Production and characterization of poly‐3‐hydroxybutyrate from biodiesel‐glycerol by Burkholderia cepacia ATCC 17759

Glycerol, a byproduct of the biodiesel industry, can be used by bacteria as an inexpensive carbon source for the production of value‐added biodegradable polyhydroxyalkanoates (PHAs). Burkholderia cepacia ATCC 17759 synthesized poly‐3‐hydroxybutyrate (PHB) from glycerol concentrations ranging from 3% to 9% (v/v). Increasing the glycerol concentration results in a gradual reduction of biomass, PHA yield, and molecular mass (M_n and M_w) of PHB. The molecular mass of PHB produced utilizing xylose as a carbon source is also decreased by the addition of glycerol as a secondary carbon source dependent on the time and concentration of the addition. ¹H‐NMR revealed that molecular masses decreased due to the esterification of glycerol with PHB resulting in chain termination (end‐capping). However, melting temperature and glass transition temperature of the end‐capped polymers showed no significant difference when compared to the xylose‐based PHB. The fermentation was successfully scaled up to 200 L for PHB production and the yield of dry biomass and PHB were 23.6 g/L and 7.4 g/L, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Production of PHB from Crude Glycerol

Crude glycerol – a by‐product of the large scale production of diesel oil from rape – is examined for its possible use as a cheap feedstock for the biotechnological synthesis of poly(3‐hydroxybutyrate) (PHB). The glycerol samples of various manufacturers differ in their contamination with salts (NaCl or K₂SO₄), methanol or fatty acids. At high cell density fermentation these pollutants could possibly accumulate to inhibiting concentrations. The bacteria used were Paracoccus denitrificans and Cupriavidus necator JMP 134, which accumulate PHB from pure glycerol to a content of 70 % of cell dry mass. When using crude glycerol containing 5.5 % NaCl, a reduced PHB content of 48 % was observed at a bacterial dry mass of 50 g/L. Furthermore the PHB yield coefficient was reduced, obviously due to osmoregulation. The effect of glycerol contaminated with K₂SO₄ was less pronounced. The molecular weight of PHB produced with P. denitrificans or C. necator from crude glycerol varies between 620000 and 750000 g/mol which allows the processing by common techniques of the polymer industry.     

Improvement of A21978C production in Streptomyces roseosporus by reporter-guided rpsL mutation selection

Aims:  Daptomycin, one of the A21978C factors produced by Streptomyces roseosporus, is an acidic cyclic lipopeptide antibiotic with potent activity against a variety of Gram‐positive pathogens. To increase the titre of this extensively used and clinically important antibiotic, we applied a reported‐guided rpsL mutation selection system to generate strains producing high levels of A21978C. Methods and Results:  In the reporter design, dptE was chosen as the overexpressing target, and neo‐encoding neomycin phosphotransferase as the reporter. Using this reporter‐guided selection system, 20% of the selected, streptomycin‐resistant mutants produced greater amounts of A21978C than the starting strain. The selection system increased the screening efficiency about 10‐fold with a frequency of 1·7% A21978C overproducing strains among str^r mutants. A21978C production was increased approximately 2·2‐fold in the rpsL K43N mutant. Conclusions:  The combination of ribosome engineering and reporter‐guided mutant selection generated an A21978C overproducing strain that produced about twice as much A21978C as the parental strain. Significance and Impact of the Study:  The strategies presented here, which integrated the advantages of both ribosome engineering and reporter‐guided mutation selection, could be applied to other bacteria to improve their yield of secondary metabolites.     

Purification and partial characterization of a bacteriocin produced by Eikenella corrodens

Aims: The purpose of this study was to purify and characterize a bacteriocin produced by Eikenella corrodens A32E2. Methods and Results: Peptostreptococcus anaerobius ATCC27337 was used as indicator strain in antagonistic assays for bacteriocin‐producing E. corrodens A32E2. Protein extraction was influenced by pH and buffer composition. The protein was active in the pH range 6–8. Inhibitory activity was lost by both heating and treatment with proteolytic enzymes and decreased with organic solvents. The substance is rather unstable but maintains 100% of its activity after being exposed to acetone and when stored at ?70°C. The antagonistic substance was first precipitated by ammonium sulfate and further partially purified by Mono‐Q FPLC and C‐18 HPLC. Mass spectrometry analysis showed that the molecular mass was 23 625 Da, and the sequence obtained for the N‐terminus was: Met‐Asn‐Phe‐Asp‐Glu‐Lys‐Val‐Gly‐Lys‐Val‐X‐Phe‐Lys‐Val‐Gly‐Asp. Conclusions: The evidence presented in this study supports the idea that an antagonistic substance produced by E. corrodens A32E2 isolated from a periodontal diseased site is a novel bacteriocin, which we designate corrodecin. Significance and Impact of the Study:  We anticipated that corrodecin might play an important role at the periodontal site. This compound could also be attractive in biotechnological applications as an interesting tool for oral ecosystem control.     

