements for enzymes to be used practically in laundry detergents. Here I describe the properties and possible catalytic mechanism of the hydrolytic reaction and the gene for the industrial alkaline cellulase produced by one of the isolates, Bacillus sp. KSM-635. Received: October 4, 1996 / Accepted: December 2, 1996     

Identification of facultatively alkaliphilic Bacillus sp. strain YN-2000 and its fatty acid composition and cell-surface aspects depending on culture pH

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA–DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment. Received: April 6, 2000 / Accepted: May 8, 2000     

Feasibility study for decomposition of gelatin layers on X-ray films by thermostable alkaline protease from alkaliphilic Bacillus sp.

Enzymatic decomposition of gelatin layers on X-ray films and repeated utilization of enzyme for potential industrialization were investigated using thermostable alkaline protease from the alkaliphilic Bacillus sp. B21-2. The decomposition of gelatin layers at 50 °C with the mutant enzyme (Ala187 was replaced by Pro) was higher than those of the wild-type and other mutant enzymes. In the repeated experiment for every 60 min (20 U ml^–1, 50 °C), the mutant enzyme could be satisfactorily used five times while three times for the wild-type enzyme.     

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K _cat). The enzyme was also calcium dependent and with 2 mM Ca⁺², the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca⁺², respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.