Newly developed medium and strategy for bacterial cellulose production

Bacterial cellulose (BC) has unique properties, such as high crystallinity, a high degree of polymerisation, high tensile strength and high purity, compared with native cellulose. In this study, a previously determined BC production medium was improved in static culture, and the production cost was evaluated and compared with molasses and with other defined media, such as Hestrin–Schramm, Zhou, Yamanaka and Park, using Gluconacetobacter xylinus. In addition to this analysis, because the surface area/volume ratio is an important parameter in static culture, different surface area/volume ratios were analysed in the range of 0.2–1.46. The defined medium (M1A05P5) and culture type contained glucose (10 g/L), yeast extract (10 g/L), peptone (7 g/L), acetic acid (1.5 ml/L), and ethanol (5 ml/L), and the pH was adjusted to 5.0 in static culture. The highest productivity was observed in the M1A05P5 medium that was 5-fold higher than either molasses or Park's medium. Although the molasses medium was proposed as a cost-effective medium, the production price of BC was the lowest in the M1A05P5 medium. Therefore, the newly developed medium and strategy were highly promising candidates for the industrial-scale production of BC.     

Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K₂HPO₄ at 20.0, 6.0, 0.25 g L⁻¹, respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L⁻¹ concomitant with kefiran production of 1.91 g L⁻¹.     

Protection of oxidative stress induced apoptosis in osteosarcoma cells by dihydromyricetin through down-regulation of caspase activation and up-regulation of BcL-2

Current study was aimed to investigate the effect of dihydromyricetin on hydrogen peroxide induced oxidative stress in the osteosarcoma cells. MTT assay showed that hydrogen peroxide treatment at a concentration of 100 μM caused a significant (p < 0.005) reduction in the viability of MG63 cells. However, reduction in cell viability caused by 100 μM concentration of hydrogen peroxide was completely prevented on incubation with 30 μM dose of dihydromyricetin. Treatment with 100 μM concentration of hydrogen peroxide for 24 h led to condensation of chromatin material, rounding of cell shape and detachment of cells. The results from flow cytometry using annexin V-FITC and PI double staining showed apoptosis induction in 47.84 ± 5.21% cells on treatment with 100 μM concentration of hydrogen peroxide compared to 2.32 ± 0.54% in controlcells. The apoptotic alterations in MG63 cell morphology were prevented significantly on pre-treatment with 30 μM doses of dihydromyricetin for 48 h. Annexin V-FITC and PI staining showed reduction of hydrogen peroxide induced apoptotic cell percentage to 3.07 ± 0.86% on pre-treatment of MG63 cells with 30 μM dose of dihydromyricetin. Western blot analysis showed a significant increase in the activation of caspase-3 and -9 on treatment of MG63 cells for 24 h with 100 μM concentration of hydrogen peroxide. The expression level of Bcl-2 was decreased significantly by 100 μM concentration of hydrogen peroxide in MG63 cells. However, pre-treatment of MG63 cells with 30 μM dose of dihydromyricetin for 48 h significantly prevented hydrogen peroxide induced increase in caspase-3 and -9 levels and reduction in Bcl-2 level. Thus dihydromyricetin prevents hydrogen peroxide induced reduction in viability and induction of apoptosis in MG63 cells through down-regulation of caspase activation and up-regulation of Bcl-2 levels.     

Two-step acid and alkaline ethanolysis/alkaline peroxide fractionation of sugarcane bagasse and rice straw for production of polylactic acid precursor

Sugarcane bagasse and rice straw were subjected to acid and alkaline ethanolysis and sequential enzymatic hydrolysis to produce glucose for lactic acid production. Influence of physico-chemical treatments using ultrasonic bath and ultrasonic probe was studied compared with mechanical stirring. The results showed that the highest glucose yield with least contamination of xylose was obtained from acid ethanolysis fractionation (5 N H₂SO₄ + 50%, v/v ethanol) when stirred at 90 °C for 4 h. Alkaline ethanolysis accomplished high amount of both glucose and xylose released, however it was not favorable substrate for homofermentative lactic acid bacteria. In order to enhance enzymatic hydrolysis of acid ethanolysis fractionated samples, lignin was subsequently removed by the second step alkaline/peroxide delignification. The maximum lactic acid was obtained at 23.6 ± 0.2 g/L from Lactobacillus casei fermentation after 72 h when hydrolysate from two-step acid hydrolysis and alkaline/peroxide fractionated sugarcane bagasse containing 24.6 g/L initial glucose concentration was used as substrate.     

Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes

The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD = 5.0 pmol/assay and LLQ = 30 pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09 nmol hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31 nmol of hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.     

