Ammonium Sulfate Precipitation Research Articles

Purification and characterization of Antarctic krill chitinase and its role on free fluoride release from Antarctic krill cuticle

To investigate factors that influence free fluoride release in Antarctic krill cuticle, the enzyme properties of a chitinase from Antarctic krill were analyzed to identify its role on free fluoride release from Antarctic krill cuticle. The chitinase was purified by ammonium sulfate precipitation,ion and gel chromatography, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The molecular mass of the chitinase purified from Antarctic krill was 59.0 kDa and its peptide sequences were similar to chitinase precursor sequences from Penaeus vannamei. The optimal pH of the chitinase was pH 6.5 and temperature 45 °C and exhibited high stability within pH 5.0-6.0 and temperatures below 35 °C. The chitinase activity was increased by Ca2+ and Mg2+, while inhibited by Fe2+, Cu2+ and Co2+. The purified chitinase had Km and Vmax values of 5.88 mmol/L and 2.11 μmol/min·mL, respectively. The endogenous chitinase purified from Antarctic krill could degrade chitin fibers and induced the release of free fluoride from Antarctic krill cuticle. Under experimental conditions, the free fluoride release law in Antarctic krill cuticle can be described by equation: CW=1−0.98e−0.0111t−0.02e0.15t×175.52 +10.80.

Indigenous production, characterization and evaluation of polyclonal antibody against Camelus dromedarius immunoglobulin.

No diagnostic kits and reagents are available in the market to detect and evaluate camel immune responses to different pathogens. This study aimed to produce sheep anti-camel (Camelus dromedarius) polyclonal antibodies (pAbs) and to determine the specificity with other species immunoglobulin. Immunoglobulins (Igs) from camel serum samples were purified using ammonium sulfate precipitation (40.00% saturated ammonium sulfate). Purity of the camel Igs was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis. PAbs against (Camelus dromedarius) immunoglobulins were generated by immunizing sheep with purified Igs. Anti- camel Ig polyclonal antibodies titer and specificity were determined using ELISA and Western blot techniques. Polyclonal antibodies specific to camel Igs were significantly high in immunized sheep which confirmed the immunization procedure. PAbs reacted specifically with camel serum immunoglobulin and did not react with other species immunoglobulin of horse and chickens. Polyclonal antibodies produced in this study can be regarded as a valuable tool to be used for immune-diagnostic purposes in camel population world-wide.

Functional expression of the sweet-tasting protein brazzein in transgenic tobacco

The sweet-tasting protein, brazzein, has potential as a low-calorie sugar substitute owing to its high sweetness, stability, and water solubility. In this study, the synthetic brazzein gene was expressed in the tobacco plant, Nicotiana tabacum. Three types of expression cassettes containing the brazzein gene were constructed to examine the expression and purification efficiency of the brazzein: pBI-BZ1 containing a signal sequence and His-tag, pBI-BZ2 containing a signal sequence, and pBI-BZ3 containing only the brazzein gene. Brazzein expression confirmed by ELISA was purified using ammonium sulfate precipitation, heat treatment, and CM-sepharose chromatography. The purity and conformational state of the brazzein were confirmed using SDS-PAGE, HPLC, and circular dichroism. The identity of the brazzein was confirmed by N-terminal amino acid analysis, ESI-MS/MS, and sweetness analysis. We successfully generated brazzein overexpression tobacco plants, suggesting that this method could be used as a brazzein production platform to provide an alternative to currently produced sweeteners.

