Showing papers in "Preparative Biochemistry & Biotechnology in 2015"

Continuous Processing for Production of Biopharmaceuticals

Abstract: The merits of continuous processing over batch processing are well known in the manufacturing industry. Continuous operation results in shorter process times due to omission of hold steps, higher productivity due to reduced shutdown costs, and lowers labor requirement. Over the past decade, there has been an increasing interest in continuous processing within the bioprocessing community, specifically those involved in production of biotherapeutics. Continuous operations in upstream processing (perfusion) have been performed for decades. However, recent development of continuous downstream operations has led the industry to envisage an integrated bioprocessing platform for efficient production. The regulators, key players in the biotherapeutic industry, have also expressed their interest and willingness in this migration from the traditional batch processing. This paper aims to review major developments in continuous bioprocessing in the past decade. A discussion of pros and cons of the different proposed approaches has also been presented.

Detergent-compatible proteases: microbial production, properties, and stain removal analysis.

Abstract: Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods.

Enhancement of Mechanical Properties, Microstructure, and Antimicrobial Activities of Zein Films Cross-Linked Using Succinic Anhydride, Eugenol, and Citric Acid

Abstract: Zein constitutes about half of the endosperm proteins in corn. Recently, attempts have been made to utilize zein for food coatings and biodegradable materials, which require better physical properties, using chemical modification of zein. In this study, zein proteins were modified using citric acid, succinic anhydride, and eugenol as natural cross-linking agents in the wet state. The cross-linkers were added either separately or combined in increment concentrations (0.1, 0.2, 0.3, and 0.4%). The effects of those agents on the mechanical properties, microstructure, optical properties, infrared (IR) spectroscopy, and antibacterial activities of zein were investigated. The addition of cross-linking agents promoted changes in the arrangement of groups in zein film-forming particles. Regarding the film properties, incorporation of cross-linking agents into zein films prepared in ethanol resulted in two- to three-fold increases in tensile strength (TS) values. According to the Fourier-transform infrared (FTIR) spectra and Hunter parameters there were no remarkable changes in the structure and color of zein films. Transparency of zein films was decreased differentially according to the type and cross-linker concentration. The mechanical and optical properties of zein films were closely related to their microstructure. All cross-linked films showed remarkable antibacterial activities against Bacillus cereus ATCC 49064 and Salmonella enterica ATCC 25566. Food spoilage and pathogenic bacteria were affected in a film-dependent manner. Our experimental results show that even with partial cross-linking the mechanical properties and antipathogen activities of zein films were significantly improved, which would be useful for various industrial applications.

Optimization of medium composition for erythritol production from glycerol by Yarrowia lipolytica using response surface methodology.

Abstract: Several factors affecting erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 strain were examined in batch fermentations. Ammonium sulfate, monopotassium phosphate, and sodium chloride were identified as critical medium components that determine the ratio of polyols produced. The central composite rotatable experimental design was used to optimize medium composition for erythritol production. The concentrations of ammonium sulfate, monopotassium phosphate, and sodium chloride in the optimized medium were 2.25, 0.22, and 26.4 g L−1, respectively. The C:N ratio was found as 81:1. In the optimized medium with 100 g L−1 of glycerol the Wratislavia K1 strain produced 46.9 g L−1 of erythritol, which corresponded to a 0.47 g g−1 yield and a productivity of 0.85 g L−1 hr−1. In the fed-batch mode and medium with the total concentration of glycerol at 300 g L−1 and C:N ratio at 81:1, 132 g L−1 of erythritol was produced with 0.44 g g−1 yield and a productivity of 1.01 g L−1 hr−1.

Purification and characterization of polyphenol oxidase from waste potato peel by aqueous two-phase extraction.

Abstract: Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up ...

Biosynthesis of Poly-3-Hydroxybutyrate (PHB) from Glycerol by Paracoccus denitrificans in a Batch Bioreactor: Effect of Process Variables

Abstract: In this study, the kinetics of poly-3-hydroxybutyrate (PHB) biosynthesis from glycerol by Paracoccus denitrificans DSMZ 413 were explored in a batch bioreactor. Effects of inorganic and organic nitrogen source, carbon to nitrogen ratio, and other process variables such as pH, aeration, and initial glycerol concentration on PHB production were investigated in a 2.5-L bioreactor. Yeast extract was found to be the best nitrogen source compared to several organic nitrogen sources tested. At pH 6, specific growth rate, product formation rate, and accumulation of PHB within the cell were maximum. Specific growth rate increased with increase in oxygen transfer rate, but moderate oxygen transfer rate promoted PHB production. High glycerol concentration inhibited specific product formation rate but not growth. High initial carbon/nitrogen (C/N) ratio favored PHB accumulation and its productivity. At a C/N ratio of 21.4 (mol mol−1), 10.7 g L−1 of PHB corresponding to 72% of cell dry weight was attained.

