Studies of Hemicellulolytic Enzymes of Bacillus subtilis

Many strains of Bacillus subtilis were found to secrete several hemicellulolytic enzymes such as arabinoxylanase, galactomannanase, arabinogalactanase, etc. Chemical and enzymatic properties of certain strains of the bacterium were comparatively investigated. An arabinogalactanase was purified and obtained in a crystalline state. Its molecular weight and isoelectric point were estimated to be 3.7×10⁴ and 8.39, respectively. The enzyme showed an optimal pH for reactions at 6.0, and was stable in a pH range of 5.0 to 9.5 at 30°C. It required no metallic ions for its activity and hydrolyzed soybean arabinogalactan, forming galactobiose as the main product. No liberation of arabinose was observed in the hydrolysate. Also, the enzyme did not attack coffee bean arabinogalactan. Some implications of the experimental results are discussed.     

Purification and Properties of Two Galactanases from Penicillium citrinum

Two galactanases (I and II) were purified to homogeneous states from water extracts of a wheat bran culture of Penicillium citrinum. Although these enzymes were separable by affinity chromatography and distinct on Polyacrylamide gel electrophoresis, they were similar in physical and enzymatic nature. They had almost the same molecular weight (3.5 } 10⁴) and isoelectric point (pH 4.2), and similar amino acid compositions and carbohydrate contents (3%). Alanine and leucine were detected for both enzymes as the N- and C-terminal amino acids, respectively.

These enzymes were most active at pH 4.5 and 55%C, and were stable between pH 4 and 10 (at 15%C for 24hr), and below 55%C (at pH 5.5 for 10min). The enzymes hydrolyzed soybean arabinogalactan to produce galactose and several galac-tooligosaccharides, but did not attack coffee bean arabinogalactan. Therefore, these enzymes were suggested to be endo-l,4-²-D-galactanases.

The enzymes also attacked ONPG and PNPG, and lag phases were observed at the beginning of the reactions.

Among the compounds examined, Hg²⁺ and Fe³⁺ inhibited the enzyme actions. ONPG-hydrolyzing activities of the enzymes were inhibited by some sugars such as lactose, galactose, arabinose, glucose and xylose.

Galactobiose, -triose and -tetraose prepared from soybean arabinogalactan with purified galactanase I were found to be further hydrolyzed by the enzymes. A lag phase was also observed in the time course of hydrolysis of galactobiose.     

Purification and characterization of an exo-1,4-beta-galactanase from a strain of Bacillus subtilis

An arabinogalactan 4-beta-D-galactanohydrolase was purified to a homogeneous state from the culture filtrate of a strain of Bacillus subtilis. The enzyme have a molecular mass of 36 kDa and an isoelectric point of pH 7.9. The enzyme is most active at around pH 6.5-7 and at 60 degrees C, and is stable between pH 6-10 and below 55 degrees C. Hg2+ and Cu2+ inhibit the activity. The enzyme hydrolyze soybean arabinogalactan which contains beta-1,4-galactosidic linkages in its main chain structure, but not other polysaccharides with beta-1,3-galactosidic linkages. The hydrolysis products from soybean arabinogalactan are predominantly galactobiose with a small amount of galactotetraose. The enzyme is an exo-enzyme and the ability to transfer galactobiose to other galactobiose molecules is indicated by the formation of galactotetraose.     

Complete Growth Inhibition of Bacillus subtilis by Nisin-Producing Lactococci in Fermented Soybeans

The growth inhibition by nisin-producing lactococci against Bacillus subtilis and its application to soybean miso fermentation were investigated. Lactococcus lactis subsp. lactis IFO12007 (nisin-producing, salt-intolerant) rapidly proliferated to more than 10⁹ cells/g in cooked soybeans without any excessive pH decrease. In spite of the mild decrease in pH, the growth of B. subtilis was completely inhibited; no living cells were detected in a soybean sample inoculated with 10⁶ cells/g and incubated for 24 to 72 h. This Lc. lactis was applied to soybean miso fermentation as a starter culture. It produced high nisin activity (1.28×10⁵ AU/g) in cooked soybean, resulting in the complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation, throughout the process of miso production. Over-acidification, which is undesirable for miso quality, was successfully prevented simply by adding salt which killed the salt-intolerant Lc. lactis. Furthermore, the nisin activity in miso disappeared with aging.     

Studies on Biologically Active Substances Formed by Bacilli

The products of several Bacillus strains were investigated on rabbit serum calcium decreasing, oxytocic and toad heart function promoting activities. These products were obtained from the clear supernatant fluid of the culture medium after the cells were removed by centrifugation.

For the production of rabbit serum calcium decreasing substance, Bacillus subtilis K and Bacillus natto No. 8 were found to be usefull, Bacillus megaterium KM, Bacillus cereus var. mycoides and Bacillus subtilis K produced oxytocic principle. Bacillus subtilis K, Bacillus brevis and Bacillus megaterium KM also produced toad heart function promoting factor.

