Tannase production by Bacillus licheniformis

Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21 U ml^–1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1 g l^–1) after 18–21 h growth though the organism did not attain maximum growth until 36 h.     

Isolation and characterization of a new keratinolytic Bacillus licheniformis strain

A keratin-degrading bacterium strain (K-508) was isolated from partially degraded feathers and characterized. This isolate exhibited a high chicken feather-degrading activity when cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 °C. On the basis of its phenotypic characteristics (quickly moving, Gram-positive rods), the results of metabolic tests and rDNA sequence analysis, it was identified as Bacillus licheniformis. Its fermentation broth showed activity on N-Bz-l-Phe-l-Val-l-Arg-p-nitroanilide, N-Suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide, N-CBZ-Gly-Gly-l-Leu-p-nitroanilide and N-CBZ-l-Ala-l-Ala-l-Leu-p-nitroanilide as chromogenic protease substrates at near neutral pH. Both trypsin-like and chymotrypsin-like proteases were constitutively secreted by this strain.     

Fermentation characteristics and secretion of proteases of a new keratinolytic strain of Bacillus licheniformis

A keratin-degrading strain of Bacillus licheniformis (K-508) was isolated from partially-degraded feathers and characterised. It had high chicken feather-degrading activity when cultured in feather-containing broth, with a growth optimum at pH 7 and 47 °C. Broth filtrates were active towards N-Bz-Phe-Val-Arg-p-nitroanilide and N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide, as chromogenic protease substrates at pH 8. Strain K-508 displays keratinolytic activity against native feather keratin (without any pretreatment) in the presence of SH-reducing compounds. It constitutively secreted both trypsin-like and chymotrypsin-like proteases.     

Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis

Strains of thermophilic bacilli were screened for cellulolytic activity by gel diffusion assay on selective medium at 55°C. Strain B-41361, identified as a strain of Bacillus licheniformis, displayed activity against carboxymethylcellulose. Zymogram analysis demonstrated several catalytically active polypeptides with the most prominent species having a mass of 37 kDa. The enzyme was purified 60-fold with a 17% yield and specific activity of 183 U/mg. The amino terminal sequence was homologous to members of glycoside hydrolase family 5. Optimal temperature was 65°C (measured over 30 min), but the enzyme was most stable at 60°C, retaining greater than 90% activity after one hour. The enzyme had a broad pH range, with maximal activity at pH 6.0, 75% maximal activity at pH 4.5, and 40% at pH 10. The enzyme hydrolyzed p-nitrophenylcellobioside, barley β-glucan, and lichenan, but no activity was detected against avicel or acid-swollen cellulose.Mention of a trade name or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Effect of Cultivation Conditions on Growth and Adhesion of Bacillus licheniformis

Adhesion to glass of actively growing cells of the thermophilic Bacillus licheniformis, isolated from the Medyaginskaya test borehole (Yaroslavl' oblast), was studied. The reversible adhesion (RA) manifests itself in a decline of cell density (without cell lysis) in the liquid culture over the first 20–40 min of growth followed by normal exponential growth. The RA is minimal under favorable growth conditions but increases when cells are transferred to a new medium, especially one with a pH, temperature, salinity, or concentration of Ca²⁺ ions nonoptimal for the given species. Under unfavorable growth conditions, the adhesion becomes irreversible. The obtained data suggest that RA represents an adaptation mechanism important for population survival.     

Cloning and over-expression of an alkaline protease from Bacillus licheniformis

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.     

NAD kinase from Bacillus licheniformis: inhibition by NADP and other properties

NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent K _m for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn²⁺ and Ca²⁺ could be substituted for Mg²⁺. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a K _i of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP.This paper is dedicated to Sydney C. Rittenberg on the occassion of his retirement, with respect and much affection, in appreciation for his friendship and years of distinguished service as a teacher and scientist     

Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis

An extracellular alkaline protease produced by Bacillus licheniformis AP-1 was purified 76-fold, yielding a single 28 kDa band on SDS-PAGE. It was optimally active at pH 11 and at 60 degrees C (assayed over 10 min). The protease was completely inhibited by phenylmethylsulfonyl fluoride and diodopropyl fluorophosphate, with little increase upon Ca2+ and Mg2+ addition.     

Functional expression of keratinase (kerA) gene from Bacillus licheniformis in Pichia pastoris

A 1.2 kb DNA fragment coding for the pro-peptide and mature keratinase from Bacillus licheniformis PWD-1 (kerA) was cloned into vectors pPICZA and pGAPZA for extracellular expression in the methylotrophic yeast, Pichia pastoris. Recombinant keratinase was secreted by the pPICZA-kerA transformants 24 h after methanol induction of shake-flask cultures, and reached a final yield of 124 mg l^–1 (285 U ml^–1) 144 h after the induction. The recombinant keratinase was glycosylated ( 39 kDa), and was optimal between pH 8.5–9.5 and between 55°C –60°C using azokeratin as substrate. The enzyme degraded bovine serum albumin, collagen, and soy protein concentrate. In conclusion, P. pastoris can be used as an efficient host to express keratinase for nutritional and environmental applications.     