Response surface optimization of lyoprotectant for Lactobacillus bulgaricus during vacuum freeze-drying.

Abstract: The individual and interactive effects of skimmed milk powder, lactose, and sodium ascorbate on the number of viable cells and freeze-drying survival for vacuum freeze-dried powder formulation of Lactobacillus bulgaricus were studied by response surface methodology, and the optimal compound lyoprotectant formulations were gained. It is shown that skim milk powder, lactose, and sodium ascorbate had a significant impact on variables and survival of cultures after freeze-drying. Also, their protective abilities could be enhanced significantly when using them as a mixture of 28% w/v skim milk, 24% w/v lactose, and 4.8% w/v sodium ascorbate. The optimal freeze-drying survival rate and the number of viable cells of Lactobacillus bulgaricus were observed to be (64.41±0.02)% and (3.22±0.02)×10(11) colony-forming units (CFU)/g using the optimal compound protectants, which were very close to the expected values 64.47% and 3.28×10(11) CFU/g.

Therapeutic Alpha-Interferons Protein: Structure, Production, and Biosimilar

Abstract: In 2007, the world solemnized the golden jubilee of the discovery of interferon (IFN). Interferon is a small protein messenger called a pluripotent cytokine, produced by several cells of the host in response to various biological as well as synthetic stimuli. There are three major classes of interferons in humans: IFN-alpha, IFN-beta, and IFN-gamma. As a treatment option, interferon-alpha (IFN-α) is the most effective one. IFN-α has proved to be effective as an antiviral therapy and tumor-fighting drug in the past two decades. Meanwhile, great progress has been achieved in establishing IFN-α as the first choice of antiviral therapy for chronic hepatitis C virus (HCV) patients. Recently, novel pegylated IFN-α2 products with extended in vivo half-lives and consensus interferon, an artificially engineered type I interferon, have been developed to substantially improve treatment regimes for HCV patients. Undesirable acute and chronic side effects in addition to immunogenicity of therapeutic IFN products remain constraints to conquer for further improvements in clinical applications of IFN. It is certainly expected that more research will be conducted in the future, not only to face these challenges but also to extend the range of IFN products and their clinical targets. The objective herein is to review the current therapeutic alpha-interferons production, formulation technologies, and prospective future for the original entity and its biogeneric version.

Optimization and kinetic modeling of cell-associated camptothecin production from an endophytic Fusarium oxysporum NFX06.

Abstract: The production of cell-associated camptothecin (CPT) from an endophytic fungus Fusarium oxysporum NFX06 isolated from Nothapodytes foetida and its kinetics studies were proposed. Response surface methodology (RSM) based on central composite design (CCD) was used to construct a model to describe the effects of substrate concentration. Three independent variables (dextrose, peptone, and MgSO4) were successfully employed to study the yield of CPT under submerged fermentation. The maximum yield of CPT obtained from CCD was about 598.0 ng/g biomass. The model-validated optimum predicted CPT yield and experimental CPT yield from the biomass were found to be 628.08 ng/g and 610.09 ng/g at the concentrations of dextrose 42.64 (g/L), peptone 9.23 (g/L), and MgSO4 0.26 (g/L) respectively. The predicted yield of CPT was 4.90% higher than the value obtained from CCD and 2.85% higher than the value obtained from experiment conducted at optimum conditions. The kinetic parameters, maximum specific growth rate μmax=1.212 day(-1), growth-associated CPT production coefficient (α=29.35 ng/g biomass), and non-growth-associated CPT production coefficient (β=0.03 ng CPT/g biomass-day) were obtained. The logistic model was found suitable to predict mycelial growth with a high determination coefficient (R2). Luedeking-Piret and modified Luedeking-Piret models were employed to represent the product kinetics and substrate consumption kinetics. A good concurrence was found between the experimental and predicted values, representing that the unstructured models were able to illustrate the fermentation profile effectively.

Improving of catalase stability properties by encapsulation in alginate/Fe3O4 magnetic composite beads for enzymatic removal of H2O2.

Abstract: The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in...