Characterization and high‐yield production of non‐N‐glycosylated recombinant human BCMA‐Fc in Pichia pastoris

B‐cell maturation antigen (BCMA) fused at the C‐terminus to the Fc portion of human IgG1 (BCMA‐Fc) blocks B‐cell activating factor (BAFF) and proliferation‐inducing ligand (APRIL)‐mediated B‐cell activation, leading to immune disorders. The fusion protein has been cloned and produced by several engineering cell lines. To reduce cost and enhance production, we attempted to express recombinant human BCMA‐Fc (rhBCMA‐Fc) in Pichia pastoris under the control of the AOX1 methanol‐inducible promoter. To produce the target protein with uniform molecular weight and reduced immunogenicity, we mutated two predicted N‐linked glycosylation sites. The secretory yield was improved by codon optimization of the target gene sequence. After fed‐batch fermentation under optimized conditions, the highest yield (207 mg/L) of rhBCMA‐Fc was obtained with high productivity (3.45 mg/L/h). The purified functional rhBCMA‐Fc possessed high‐binding affinity to APRIL and dose‐dependent inhibition of APRIL‐induced proliferative activity in vitro through three‐step purification. Thus, this yeast‐derived expression method could be a low‐cost and effective alternative to the production of rhBCMA‐Fc in mammalian cell lines.     

Extraction of natural red colorants from the fermented broth of Penicillium purpurogenum using aqueous two‐phase polymer systems

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two‐phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG‐rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (K_C) was obtained in the presence of NaCl 0.1 M (K_C = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na₂SO₄ 0.5 M showed the highest K_C (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%η_TOP) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0–3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1295–1304, 2015     

Improving of red colorants production by a new Penicillium purpurogenum strain in submerged culture and the effect of different parameters in their stability

There is a worldwide interest in the development of processes for colorants production from natural sources such as microorganism. The aim of this study was to optimize red colorants production by Penicillium purpurogenum DPUA 1275 and to evaluate the effect of pH, temperature, salts and polymers on the stability of these colorants. Under optimized conditions, a 78% increase in red colorants production was achieved. The best pH and temperature conditions were obtained at pH 8.0 and 70°C, respectively. In the presence of salts NaCl and Na₂SO₄, both at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 8.0), the red colorants showed good stability. In the presence of both polymers polyethylene glycol and sodium polyacrylate, the red colorants kept their color intensity. Thus, this study presents characteristics of red colorants produced by P. purpurogenum that can be applied in different industries after toxicological examination. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:778–785, 2013     

Inhibitory effects of enterococci on the production of hydrogen sulfide by hydrogen sulfide-producing bacteria in raw meat

Aims: Applying competitive exclusion micro‐organisms to control hydrogen sulfide (H₂S) gas produced by hydrogen sulfide–producing bacteria (SPB) in chicken meat. Methods and Results: Five SPB strains, isolated from animal by‐products, were used for screening lactic acid bacteria (LAB) that can inhibit the production of H₂S by SPB in trypticase soy broth supplemented with l ‐cysteine (TSB‐l ‐cys). A sensitive and accurate test strip method was developed for H₂S determination in real time. One LAB strain, isolate L86, from cheese whey, demonstrated the highest inhibitory activity against the production of H₂S by SPB. The isolate L86 was confirmed as Enterococcus faecium that does not possess genes encoding for vancomycin resistance based on PCR analysis. Enterococcus faecium strain L86 reduced (P < 0·05) the yield of H₂S upto 51·2% in 10 h at 35°C in TSB‐l ‐cys medium. In fresh chicken meat, the yield of H₂S produced by the artificially inoculated SPB was reduced (P < 0·05) by 48·6, 49·7 and 69·8% in 10 h at 35, 30 and 25°C, respectively. Enterococcus faecium strain L86 also reduced (P < 0·05) by 53·8% on the yield of H₂S produced by the indigenous SPB in partially spoiled chicken meat at 35°C for 10 h. Conclusions: Enterococcus faecium strain L86 is effective on inhibiting the production of H₂S by SPB. Significance and Impact of the Study: The application of this biological agent to raw animal by‐products will provide a safer working environment in rendering processing plants and produce higher‐quality rendered products.     

Identification of a Passiflora alata Curtis dimeric peptide showing identity with 2S albumins

Antifungal proteins and peptides, essential compounds for plant defense, have been isolated from several tissues of various plants. These proteins could be used as a natural alternative to control phytopathogenic fungi. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptidic molecular masses of approximately 4500 Da and 7000 Da, in the presence of β-mercaptoethanol, while by removing the reducing agent a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimer mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporioides, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi.     