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

Halocin SH10 production by an extreme haloarchaeon Natrinema sp. BTSH10 isolated from salt pans of South India

Halobacteria, members of the domain Archaea that live under extremely halophilic conditions, are often considered as dependable source for deriving novel enzymes, novel genes, bioactive compounds and other industrially important molecules. Protein antibiotics have potential for application as preserving agents in food industry, leather industry and in control of infectious bacteria. Halocins are proteinaceous antibiotics synthesized and released into the environment by extreme halophiles, a universal characteristic of halophilic bacteria. Herein, we report the production of halocin (SH10) by an extremely halophilic archeon Natrinema sp. BTSH10 isolated from salt pan of Kanyakumari, Tamilnadu, India and optimization of medium for enhanced production of halocin. It was found that the optimal conditions for maximal halocin production were 42 °C, pH 8.0, and 104 h of incubation at 200 rpm with 2% (V/V) inoculum concentration in Zobell’s medium containing 3 M NaCl, Galactose, beef extract, and calcium chloride as additional supplements. Results indicated scope for fermentation production of halocin for probable applications using halophilic archeon Natrinema sp. BTSH10.     

Biobleaching of loblolly pine kraft pulp with Trametes trogii culture fluids followed by a peroxide stage. Application of Doehlert experimental design to evaluate process parameters

Loblolly pine kraft pulp was bleached in a totally chlorine-free sequence that involved treatment with culture supernatants from the white-rot fungus Trametes trogii followed by a peroxide stage. The whole process was performed at 28 °C, and did not require mediator addition in the enzymatic stage. Different operating conditions in the peroxide stage (pH, peroxide concentration and treatment time), were tested by using response surface methodology based on a Doehlert experimental design, in order to describe their effects and normalize a biobleaching protocol. The results showed that all three independent variables had significant effect on the luminance (L*) and Chroma (C*) of the enzyme-treated pulp. Best results were obtained after 1 h of enzyme incubation (352 U laccase, 2 U Mn-peroxidase per g of oven-dry pulp), followed by 96 h treatment with 2.5% hydrogen peroxide in sodium succinate buffer pH 6 (5% consistency). We obtained a noteworthy increase in L* = 94.45 (compared with 94.5 of the white reference standard (titanium oxide), 69.94 of the initial pulp, and 83.11 of the peroxide-bleached control), a decrease in C* (9.85), with minor pulp yield loss (less than 5%), under essentially mild conditions, using a low-cost source of enzyme.     

Synthesis of structured lipids by two enzymatic steps: Ethanolysis of fish oils and esterification of 2-monoacylglycerols

This paper studies the synthesis of structured triacylglycerols (STAGs), rich in polyunsaturated fatty acids (PUFAs) by a two-step enzymatic process: (i) alcoholysis of fish oils (cod liver and tuna oils) with ethanol to obtain 2-monoacylglycerols (2-MAGs), catalyzed by 1,3 specific lipases and (ii) esterification of these 2-MAGs with caprylic acid (CA, 8:0), also catalyzed by a 1,3 specific lipase, to produce STAGs of structure CA–PUFA–CA. As regards the alcoholysis reaction, three factors have been studied: the influence of the type of lipase used (lipase D from Rhizopus oryzae, immobilized on Accurel MP1000, and Novozym 435 from Candida antarctica), the operational mode of a stirred tank reactor (STR operating in discontinuous and continuous mode) and the intensity of treatment (IOT = lipase amount × reaction time/oil amount). Although higher 2-MAG yields were obtained with lipase D, Novozym 435 was selected due to its greater stability in the operational conditions. The highest 2-MAG yield (63%) was attained in the STR operating in discontinuous mode at an IOT of 1 g lipase × h g oil^?1 (at higher IOT the 2-MAGs were degraded to glycerol). This system was scaled up to 100 times the initial volume, achieving a similar yield (65%) at the same IOT. The 2-MAGs in the final alcoholysis reaction mixture were separated from ethyl esters by solvent extraction using solvents of low toxicity (ethanol and hexane); the 2-MAG recovery yield was over 90% and the purity was approximately 87–90%. Regarding the esterification of the 2-MAGs, the following factors were studied: the influence of the lipase type used, the presence or absence of solvent (hexane) and the reaction time or intensity of treatment (IOT = lipase amount × reaction time/2-MAG amount). Of the five lipases tested, the highest STAG percentages (over 90%) were attained with lipases D and DF, immobilized on Accurel MP1000. These STAGs contain 64% CA, of which 98% is at positions 1 and 3. Position 2 contains 5% CA and 45% PUFAs, which means that all the PUFAs that were located at position 2 in the original oil remain in that position in the final STAGs. The lipase D immobilized on Accurel MP1000 is stable in the operational conditions used in the esterification reaction. Finally the purification of STAGs was carried out by neutralization of free fatty acids with hydroethanolic solution of KOH and extraction of STAGs with hexane. By this method purity was over 95% and separation yields were about 80%.     