Homologies among three Bacillus licheniformis plasmids and molecular characterization of their replication module

To assess to what extent three Bacillus licheniformis plasmids had the same molecular organization a physical map of the 9.34, 8.40 and 7.90 kb plasmids was achieved by using seventeen restriction enzymes. Southern hybridization was performed on plasmids using restriction fragments of the smallest plasmid as probes. Data from different hybridization patterns show a close homology among the three plasmids hypothesizing a similar molecular organization. The lack of plasmid diversity observed, seem to support the hypothesis of a similar phylogeny among these plasmids. This investigation provides more information concerning phylogeny, interrelationships and level of diversity among Bacillus plasmids and a molecular characterization of three plasmids useful for the construction of cloning vectors.     

Extractive fermentation for enhanced production of alkaline phosphatase from Bacillus licheniformis MTCC 1483 using aqueous two-phase systems

A study was made to find out maximum partitioning of Bacillus licheniformis alkaline phosphatase in different ATPSs composed of different molecular weight of PEG X (X = 2000, 4000, 6000) with salts (magnesium sulphate, sodium sulphate, sodium citrate) and polymers (dextran 40, dextran T500). Physicochemical factors such as effect of system pH, system temperature and production media were evaluated for partitioning of alkaline phosphatase. PEG 4000 [9.0% (w/v)] and dextran T500 [9.6% (w/v)] were selected as most suitable system components for alkaline phosphatase production by B. licheniformis based on greater partition coefficient (k = 5.23). The two-phase system produced fewer enzymes than the homogeneous fermentation (control) in early stage of fermentation, but after 72 h the enzyme produced in the control system was less than that in the ATPS. Total alkaline phosphatase yield in ATPS fermentation was 3907.01 U/ml and in homogeneous fermentation 2856.50 U/ml.     

Purification and characterization of a cellulase-free xylanase of a moderate thermophile Bacillus licheniformis A99

Two cellulase-free xylanases were secreted by a thermophile, Bacillus licheniformis A99. Of the two, the predominant one was purified to homogeneity. The enzyme was optimally active at 60 °C, pH 6–7.5, and had a molecular weight of about 45 KDa and isoelectric point of 7.0 ± 0.2. The K _m (for birchwood xylan) and V _max were 3.33 mg/ml and 1.111 mmols mg^–1 protein min^–1 respectively. The half-life of the enzyme was 5 h at 60 °C. All cations except Hg²⁺ and Ag⁺ as well as EDTA were well tolerated and did not adversely affect xylanase activity. However, SDS inhibited the enzyme activity. The release of reducing sugars from unbleached commercial pulp sample on treatment with the enzyme indicated its potential in prebleaching of paper pulp. The enzyme caused saccharification of lignocellulosics such as wheat bran, wheat straw and sawdust. This is the first report on purification and characterization of cellulase-free xylanase from a moderate thermophile Bacillus licheniformis.     

Application of crude xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft pulp

Alkalophilic Bacillus licheniformis 77-2 produced an extracellular alkali-tolerant xylanase with negligible cellulase activity in medium containing corn straw. The effectiveness of crude xylanase on treatment of eucalyptus Kraft pulp was evaluated. A biobleaching experiment was carried out to compare the chlorine saving with pulp treated and untreated by the enzyme. Two-stage bleaching was employed, using a ClO₂ chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of Kappa number and brightness, respectively 28.5 and 30% less ClO₂ was required in comparison to the enzymatically untreated samples.     

Physical mapping of LP51 and LP52 prophages of lysogenic strains of Bacillus licheniformis

Summary The physical maps of the LP51 and LP52 prophages in lysogenic strains of Bacillus licheniformis were constructed on the basis of data obtained by hybridization of phage DNA probes with Southern blots of restricted DNA of the lysogens. The data were compatible with the Campbell model for chromosomal integration; the attP site was mapped at 58.7–61.8 map units of the genomes of both phages. Identification of prophage-host DNA junction fragments indicated the presence of a unique attB site on the bacterial chromosome; the set of junction fragments in the strain B. licheniformis ATCC 10716 was identical to that of ATCC 11946, but different from ATCC 8187. Both the LP51 and LP52 phages used the same integration sites. Upon reinfection with either phage, the cured strains UM12 and UM18 (i.e. 10716 and 11946 cured of LP52 or LP51, respectively) turned out to be integration deficient. In surface cultures the reinfected bacteria could be maintained in the lysogenic state without, however, integrating the phage genome; when these bacteria were passaged in submerged cultures, several modes of anomalous integration were observed, and the phage segregated into a variety of forms, discernible by virulence and plaque morphology. In liquid cultures of UM12(LP51) or UM12(LP52) lytic forms finally predominated, while most lysogenized UM18 were converted into defective lysogens which contained a defective prophage in a stably integrated form.     

