Sensitive and Simple Methods for Determination of l-Lysine with l-Lysine α-Oxidase

l-Lysine could be determined satisfactorily with a new fungal enzyme, l-Iysine α-oxidase (EC 1.4.3). The method consists of the oxidative deamination of l-Iysine with l-lysine α-oxidase and the spectrophotometric determination of one of the reaction products: α-keto-ε-aminocaproate, its intramolecular dehydrated form, Δ¹-piperideine-2-carboxylate or hydrogen peroxide. The method on the basis of the color reaction of hydrogen peroxide formed from l-lysine with 4-aminoantipyrine and phenol in the presence of peroxidase was most sensitive and simple. The method could be used for the direct assay of l-lysine levels in serums from several animals without pretreatments.     

Parametric analysis of metabolic fluxes of α-amylase and protease-producing Bacillus subtilis

Parametric analysis was applied for a metabolic flux model for the fed-batch culture of Bacillus subtilis producing recombinant α-amylase and protease. The metabolic flux model was formulated as a linear programming problem consisting of 49 reactions (decision variables) and 50 metabolites (equality constraints). This study was aimed to determine the response of the metabolic fluxes and objective function value of minimizing the difference between ATP consumption and ATP production (ATP balance). With regard to intracellular metabolite accumulation, the objective function value was least sensitive to variation in succinate and most sensitive to variation in malate. Amongst the variations in the accumulation rates of extracellular metabolites, the objective function value was least sensitive to variation in glutamate and most sensitive to variation in starch hydrolysis and triglyceride synthesis. A 10% variation in metabolite accumulation rates caused a maximum of 13.8% variation (standard error = 3.8%) in the objective function value.     

Reversibility of Acid-Inactivation of Bacillus subtilis’ α-Amylase

The inactivation of Bacillus subtilis’ α-amylase by acid was shown to be reversible. In the experiment, two different Bac. subtilis’ α-amylases, saccharifying and liquefying types, were used and the reversibility was investigated deviding into two processes of inactivation and reactivation. Both amylases showed the reversibility in a similar degree and in general the inactivated enzymes by acid were reactivated only by adjusting the pH to slightly alkaline values followed by incubation under certain conditions. However, the reversibility, especially, the reactivation was greatly influenced by several chemicals, the effect of certain chemicals being different according to the type of the bacterial amylase. Contrary to liquefying amylase, saccharifying amylase was insensitive to metal chelators but, nevertheless, the reactivation of the amylase was prevented by metal chelators. Also the reactivation of saccharifying amylase was inhibited by sulfhydryl reagents, although the native enzyme was quite insensitive to the chemicals. In the acid-inactivation and reactivation process, a reversible change in the ultraviolet absorption spectra of the enzymes was observed, and some discussion of the implication was presented.     

Extracellular Production of l-Lysine α-Oxidase in Wheat Bran Culture of a Strain of Trichoderma viride

A mold strain Y244-2 capable of producing l-lysine α-oxidase, a new enzyme catalyzing the α-oxidative deamination of l-lysine, was identified as Trichoderma viride. Among strains belonging to the genus Trichoderma tested, only Trichoderma viride Y244-2 produced the enzyme in wheat bran culture. The maximum enzyme production by the mold grown on wheat bran was observed after 10 and 14 days incubation with and without NaN0₃, respectively. Addition of NaN0₃, NH₄N0₃, adenine, purine nucleosides, l-histidine, glycine or l-glutamine to wheat bran stimulated the production of the enzyme. In the liquid culture, the enzyme was produced extracellulary under the aerobic conditions, although the production was much lower than that in the wheat bran culture.     

Reversibility of Heat-Inactivation of Bacillus subtilis’ α-Amylase

Inactivation of Bacillus subtilis’ α-amylase by heat was found to be reversible under a certain condition, and the factors affecting there were investigated, distinguishing into two groups: those influencing on the inactivation process by heat and those on the reactivation at the subsequent incubation after heating. Generally, the amylase heated in borate buffer solution was best in the reactivation degree. For reactivation of the heat-inactivated enzyme there was found an optimum in temperature, pH and concentration of enzyme, respectively. The reactivation was temporarily prevented by urea, but irreversibly inhibited by either calcium salts or calcium binding agents. In the reversible heat-inactivation of the enzyme was also found a reversible change in the absorption spectra as well as in the behavior of the enzyme toward proteinase.     