Protective role of probiotic lactic acid bacteria against dietary fumonisin B1-induced toxicity and DNA-fragmentation in sprague-dawley rats.

Abstract: The genus Fusarium, especially F. verticillioides and F. proliferatum, has been found in several agricultural products worldwide, especially in maize. Regardless the occurrence of symptoms, the presence of Fusarium in maize constitutes an imminent risk due to its ability to produce fumonisins, mycotoxins with proven carcinogenic effect on rats, swine, and equines and already classified as possible carcinogens to humans. The toxicity of incremental levels of fumonisin B1 (FB1), that is, 50, 100, and 200 mg FB1/kg diet, and the role of Lactobacillus delbrueckii subsp. lactis DSM 20076 (LL) and Pediococcus acidilactici NNRL B-5627 (PA) supplementation in counteracting the FB1 effects in intoxicated rats were monitored over a period of 4 weeks. Effects on the feed intake and body weight gain were noticed. A significant (p ≤ 0.05) increase in the level of liver and kidney functions markers and DNA fragmentation was also noticed in rat groups T100 and T200. The lactic acid bacteria (LAB) supplementation could b...

Astaxanthin induction in Microalga H. pluvialis with flat panel airlift photobioreactors under indoor and outdoor conditions.

Abstract: Astaxanthin was induced from Haematococcus pluvialis (NIES-144) under indoor and outdoor conditions using 17-, 50-, and 90-L flat-panel airlift photobioreactors (FP-APBRs). Preliminary experiments in 1.5-L bubble column photobioreactors (BC-PBRs) revealed that sterilized clean water with 3% CO2 aeration (1.47 cm3 s−1 CO2 loading) could best encourage astaxanthin accumulation at 18.21 g m−3 (3.63% by weight). Operating 17-L FP-APBRs with these bubble column parameters under indoor conditions could further enhance astaxanthin to 26.63 g m−3 (5.34% by weight). This was potentially due to the inherited up-lift force from the reactor that helped avoid cell precipitation by allowing the cells to be circulated within the reactor. In addition, the various sizes of FP-APBRs exhibited similar performance, implying a potential scale-up opportunity. However, similar operation under outdoor condition exhibited slightly poorer performance due to the light inhibition effect. The best outdoor performance was obtained wit...

Isolation, Screening, and Identification of Potential Cellulolytic and Xylanolytic Producers for Biodegradation of Untreated Oil Palm Trunk and Its Application in Saccharification of Lemongrass Leaves

Abstract: This study presents the isolation and screening of fungi with excellent ability to degrade untreated oil palm trunk (OPT) in a solid-state fermentation system (SSF). Qualitative assay of cellulases and xylanase indicates notable secretion of both enzymes by 12 fungal strains from a laboratory collection and 5 strains isolated from a contaminated wooden board. High production of these enzymes was subsequently quantified in OPT in SSF. Aspergillus fumigates SK1 isolated from cow dung gives the highest xylanolytic activity (648.448 U g−1), generally high cellulolytic activities (CMCase: 48.006, FPase: 6.860, beta-glucosidase: 16.328 U g−1) and moderate lignin peroxidase activity (4.820 U/g), and highest xylanolytic activity. The xylanase encoding gene of Aspergillus fumigates SK1 was screened using polymerase chain reaction by a pair of degenerate primers. Through multiple alignment of the SK1 strain's xylanase nucleotide sequences with other published xylanases, it was confirmed that the gene belonged to th...

Optimization of ellagitannase production by Aspergillus niger GH1 by solid-state fermentation

Abstract: Ellagic acid is one of the most bioactive antioxidants with important applications in pharmaceutical, cosmetic, and food industries. However, there are few biotechnological processes developed for its production, because it requires precursors (ellagitannins) and the corresponding biocatalyst (ellagitannase). The aim of this study was to optimize the culture conditions for ellagitannase production by Aspergillus niger in solid-state fermentation (SSF). The bioprocess was carried out into a column bioreactor packed with polyurethane foam impregnated with an ellagitannins solution as carbon source. Four strains of Aspergillus niger (PSH, GH1, HT4, and HC2) were evaluated for ellagitannase production. The study was performed in two experimental steps. A Plackett–Burman design was used to determine the influencing parameters on ellagitannase production. Ellagitannins concentration, KCl, and MgSO4 were determined to be the most significant parameters. Box–Behnken design was used to define the interaction of th...

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

Abstract: In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH4)2SO4, KH2PO4, or Mg2+ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the ...

