Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

The anaerobic pathway for the biosynthesis of cobalamin (vitamin B₁₂) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH₆₀, that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH₆₀ was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH₆₀ demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.     

Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.     

One pathway can incorporate either adenine or dimethylbenzimidazole as an alpha-axial ligand of B12 cofactors in Salmonella enterica

Corrinoid (vitamin B₁₂-like) cofactors contain various α-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form “complete” corrinoids aerobically by importing an “incomplete” corrinoid, such as cobinamide (Cbi), and adding appropriate α- and β-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B₁₂, but it can make B₁₂ from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B₁₂ cofactors (having adenine as an α-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an α-ligand. When DMB is provided to these mutants, synthesis of pseudo-B₁₂ cofactors ceases and B₁₂ cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an α-ligand. Wild-type cells make pseudo-B₁₂ cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B₁₂ for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B₁₂ cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B₁₂. Models are described for the mechanism and control of α-axial ligand installation.     

High yield production of extracellular recombinant levansucrase by Bacillus megaterium

In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH?6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg?L^?1) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U?mL^?1 on fructose and 17.2 U?mL^?1 on glycerol). This was further increased in high cell density fed-batch processes up to 55 U?mL^?1, reflecting a levansucrase concentration of 0.52 g?L^?1. This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.     

Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium

Cobalamin (vitamin B₁₂) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B₁₂ biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG^AcbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B₁₂ riboswitch upstream of the cbiXJCDETLFGAcysG^AcbiYbtuR operon and the recombinant production of three different vitamin B₁₂ binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B₁₂-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B₁₂ concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.     

Influence of 5,6-dimethylbenzimidazole (DMB) on vitamin B12 biosynthesis by strains of Propionibacterium

Work on vitamin B₁₂ biosynthesis in whey permeate using 5,6-dimethylbenzimidazole (DMB) as a precursor has often been carried out, but with no reference to the stage of fermentation at which it is to be added to the fermentation medium. In the present paper we report 168 h incubation as the optimum time for vitamin B₁₂ biosynthesis and the effect of DMB addition at four phases of fermentation, on vitamin B₁₂ productivity, growth and substrate utilization by three strains of Propionibacterium. The infusion of DMB at the end of 144 h of incubation (24 h before the end of fermentation) has been found optimum for maximum metabolic efficiency of the cultures.     

The Glucomannokinase of the Gram-Negative Ruminal Bacterium,Prevotella bryantii B14, and its Sequence Conservation with Regulatory Glucokinases of Gram-Positive Bacteria

The glucomannokinase gene of Prevotella bryantii B₁4, a strictly anaerobic ruminal bacterium, was amplified with degenerate PCR primers. The degenerate PCR primers were based on the N-terminal amino acid residues of the purified glucomannokinase and the C-terminus of other bacterial glucokinases. The PCR product had a molecular weight of approximately 550 bp. Because the purified glucomannokinase was composed of dimers with a monomer molecular weight of 34.5 kDa, the PCR product accounted for approximately 60% of the glucomannokinase DNA sequence. The B₁4 sequence had significant similarity with the glucokinases of several aerobic, Gram-positive bacteria (Streptomyces coelicolor,Staphylococcus xylosus , Bacillus subtilis, Bacillus megaterium). Previous work indicated that the Gram-positive glucokinases and the B₁4 glucomannokinase have a role in catabolite repression. These presumptive regulatory kinases formed a cluster that was distinct from other bacterial glucokinases. Amino acid sequence analyses indicated that the regulatory glucokinase genes (including the P. bryantii B₁4 glucomannokinase gene) had a mean sequence similarity of 42 ± 2.4%. The regulatory kinases had some highly conserved regions with other bacterial glucokinases, but the mean similarity was lower (17 ± 2.0%). The role of regulatory kinases in catabolite repression has not been precisely defined, but sequence comparisons suggest a common theme may exist. Because P. bryantii B₁4 is a Gram-negative bacterium, it appears that regulatory glucokinases may be widely distributed in eubacteria and not restricted to Gram-positive species.     

