Isolation and characterization of feather degrading enzymes from Bacillus megaterium SN1 isolated from Ghazipur poultry waste site

The SN1 strain of Bacillus megaterium, isolated from soil of Ghazipur poultry waste site (India) produced extracellular caseinolytic and keratinolytic enzymes in basal media at 30°C, 160 rpm in the presence of 10% feather. Feathers were completely degraded after 72 h of incubation. The caesinolytic enzyme was separated from the basal media following ammonium sulphate precipitation and ion exchange chromatography. We report 29.3-fold purification of protease after Q Sepharose chromatography. The molecular weight of this enzyme was estimated to be 30 kDa as shown by SDS-PAGE and zymography studies. Protease activity increased by 2-fold in presence of 10 mM Mn²⁺ whereas Ba²⁺ and Hg²⁺ inhibited it. Ratio of milk clotting activity to caseinolytic activity was found to be 520.8 for the 30–60% ammonium sulphate fraction in presence of Mn²⁺ ion suggesting potential application in dairy industry. Keratinase was purified to 655.64 fold with specific activity of 544.7 U/mg protein and 12.4% recovery. We adopted the strategy of isolating the keratinolytic and caesinolytic producing microorganism by its selective growing in enriched media and found that feather protein can be metabolized for production of animal feed protein concentrates.     

Isolation and characterization of forespores from Bacillus megaterium

A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed. The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol. The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation. The fine structure of the forespores prepared at 6 hr (t6) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger. The density, determined by density gradient centrifugation, of the forespores isolated at t6, t10, t12, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t6 and t8 were extremely heat labile (D80 of 9.5 and 21.5 min, respectively) relative to mature spores (D80 of 277.8 min). These forespores were also less resistant to organic solvents. Germination of the forespores as well as mature spores was induced by KNO3, D-glucose, and L-leucine. Forespores at t6 were more sensitive to KNO3-induced germination than those at t10, t12, and mature spores when measured by reduction in the optical density of cell suspension.     

Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2?U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2?U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65?°C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60?°C for almost 90?min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.     

Dielectric characterization of forespores isolated from Bacillus megaterium ATCC 19213

Isolated stage III forespores of Bacillus megaterium ATCC 19213 in aqueous suspensions were nearly as dehydrated as mature spores, as indicated by low dextran-impermeable volumes of ca. 3.0 ml per g (dry weight) of cells compared with values of ca. 2.6 for mature spores and 7.3 for vegetative cells. The forespores lacked dipicolinate, had only minimal levels of calcium, magnesium, manganese, potassium, and sodium, and were more heat sensitive than vegetative cells. The effective homogeneous conductivities and dielectric constants measured over a frequency range of 1 to 200 MHz indicated that the inherent conductivities of the forespores were unusually low, in keeping with their low mineral contents, but that the forespores could be invaded by environmental ions which could penetrate dielectrically effective membranes. Overall, our findings support the view that the dehydration of a forespore during stage III of sporogenesis may be the result of ion movements out of the forespore into the sporangium.     

Isolation and characterization of the siderophore N-deoxyschizokinen from Bacillus megaterium ATCC 19213

N-Deoxyschizokinen, a novel siderophore, was isolated from stationary phase cultures of Bacillus megaterium ATCC 19213 and identified as 4-[(3(acetylhydroxyamino)propyl)amino]-2-[2-[(3-(acetylamino)propyl)amino]-2-oxoethyl]-2-hydroxy-4-oxo-butanoic acid. The siderophore was purified by HPLC and its structure determined using ¹H and ¹³C NMR, ¹H-¹H COSY and electrospray mass spectroscopy. The monohydroxamate siderophore has the same carbon skeleton as schizokinen but the hydroxyl group on one hydroxamate is replaced by a hydrogen. A detailed ¹H NMR study of schizokinen, N-deoxyschizokinen and their imides, schizokinen A and N-deoxyschizokinen A is presented.     