A novel mono- and diacylglycerol lipase highly expressed in Pichia pastoris and its application for food emulsifier preparation

A mono- and diacylglycerol lipase (MDL) was cloned from Penicillium cyclopium and expressed in Pichia pastoris strain GS115. The recombinant enzyme was named Lipase GH1. High cell density fermentation was performed by culture in a 7.5-L fermenter using BSMG medium, in which the phosphate in basal salt medium was replaced by sodium glycerophosphate (Na₂GP). The maximal lipase activity detected was 18,000 U per mL, and total protein content in the fermentation supernatant was 3.94 g per L. The activity of the liquid enzyme remained stable under alkaline conditions at 4 °C for 6 months and was 50% after one year. Lipase GH1 was used for the synthesis of mono- and diacylglycerols (MAGs and DAGs), which are commonly used emulsifiers for industrial applications. A conversion rate of 84% after 24 h of reaction was obtained using glycerol/oleic acid molar ratio 11:1, water content 1.5 wt%, enzyme dosage 80 U per g, and reaction temperature 35 °C. Lipase GH1 was more efficient for the synthesis of MAGs and DAGs than was Lipase G50 (a similar, commercially available lipase derived from Penicillium camemberti) when oleic acid was used as an acyl donor. Lipase GH1 has potential for food emulsifier preparation.     

Production of human milk fat substitutes enriched in omega-3 polyunsaturated fatty acids using immobilized commercial lipases and Candida parapsilosis lipase/acyltransferase

In human milk fat (HMF), palmitic acid (20–30%), the major saturated fatty acid, is mostly esterified at the sn-2 position of triacylglycerols, while unsaturated fatty acids are at the sn-1,3 positions, conversely to that occurring in vegetable oils.This study aims at the production of HMF substitutes by enzyme-catalyzed interesterification of tripalmitin with (i) oleic acid (system I) or (ii) omega-3 polyunsaturated fatty acids (omega-3 PUFA) (system II) in solvent-free media. Interesterification activity and batch operational stability of commercial immobilized lipases from Rhizomucor miehei (Lipozyme RM IM), Thermomyces lanuginosa (Lipozyme TL IM) and Candida antarctica (Novozym 435) from Novozymes, DK, and Candida parapsilosis lipase/acyltransferase immobilized on Accurel MP 1000 were evaluated. After 24-h reaction at 60 °C, molar incorporation of oleic acid was about 27% for all the commercial lipases tested and 9% with C. parapsilosis enzyme. Concerning omega-3 PUFA, the highest incorporations were observed with Novozym 435 (21.6%) and Lipozyme RM IM (20%), in contrast with C. parapsilosis enzyme (8.5%) and Lipozyme TL IM (8.2%). In system I, Lipozyme RM IM maintained its activity for 10 repeated 23-h batches while for Lipozyme TL IM, Novozym 435 and C. parapsilosis enzyme, linear (half-life time, t_1/2 = 154 h), series-type (t_1/2 = 253 h) and first-order (t_1/2 = 34.5 h) deactivations were respectively observed. In system II, Lipozyme RM IM showed linear deactivation (t_1/2 = 276 h), while Novozym 435 (t_1/2 = 322 h) and C. parapsilosis enzyme (t_1/2 = 127 h), presented series-type deactivation. Both activity and stability of the biocatalysts depended on the acyl donor used.     

Production of triacylglycerols rich in palmitic acid at position 2 as intermediates for the synthesis of human milk fat substitutes by enzymatic acidolysis