Isolation and Properties of Bacillus subtilis Strains Lysogenized by a Clear Plaque Mutant of Bacteriophage φ105

A clear plaque mutant of the temperate Bacillus phage phi105 lysogenized a small fraction of infected cells forming an integrated prophage at or near the normal phi105 insertion site. These lysogens exhibited a spontaneous induction rate approximately 1,000-fold lower than wild type and were noninducible (ind(-)) by mitomycin C. Prophage was induced, however, when competent cultures were incubated with transforming DNA. The ind(-) phenotype could not be attributed solely to the clear plaque mutation and appears to involve a cell-specific factor. Lysogenization by the clear plaque mutant, in contrast to wild-type phage, did not cause a marked reduction in transformation efficiency.     

Integration,amplification and expression of the Bacillus licheniformis α-amylase gene in Bacillus subtilis chromosome

We have introduced the α-amylase gene from Bacillus licheniformis (amy gene) in a non-replicative plasmid which can be conveniently integrated and amplified at a specific site of the B. subtilis chromosome. Although we were able to select spontaneous and stable gene amplification of about 20 integrated copies, the amylase secretion remained very low. A DNA fragment presenting a high promoter activity in B. subtilis was therefore inserted upstream from the amy gene coding sequence, leading to a significant increase of amylase production. However, the amplified structures obtained with this construction were found to contain no more than 12 copies of the amy gene and to be rather unstable when cells were grown under non-selective conditions.     

Properties of the competence-inducing factor of Bacillus subtilis 168I−

It has been shown that the competence-inducing factor of Bacillus subtilis 168I^? exhibits lytic activity toward isolated cell walls and nuclease activity toward transforming DNA. It has been shown that the competence factor covalently bound to CNBr-Sepharose exhibits the same enzymatic properties. A mechanism for the transformation process is proposed which advances the mechanism previously proposed by this laboratory.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

5′-Nucleotidase Activity in Improved Inosine-producing Mutants of Bacillus subtilis

An inosine- and guanosine-producing strain, AJ11100, of Bacillus subtilis could not grow in the minimum medium supplemented with 50 µg of sulfaguanidine per ml. When sulfaguanidine resistant mutants were derived from AJ11100, the sulfaguanidine resistance was frequently accompanied by xanthine requirement. All the xanthine auxotrophic mutants required a large amount of xanthine for cell growth and inosine accumulation. Revertants were then derived from one of the xanthine auxotrophic mutants, AJ11101, and improved inosine producers were obtained. The best mutant, AJ11102, accumulated 20.6 g of inosine per liter.

Furthermore, enzyme activities of inosine 5′-monophosphate (IMP) dehydrogenase, 5′-nucleotidase and phosphoribosyl pyrophosphate (PRPP) amidotransferase were assayed to investigate why AJ11102 accumulated an increased amount of inosine. The results showed that the increase of specific activity of 5′-nucleotidase contributed much to the increased accumulation of inosine.     

Hydrolytic Activity of α-Mannosidase against Deoxy Derivatives of p-Nitrophenyl α-d-Mannopyranoside

Deoxy derivatives of p-nitrophenyl (PNP) α-d-mannopyranoside, PNP 2-deoxy-α-d-arabino-hexopyranoside, 3-deoxy-α-d-arabino-hexopyranoside, 4-deoxy-α-d-lyxo-hexopyranoside, and α-d-rhamnopyranoside, were synthesized and hydrolytic activities of jack bean and almond α-mannosidases against them were investigated. These α-mannosidases scarcely acted on the 2-, 3-, and 4-deoxy derivatives, while the 6-deoxy one was hydrolyzed by the enzymes as fast as PNP α-d-mannopyranoside, which is a common substrate for α-mannosidase. These results indicate that the hydroxyl groups at C-2, 3, and 4 of the mannopyranoside are necessary to be recognized as a substrate by these enzymes, while that at C-6 does not have so a crucial role in substrate discrimination. Values of K_m and V_max of the enzymes on the hydrolysis of PNP α-d-rhamnopyranoside were obtained from kinetic studies.     