A procedure was developed to obtain the electrophoretically homogenous rabbit serum calcium decreasing substance from culture filtrate of Bacillus subtilis K. The crude substance was obtained as isoelectric precipitate by adjusting the culture filtrate to pH 3.0. The crude substance was purified by gel filtration on a Sephadex G-75 column, isoelectric fractionation and chromatography on DEAE-cellulose column. The purified preparation was shown to be homogenous by Tiselius electrophoresis but was separated into two bands by polyacrylamide electrophoresis. The chemical analysis of this biologically active substance indicated this substance to be a lipoprotein. The substance decreased rabbit serum calcium level about 12% at 6~8hr after intravenous injection (dose; 0.5 mg/kg body weight).     

Design of an expression vector and its application to heterologous protein expression in Bacillus subtilis

An expression vector, pUBEX, was constructed for extracellular production of heterologous proteins in Bacillus subtilis using a polyhistidine tag on the C-terminal sequence, providing an efficient and easy purification of the protein. A CII protein, a member of Bowman–Birk protease inhibitors, which was expressed as an inactive protein in Escherichia coli, was successfully expressed in Bacillus subtilis using the pUBEX vector and was purified to 6.4 mg l^–1 by the immobilized metal affinity chromatography.     

Mycelial β-N-Acetylglucosaminidase of Streptomyces sp. Having β-N-Acetylgalactosaminidase Activity

Formation of transfer products from soybean arabinogalactan and glycerol by endo-1,4-β-d-galactanase from Penicillium citrinum was described. The amount of transfer products depended on the glycerol concentration. About 50% of the galactose residues which could be liberated from the polysaccharide by the enzyme were transferred to glycerol at an acceptor concentration of 2.5% (w/v). Transfer products with various polymerization degrees were accumulated at the beginning of the reaction and then those with higher polymerization degrees were degraded gradually. At a final stage of the reaction, two transfer products in addition to two hydrolysis products (galactose and galactobiose) were mainly accumulated. The two transfer products were isolated and their structures were examined. They were 2-O-β-d-galactosyl glycerol and O-β-d-galactosyl-(1 → 4)-O-β-d-galactosyl-(1 → 2)glycerol.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53

The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Bacillus subtilis subsp. subtilis by analyses of 16S rDNA and partial sequences of the gyrA gene, and named as B. subtilis subsp. subtilis A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56 kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and p-nitrophenyl-β-d-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50 °C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40 °C after 24 h incubation at 50 °C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K⁺, Ni²⁺, Sr²⁺, Pb²⁺, and Mn²⁺, but inhibited by Co²⁺ and Hg²⁺.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">polyfermenticus</Emphasis> isolated from <Emphasis Type="Italic">Meju,</Emphasis> Korean soybean fermentation starter

Bacteria of the Bacillus species have been reported as an important microorganism in fermented soybean products. In the present study, thirty Bacillus isolates were screened from Meju, a Korean soybean fermentation starter. The comparative analysis of 16S rDNA sequences, 16S-23S internal transcribed spacer sequences, phenotypic, and biochemical characterizations revealed three phylogenetically distinct groups namely Bacillus atrophaeus, Bacillus polyfermenticus and Bacillus subtilis. The isolates were assayed for poly-γ-glutamate production and fibrinolytic activity. Among the isolates, B. polyfermenticus exhibited maximum poly-γ-glutamate production and fibrinolytic activity. Moreover, the soybean products fermented by B. polyfermenticus have increased the time taken for coagulation and hemorrhage in mice. The results of the present study clearly indicate the functional role of B. polyfermenticus in fermented soybean products.     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

High-level expression,purification and characterization of a recombinant medium-temperature <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Alpha-amylases are important industrial enzymes with a wide range of applications. Although medium-temperature alpha amylase (AmyE) has some practical advantages, its low yield has limited its applications. When an amyE gene from Bacillus subtilis BF768 was cloned into vector pWB980 and over-expressed in B. subtilis WB600, high activities (723 U ml⁻¹) of secreted AmyE were produced. Recombinant AmyE was purified to a specific activity of 36 U mg⁻¹ having optimal activity at pH 6.0 and 60°C.     

NMR assignment of the N-terminal repeat domain of Bacillus subtilis ClpC

The HSP100/AAA+ superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. We present here the backbone and side-chain assignments of the N-terminal repeat domain (residues 1–145) of ClpC from Bacillus subtilis. Douglas J. Kojetin and Patrick D. McLaughlin have equally contributed.     

n-Terminal Amino Acid Sequences of Neutral Proteases from Bacillus amyloliquefaciens and Bacillus subtilis: Identification of a Neutral Protease Gene Cloned in Bacillus subtilis

Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures.

The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini.

Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.     