Production, purification, and antibiofilm activity of a novel exopolysaccharide from Arthrobacter sp. B4.

Abstract: A novel exopolysaccharide (EPS), namely, B4-EPS, is produced by Arthrobacter sp. B4. Response surface methodology (RSM) was employed to optimize the fermentation medium for increasing B4-EPS production. Based on Plackett–Burman design (PBD), glucose, yeast extract, and KH2PO4 were selected as significant variables, which were further optimized by a central composite design (CCD). According to response surface and canonical analysis, the optimal medium was composed of 16.94 g/L glucose, 2.33 g/L yeast extract, and 5.32 g/L KH2PO4. Under this condition, the maximum yield of B4-EPS reached about 8.54 g/L after 72 hr of batch fermentation, which was pretty close to the predicted value (8.52 g/L). Furthermore, B4-EPS was refined by column chromatography. The main homogeneous fraction (B4-EPS1) was collected and applied to assay of antibiofilm activity. B4-EPS1 exhibited a dose-dependent inhibitory effect on biofilm formation of Pseudomonas aeruginosa PAO1 without antibacterial activity. About 86.1% of biofilm ...

Beneficial Effect of Corncob Acid Hydrolysate on the Lipid Production by Oleaginous Yeast Trichosporon dermatis

Abstract: In this work, corncob acid hydrolysate and its simulated medium whose sugar composition was the same as the corncob acid hydrolysate were used as fermentation substrate for lipid production by oleaginous yeast Trichosporon dermatis. On the corncob acid hydrolysate, after 7 days of fermentation, the biomass, lipid content, lipid yield, and lipid coefficient of T. dermatis were 17.3 g/L, 40.2%, 7.0 g/L, and 16.5%, respectively. Interestingly, during the lipid fermentation on the corncob acid hydrolysate, glucose, xylose, arabinose, and even acetic acid could be well utilized as carbon sources by T. dermatis. Surprisingly, the lipid yield (7.0 g/L) of T. dermatis on the corncob acid hydrolysate was much higher than that (3.8 g/L) on the simulated medium, in spite of the fact that the lipid coefficient (17.4%) on the simulated medium was a little higher. This phenomenon further showed that lignocellulosic acid hydrolysate was a suitable substrate for lipid fermentation by T. dermatis. This work would help the...

Identification of Newly Isolated Talaromyces pinophilus and Statistical Optimization of β-Glucosidase Production Under Solid-State Fermentation

Abstract: Fungi able to degrade agriculture wastes were isolated from different soil samples, rice straw, and compost; these isolates were screened for their ability to produce β-glucosidase. The most active fungal isolate was identified as Talaromyces pinophilus strain EMOO 13-3. The Plackett-Burman design is used for identifying the significant variables that influence β-glucosidase production under solid-state fermentation. Fifteen variables were examined for their significances on the production of β-glucosidase in 20 experimental runs. Among the variables screened, moisture content, Tween 80, and (NH4)2SO4 had significant effects on β-glucosidase production with confidence levels above 90% (p < 0.1). The optimal levels of these variables were further optimized using Box-Behnken statical design. As a result, the maximal β-glucosidase activity is 3648.519 U g(-1), which is achieved at the following fermentation conditions: substrate amount 0.5 (g/250 mL flask), NaNO3 0.5 (%), KH2PO4 0.3 (%), KCl 0.02 (%), MgSO4 · 7H2O 0.01 (%), CaCl2 0.01 (%), yeast extract 0.07 (%), FeSO4 · 7H2O 0.0002 (%), Tween 80 0.02 (%), (NH4)2SO4 0.3 (%), pH 6.5, temperature 25°C, moisture content 1 (mL/g dry substrate), inoculum size 0.5 (mL/g dry substrate), and incubation period 5 days.

Statistical optimization of alkaline protease production from Penicillium citrinum YL-1 under solid-state fermentation.

Abstract: Proteases from halotolerant and halophilic microorganisms were found in traditional Chinese fish sauce In this study, 30 fungi were isolated from fermented fish sauce in five growth media based on their morphology However, only one strain, YL-1, which was identified as Penicillium citrinum by internal transcribed spacer (ITS) sequence analysis, can produce alkaline protease This study is the first to report that a protease-producing fungus strain was isolated and identified in traditional Chinese fish sauce Furthermore, the culture conditions of alkaline protease production by P citrinum YL-1 in solid-state fermentation were optimized by response surface methodology First, three variables including peptone, initial pH, and moisture content were selected by Plackett–Burman design as the significant variables for alkaline protease production The Box–Behnken design was then adopted to further investigate the interaction effects between the three variables on alkaline protease production and determine

Effect of phosphoric acid pretreatment of corncobs on the fermentability of Clostridium beijerinckii TISTR 1461 for biobutanol production.