This paper studies the synthesis of triacylglycerols (TAGs) rich in palmitic acid (PA) at position 2, from palm oil stearin (POS), a vegetable oil highly rich in this acid (60%). The developed process consists of two steps: (1) obtaining PA enriched free fatty acids (FFAs), and (2) enrichment of POS in PA by acidolysis of this oil with PA enriched FFAs, catalyzed by lipase Novozym 435. In step (1) two PA enriched FFA mixtures were obtained: one by saponification of POS, and a PA concentrate (75.1% PA) obtained by crystallization at low temperature in solvents. The latter was obtained carrying out two crystallizations in acetone at −24 and −20 °C, from which PA was recovered in the solid phases with a total yield of 84%. These PA enriched FFA mixtures were used in step (2) of acidolysis of POS, along with commercial PA (98% PA). In this acidolysis step four factors were studied: temperature, hexane/reaction mixture ratio, FFA/POS molar ratio and the intensity of treatment (IOT = lipase amount × reaction time/POS amount). The best results (TAGs with 79% PA and 75% PA at position 2) were obtained with commercial PA, at 37 °C, 10 mL hexane/g reaction mixture, a FFA/POS molar ratio 3:1 (1:1, w/w) and an IOT = 9.6 g lipase × h/g POS (for example 48 h, 10 g lipase and 50 g POS). PA enriched TAGs were purified neutralizing the FFAs by KOH hydroethanolic solutions and extracting the TAGs with hexane. In this way 99% pure acylglycerols (TAG + DAG) were obtained; the recovery yield of this purification step was 95%. The experiments carried out with POS demonstrated that it is possible to use only this oil (60% PA, 23% PA at position 2) as a source of PA to obtain a TAG with 70.7% PA and 70.5% PA at position 2. This process consists of four steps: (1) saponification of POS, (2) crystallization of FFAs to obtain PA enriched FFAs (75.1% PA), (3) acidolysis of POS with these FFAs, catalyzed with Novozym 435, to produce PA enriched TAGs at position 2 (70.5% PA) and (4) purification of TAGs to obtain approximately 95% purity and yield. These PA enriched TAGs could be used to obtain structured TAGs rich in PA at position 2 and in oleic acid at positions 1 and 3 (OPO), which is the principal TAG of human milk fat.     

Descriptive analysis of kinematics and kinetics of catchers throwing to second base from their knees

In order to decrease the amount of time that it takes the catcher to throw the ball, a catcher may chose to throw from the knees. Upper extremity kinematics may play a significant role in the kinetics about the elbow observed in catchers throwing from the knees. If relationships between kinematics and kinetics exist then the development of training and coaching instruction may help in reduced upper extremity injury risk. Twenty-two baseball and softball catchers (14.36 ± 3.86 years; 165.11 ± 17.54 cm; 65.67 ± 20.60 kg) volunteered. The catchers exhibited a less trunk rotation (5.6 ± 16.2°), greater elbow flexion (87.9 ± 21.4°) and decreased humeral elevation (71.1 ± 12.3°) at the event of maximum shoulder external rotation as compared to what has previously reported in catchers. These variables are important, as they have previously been established as potential injury risk factors in pitchers, however it is not yet clear the role these variables play in catchers’ risk of injury. A positive relationship between elbow varus torque during the deceleration phase and elbow flexion at MIR was observed (r = 0.609; p = 0.003). Throwing from the knees reduces a catcher’s ability to utilize the proximal kinetic chain and this may help to explain why their kinematics and kinetics differ from what has previously been presented in the literature.     

Preparation and characterization of mixed-mode magnetic adsorbent with p-amino-benzamidine ligand: Operated in a magnetically stabilized fluidized bed reactor for purification of trypsin from bovine pancreas

The magnetic beads were synthesized using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. A multimodal ligand (i.e., p-amino-benzamidine) was covalently immobilized onto magnetic beads after glutaraldehyde activation, and consequently used for purification of the trypsin from bovine pancreas. The p-amino-benzamidine ligand immobilized magnetic beads were characterized by FTIR, VSM, SEM, and analytical methods. Trypsin adsorption experiments were investigated under different experimental conditions (i.e., medium pH, initial trypsin concentration, temperature, and ionic strength) in a batch system. Maximum trypsin adsorption capacity was found to be 75.9 ± 2.6 mg/g beads. Adsorbed trypsin was eluted by using (0.1 M acetate buffer, pH 3.0) with a 97% recovery. The purification factor of trypsin from crude pancreas extract was 8.7 folds. The purity of the eluted trypsin from p-amino-benzamidine functionalized magnetic beads was determined as 86% by HPLC. The method developed in this report was successfully applied for purification of the trypsin from crude pancreas extract in a magnetically stabilized fluidized bed reactor.     