Frequency of the Insertion Sequence IS4Bsu1 among Bacillus subtilis Strains Isolated from Fermented Soybean Foods in Southeast Asia

Among 45 Bacillus subtilis strains isolated from non-salted types of fermented soybeans produced in several Southeast Asian countries, 20 had the insertion sequence IS4Bsu1 in the chromosome. In contrast, none of 49 B. subtilis strains of non-food origin contained IS4Bsu1. Frequent occurrence of this mobile DNA element in the soybean-fermenting B. subtilis would reflect the fact that few strains flourish on soybeans and thereby contribute to soybean fermentation.     

Phenotypic diversity and technological properties of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> species isolated from <Emphasis Type="Italic">okpehe</Emphasis>, a traditional fermented condiment

The Bacillus subtilis wild strains isolated from okpehe, a traditional fermented condiment used as seasoning in Nigeria, the reference and typed strains were investigated for their phenotypic diversity and their technological parameters with a view to obtain adequate data that would enable selection of appropriated starter cultures for vegetable protein fermentation in West Africa. All the 7 strains studied demonstrated diverse phenotypic characteristics and they were identified as Bacillus subtilis, based on the API 50 CHB combined with API 20E profile. Specific sugars that indicated a good hydrolytic potential of the wild strains were fermented. The highest proteinase activity of 90 AU/ml determined quantitatively was observed in the strain Bacillus subtilis BFE 5372, the proteinase was identified by the APIZYM gallery as chymotrypsin. Highest amylase activity of 13 AU/ml was noticed in strain Bacillus subtilis DSM 347 while only 4 strains produced polyglutamic acid with the strain Bacillus subtilis BFE 5359 producing the highest polyglutamate activity of 2.5 mm. Although strain Bacillus subtilis BFE 5301 did not release detectable polyglutamate, the strain demonstrated antagonism against different bacteria and the antimicrobial substance produced by strain Bacillus subtilis BFE 5301 was confirmed as a bacteriocin since its activities were lost after treatment with chymotrypsin and pepsin. The data generated showed the technological parameters that can aid selection of wild strains such as Bacillus subtilis BFE 5301, BFE 5359 and BFE 5372 for optimization of condiment production.     

Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I,from Bacillus subtilis – Bsu121I,a type II restriction endonuclease from Bacillus subtilis

A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified. The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease. The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp. The endonuclease activity required Mg⁺² as cofactor like other type II endonucleases.     

Purification and kinetics of a protease-resistant,neutral, and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 ameliorating food nutrition

Abstract

A novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca⁺², K⁺, and Co⁺² and it was inhibited by SDS and Mg⁺², Al⁺², and Fe⁺². Purified enzyme showed specificity to different salts of phytic acid and values of K_m and V_max were 0.293?mM and 11.49 nmoles s^?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein.     

Expression and Secretion of an Acid-Stable <Emphasis Type="Italic">α</Emphasis>-Amylase Gene in Bacillus Subtilis by <Emphasis Type="Italic">SacB</Emphasis> Promoter and Signal Peptide

Alpha amylase gene from Bacillus licheniformis was mutated by site-directed mutagenesis to improve its acid stability. The mutant gene was expression in Bacillus subtilis under the control of the promoter of sacB gene which was followed by either the α-amylase leader peptide of Bacillus licheniformis or the signal peptide sequence of sacB gene of Bacillus subtilis. Both peptides efficiently directed the secretion of α-amylase from the recombinant B. subtilis cells. The extracellular α-amylase activities in two recombinants were 1001 and 2012 U ml⁻¹, respectively. The purity of the recombinant product was confirmed by SDS-PAGE.     

The distinct PhoPR mediated responses to phosphate limitation in Bacillus subtilis subspecies subtilis and spizizenii stem from differences in wall teichoic acid composition and metabolism

The PhoPR‐mediated response to phosphate limitation (PHO response) in Bacillus subtilis subsp subtilis is amplified and maintained by reducing the level of Lipid V^G composed of poly(glycerol phosphate), a wall teichoic acid (WTA) biosynthetic intermediate that inhibits PhoR autokinase activity. However, the reduction in Lipid V^G level is effected by activated PhoP～P, raising the question of how the PHO response is first initiated. Furthermore, that WTA is composed of poly(ribitol phosphate) in Bacillus subtilis subsp spizizenii prompted an investigation of how the PHO response is regulated in that bacterium. We report that the PHO responses of B. subtilis subsp subtilis and subsp spizizenii are distinct. The PhoR kinases of the two B. subtilis subspecies are functionally equivalent and are activated either by the TagA/TarA or TagB/TarB enzyme product. However, they are inhibited by Lipid V^G composed of poly(glycerol phosphate) but not by Lipid V^R composed of poly(ribitol phosphate). Therefore, the distinctive PHO responses of these B. subtilis subspecies stem from the differential sensitivity of PhoR kinases to the polyol composition of Lipid V and from the genomic organization of WTA biosynthetic genes and the regulation of their expression.