Abstract: Corncobs pretreated with H2SO4, HNO3, and H3PO4 were compared to evaluate the fermentation ability of Clostridium beijerinckii TISTR 1461 to produce biobutanol via acetone–butanol–ethanol (ABE) fermentation. It was found that the hydrolysate from H3PO4 pretreatment could be used as a substrate without any inhibitor removal methods. However, in terms of sugar yield, it gave the lowest total sugars in both pretreatment and enzymatic hydrolysis. Response surface methodology was applied to optimize enzymatic hydrolysis of the pretreated corncobs. The optimized conditions reduced the consumption of enzymes and hydrolysis time to 7.68 FPU/g biomass and 63.88 hr, respectively, and yielded 51.82 g/L reducing sugars. The Celluclast 1.5 L and Novozyme 188 enzyme ratio were varied to maximize the hydrolyzed sugars. The ABE fermentation, using substrate from phosphoric acid pretreatment of corncobs, with 10 g/L glucose supplementation produced 11.64 g/L of total ABE, which was close to the control experiment using sy...

Partial purification and characterization of chromate reductase of a novel Ochrobactrum sp. strain Cr-B4.

Abstract: Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (Ea) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (Ed) was found to be 79.6 kJ/mol over a temperature range of 50-80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s(-1), and for deactivation of chromate reductase it was found to be 265.66 × 10(3) s(-1). The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN3, Pb(2+), Ni(2+), Zn(2+), and Cd(2+)) inhibited the enzyme activity, while metals like Cu(2+) and Fe(3+) significantly enhanced the reductase activity. The enzyme followed Michaelis-Menten kinetics with Km of 104.29 µM and a Vmax of 4.64 µM/min/mg.

Purification and characterization of a thermo- and organic solvent-tolerant alkaline protease from Bacillus sp. JER02.

Abstract: Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. Km and Vmax of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H2O2 (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.

A novel method for separation of caseins from milk by phosphates precipitation.

Abstract: Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze-thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.

Biodegradation of isopropanol by a solvent-tolerant Paracoccus denitrificans strain.

Abstract: The biodegradation of high concentration isopropanol (2-propanol, IPA) at 16 g/L was investigated by a solvent-tolerant strain of bacteria identified as Paracoccus denitrificans for the first time by 16S rDNA gene sequencing. The strain P. denitrificans GH3 was able to utilize the high concentration of IPA as the sole carbon source within a minimal salts medium with a cell density of 1.5 × 108 cells/mL. The optimal conditions were found as follows: initial pH 7.0, incubation temperature 30°C, with IPA concentration 8 g/L. Under the optimal conditions, strain GH3 utilized 90.3% of IPA in 7 days. Acetone, the major intermediate of aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Both IPA and acetone were completely removed from the medium following 216 hr and 240 hr, respectively. The growth of strain GH3 on IPA as a sole carbon and energy source was well described by the Andrews model with a maximum growth rate (μ max ) = 0.0277/hr, a saturation constant (K S ) =...

Optimization of ethanol production from microfluidized wheat straw by response surface methodology.

Abstract: In this study, wheat straw was pretreated with a microfluidizer to improve its enzymatic hydrolysis and ethanol yields. The pretreatment was performed at various pressures (500, 1000, and 1500 bar) and solid loadings (1, 2, and 3%). The microfluidized biomass was then subjected to hydrolysis and simultaneous saccharification and co-fermentation (SSCF) experiments at different enzyme loadings (5, 10, and 15 FPU/g dry wheat straw) using a mutant yeast. The results indicated that the microfluidization method alters the structure of biomass and leads to a reduction in lignin content. The samples pretreated at 1% solid loading contained the minimum lignin concentration and provided the maximum sugar and ethanol yields. These results signified that the microfluidization method is more effective on biomass at low solid loadings. The process conditions were optimized for higher ethanol and sugar yields using response surface methodology (RSM). The optimum pressure and solid and enzyme loadings were found as 1500 ...

