Bacillus amyloliquefaciens 35 M can exclusively produce and secrete proteases when cultured in soybean-meal-based medium.

Some microbial strains are ideal producers of extracellular enzymes that can be used in various industries. However, in many fields, especially in the pharmaceutical field, these enzymes need to be recovered and purified through multistep processes and tedious procedures before they can be used. The recovery process is difficult and increases the cost of enzyme production. Therefore, reducing purification steps will greatly benefit the utilization of microbial enzymes. The 35 M strain of Bacillus amyloliquefaciens, which has high extracellular protease production, was isolated from a phosphate mine. When cultured in a medium with soybean meal as the main component, the maximum activity of extracellular protease reached 16,992 U/mL. SDS-PAGE showed that there were two main proteins in the fermentation supernatant, with a paucity of other defined protein bands. Mass spectrometry and zymogram analysis showed that the two main bands were two proteases, corresponding to alkaline protease (AprM) and neutral protease (NprM), respectively. Gene cloning, sequencing, and further comparisons were used to confirm AprM and NprM correspond to these proteases from B. amyloliquefaciens. Notably, SDS-PAGE and zymogram analysis showed that NprM had obviously higher catalytic efficiency toward casein than did AprM. Strain 35 M is a promising protease producer with great potential for applications in industrial protease production. Additionally, this study demonstrates strain 35 M may be particularly well suited to use in degrading anti-nutritional factors in soybean meal, so as to improve the nutritional value of soybean meal.

Nocardioides phosphati sp. nov., an actinomycete isolated from a phosphate mine.

A Gram-staining-positive, non-spore-forming and non-motile strain, designated WYH11-7T, was isolated from a phosphate mine in Yunnan Province, PR China. The taxonomic position of WYH11-7T was investigated by polyphasic approaches. Phylogenetic analyses based on 16S rRNA gene sequences indicated that WYH11-7T represents a member of the genus Nocardioides. WYH11-7T was closely related to Nocardioidesjensenii DSM 20641T, Nocardioidesdubius DSM 19084T and Marmoricolaterrae DSM 27141T, and had pairwise 16S rRNA gene sequence similarities of 97.4, 97.2 and 97.0 %, respectively. DNA-DNA relatedness values between WYH11-7T and related type strains N. jensenii DSM 20641T and N. dubius DSM 19084T were found to be 17.6±4.9 and 14.6±3.1 %, respectively. The respiratory menaquinone of WYH11-7T was MK-8 (H4) while the major fatty acids were C18 : 1ω9c, C16 : 0, C17 : 0, C17 : 1ω8c, C18 : 1 10-methyl and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unidentified phospholipids. Whole-cell hydrolysates contained mannose, ribose, glucose and galactose along with ll-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The DNA G+C content was 71.2 mol%. Phenotypic, phylogenetic and chemotaxonomic data indicated that strain WYH11-7T represents a novel species of the genus Nocardioides, for which the name Nocardioidesphosphatisp. nov. is proposed. The type strain is WYH11-7T (=CGMCC 4.7371T=DSM 104026T).

Comamonas phosphati sp. nov., isolated from a phosphate mine.

A Gram-stain-negative, facultatively anaerobic, non-pigmented, non-sporulating, rod-shaped bacterial strain (WYH 22-41T) was isolated from a phosphate mine in Yunnan Province, China. The cells were motile with a single polar flagellum. The 16S rRNA gene of strain WYH 22-41T was phylogenetically related to the corresponding gene of Comamonas terrae DSM 27221T (98.4 % 16S rRNA gene sequence similarity), Comamonas odontotermitis LMG 23579T (97.6 %) and Comamonas aquatica LMG 2370T (97.4 %). DNA-DNA hybridizations of strain WYH 22-41T with these three strains showed relatedness values of 33.2 %, 20.5 % and 27.7 %, respectively. The DNA G+C content of strain WYH 22-41T was 62.4 mol%. The predominant respiratory quinone was ubiquinone-8. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major fatty acids of strain WYH 22-41T were C16 : 0, C17 : 0 cyclo, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c). On the basis of phenotypic properties, phylogenetic characteristics, DNA-DNA hybridization, as well as whole-cell fatty acid composition, strain WYH 22-41T represents a novel species of the genus Comamonas, for which the name Comamonas phosphati sp. nov. is proposed. The type strain is WYH 22-41T ( = CGMCC 1.12294T = DSM 26017T).