A jacalin-related lectin domain-containing lipase from chestnut (Castanea crenata): Purification, characterization, and protein identification

A novel lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was discovered from Korean chestnut (Castanea crenata). The lipase was isolated and purified by ammonium sulfate precipitation and a fast protein liquid chromatography system equipped with HiTrap DEAE-Sepharose Fast Flow, HiTrap Q-Sepharose Fast Flow, and HiPrep Sephacryl S-100 Hi-Resolution columns. The purified C. crenata lipase showed a 15.8% yield, purification fold number of 465.8, and specific activity against triolein of 88.5 mU/mg. The enzyme exhibited hydrolytic activity toward tributyrin, trilaurin, and triolein, and was maximally active at pH 8.0 and 35 °C, with triolein used as the substrate. The activation energy (Ea) and deactivation energy (Ed) of triolein hydrolysis were 38.41 and 83.35 kJ/mol, respectively. In the enzyme kinetic study, Vmax, Km, and kcat were 110.58 mU/mg, 0.11 mM, and 0.221 min−1, respectively. The relatively low Km value indicated that the lipase has high affinity for its substrate. Moreover, Mg2+ and Ca2+ increased the lipase activity to 115.4% and 108.3%, respectively. The results of peptide fingerprinting revealed that the C. crenata lipase with a molecular weight of 33.3 kDa was structurally similar to the mannose-binding lectin of the jacalin-related lectin domain superfamily, implying that it has potential as a therapeutic agent for use in the biomedical industry.

Isolation and partial characterization of a novel bacteriocin from Pseudomonas azotoformans with antimicrobial activity against Pasterella multocida.

In this study, a bacteriocin PA996 isolated from Pseudomonas azotoformans (P. azotoformans) was purified to homogeneity by ammonium sulphate precipitation and SP-Sepharose column chromatography. P. azotoformans began to grow at 6h, reached exponential phase at 12-18h. Bacteriocin PA996 was produced at 18h and reached a maximum level of 2400AU/mL. The molecular mass of purified bacteriocin PA996 was estimated by SDS-PAGE and its molecular mass was approximately 50kDa. By screening in vitro, the bacteriocin PA996 showed an antimicrobial activity against Pasteurella multocida (P. multocida). The bacteriocin PA996 showed antibacterial activity in the range of pH2-10 and it was heat labile. The inhibitory activities were diminished after treatment with proteinase K, trypsin and papain, respectively, while catalase treatment was ineffective. The minimal inhibitory concentration (MIC) and bactericidal kinetics curves showed that the bacteriocin PA996 had a good inhibitory ability against P. multocida. Our data indicate that bacteriocin PA996 could inhibit the growth of P. maltocida and it may have the potential to apply as an alternative therapeutic drug.

Production, Purification and Characterization of Bacteriocin Produced by Novel L. PentosusMW857478 for Enhancement of Food Safety and Shelf-Life of Paneer

This research paper is based upon the production, purification and characterization of bacteriocin by L.pentosusMW857478 followed by its enhancement of food safety and shelf life of paneer. In recent year LAB (Lactic acid bacteria) produced bacteriocinattract a great attention of researcher due to their many potential applications. This paper focused on isolation, identification, evaluation of broad spectrum inhibitory activity, production and purification, characterization and evaluation of food safety and shelf life of paneer. Bcateriocin produced byL. pentosusshowedantagonistic activity against food spoiling pathogens in broad range ofBacteriocin production parameter was optimized with pH 5.5 incubated at 35°C forL. pentosus. Bacteriocin was purified by ammonium sulphate precipitation and purified bacteriocin with single band on SDS-PAGE for molecular weight. The purified bacteriocin stableat 2-10 pH and 30-75°C temperatures, suggesting L. pentosusa potent candidates for safety and extending shelf life of paneer for 15 days.

Isolation, Optimization and Characterization of Lipase Producing Bacteria from Abbatoir Soil

Microorganisms can easily degrade agro-industrial wastes in a solid-state fermentation process thereby synthesizing many important commercial bio compounds including lipolytic enzymes. Screening different habitats for bacteria with lipase activity and optimizing its fermentation parameters with facilities effective production and usage in industries. In this study, soil samples from different Abattoirs in Kaduna metropolis were screened for presence of lipase positive strains culture medium parameters such as carbon source, inducers, nitrogen source, pH, temperature, inoculums level and incubation period were varied for the purpose of optimization. Maximum lipase activity was recorded at 48hours incubation, this activity occurred in media containing groundnut peel and rice husk, supplemented with ammonium nitrate and urea which are the best carbon and nitrogen sources. The isolated bacteria were identified as Pseudomonas biochemically, the production, partial purification and characterization of lipase activity at different pH, determination of molecular weight analysis. The lipase was partially purified up to saturation using ammonium sulphate precipitation spectrophotometric lipase assay was used for enzyme characterization. Lipases produced by pseudomonas have been used widely in pharmaceutical, food, pulp, textile, paper, leather industries among others due to their stability, selectivity and broad substrate specificity.