废次烟叶提取液源木质素降解菌Bacillus subtilis SM降解特性

【目的】目前造纸法再造烟叶工艺已经成为我国重要的废烟叶处理和利用方式,该工艺中烟梗中高木质素的降解是个挑战性的需解决问题。从废次烟叶提取液(Tobacco waste extract,TWE)中筛选木质素的降解微生物用来直接处理烟梗或烟末提取液,可实现对木质素含量的调控。【方法】将废次烟叶提取液(TWE)浓缩液中分离出的Bacillus subtilis SM接种到以Kraft木质素为唯一碳源的无机盐培养基中,在pH 7.0、30°C培养基中培养4 d来检测菌株对木质素的降解效果。通过HPLC、TOC、GPC和色度来表征SM对木质素的降解,并采用烟梗无机盐培养基在pH 7.0、30°C培养4 d检测SM对烟梗木质素的降解。【结果】HPLC结果显示SM在以木质素磺酸钠为唯一碳源的无机盐培养基中可全部降解分子质量为534.5的木质素磺酸钠,而对Kraft木质素降解不明显,仅观察到组分的变化。脱色结果显示脱色率达到40.7%,但在对Kraft木质素矿化方面矿化率只能达到5.4%。SM在烟梗无机盐培养基中可使烟梗失重率分别达到50%以上(对照组为18.9%),烟梗中木质素含量减少了70%左右。【结论】来源于废次烟叶提取液(TWE)的Bacillus subtilis SM能够以Kraft木质素为唯一碳源生长,也能够有效降解烟梗中的木质素,可应用于烟草废弃物原料中木质素的降解。     

Isolation and characterization of outermost layer deficient mutant spores of Bacillus megaterium

Outermost layer deficient mutant spores of Bacillus megaterium ATCC 12872 were isolated by Urografin density gradient centrifugation after mutagenesis with ethyl methanesulfonate. Although the composition of the cortex peptidoglycan was the same as that of the parent spores, three major proteins (48, 36, and 22 K daltons) were missing, suggesting that these proteins are components of the outermost layer. All mutant spores were also found to have very hydrophobic surface by 'salt aggregation test,' which would facilitate selection of such mutants.     

Isolation and characterization of a unique division mutant of Bacillus megaterium.

A filamentous division mutant, PV302, of Bacillus megaterium QM B1551 was isolated while screening for sporulation-defective mutants after nitrosoguanidine mutagenesis. Both phase-contrast and electron microscopy revealed that the mutant produced small spherical cells as well as filaments. It also accumulated large amounts of poly-beta-hydroxybutyrate. Poly-beta-hydroxybutyrate accounted for 16% of the dry weight of the mutant strain even after 28 h growth. In comparison to the parental strain, the division mutant also showed both an inability to sporulate and a reduced growth rate. All these phenotypes transduced together. Revertants gained the ability to sporulate, divide, and grow normally. Transductional mapping of the mutation, designated div-1, established a new linkage group for B. megaterium consisting of div-1 and the pyrimidine biosynthesis genes pyrD BCF. The spherical cells were separated from filaments by sucrose gradients and were tested for nucleic acid content and viability. The purified spherical cell fraction contained one-fifth the amount of DNA per mg protein as compared with the filamentous cell fraction and was shown to contain both non-viable minicells and some cells capable of growing after a lag of about 4 h. This suggests that the mutation not only causes defects in septum placement and sporulation, but may possibly affect DNA partitioning.     

Isolation and properties of membranes from Bacillus megaterium spores.

Membranes from dormant and heat-activated spores of Bacillus megaterium QM B1551 were isolated and purified by gentle lysis procedures followed by differential and sucrose density gradient centrifugations. The purified membranes were enriched for inner membranes and were characterized by their density and content of proteins, phospholipids, enzymes, cytochromes, and carotenoids. These purified spore membranes could be used to investigate their role in the triggering of germination.     

Proline iminopeptidase from Bacillus megaterium: purification and characterization

Proline iminopeptidase [EC 3.4.11.5] was purified about 1,700-fold from cell free extract of Bacillus megaterium by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose and hydroxyapatite, and gel filtration on Toyopearl FW-55. The purified enzyme still contained a minor contaminant as judged by disc gel electrophoresis. The enzyme was most active at pH 7.0 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminal. The enzyme was completely inactivated by p-chloromercuribenzoate (PCMB), but was not inhibited by metal chelators, diisopropylphosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by adding 2-mercaptoethanol. From this result and the chromatographic profile on PCMB-T-Sepharose, the enzyme seems to be a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0. The molecular weight of the enzyme was estimated to be 58,000 by gel filtration on Toyopearl and 60,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is a monomer.     