Novosphingobium kunmingense sp. nov., isolated from a phosphate mine.

A yellow-pigmented, Gram-stain-negative, strictly aerobic, rod-shaped, round-ended bacterium, designated strain 18-11HK(T), was isolated from a phosphate mine situated in the suburb of Kunming in Yunnan province in south-western China. The taxonomic status of this strain was evaluated by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity, strain 18-11HK(T) was shown to belong to the genus Novosphingobium, showing the highest levels of sequence similarity with respect to 'Novosphingobium ginsenosidimutans' FW-6 (97.2%), Novosphingobium subterraneum DSM 12447(T) (96.7%), Novosphingobium aromaticivorans DSM 12444(T) (96.7%) and Novosphingobium tardaugens DSM 16702(T) (96.3%). Strain 18-11HK(T) had a genomic DNA G+C content of 65.3 mol% and Q-10 as the predominant respiratory quinone. DNA-DNA hybridizations of strain 18-11HK(T) with N. subterraneum DSM 12447(T), N. aromaticivorans DSM 12444(T) and N. tardaugens DSM 16702(T) showed low relatedness values of 29.6, 33.5 and 32.3%, respectively. The predominant fatty acids of strain 18-11HK(T) were summed feature 8 (C18 : 1ω7c and/or C18: 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0, and the major 2-hydroxy fatty acid was C14 : 0 2-OH. The polar lipid profile revealed the presence of sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and some unidentified lipids. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain 18-11HK(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium kunmingense sp. nov. is proposed. The type strain is 18-11HK(T) ( = CGMCC 1.12274(T) = DSM 25975(T)).

Pseudomonas kunmingensis sp. nov., an exopolysaccharide-producing bacterium isolated from a phosphate mine.

A Gram-stain-negative, rod-shaped, exopolysaccharide-producing, strictly aerobic bacterium with a single polar flagellum, designated strain HL22-2(T), was isolated from a phosphate mine situated in a suburb of Kunmming in Yunnan province in south-western China. The taxonomic status of this strain was evaluated by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HL22-2(T) was related to members of the genus Pseudomonas. 16S rRNA gene sequence similarities between strain HL22-2(T) and Pseudomonas xanthomarina KMM 1447(T), Pseudomonas alcaliphila AL15-21(T) and Pseudomonas stutzeri ATCC 17588(T) were 98.9, 98.10% and 98.06%, respectively. The major cellular fatty acids were C(18 : 1)ω7c, C(16 : 0) and summed feature 3 (C(16 : 1)ω7c and/or C(16 : 1)ω6c). The DNA G+C content was 60.3 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness values, strain HL22-2(T) represents a novel species of the genus Pseudomonas, for which the name Pseudomonas kunmingensis sp. nov. is proposed. The type strain is HL22-2(T) ( = CGMCC 1.12273(T) = DSM 25974(T)).

[Proteomic analysis of Bacillus subtilis 168 transforming cis-propenylphosphonic acid to fosfomycin].