A Novel Antimicrobial Peptide from <Emphasis Type="Italic">Crotalaria</Emphasis> <Emphasis Type="Italic">pallida</Emphasis> Seeds with Activity Against Human and Phytopathogens

An actual severe problem in agriculture consists of an expressive increase of economical losses caused by fungi and resistant bacteria toward antibiotics. In order to find a solution to this problem, several studies have been concentrating on the screening of novel plant defense peptides with antimicrobial activities. These peptides are commonly characterized by having low molecular masses and cationic charges. The present work reports the purification and characterization of a novel plant peptide with molecular mass of 5340 Da, named Cp-AMP, from seeds of C. pallida, a typical plant from Caatinga biome. Purification was achieved using a size exclusion S-200 column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Cp-AMP was able to inhibit the development of filamentous fungi Fusarium oxysporum as well as the gram-negative bacterium Proteus sp. The identification of Cp-AMP could contribute, in the near future, to the development of biotechnological products, such as transgenic plants with enhanced resistance to pathogenic fungi and/or of antibiotics production derived from plant sources in order to control bacterial infections.     

Polylysine Produced by Streptomyces

The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS–PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.     

Increase in hyaluronic acid production by Streptococcus equi subsp. zooepidemicus strain deficient in β-glucuronidase in laboratory conditions

Streptococcus equi subsp. zooepidemicus is known to produce a hyaluronic acid capsule to resist the host immune defense. As the structure of the polysaccharide is identical to the one produced by humans, the bacteria S. equisubsp. zooepidemicusis used in biotechnological production of hyaluronic acid. In our laboratory we prepared mutated strains that are β-glucuronidase deficient. Comparing the wild-type strain, which is positive in β-glucuronidase activity, with the mutated strains named clone1 and clone2 in laboratory conditions, we observed that β-glucuronidase influences the production of hyaluronic acid considerably and the molecular weight of hyaluronan slightly. The production of hyaluronic acid by the mutated strains is higher by approximately 20% and the molecular weight is larger by about 2%. The significant increase in the production of hyaluronic acid and the slight increase in the molecular weight are probably caused by an absence of free β-glucuronic acid, due to its removal from the non-reducing termini of the polysaccharide by β-glucuronidase. The presence of free β-glucuronic acid would likely induce the expression of the β-glucuronic-acid-utilizing operon, which in turn would reflect into a misuse of energy in the glucose-rich media.     

Probiotic potential of Staphylococcus hominis MBBL 2–9 as anti‐Staphylococcus aureus agent isolated from the vaginal microbiota of a healthy woman

Aims: To isolate and characterize an antagonist for use as probiotic agent in the biocontrol of Staphylococcus aureus. Methods and Results: Bacteria that exhibited antimicrobial activity against Gram‐positive bacteria including Staph. aureus were isolated from 12 healthy women, with Staphylococcus hominis MBBL 2–9 showing the strongest activity. The bacteriocin produced by Staph. hominis MBBL 2–9 was purified by 60% ammonium sulfate saturation, ultrafiltration, HLB cartridge and reverse‐phase HPLC. The molecular weight was estimated as 2038·2 Da by MALDI–TOF mass spectrometry. The antagonist survived up to 2 h in artificial gastric juice (pH 2·5) and grew in the presence of 1% porcine bile extract. In addition, Staph. hominis MBBL 2–9 adhered effectively to HT‐29 epithelial cell line. Conclusion: Staphylococcus hominis MBBL 2–9 exhibited desirable probiotic traits such as acid tolerance, bile resistance and adherence to epithelial cell line. The bacterium also produced a bacteriocin with unique molecular weight and high antimicrobial activity similar to traditional antibiotics. Significance and Impact of the Study: This study is the first report of a bacteriocin‐producing Staph. hominis MBBL 2–9 that has potential for use as a probiotic agent against Staph. aureus.     

The use of an acetoacetyl‐CoA synthase in place of a β‐ketothiolase enhances poly‐3‐hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C₄ plants for the production of poly[(R)‐3‐hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield‐potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing β‐ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl‐CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB‐producing sugarcane containing the β‐ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10⁶ Da. These results are a major step forward in engineering a high biomass C₄ grass for the commercial production of PHB.     