Chitobiose production by using a novel thermostable chitinase from Bacillus licheniformis strain JS isolated from a mushroom bed

The thermophilic Bacillus licheniformis strain JS was isolated from a bed of mushrooms, Pleurotus sajor-caju. The organism could produce a novel, single-component, thermostable chitinase that was purified by ion-exchange chromatography using DEAE-cellulose in 7.64% yield and in an 8.1-fold enhancement in purity. Its molecular weight is 22 kDa. The enzyme is a chitobiosidase, since the chitin hydrolysate is N^I,N^II-diacetylchitobiose. The optimum temperature for enzyme activity is 55 °C, and the optimum pH is 8.0. It was completely inhibited by Hg²⁺ ions whereas Co²⁺ ions served as an activator. The thermostability of this enzyme is important in the bioconversion of chitinous waste and for the production of chitooligosaccharides.     

Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations

This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest k _cat /Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The k _cat /Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.     

Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37

A protease was purified from the cell-free supernatant of Bacillus licheniformis RSP-09-37, a mutant from a thermophilic bacterial strain, B. licheniformis RSP-09, using affinity chromatography with alpha-casein agarose resin. The protease was purified 85-fold to electrophoretic homogeneity. The apparent molecular mass of purified protease was 55 kDa using gel filtration in high-performance liquid chromatography, which is in agreement with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a monomeric nature of the protein. The purified protease revealed temperature optima of 50 degrees C and pH optima of 10.0 and was classified as serine protease based on its complete inhibition with phenyl methyl sulfonyl fluoride. The purified protease exhibited tolerance to both detergents and organic solvent. The synthetic activity of the protease was tested using the transesterification reaction between N-acetyl-L-phenylalanine-ethyl ester and n-propanol in organic solvents varying in their log P values and the kinetic parameters of the enzyme in these organic solvents were studied. The enzyme has potential to be employed for synthetic reactions and in detergent formulations.     

A continuous culture study of the bioenergetic aspects of growth and production of exocellular protease in Bacillus licheniformis

Summary Bacillus licheniformis S 1684 is able to produce an alkaline serine protease exocellularly. In glucose-limited chemostat cultures the specific rate of protease production was maximal at a -value of 0.22. Above this growth rate protease production was repressed. Dependent on 10–20% of the glucose input was used for exocellular product formation. The degree of reduction of exocellular products was 4.1.Maximum molar growth yields were high and indicate a high efficiency of growth. The values of Y _glu ^max and YO ₂ ^max were 83.8 and 53.3, respectively. When Y _glu ^max was corrected for the amount of glucose used for product formation a value of 100.3 was obtained. These high maximum molar growth yields are most probably caused by a high Y _ATP ^max . Anaerobic batch experiments showed a Y _ATP of 14.6.Sometimes the used strain was instable in cell morphology and protease production. Non-protease producing cells most probably develop from producing cells by mutation in the rel-gene. Producing cells most probably are relaxed (rel ^-) and non-producing cells stringent (rel ⁺).Glossary specific growth rate (h^-1) - Y _sub growth yield permol substrate (g biomass/mol) - Y ^max maximum molar growth yield, corrected for maintenance requirements (g biomass/mol) - Y ^max(corr) Y ^max corrected for product formation (g biomass/mol) - m _sub maintenance requirements (mol/g biomass·h) - m _sub(corr) maintenance requirements corrected for product formation (mol/g biomass·h) - Y _c fraction of organic substrate converted in biomass - z fraction of organic substrate converted in exocellular products - d fraction of organic substrate converted in CO₂ (g mol/g atom C) - Crec% carbon recovery % - average degree of reduction of exocellular products - P/O amount of ATP produced during electron-transport of 2 electrons to oxygen     

Effect of environmental phosphate concentration on protein export by Bacillus licheniformis NCIB 6346

Bacillus licheniformis NCIB 6346 was grown under phosphate or magnesium limitation in continuous culture and forward and reverse transitions carried out to examine their effects upon extracellular enzyme production. Analysis of the observed kinetics allowed a distinction to be made between the effects of phenotypic variation and of genetic selection upon exoenzyme production. Furthermore, increases in the phosphate concentration (0.3–30 mM) were directly related to increased phenotypic expression of alpha-amylase. By contrast, penicillinase production was reduced under these conditions but analysis revealed that its phenotypic expression was only indirectly influenced by the concentrations of either magnesium or phosphate present during the transitions.