Purification, characterisation and mutagenic enhancement of a thermoactive α-amylase from Bacillus subtilis

Bacillus subtilis was isolated from flour mill wastes. It produced a thermostable α-amylase in complex media containing starch. Amylase activity was optimal at the exponential phase and was more strongly expressed with sorghum, yam peel and corn starch than soluble potato starch. The enzyme was purified 24-fold to a specific activity of 2200 U mg⁻¹, with a yield of 10%. It yielded a single band when subjected to SDS-PAGE and an apparent molecular mass of 54780 was determined by mass spectrometry. The enzyme, which was optimally active at 80°C and pH 5.6, released saccharides with a polymerisation degree of 1–6 following hydrolysis of yam peel, sorghum and corn starch. Cells of B. subtilis were exposed to ultraviolet irradiation and N-methyl-N′-nitro-N-nitrosoguanidine. Hyperproductive mutants were obtained by these treatments. Received 14 February 1997/ Accepted in revised form 13 August 1997     

Stereoselective Reduction of α- and β-Keto Esters with Aerobic Thermophiles,Bacillus Strains

The first example of stereoselective reduction with aerobic thermophiles is reported. Various α- and β-keto esters were reduced stereoselectively to the corresponding alcohols by the aerobic thermophiles, Bacillus strains. In particular, the reduction of ethyl 3-methyl-2-oxobutanoate with B. stearothermophilus DSM 297 gave the corresponding (R)-alcohol with high yield in excellent enan-tioselectively (> 99% e.e.). The conversions of keto esters to the corresponding hydroxy esters with Bacillus strains were increased by introduction of glycerol in the reaction mixture as an additive.     

Antibacterial and Anticancer activity of ε -poly-L-lysine (ε -PL) produced by a marine Bacillus subtilis sp

A marine Bacillus subtilis SDNS was isolated from sea water in Alexandria and identified using 16S rDNA sequence analysis. The bacterium produced a compound active against a number of gram negativeve bacteria. Moreover, the anticancer activity of this bacterium was tested against three different human cell lines (Hela S3, HepG2 and CaCo). The highest inhibition activity was recorded against Hela S3 cell line (77.2%), while almost no activity was recorded towards CaCo cell line. HPLC and TLC analyses supported evidence that Bacillus subtilis SDNS product is ?;-poly-L-lysine. To achieve maximum production, Plackett-Burman experimental design was applied. A 1.5 fold increase was observed when Bacillus subtilis SDNS was grown in optimized medium composed of g/l: (NH(4) )(2) SO(4) , 15; K(2) HPO(4) , 0.3; KH(2) PO(4) , 2; MgSO(4) · 7 H(2) O, 1; ZnSO(4) · 7 H(2) O, 0; FeSO(4) · 7 H(2) O, 0.03; glucose, 25; yeast extract, 1, pH 6.8. Under optimized culture condition, a product value of 76.3 mg/l could be obtained. According to available literature, this is the first announcement for the production of ?;-poly-L-lysine (?;-PL) by a member of genus Bacillus. (? 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

Influence of a Cell-Wall-Associated Protease on Production of α-Amylase by Bacillus subtilis

AmyL, an extracellular α-amylase from Bacillus licheniformis, is resistant to extracellular proteases secreted by Bacillus subtilis during growth. Nevertheless, when AmyL is produced and secreted by B. subtilis, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the B. subtilis wprA gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible wprA gene, we show that a product of wprA, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in which wprA is not expressed exhibits an increased yield of α-amylase. The potential role of wprA in protein secretion is discussed, together with implications for the use of B. subtilis and related bacteria as hosts for the secretion of heterologous proteins.The cell envelope of the gram-positive bacterium Bacillus subtilis consists of a single (cytoplasmic) membrane surrounded by a relatively thick cell wall consisting of similar proportions of peptidoglycan and covalently attached anionic polymers. The absence of an outer membrane means that there is no equivalent of the membrane-enclosed periplasm found in gram-negative bacteria. However, by virtue of its thickness and high density of negative charge, the cell wall may perform some of the roles of the periplasm in gram-positive bacteria.The absence of an outer membrane in gram-positive bacteria also simplifies the secretion pathway, and, consequently, B. subtilis and its close relatives have the potential to secrete proteins directly into the growth medium, at concentrations in excess of 5 grams per liter (4). Despite its extensive use in the production of commercially important Bacillus enzymes (e.g., α-amylases and alkaline proteases), attempts to exploit B. subtilis for the production of heterologous proteins at high concentrations have proved disappointing (8). One reason for this failure is the production and release into the culture medium of several extracellular proteases (24, 28, 37). Although native Bacillus proteins are generally resistant to these proteases, heterologous proteins are often rapidly degraded in their presence. As a result, strains of B. subtilis that are multiply deficient in extracellular proteases have been developed (11, 37). The more developed of these strains have less than 1% of the proteolytic activity of the wild type (37). To date, efforts have concentrated mainly on the proteases which reside in a truly extracellular location, while those which remain cell associated have been largely overlooked.Although strains deficient in extracellular proteases have improved the productivity of B. subtilis for the production of heterologous proteins, they have only partially overcome problems of unexpectedly low yields. We and others have recently shown (22, 31) that significant amounts of secretory protein are degraded within minutes of being synthesized. This degradation is observed even for Bacillus proteins that are highly resistant to proteases released into the culture medium, suggesting that a component of this degradation is cell associated.Margot and Karamata recently reported the identification of a cell-wall-associated protease encoded by the wprA gene (21). The primary product of this gene is a 96-kDa polypeptide that is processed into two previously identified cell wall proteins, namely, CWBP52 and CWBP23. The processing of the WprA precursor during secretion accompanies the targeting of CWBP52 and CWBP23 to the cell wall and is analagous to the processing of another B. subtilis cell-wall-bound protein, namely, WapA (5). The amino acid sequence of CWBP52 shows a high degree of similarity with serine proteases of the subtilisin family, and phenylmethylsulfonile fluoride (PMSF)-sensitive protease activity was detected in proteins extracted from the cell wall of a wprA⁺ strain, but not one in which this gene had been insertionally inactivated (21). In the absence of homology to proteins in the databases, the N-terminal CWBP23 moiety was presumed to function as a chaperone-like propeptide that is proteolytically processed on the trans side of the membrane. In this paper, we report on a potential role of products of wprA in the integrity of secretory proteins during late stages in the secretion pathway. We also discuss the potential of wprA mutants to increase the productivity of B. subtilis for secretory proteins.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