Identification and characterization of two novel antimicrobial peptides,temporin‐Ra and temporin‐Rb,from skin secretions of the marsh frog (Rana ridibunda)

In this study, two novel antimicrobial peptides from the skin secretions of the marsh frog, Rana ridibunda, named temporin‐Ra and temporin‐Rb, were identified and purified using RP‐HPLC. Temporin‐Ra and temporin‐Rb are composed of 14 and 12 amino acids, respectively. Our results show that these peptides have inhibitory effects on both gram‐negative and gram‐positive bacteria, especially antibiotic resistant strains prevalent in hospitals, such as Staphylococcus aureus and Streptococcus agalactiae. The sequences and molecular weights of these peptides were determined using tandem MS. The molecular masses were found to be 1242.5 Da for temporin‐Rb and 1585.1 Da for temporin‐Ra. Human red blood cells tolerated well exposure to temporin‐Ra and temporin‐Rb, which, at a concentration of 60 µg/ml, induced 1.3% and 1.1% hemolysis, respectively. MIC values of these peptides are suitable for potent antimicrobial peptides. The low hemolytic effect and wide‐spectrum antimicrobial activity suggest a possible therapeutic application of these novel peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Multiple Bacteriocin Production by Leuconostoc mesenteroidesTA33a and Other Leuconostoc/Weissella Strains

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins. Received: 11 April 1997 / Accepted: 10 June 1997     

Sequencing and phylogenetic analyses of talaromyces amestolkiae from amazon: A producer of natural colorants

The population interest in health products is increasing day-by-day. Thus, the demand for natural products to be added in food and pharmaceutical commodity is also rising. Among these additives, colorants, which provides color to products, can be produced by microorganism through bioprocess. Looking for new source of natural colorants, fungi have been employed to this purpose producing novel and safer natural colorants. So, the main goal of this study was to describe a Talaromyces species able to produce natural colorants and investigate nutritional parameters of colorants production using statistical tool. The taxonomy classified the microorganism as Talaromyces amestolkiae. The statistical design evaluated pH and glucose, meat extract and meat peptone concentration as independent variables, and red colorants production as main response. Under the best condition (g/L: glucose 30, meat extract 1, meat peptone 10, and initial pH of 7.0) an increase of 229% in the red colorant production was achieved as compared with the initial media used. The dried fermented broth containing red colorants showed low cytotoxicity against fibroblasts cells (IC₅₀ > 187.5 g/L) and effective antimicrobial activity against S. aureus (MIC of 2.5 g/L). Thus, T. amestolkiae colorants can be attractive to food and pharmaceutical applications as it does not produce toxic compounds and can promote protection against microorganism contaminants. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2684, 2019     

Polyhydroxybutyrate production by Saccharophagus degradans using raw starch as carbon source

Biosynthesis of poly(3‐hydroxybutyrate) (PHB) from raw starch as the carbon source by the polysaccharide‐digesting bacteria Saccharophagus degradans was investigated in a fed‐batch culture. The production and properties of the PHB synthesized from starch were compared to those obtained using glucose as carbon source. In fed‐batch cultures, S. degradans accumulated 21.35 and 17.46% of PHB, using glucose or starch as carbon source, respectively. The physical properties of the biopolymer produced from each carbon source were similar between them. Molecular mass, melting temperature and heat of fusion were 54.23 kDa, 165.61°C and 59.59 J/g, respectively, using glucose; and 57.07 kDa, 174.31°C and 67.66 J/g, respectively, using starch. This is the first work describing the capability of S. degradans to utilize raw starch as the sole carbon source for the production of PHB.     

Isolation of keratinase from bacterial isolates of poultry soil for waste degradation

Isolation of two keratinolytic bacterial strains from poultry soil as well as purification and properties of keratinase were investigated. Isolates were designated as KI8101 and KI8102 (KI, keratin isolates) and were identified as Bacillus subtilis and B. licheniformis respectively. The purified enzyme from KI8102 exhibited a high specific activity of 500 U/mg with 71‐fold purification and 41% yield. SDS‐PAGE analysis indicated that the purified keratinase had a molecular mass of 32 kDa. The optimum temperature and pH were 50°C and 7.5, respectively. Its K_m was 83.3 μM and V_max was 71.4 μmol/mL min. The bacterium could potentially degrade keratin waste such as human hair, nails, bovine hair and wool. Therefore, the enzyme could improve the nutritional value of meat and poultry‐processing waste containing keratin and could be a potential candidate for biotechnological processing involving keratin hydrolysis.     

4株羊瘤胃功能性细菌的筛选

通过DNS法测定羊瘤胃源功能性细菌产生的纤维素酶和淀粉酶的活力,福林酚法测定产生的蛋白酶的活力,检测细菌产生酶的特性。同时检测菌株的发酵液对大肠埃希菌(ATCC25922)、副溶血弧菌(ATCC17802)、藤黄八叠球菌(HY78)和产气杆菌(AS1489)等指示菌的抑制能力,分析它们的抑菌活性。结果表明,羊瘤胃源细菌C13产生的纤维素酶活力最高,产酶量也最高;而细菌C5产淀粉酶活力和蛋白酶活力最高,产生淀粉酶和蛋白酶的能力也最高。抑菌活性检测发现,细菌C9对副溶血弧菌(ATCC17802)有很高的抑制作用,而细菌C12对大肠埃希菌(ATCC25922)的抑制能力最明显。