Genome-assisted Identification, Purification, and Characterization of Bacteriocins.

Bacteriocins are antimicrobial peptides with activity against antibiotic resistant bacterial pathogens. Here, we describe a set of methods aimed at purifying, identifying, and characterizing new bacteriocins. The purification consists of ammonium sulphate precipitation, cation-exchange chromatography, and reversed-phase chromatography. The yield of the bacteriocin is quantified by bacteriocin antimicrobial activity in a microtiter plate assay after each purification step. The mass of the purified bacteriocin is assessed by MALDI TOF MS analysis of the active fractions after reversed-phase chromatography. The mass is compared with the theoretical mass based on genetic information from the whole genome sequencing of the bacteriocin producer strain. Physicochemical characterization is performed by assessing antimicrobial activity following heat and protease treatments. Fluorescent techniques are used to examine the capacity of the bacteriocin to disrupt membrane integrity. Herein a set of protocols for purification and characterization of the bacteriocin nisin Z is used as a typical example in this paper.

Cytotoxic and apoptotic induction activity of protein isolated from blood clam (Anadara (tegillarca) granosa, Linnaeus 1758) Against breast cancer t47d cell line

Background: Cancer has become a major cause of death today. These risk factors are also balanced with the development of drugs, one of which comes from nature. Medical products derived from natural resources such as blood clam (Anadara (tegillarca) granosa) have been already developed. Crude protein extracted from blood calm contains a 20-kDa protein which had been known to be able to inhibit the growth of HT-29 cell line. Purpose: This study aimed to determine cytotoxic activity and apoptotic effect of crude protein which is isolated from blood calm against breast cancer T47d cell line. Materials and Methods: This study used the ammonium sulfate precipitation method for the isolation of a crude protein of blood clams. MTT assay method was performed to determine cytotoxicity and to know the value of IC50, apoptosis test qualitatively and quantitatively was done by double-staining using ethidium bromide and acridine orange as well as flow cytometric dye using annexin V dyes and propidium iodide. Findings: Crude protein clams of blood have IC50 value of 11.11μg/ml and can induce apoptosis of breast cancer cell line (T47D) at 15 μg/ml concentration by double-staining method, but the calculation with flow cytometry method still shows different results in this study. Research Limitations: This study observed the inhibitory concentration of crude extract consisting of proteins with various molecular weights against the t47d cell line. Originality: The results of this study indicate that crude protein clams are cytotoxic and can induce apoptosis in breast cancer cell lines (T47D).

Human Auto-IgG Purification from High Volume Serum Sample by Protein G Affinity Purification.

Immunoglobulins are proteins produced by the immune system, which bind specifically to the antigen that induced their formation and target it for destruction. Highly purified human immunoglobulins are commonly used in research laboratories for several applications, such as in vitro to obtain hybridomas and in vivo animal immunisation. Several affinity purification methods are used to purify immunoglobulins from human serum, such as protein A/G Sepharose beads, polyethylene glycol, and caprylic acid ammonium sulphate precipitation. Here, we provide a detailed protocol for purification of high-quality IgG from human serum, using affinity chromatography with protein G. The protocol is divided into four main steps (column preparation, serum running, wash, and elution) for IgG purification, and two extra steps (protein dialysis and sucrose concentration) that should be performed when buffer exchange and protein concentration are required. Several IgG affinity purification methods using protein A or G are available in the literature, but protein A has a higher affinity for rabbit, pig, dog, and cat IgG, while protein G has a higher affinity for mouse and human IgG. This affinity-based purification protocol uses protein G for a highly specific purification of human IgG for animal immunization, and it is particularly useful to purify large amounts of human IgG. This protocol was validated in: Pain (2019), DOI: 10.1097/j.pain.0000000000001662 Graphical abstract IgG purification protocol. The IgG purification protocol consists of four main steps (column preparation, serum running, wash, and elution) and two extra steps (protein dialysis and concentration). a. Diluted serum is added to the protein G beads and IgG binds to the Fc receptors on protein G beads. b. Beads are washed in Hartman's solution to fully remove the complex protein mixture (multicolour shapes, as depicted in the graphical abstract). c. IgG (orange triangles, as depicted in the graphical abstract) are removed from protein G with glycine and collected in Tris buffer. d. The IgG is transferred into a semi-permeable membrane ('snake skin') and allowed to dialyse overnight for buffer exchange with a physiological solution (Hartmann's).