Studies on a gram-positive hydrogen bacterium,Nocardia opaca strain 1b

Summary A new hydrogen bacterium has been isolated by enrichment culture on propane. It is a strictly aerobic, Gram-positive, non acid-fast bacterium, characterized by filamentous growth, and has been tentatively assigned to Nocardia opaca (strain 1 b).It grows heterotrophically, on many organic compounds (71 out of 138 tested substrates including organic acids and sugars), on hydrocarbons (C₁₁–C₁₈) as well as under autotrophic conditions (under an atmosphere of hydrogen, oxygen, and carbon dioxide=8:1:1) In the absence of a nitrogen source storage materials, mainly carbohydrates, are accumulated.Hydrogenase is an inducible enzyme. Under appropriate growth conditions the specific hydrogenase activity reaches high values: 2700 enzyme units/g cell protein. The formation of hydrogenase is repressed by fructose. With increasing oxygen concentrations during growth the specific hydrogenase activity decreases. In resting cell oxygen progressively inhibits the oxyhydrogen reaction.Cell-free extracts of autotrophically grown cells are able to reduce oxygen benzyl-and methyl viologen, dichlorphenolindophenol, methylene blue and nicotinamide adeninedinucleotide with hydrogen.     

Development of a deletion mutant of Pseudomonas denitrificans that does not degrade 3-hydroxypropionic acid

Pseudomonas denitrificans is a gram-negative bacterium that can produce vitamin B₁₂ under aerobic conditions. Recently, recombinant strains of P. denitrificans overexpressing a vitamin B₁₂-dependent glycerol dehydratase (DhaB) were developed to produce 3-hydroxypropionic acid (3-HP) from glycerol. The recombinant P. denitrificans could produce 3-HP successfully under aerobic conditions without an exogenous supply of vitamin B₁₂, but the 3-HP produced disappeared during extended cultivation due to the 3-HP degradation activity in this strain. This study developed mutant strains of P. denitrificans that do not degrade 3-HP. The following eight candidate enzymes, which might be responsible for 3-HP degradation, were selected, cloned, and studied for their activity in Escherichia coli: four (putative) 3-hydroxyisobutyrate dehydrogenases (3HIBDH), a putative 3-HP dehydrogenase (3HPDH), an alcohol dehydrogenase (ADH), and two choline dehydrogenases (CHDH). Among them, 3HIBDHI, 3HIBDHIV, and 3HPDH exhibited 3-HP degrading activity when expressed heterologously in E. coli. When 3hpdh alone or along with 3hibdhIV were disrupted from P. denitrificans, the mutant P. denitrificans exhibited greatly reduced 3-HP degradation activity that could not grow on 3-HP as the sole carbon and energy source. When the double mutant P. denitrificans Δ3hpdhΔ3hibdhIV was transformed with DhaB, an improved 3-HP yield (0.78 mol/mol) compared to that of the wild-type counterpart (0.45 mol/mol) was obtained from a 24-h flask culture. This study indicates that 3hpdh and 3hibdhIV (to a lesser extent) are mainly responsible for 3-HP degradation in P. denitrificans and their deletion can prevent 3-HP degradation during its production by recombinant P. denitrificans.     

Vitamin B12-dependent Methionine Biosynthesis and Its Metabolic Role in Corynebacterium simplex ATCC 6946, a Vitamin B12-producing and Hydrocarbon-utilizable Bacterium

A vitamin B₁₂-dependent N⁵-methyltetrahydrofoIate-homocysteine methyltransferase was found in cell-free extracts of Corynebacterium simplex ATCC 6946 grown aerobically in a medium containing hydrocarbon as a sole carbon source and the enzyme was partially purified. Absolute requirements for S-adenosylmethionine and an appropriate reducing system were observed for the transmethylation from N⁵-methyltetrahydrofolate. The same preparation catalyzed also the formation of methionine from homocysteine and methyl-B₁₂ under both aerobic and anaerobic conditions. The concentration of cobalt ion in the growth medium had a pronounced effect on the intracellular vitamin B₁₂ level and the activity of the vitamin-dependent methionine-synthesizing system in the bacterium. The relationship between the methionine synthesis and the methyl branched-chain fatty acid formation was discussed.     

Dependence of Betaine Stimulation of Vitamin B12 Overproduction on Protein Synthesis

The betaine-stimulated differential synthesis of vitamin B₁₂, i.e., the increase in B₁₂ per increase in dry cell weight, by Pseudomonas denitrificans was inhibited by rifampin and chloramphenicol but not by benzylpenicillin and carbenicillin at concentrations of antibiotic that inhibit growth. The level of the first enzyme of corrin (and porphyrin) biosynthesis, δ-aminolevulinic acid synthetase, was decreased to a much greater degree by rifampin and chloramphenicol than by the penicillins. These data support the concept that betaine stimulation of B₁₂ synthesis is a result of its stimulation of synthesis of δ-aminolevulinic acid synthetase, a labile and presumably rate-limiting enzyme of corrin formation requiring continuous induction. In further support of this hypothesis, it was found that chloramphenicol immediately interfered with both vitamin B₁₂ and δ-aminolevulinic acid synthetase formation, no matter when it was added to the system.     