Optimizing Ultrasound-Assisted Extraction of Prodigiosin by Response Surface Methodology

Abstract: Prodigiosin extraction from dried Serratia marcescens jx1 cells using ultrasound-assisted extraction was optimized. The experiment was carried out in accordance with a central composite design (CCD) three-level and single-variable approach. The extraction time, extraction temperature, and solute to solvent ratio with the application of ultrasonication were optimized using response surface methodology (RSM) to maximize the extraction of prodigiosin from dried S. marcescens jx1 cells. The response of prodigiosin was determined using spectrophotometry. A quadratic model was established to predict the prodigiosin extraction yield. The analysis of variance showed that the quadratic model significantly contributed to the response of prodigiosin. The optimal extraction parameters were an extraction time of 17.5 min, an extraction temperature of 23.4°C, and a solvent-to-solute ratio of 1:27.2. Under these optimum conditions, the average prodigiosin yield was 4.3 g±0.02 g from 100 g of dried cells, which matches the predicted values. The obtained optimum conditions for prodigiosin extraction provide a scientific basis for the economical large-scale production of prodigiosin.

A simplified chromatographic approach to purify commercially available bovine submaxillary mucins (BSM).

Abstract: In this study, a simple purification protocol is developed to reduce the bovine serum albumin (BSA) content in commercially available bovine submaxillary mucin (BSM). This involved purification of the BSM by one-column anion-exchange chromatography protocol resulting in BSM with greatly reduced BSA content and homogeneously distributed size, and in a high yield of ∼43% from BSM as received from the manufacturer. The purity and composition of commercially acquired BSM were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry, which verified that BSA is the most abundant nonmucinous protein component. The purification effect was evident from a significantly altered circular dichroism (CD) spectrum of BSM after anion-exchange chromatography.

Optimization of β-Glucosidase Production by Aspergillus terreus Strain EMOO 6-4 Using Response Surface Methodology Under Solid-State Fermentation

Abstract: Forty-two morphologically different fungal strains were isolated from different soil samples and agricultural wastes and screened for β-glucosidase activity under solid-state fermentation. Eight species were chosen as the most active β-glucosidase producers and were subjected to primary morphological identification. β-Glucosidase was highly produced by Aspergillus terreus, which showed the highest activity, and was subjected to full identification using scanning electron microscopy and molecular identification. Initial screening of different variables affecting β-glucosidase production was performed using Plackett-Burman design and the variables with statistically significant effects were identified. The optimal levels of the most significant variables with positive effect and the effect of their mutual interactions on β-glucosidase production were determined using Box-Behnken design. Fifteen variables including temperature, pH, incubation time, inoculum size, moisture content, substrate concentration, Na...

Modeling of process parameters for enhanced production of coenzyme Q10 from Rhodotorula glutinis.

Abstract: Coenzyme Q10 (CoQ10) plays an indispensable role in ATP generation through oxidative phosphorylation and helps in scavenging superoxides generated during electron transfer reactions. It finds extensive applications specifically related to oxidative damage and metabolic dysfunctions. This article reports the use of a statistical approach to optimize the concentration of key variables for the enhanced production of CoQ10 by Rhodotorula glutinis in a lab-scale fermenter. The culture conditions that promote optimum growth and CoQ10 production were optimized and the interaction of significant variables para-hydroxybenzoic acid (PHB, 819.34 mg/L) and soybean oil (7.78% [v/v]) was studied using response surface methodology (RSM). CoQ10 production increased considerably from 10 mg/L (in control) to 39.2 mg/L in batch mode with RSM-optimized precursor concentration. In the fed-batch mode, PHB and soybean oil feeding strategy enhanced CoQ10 production to 78.2 mg/L.

Characterization, kinetic, and thermodynamic studies of marine pectinase from Bacillus subtilis.

Abstract: Characterization, kinetic and thermodynamic parameters of purified pectinase from Bacillus subtilis, isolated from a marine sediment sample collected from Chinchani beach at Tarapore, India, were studied. Marine pectinase produced under submerged growth conditions was purified by ammonium sulfate precipitation followed by gel filtration chromatography using DEAE cellulose. Partial characterization of the marine pectinase was carried out in terms of effect of pH, temperature, substrate concentration, and metal ions. It was found that pectinase from marine B. subtilis showed maximal activity in alkaline buffer at pH 9.0 and at 40°C. It was also found that metal ions, namely, Mn(2+) and Fe(2+), stimulate pectinase activity. Marine pectinase followed Michaelis-Menten kinetics. The kinetics and thermodynamic parameters of the purified marine pectinase from B. subtilis were studied as the characterization of the enzyme is vital for its use in industrial processes.