Review on Microbial Sources, Production, Purification and Potential Industrial Applications of Laccases

Laccase (EC 1.10.3.2) is a multicopper blue oxidase which are involved in the oxidation of a broad range of organic substrates, including phenols, polyphenols, anilines, and even certain inorganic compounds by a one-electron transfer mechanism. Laccases are widely distributed in bacteria, fungai, insects and higher plants. There are mainly two production techniques for cultivation of laccase such as submersed fermentation and solid- state fermentation. This paper briefly discuss the effect of carbon source, effect of nitrogen source, effect of inducers, effects of surfactants, effect of agitator, influence of metal ions and use of agro-industrial waste in production medium. The paper also discussed the purification techniques such as ammonium sulphate precipitation for extraction purpose followed by dialysis and ion-exchange chromatography as well characterization techniques. Laccases are known to show application ranging from pharmaceutical industries to textile sector as well as in biosensor development.

Antimicrobial activity of bacteriocin produced by a new Latilactobacillus curvatus sp.LAB-3H isolated from traditional yogurt.

In recent years, the use of bacteriocin-producing Lactobacillus species has received much attention in different areas, including using as probiotics, food preservation, and as broad antimicrobial spectrum activity. In this study, a bacteriocin-producingLactobacillusstrain was isolated from traditional yogurt. The isolate was identified by morphological, biochemical, 16S rRNA analyses, and designated asLatilactobacillus curvatusLAB-3H. The primary antimicrobial activity of the isolate was evaluated onMicrococcus luteusPTCC 1408 by the agar gel diffusion method. The growth ofL. curvatusLAB-3H in MRS broth at 37°C and its antimicrobial activity againstLactobacillus caseiATCC 39,392 was evaluated. In batch culture analysis, bacteriocin production starts at the early exponential growth phase and continues to the middle of the stationary phase about (660 AU/ml) at 28h and pH 3.8. Bacteriocin produced byL. curvatusLAB-3H showed antibacterial activity against some selected foodborne pathogens, includingListeria monocytogenesPTCC1294,Bacillus cereusPTCC1857,Staphylococcus aureusPTCC1917, andEscherichia coliPTCC1276. For purification of bacteriocin, ammonium sulfate precipitation and cation exchange chromatography methods were used. The maximum antimicrobial activity observed was about 3985.15 AU/mg of protein, which was a 249.22-fold increase, and 5.21% yield compared with that in the cell-free supernatant. The molecular weight of bacteriocin was approximately 55kDa by SDS-PAGE. The obtained results in this study demonstrate that bacteriocin fromL. curvatusLAB-3H is a potential candidate for controlling microbial contaminations and can be used in different sectors, such as food industries.

Purification and Characterization of Thermostable Alkaline Xylanase by Aspergillus terreus from Elephant Dung