Isolation and characterization of two distinct fractions from the inner membrane of dormant Bacillus megaterium spores

Two distinct membrane bands were obtained after sucrose velocity gradient centrifugation of crude inner membranes from dormant Bacillus megaterium spores disrupted under conditions which minimized endogenous enzyme action. These two inner membrane fractions (termed LD and HD) contained similar amounts of total and individual phospholipid species. However, LD and HD differed significantly in phospholipid/protein ratios (4.3 and 0.47 mg/mg, respectively), equilibrium densities (1.12 and 1.18 g/cm3), NADH oxidase specific activity (less than 0.01 and 0.13 mumol/min X mg), and content of specific proteins. In contrast, crude membranes prepared in identical fashion from germinated spores gave only a single inner membrane band (termed G) on sucrose velocity gradients. G had a phospholipid/protein ratio of 0.98 mg/mg, an equilibrium density of 1.16 g/cm3, and an NADH oxidase specific activity of 2.1 mumol/min X mg. Essentially all of the proteins present in LD or HD or both were found in G, consistent with the latter membrane being derived from a mixture of LD and HD. No evidence was found suggesting that there is significant degradation of dormant spore inner membrane protein upon spore germination.     

Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes

A thermophilic Bacillus strain NG80-2 growing within the temperature range of 45–73°C (optimum at 65°C) was isolated from a deep subterranean oil-reservoir in northern China. The strain was able to utilize crude oil and liquid paraffin as the sole carbon sources for growth, and the growth with crude oil was accompanied by the production of an unknown emulsifying agent. Further examination showed that NG80-2 degraded and utilized only long-chain (C15–C36) n-alkanes, but not short-chain (C8–C14) n-alkanes and those longer than C40. Based on phenotypic and phylogenic analyses, NG80-2 was identified as Geobacillus thermodenitrificans. The strain NG80-2 may be potentially used for oily-waste treatment at elevated temperature, a condition which greatly accelerates the biodegradation rate, and for microbial enhancing oil recovery process.Lei Wang, Yun Tang and Shuo Wang contributed equally to this study.     

Isolation, characterization and biological activities of a toxin from Bacillus megaterium (B-23)

Fungitoxic substance was isolated from the culture filtrate of B. megaterium (B-23). Age of culture and pH of medium influence the fungitoxicity of its culture filtrate. Partially purified toxin was thermolabile, non-dialysable, ethyl acetate soluble, vanillin-sulphuric acid positive and effective within a range of pH 5-9. It exhibited maximum UV absorption at 224 nm. Its melting point was 242 degrees C. The efficacy of this compound was tested on 4 jute parasites namely, C. corchori, C. gloeosporioides, M. roridum and A. citri, of which M. roridum and C. corchori were least and most sensitive to the toxin respectively.     

Purification and characterization of an efficient poultry feather degrading-protease from Myrothecium verrucaria

The purpose of this work was to characterize an alkaline protease from the filamentous fungus Myrothecium verrucaria and to explore its capability to degrade native poultry feathers. The enzyme was purified to homogeneity using a single chromatographic step. Recovery was high, 62%, with a specific activity of 12,851.8 U/mg protein. The enzyme is a small monomeric protein with a molecular mass of 22 ± 1.5 kDa. It presented pH optimum of 8.3 and was stable over a broad pH range (5.0–12.0). The temperature optimum was 37°C, with thermal stability at temperatures up to 45°C. The enzyme presented an efficiency of 80.3% in the degradation of poultry feather meal, releasing amino acids and soluble peptides. It was able to hydrolyze β-keratin without necessity of chemical or enzymatic reduction of the disulphide bonds. Considering that, everyday, poultry-processing plants produce feathers as a waste products, this protease can be useful in biotechnological processes aiming to improve the transformation of poultry feathers through solubilization of β-keratin into usable peptides. Furthermore, it can also be useful in processes aiming to reduce the environmental pollution caused by the accumulation of feathers.     

Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.     

Isolation,characterization, and metabolic activities of Bacillus brevis degrading isonicotinic acid

An organism isolated from soil by enrichment on isonicotinic acid (INA) was characterized as Bacillus brevis. It used sugars more readily than amino acids as growth substrates. The organism also used isoniazid, 2-hydorxypyridine, and benzoic and p-hydroxybenzoic acids. This bacterium did not metabolize 2-hydroxy-INA, citrazinic acid, or other mono- and dihydroxypyridine compounds as well as intermediates of the maleamate pathway. Accumulation of hydroxylated pyridine compounds was not detected during fermentation, or incubation of INA with resting cells in the presence or absence of inhibitors. Succinic semialdehyde was isolated and characterized as a key intermediate and was rapidly oxidized by INA-adapted cells. Formate was detected as a product of INA metabolism, and formate but not formamide was oxidized by INA-adapted cells; γ-aminobutyrate or γ-aminocrotonate were oxidized. A pathway for INA degradation involving oxygenative cleavage of a partially reduced pyridine ring is proposed.     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans , five were B. circulans and four were B. licheniformis . Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65°C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70°C for 30 min.     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans, five were B. circulans and four were B. licheniformis. Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65 degrees C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70 degrees C for 30 min.