In this study, we investigated the mechanism of transformation by Bacillus subtilis strain 168 by proteomic analysis. B. subtilis strain 168 was able to stereoselectively transform cis-propenylphosphonic acid (cPPA) to fosfomycin. The maximal fosfomycin production was 816.6 microg/mL after two days cultivation, with a conversion rate of 36.05%. We separated the whole cellular proteins by two-dimensional gel electrophoresis (2-DE) method, and 562 protein spots were detected in the presence of cPPA in the medium, while 527 protein spots were detected in the absence of cPPA. Of them, 98 differentially expressed protein spots were found. Among them, 52 proteins were up-regulated whereas 20 were down-regulated in the presence of cPPA in the medium, and 26 induced at the presence of cPPA. The differentially expressed proteins were analyzed by combined MS and MS/MS methods. Eighty protein spots, including 45 up-regulated proteins, 17 down-regulated proteins, and 18 induced by cPPA were identified. Based on the results of proteomic analysis, we postulated two steps of transformation: in the first step, cPPA was hydrated to 2-hydroxypropylphosphonic acid; in the second step, 2-hydroxypropylphosphonic acid was transformed to fosfomycin via a dehydrogenation reaction.

Purification and characterization of four keratinases produced by Streptomyces sp. strain 16 in native human foot skin medium.

Four extracellular keratinases (designated KI, KII, KIII, and KIV) were produced during submerged aerobic cultivation in a medium containing native human foot skin (NHFS) for enzyme synthesis. The molecular weights, determined by SDS-PAGE, were 25, 50, 34, and 19 kDa, respectively. Gel filtration of the four purified enzymes in native conditions indicated that active keratinase KI is a novel homo-octamer, KII a homo-dimer, and KIII and KIV monomers. All four keratinases exhibited high activities at pH 8.0-10.0 with an optimal pH of 9.0. The optimal temperature for keratinolytic activity of KI, KII, and KIII was approximately 50, and 60 degrees C for KIV. One millimolar of PMSF completely inhibited the keratinolytic activities of the four enzymes. The N-terminal sequences of KI, KII, and KIII showed that they were different from previously described enzymes, whereas KIV shared an identical N-terminal sequence with two other peptidases from Streptomyces.

Stereoselective epoxidation of cis-propenylphosphonic acid to fosfomycin by a newly isolated bacterium Bacillus simplex strain S101.

In industry, fosfomycin is mainly prepared via chemical epoxidation of cis-propenylphosphonic acid (cPPA). The conversion yield of fosfomycin is less than 50% in the whole process and a large quantity of waste is produced. Biotransformation by microorganisms is an alternative method of preparation. This kind of conversion is more delicate, environmentally friendly, and the conversion yield of fosfomycin would be higher. In this work, an aerobic bacterium capable of transforming cPPA to fosfomycin was isolated. The organism, designated as strain S101, was identified as Bacillus simplex by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. Fosfomycin was assayed by two means, bioassay and gas chromatography (GC). Glycerol was a good carbon source for growth and cPPA conversion of strain S101. When cPPA was used as the sole carbon source, neither growth nor conversion to fosfomycin occurred. The optimum cPPA concentration in the conversion medium was 2,000 microg ml(-1). After 6 days of incubation, the concentration of fosfomycin reached its maximum level (1,838.2 microg ml(-1)), with a conversion ratio of 81.3%. Air was indispensable for the growth but not for the conversion to fosfomycin. Furthermore, vanadium ions were found to be essential for the conversion. High concentrations of cPPA had fewer inhibitory effects on the growth of strain S101.

Purification, characterization, and substrate specificity of two 2,3-dihydroxybiphenyl 1,2-dioxygenase from Rhodococcus sp. R04, showing their distinct stability at various temperature.