Aims: Purification and characterization of xylanase by Aspergillus terreus isolated from elephant dung.
 Study Design: Xylanase enzyme produced by A. terreus was extracted from the fermented solid medium using solid state fermentation and purified using chromatographic techniques. The purified enzyme was characterized for physio-chemical and kinetic properties.
 Place and Duration of Study: Experiments were performed at the School of Biotechnology, Devi Ahilya University, Indore, INDIA and Maharaja Ranjit Singh College of Professional Sciences, Indore, INDIA, between June, 2019 and September, 2021.
 Methodology: The enzyme was extracted and purified using ammonium sulphate precipitation, ion exchange chromatography using DEAE cellulose and gel filtration chromatography (GFC) using Sephadex G-200. The molecular weight of the purified enzyme was determined using native poly acrylamide gel electrophoresis (Native PAGE) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme was characterized to determine pH and temperature optima. Thermostability, pH stability and substrate kinetics were studied for purified xylanase. Effect of several metal ions and detergents were also studied.
 Results: The alkali-thermo xylanase of Aspergillus terreus was purified to 14.6-fold with a 39.48% recovery through IEC on DEAE-cellulose and GFC using Sephadex G-200. The purified enzyme had a specific activity, 111.6 U/mg. The SDS-PAGE revealed that the enzyme was monomeric with a molecular weight of 87 kDa. The optimum pH and temperature were 8 and 60⁰C, respectively. The Km and Vmax values of the partially purified xylanase were found to be 0.016 mg/l and 102.64 U/mg protein, respectively with birch wood xylan as substrate.
 Conclusion: The enzyme was active over a wide pH range of 6-11 and temperature range of 40⁰C to 80ºC, indicating its alkali tolerant and thermostable characteristics which is the requirement of different industries. Several ions namely Zn2+ enhanced xylanase activity by 187% followed by Ca2+ (129%), Mg2+ (123%), NH4+ (146%) and Fe3+ (118%) at 10 mM concentration. However, Cu2+ and Hg2+ completely inhibited xylanase activity. This research provides the basis for application of xylanase and improves its biotechnological potential in mainly in paper pulp industry, biofuel generation and in bread baking.

The role of endogenous serine proteinase on disintegration of collagen fibers from grass carp (Ctenopharyngodon idellus)

Softening, causing by the degradation of collagen, leads to serious quality deterioration of fish fillets during post-mortem storage. In order to study the role of endogenous enzymes in softening of fish muscle, collagen fibers from grass carp (Ctenopharyngodon idellus) were degraded by serine proteinase. The structural and biochemical changes of collagen fibers with serine proteinase digestion were investigated in terms of scanning electron microscopy, fourier transform infrared spectroscopy, electrophoresis and chemical analysis. In the present study, an endogenous proteinase (approximately 250 kDa) with gelatinolytic activity was isolated from grass carp muscle by ammonium sulfate precipitation and column chromatographies, and identified as serine proteinase. Collagen fibers were partly disaggregated into collagen fibrils after serine proteinase treatment for 48 h, coinciding with a release of hydroxyproline (5.56 ± 0.02%) and glycosaminoglycans (9.04 ± 0.03%). However, almost all gelatin and 40% of acid-soluble collagen were degraded within 1 h and 6 h, respectively. Meanwhile, serine proteinase-treated collagen fibers maintained the triple-helical structure of collagen molecule, while their thermal stability was gradually decreased with digestion time. All results indicated that disintegration of collage fibers by serine proteinase might be due to both the degradation of proteoglycans and collagen molecules during textural deterioration of chilled grass carp fillets.

Characterisation of alpha-amylase inhibitor from Streptomyces xinghaiensis AAI2 in solid substrate

Alpha-amylase inhibitors, which play metabolic roles in the organisms that produce them, by impeding the activity of alpha-amylase enzyme, have been isolated from many sources including plants and microorganisms. They vary in characteristics and applications based on their sources though there are characters that appear similar despite the source. Alpha-amylase inhibitor was produced by Streptomyces xinghaiensis AAI2 cultured on wheat bran substrate supplemented with a nutrient basal medium which was also used to adjust both the moisture content (73%) and initial pH (6.48) of the medium. The extract obtained was purified using standard purification techniques (ammonium sulphate precipitation, dialysis and column chromatography). Characteristics such as effects of pre-incubation and pH, total protein, specific activity, total activity, percentage yield, molecular weight and kinetics were determined. The alpha-amylase inhibitor produced was found to occur in isoforms having molecular weights of 14.2 and 56 kDa. Total protein, specific activity and percent yield obtained were 0.19 mg/mL, 745.11 AIU and 2.27% respectively. The optimum activity was obtained at pH 8.0 and in the presence of Cobalt and Calcium ions while pre-incubation for 10 min was best with no further improvement in the activity beyond this time. The inhibitor exerted a non-competitive type of inhibition.

Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

Purification and characterisation of uricase from Bacillus subtilis SP6

Uricase enzyme was purified to homogeneity from the culture filtrates of Bacillus subtilis SP6 in a two-step process involving ammonium sulphate precipitation and ion-exchange chromatography. The purified uricase was 8.65-fold pure with 26.35 % yield and had a molecular weight of 44 kDa. The purified uricase had an optimal pH of 9.0 and was stable at a pH range of 6.0–9.0. The optimum temperature for uricase was 37 °C and exhibited thermostability in the range of 10 °C–37 °C. The uricase activity was induced moderately by CuSO4 while SDS, EDTA, Triton X-100, HgI and K-ferricyanide significantly diminished the activity. The kinetic parameters (Km and Vmax) for the purified uricase were 0.17 mM and 1.5 × 10−4mol l-1 min-1, respectively. Biophysical characterisation of uricase revealed that the enzyme has 35.7 % α-helix, 41.8 % β-sheet and 3.8 % turns and Tm 42 °C. The byproduct allantoin generated by the action of uricase on uric acid was identified by LCMS. Thus, this study highlights the potential of uricase from B. subtilis SP6 which displayed significant activity at normal physiological conditions. This may accommodate for alluring applications in pharma industries, while its efficacy to generate the byproduct ‘allantoin’ also opens an avenue for its use in pharma-cosmeceutical industries.

Eruca sativa seed napin structural insights and thorough functional characterization

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC–MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

Pilot scale production, extraction and purification of a thermostable phycocyanin from Synechocystis sp. PCC 6803

Phycocyanin (PC) is a soluble blue pigment-protein primarily harvested from the cyanobacterium Arthrospira platensis. PC is in high demand from several industries, but its narrow stability range limits potential applications. Here, a pilot scale (120 L total) batch production, extraction and purification process for thermostable PC (Te-PC) from a Synechocystis sp. PCC 6803 'Olive' strain expressing the PC operon cpcBACD from Thermosynechococcus elongatus BP-1 on a self-replicating vector is presented. Batch cultivation without antibiotics had no impact on growth or Te-PC production and optimisation of growth conditions resulted in Te-PC contents of 75.3 ± 1.7 mg g DW−1. Wet biomass was harvested following chitosan-based flocculation with a 97 ± 2% efficiency, and Te-PC was extracted by high pressure homogenisation. Subsequent purification by heat-treatment and two-step ammonium sulfate precipitation removed chlorophyll and allophycocyanin contamination, resulting in Te-PC purities of 2.9 ± 0.7 and a mean Te-PC recovery of 84 ± 12%.

Heterologous expression of bovine lactoferrin C-lobe in Bacillus subtilis and comparison of its antibacterial activity with N-lobe

The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difficult. Here, we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the Pveg promoter in Bacillus subtilis 168 at 20 °C. The yield was 7.5 mg/L, and 90.6% purity was achieved using ammonium sulfate precipitation, Ni–NTA and molecular exclusion. The C-lobe at 10 mg/mL completely inhibited cell growth of Escherichia coli JM109 (DE3) and Pseudomonas aeruginosa CGMCC 1.6740, and 48.4% of growth of Staphylococcus aureus CGMCC 1.282, the result is similar to that of 200 ng/mL N-lobe. The minimum inhibitory concentrations of C-lobe were 4, 8 and 16 mg/mL, while those of N-lobe were 128, 256 and 512 μg/mL for E. coli, P. aeruginosa and S. aureus, respectively. This is the first report on bovine lactoferrin C-lobe expression and the comparative resistance of the recombinant N- and C-lobes in a food-safe strain of B. subtilis. Our findings offer the potential to study the structure–function relationship of the N- and C-lobes recombinantly produced in the same host.