Effect of polyethylene glycol on Bacillus subtilis α-amylase purification with raw and modified starch

Polyethylene glycol was found to enhance adsorption of Bacillus subtilis -amylase on starch in optimum concentration 10 % (w/w). Degree of adsorption at 12°C was increased from 83 to 98 % and from 30 to 81 % for cross-linked and raw starch, resp. Higher sorption capacity and easy desorption of -amylase without temperature or pH change was reached at 22 °C. Yield of -amylase 95 % and purification factor 8.3 were achieved on the cross-linked starch column. The method is suitable for -amylase isolation from PEG phase after its microbial production in aqueous two-phase systems.     

Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103

A maltooligosaccharide-forming α-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of α-amylase in KCC103 was not catabolite-repressed. The α-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified α-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65–70 °C, it was rapidly deactivated at 70 °C with a half-life of 7 min and at 50 °C, the half-life was 94 min. The K _m and V _max for starch hydrolysis were 2.6 mg ml⁻¹ and 909 U mg⁻¹, respectively. Ca²⁺ did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg²⁺, Ag²⁺, and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The α-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2–D4) were formed more predominantly than larger maltooligosaccharides (D5–D7). This maltooligosaccharide forming endo-α-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.     

Activity of Debaryomyces hansenii UFV-1 α-galactosidases against α-D-galactopyranoside derivatives

α-D-Galactopyranosides were synthesized and their inhibitory activities toward the Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases were evaluated. Methyl α-D-galactopyranoside was the most potent inhibitor compared to the others tested, with K(i)(') values of 0.82 and 1.12 mmolL(-1), for extracellular and intracellular enzymes, respectively. These results indicate that the presence of a hydroxyl group in the C-6 position of α-D-galactopyranoside derivatives is important for the recognition by D. hansenii UFV-1 α-galactosidases.     

Biochemical characterization and structural insights into the high substrate affinity of a dimeric and Ca2+ independent Bacillus subtilis α-amylase

An extracellular amylase (AmyKS) produced by a newly isolated Bacillus subtilis strain US572 was purified and characterized. AmyKS showed maximal activity at pH 6 and 60°C with a half-life of 10 min at 70°C. It is a Ca²⁺ independent enzyme and able to hydrolyze soluble starch into oligosaccharides consisting mainly of maltose and maltotriose. When compared to the studied α-amylases, AmyKS presents a high affinity toward soluble starch with a K_m value of 0.252 mg ml⁻¹. Coupled with the size-exclusion chromatography data, MALDI–TOF/MS analysis indicated that the purified amylase is a dimer with a molecular mass of 136,938.18 Da. It is an unusual feature of a non-maltogenic α-amylase. A 3D model and a dimeric model of AmyKS were generated showing the presence of an additional domain suspected to be involved in the dimerization process. This dimer arrangement could explain the high substrate affinity and catalytic efficiency of this enzyme.