The genes of two 2,3-dihydroxybiphenyl 1,2-dioxygenases (BphC1 and BphC2) were obtained from the gene library of Rhodococcus sp. R04. The enzymes have been purified to apparent electrophoretic homogeneity from the cell extracts of the recombinant harboring bphC1 and bphC2. Both BphC1 and BphC2 were hexamers, consisting of six subunits of 35 and 33 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzymes had similar optimal pH (pH 9.0), but different temperatures for their maximum activity (30 degrees C for BphC1, 80 degrees C for BphC2). In addition, they exhibited distinct stability at various temperatures. The enzymes could cleave a wide range of catechols, with 2,3-dihydroxybiphenyl being the optimum substrate for BphC1 and BphC2. BphC1 was inhibited by 2,3-dihydroxybiphenyl, catechol and 3-chlorocatechol, whereas BphC2 showed strong substrate inhibition for all the given substrates. BphC2 exhibited a half-life of 15 min at 80 degrees C and 50 min at 70 degrees C, making it the most thermostable extradiol dioxygenase studied in mesophilic bacteria. After disruption of bphC1 and bphC2 genes, R04DeltaC1 (bphC1 mutant) delayed the time of their completely eliminating biphenyl another 15 h compared with its parent strain R04, but R04DeltaC2 (bphC2 mutant) lost the ability to grow on biphenyl, suggesting that BphC1 plays an assistant role in the degrading of biphenyl by strain R04, while BphC2 is essential for the growth of strain R04 on biphenyl.

Screening for a new Streptomyces strain capable of efficient keratin degradation.

Keratinous wastes could be degraded by some microorganisms in nature. Native human foot skin (NHFS) was used as sole nitrogen source to screen microorganisms with keratin-degrading capability. From approximately 200 strains, a strain of Streptomyces sp. strain No.16 was found to possess the strongest keratinolytic activity, and the total activity in the culture was 110 KU/ml with specific activity of 2870 KU/mg protein (KU: keratinase unit). Substrate specificity test indicated that the crude keratinase could degrade keratin azure, human hair, cock feathers and collagen. The optimal pH of the crude keratinase ranged from 7.5 to 10 and the temperature ranged from 40 degrees C to 55 degrees C. Metal chelating agent ethylenediamine tetraacetic acid obviously stimulated the keratinolytic activity but suppressed the proteolytic activity. To our knowledge, this is the first report on specific induction of keratinases by NHFS from an actinomycete. Moreover, excellent characteristics of its crude keratinase may lead to the potential application in waste treatment and recovery, poultry and leather industry, medicine, and cosmetic development.

On-column refolding and purification of transglutaminase from Streptomyces fradiae expressed as inclusion bodies in Escherichia coli.

Since transglutaminase (TGase) have been widely used in industry, mass production of the enzyme is especially necessary. The mature TGase gene from Streptomyces fradiae was cloned into pET21a and overexpressed in Escherichia coli BL21(DE3). The recombinant TGase was formed as inclusion bodies, and its content was as high as 55% of the total protein content. The insoluble fractions were separated from cellular debris by centrifugation and solubilized with 8 M urea. With an on-column refolding procedure based on cation SP Fast Flow chromatography with dual-gradient, the active TGase protein was recovered efficiently from inclusion bodies. The final purified product was 95% pure detected by SDS-PAGE. Under appropriate experimental conditions, the protein yield and specific activity of the TGase were up to 53% and 21 U/mg, respectively. Furthermore, the refolded recombinant protein demonstrated nearly identical ability to polymerized BSA compared with that of native TGase. One hundred and five milligrams of refolded TGase protein was obtained from 3.2g wet weight cells in the 400 ml cell culture.

Cloning of transglutaminase gene from Streptomyces fradiae and its enhanced expression in the original strain.

The transglutaminase (TGase) gene of Streptomyces fradiae was cloned. It had an ORF of 1242 bp, encoding a presumed prepro-region of 82 amino acids and a mature TGase of 331 amino acids. Enhanced expression of the TGase was achieved by introducing another copy of TGase gene into the original host genome which was driven by the strong constitutive promoter, "ermE up", and shown to be expressed at the mRNA and protein levels. TGase activity in the recombinant strain (3.2 U/ml) was improved 1.3-fold when compared to that normally expressed in the original strain (2.4 U/ml). The specific enzyme activity in the recombinant strain (3.8 U/mg) was double that of the original strain (1.9 U/mg).