An aminotransferase from bacterium ATCC 55552 deaminates hydrolyzed fumonisin B<Subscript>1</Subscript>

Previous research identified several microorganisms and pathways capable of degrading the mycotoxin fumonisin B₁ (FB₁). Degradation of FB₁ by microorganisms seems to comprise two essential steps: hydrolysis to hydrolyzed fumonisin B₁ (HFB₁) and deamination of the hydrolysis product. One of the previously studied microorganisms was the Gram negative bacterium ATCC 55552. The gene corresponding to the first step of FB₁ degradation in this bacterium was identified, but the genetic basis for deamination of the hydrolyzed intermediate remained unexplained (Duvick et al. 2000, PCT patent application WO200004158). Here we report the sequence and HFB₁-deaminating activity of a novel aminotransferase encoded by the bacterium ATCC 55552. The corresponding gene was identified, sequenced, and over-expressed in Escherichia coli. Cell lysates of the recombinant E. coli strain showed distinct HFB₁-deaminating activity in the presence of pyridoxal phosphate and pyruvate, as was demonstrated by liquid chromatography–mass spectrometry. Thus, we suggest the novel enzyme to be part of the fumonisin catabolic pathway of the bacterium ATCC 55552.     

Salmonella enterica synthesizes 5,6‐dimethylbenzimidazolyl‐(DMB)‐α‐riboside. Why some Firmicutes do not require the canonical DMB activation system to synthesize adenosylcobalamin

5,6‐Dimethylbenzimidazolyl‐(DMB)‐ α ‐ribotide [ α ‐ribazole‐5′‐phosphate ( α ‐RP)] is an intermediate in the biosynthesis of adenosylcobalamin (AdoCbl) in many prokaryotes. In such microbes, α ‐RP is synthesized by nicotinate mononucleotide (NaMN):DMB phosphoribosyltransferases (CobT in Salmonella enterica), in a reaction that is considered to be the canonical step for the activation of the base of the nucleotide present in adenosylcobamides. Some Firmicutes lack CobT‐type enzymes but have a two‐protein system comprised of a transporter (i.e., CblT) and a kinase (i.e., CblS) that can salvage exogenous α ‐ribazole ( α ‐R) from the environment using CblT to take up α ‐R, followed by α ‐R phosphorylation by CblS. We report that Geobacillus kaustophilus CblT and CblS proteins restore α ‐RP synthesis in S. enterica lacking the CobT enzyme. We also show that a S. enterica cobT strain that synthesizes GkCblS ectopically makes only AdoCbl, even under growth conditions where the synthesis of pseudoCbl is favored. Our results indicate that S. enterica synthesizes α ‐R, a metabolite that had not been detected in this bacterium and that GkCblS has a strong preference for DMB‐ribose over adenine‐ribose as substrate. We propose that in some Firmicutes DMB is activated to α ‐RP via α ‐R using an as‐yet‐unknown route to convert DMB to α ‐R and CblS to convert α ‐R to α ‐RP.     

Isolation of a novel Pseudomonas species SP2 producing vitamin B12 under aerobic condition

Vitamin B₁₂ is a complex biomolecule that acts as a cofactor for a variety of enzymes in microbial metabolism. Pseudomonas denitrificans is exclusively used as an industrial strain for the production of vitamin B₁₂ under aerobic conditions. However, only a few strains of Pseudomonas have been reported to possess the capability of producing this vitamin and they are strongly patent-protected. To improve the applicability of the vitamin B₁₂-producing microorganisms, a new isolate was obtained from municipal waste samples and characterized for its biological properties. The new isolate, designated as SP2, was identified to be a Pseudomonas species based on the sequence homology of its 16S rDNA. Pseudomonas species SP2 had essential genes for vitamin B₁₂ synthesis such as cobB and cobQ and produced a similar amount of vitamin B₁₂ (10.6 ± 0.05 μg/mL) as P. denitrificans ATCC 13867 in 24 h flask culture. SP2 grew well under aerobic condition with the maximum specific growth rate (µ _max ) of 0.91 ± 0.03/h, but showed a poor growth under micro-aerobic conditions. SP2 was resistant to antibiotics like streptomycin, carbenicillin, ampicillin, cefpodoxime, colistin, nalidixic acid and sparfloxacin. The ability of SP2 to grow faster and produce vitamin B₁₂ under aerobic conditions makes it a promising host for the production of some biochemicals requiring a coenzyme B₁₂-dependent enzyme, such as glycerol dehydratase.     

