Cloning and Expression of Thermostable alpha-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable alpha-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more alpha-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the alpha-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the alpha-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80 degrees C for 60 min.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis.

The structural gene for a thermostable protease from Bacillus stearothermophilus was cloned in plasmid pTB90. It is expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about 15-fold more protease (310 U/mg of cell dry weight) than did the wild-type strain of B. stearothermophilus. Some properties of the proteases that have been purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant difference was observed among the enzyme properties studied here despite the difference in host cells. We found that the protease, neutral in pH characteristics and with a molecular weight of 36,000, retained about 80% of its activity even after treatment of 65 degrees C for 30 min.     

High Production of Thermostable beta-Galactosidase of Bacillus stearothermophilus in Bacillus subtilis

By cloning the beta-galactosidase gene of Bacillus stearothermophilus IAM11001 (ATCC 8005) into Bacillus subtilis, enzyme production was enhanced 50 times. beta-Galactosidase could be purified to 80% homogeneity by incubating the cell extract of B. subtilis at 70 degrees C for 15 min, followed by centrifugation to remove the denatured proteins. Because of its heat stability and ease of production, beta-galactosidase is suitable for application in industrial processes.     

枯草芽孢杆菌中性β—甘露聚糖酶的产生及性质

由土壤中分离出一株产中性β甘露聚糖酶的枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）,编号ＢＭ９６０２。该菌在液体培养条件下,产生中性β甘露聚糖酶。多糖能作为碳源,而单糖不能作为碳源;有机氮源优于无机氮源。产酶最适培养基组成：魔芋粉４％,牛肉蛋白胨和酵母膏各１％。产酶最适培养条件：培养基起始ｐＨ８５,３５℃,振荡培养３６ｈ。以槐豆胶为底物,培养滤液中性β甘露聚糖酶活力为９６ＩＵ／ｍＬ。酶在ｐＨ５０～１００和５０℃下稳定;作用最适条件为ｐＨ６０和５０℃;水解魔芋粉和槐豆胶均产生寡聚糖。     

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

三草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＢＭ９６０２产生的中性内切β－甘露聚糖酶（ｅｎｄｏ－β－１,４－Ｄ－ｍａｎｎａｎ ｍａｎｎａｎｏｈｙｄｒｏｌａｅｓ,ＥＣ,３．２．１．７８）经硫酸铵分级沉淀、ＤＥＡＥ－纤维素（ＤＥ２２）离子交换柱层析,得到电泳纯的样品,提纯了４５．５倍,收率为５．９％。用ＳＤＳ－ＰＡＧＥ测得该酶的分子量为３５ｋＤ。用ＰＡＧＥＩＥＦ测得其等电点ｐⅠ为４．５。酶反应的     

芽孢杆菌M50产生β—甘露聚糖酶的条件研究

从土壤中分离到９株产生β－甘露聚糖酶的芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｐ．）。Ｂａｃｉｌｌｕｓ ｓｐ．Ｍ５０２５０ｍＬ三角瓶摇瓶培养试验,以４％的魔芋粉为碳源,１．０％（ＮＨ４）２ＳＯ４为氮源,０．３５％Ｎａ２ＣＯ３,３０～３４℃培养６０ｈ产酶达到高峰。酶活力为１８０～２２０ｕ／ｍＬ。１００Ｌ罐发酵,在３０～３２℃,１：０．７５ｖｖｍ通气量,２００ｒ／ｍｉｎ条件下,发酵液酶活力高达３３０ｕ／ｍＬ。     

Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C. The enzyme was purified and concentrated in a single adsorption step onto a cation exchanger and is made of a single polypeptide with an apparent M(r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely degrades xylan to xylose and xylobiose. The pIs of the purified protein were 9 and 7 under native and denaturing conditions, respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At 65 degrees C and pH 7, the enzyme was stable for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h. Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp. strain C-125.     

Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost.

Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 3.2.1.52). Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M. The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5. Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.     

Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).     

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product.

By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolytically active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtilis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb HinfI-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. sterothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8 degrees C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostability was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B. caldolyticus.     

Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp. BG-11

An alkalophilic, chitinase-producing Bacillus sp. BG-11 was isolated which produced an extracellular chitinase and which was purified 16.5-fold, using standard purification techniques. The purified chitinase exhibited a broad pH and temperature optima of 7.5-9.0 and 45 deg C-55 deg C, respectively. The chitinase was stable between pH 6.0-9.0 and 50°C for more than 2 h. Half lives of enzyme at 60 deg C, 70 deg C and 80 deg C were 90 min, 30 min and 20 min respectively. Km value was 12 mg chitin per ml. Shelf life was 60 days at 4°C. Ca2+, Ni2+ and Triton-X-100 stimulated the activity up to 20% whereas Ag+, Hg2+, dithiothreitol, -mercaptoethanol, glutathione, iodoacetic acid and iodoacetamide inhibited the activity up to 50%.     

Gene cloning and biochemical characterizations of thermostable ribonuclease HIII from Bacillus stearothermophilus

The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst-RNase HIII) was purified and biochemically characterized. Bst-RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu-RNase HIII). The enzymatic properties of Bst-RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu-RNase HIII. However, Bst-RNase HIII was more stable than Bsu-RNase HIII, and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was 55 degrees C for Bst-RNase HIII and 35 degrees C for Bsu-RNase HIII. The optimum temperature for Bst-RNase HIII activity was also shifted upward by roughly 20 degrees C as compared to that of Bsu-RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.     

Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.     

Purification and characterization of carboxymethyl cellulase from Bacillus sp. isolated from a paddy field

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.     

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.