Mechanism of the 6-Hydroxy-3-succinoyl-pyridine 3-Monooxygenase Flavoprotein from Pseudomonas putida S16

6-Hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB), a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, catalyzes pyridine-ring β-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine. Here, we generated His₆-tagged HspB in Escherichia coli, characterized the properties of the recombinant enzyme, and investigated its mechanism of catalysis. In contrast to conclusions reported previously, the second product of the HspB reaction was shown to be succinate, with isotope labeling experiments providing direct evidence that the newly introduced oxygen atom of succinate is derived from H₂O. Phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB. The reduction of HspB (with maxima at 375 and 460 nm, and a shoulder at 485 nm) by NADH was followed by stopped-flow spectroscopy, and the rate constant for reduction was shown to be stimulated by HSP. Reduced HspB reacts with oxygen to form a C_(4a)-(hydro)peroxyflavin intermediate with an absorbance maximum at ∼400 nm within the first few milliseconds before converting to the oxidized flavoenzyme species. The formed C_(4a)-hydroperoxyflavin intermediate reacts with HSP to form an intermediate that hydrolyzes to the products 2,5-dihydroxypyridine and succinate. The investigation on the catalytic mechanism of a flavoprotein pyridine-ring β-position hydroxylase provides useful information for the biosynthesis of pyridine derivatives.     

The Pseudoenzyme PDX1.2 Boosts Vitamin B6 Biosynthesis under Heat and Oxidative Stress in Arabidopsis

Vitamin B₆ is an indispensable compound for survival, well known as a cofactor for numerous central metabolic enzymes and more recently for playing a role in several stress responses, particularly in association with oxidative stress. Regulatory aspects for the use of the vitamin in these roles are not known. Here we show that certain plants carry a pseudoenzyme (PDX1.2), which is involved in regulating vitamin B₆ biosynthesis de novo under stress conditions. Specifically, we demonstrate that Arabidopsis PDX1.2 enhances the activity of its catalytic paralogs by forming a heterododecameric complex. PDX1.2 is strongly induced by heat as well as singlet oxygen stress, concomitant with an enhancement of vitamin B₆ production. Analysis of pdx1.2 knockdown lines demonstrates that boosting vitamin B₆ content is dependent on PDX1.2, revealing that this pseudoenzyme acts as a positive regulator of vitamin B₆ biosynthesis during such stress conditions in plants.     

A high throughput method to screen companion bacterium for 2-keto-l-gulonic acid biosynthesis by co-culturing Ketogulonicigenium vulgare

Bacillus megaterium is widely used as companion bacterium in the two-step biosynthesis of 2-keto-l-gulonic acid (2-KLG) by Ketogulonicigenium vulgare. To screen efficiently target companion strains from large numbers of random mutants, a screen method based on spectrophotometry and 24-well microtiter plates was developed and validated on an integrated library of 450 transposon random insertional mutants and two sporulation-defective mutants. The co-culture processes were classified into three groups (low, intermediate and high performance) by K-mean clustering analysis. In addition, mutant m71 was successfully screened out from the library. The substrate conversion ratio of m71 and K. vulgare co-culture process after 72 h was decreased by about 38% compared with that of the wild-type co-culture process in 750 ml flasks. These results indicated that the proposed high throughput method is feasible for screening target companions for the co-culture process of 2-KLG biosynthesis.     

A Bacillus megaterium Plasmid System for the Production, Export, and One-Step Purification of Affinity-Tagged Heterologous Levansucrase from Growth Medium

A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg/liter of a His₆-tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.     

Production of single chain Fab (scFab) fragments in Bacillus megaterium

Background

The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.

Results

The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.

Conclusion

B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.     

Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium

The aim of this study was to investigate environmental conditions affecting chicken feather degradation and keratinolytic enzyme production by Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. B. megaterium F7-1 degraded whole chicken feather completely within 7 days. The bacterium grew with an optimum at pH 7.0–11.0 and 25–40 °C, where maximum keratinolytic activity was also observed. The production of keratinolytic enzyme by B. megaterium F7-1 was inducible with feather. Keratinolytic enzyme production by B. megaterium F7-1 at 0.6% (w/v) skim milk was 468 U/ml, which was about 9.4-fold higher than that without skim milk. The amount of keratinolytic enzyme production depended on feather concentrations. The degradation rate of autoclaved chicken feathers by cell-free culture supernatant was 26% after 24 h of incubation, but the degradation of untreated chicken feathers was unsuccessful. B. megaterium F7-1 effectively degraded feather meal, duck feather and human nail, whereas human hair and sheep wool showed relatively low degradation rates. B. megaterium F7-1 presented high keratinolytic activity and was very effective in feather degradation, providing potential use for biotechnological processes of keratin hydrolysis.