Rapid biochemical synthesis of 11C-labeled single chain variable fragment antibody for immuno-PET by cell-free protein synthesis

Immuno-PET is a promising approach for improved cancer diagnosis, by taking advantage of the high specificity of antibodies. Here, we present a novel cell-free protein synthesis method for preparing a positron emitter labeled-antibody. Functional anti-human EGFRvIII single chain Fv, MR1-1, was successfully labeled with carbon-11 (half-life = 20.4 min) in 5 min (36% yield) by the direct incorporation of the clinical PET tracer, l-[¹¹C]methionine. The product [¹¹C]MR1-1 was easily and rapidly isolated with high radiochemical purity (>95%) from the reaction solution, by affinity purification. This method would be widely applicable to the preparation of radiolabeled antibodies for PET imaging.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

High titer mevalonate fermentation and its feeding as a building block for isoprenoids (isoprene and sabinene) production in engineered Escherichia coli

Isoprenoids are important fine chemicals as material monomers, advanced fuels and pharmaceuticals. A variety of natural isoprenoids can be synthesized by engineered microbial strains. This work established a process by dividing the current isoprenoid pathway into the upstream fermentation process, from sugar to mevalonate (MVA), and the downstream process, from MVA to the target isoprenoids. The results showed that significant differences existed in the process conditions between the upstream and downstream fermentations. After individually optimizing the process conditions, the upstream MVA production (84.0 g/L, 34.0% and 1.8 g/ L/h) and downstream isoprene production (11.0 g/L and 0.23 g/L/h) were greatly improved in this two-step process. Flask fermentation experiments also confirmed that two-step route can significantly improve the sabinene titer to 150 mg/L (6.5-fold of the sabinene titer in an earlier flask study of our lab). Therefore, the two-step route proposed in this study may have potential benefits towards the current isoprenoids production directly from glucose. The high titer and yield of MVA indicate that MVA has great potential to be more broadly utilized as starting precursor in synthetic biology.     

Kinematic chain reactions on trunk and dynamic postural steadiness in subjects with recurrent low back pain

Although subjects with recurrent low back pain (LBP) demonstrate altered trunk control, the kinematic and kinetic responses of the trunk have not been carefully investigated. This study was conducted to compare the standing time, spine range of motion (ROM), and dynamic postural steadiness index (DPSI) based on visual condition between subjects with and without recurrent LBP during upright one leg standing. Sixty-three individuals participated in the study, including 34 control subjects and 29 subjects with recurrent LBP. The DPSI was a composite of the medio-lateral (MLSI), anterior-posterior (APSI), and vertical steadiness indices (VSI) on a force platform. The control group demonstrated longer standing time (s) during the eyes-open condition than the LBP group (26.82 ± 6.03 vs. 19.87 ± 9.36; t = 2.96, p = 0.01). Regarding spine ROM, visual condition was significantly different between groups (F = 7.09, p = 0.01) and demonstrated interactions with spine region and group (F = 5.53, p = 0.02). For the kinetic measures, there was a significant interaction between visual conditions and indices (F = 25.30, p = 0.001). In the LBP group, the DPSI was significantly correlated with the MLSI (r = 0.59, p = 0.002), APSI (r = 0.44, p = 0.03), and VSI (r = 0.98, p = 0.01) in the eyes-closed condition. Overall, the results of this study indicated that the LBP group decreased thorax and lumbar spine rotations during the eyes-closed condition. The LBP group also demonstrated positive correlations with the kinetic indices, enhancing dynamic postural steadiness in the eyes-closed condition in order to possibly avoid pain or further injury. This dynamic postural steadiness strategy is necessary to improve kinetic and kinematic chain reactions in the LBP group. This compensatory pattern supports the development of optimal postural correction strategies to prevent LBP recurrence and might represent a chain reaction to protect trunk control without visual input.     

Biochemical characterization of a lichenase from Penicillium occitanis Pol6 and its potential application in the brewing industry

The purification and characterization of an extracellular lichenase from the fungus Penicillium occitanis Pol6 were studied. The strain produced the maximum level of extracellular lichenase (45 ± 5 U ml⁻¹) when grown in a medium containing oat flour (2%, w/v) at 30 °C for 7 days. The purified enzyme EG_L showed as a single protein band on SDS–PAGE with a molecular mass of 20 kDa. Its N-terminal sequence of 10 amino acid residues was determined as LDNGAPLLNV. The purified enzyme showed an optimum activity at pH 3.0 and 50–60 °C. The half-lives of EG_L at 60 °C and 70 °C were 80 min and 21 min, respectively. Substrate specificity studies revealed that the enzyme is a true β-1,3-1,4-d-glucanase. The enzyme hydrolyzed lichenan to yield trisaccharide, and tetrasaccharide as the main products. Under simulated mashing conditions, addition of EG_L (20 U/ml) or a commercial β-glucanase (20 U/ml) reduced the filtration time (25% and 21.3%, respectively) and viscosity (10% and 8.18%, respectively). These characteristics indicate that EG_L is a good candidate in the malting and brewing industry.     

Characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli for enzyme replacement therapy of Morquio A disease

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Currently, specific therapies are not available for MPS IVA patients. In this study, a biologically active recombinant GALNS enzyme (rGALNS) produced in Escherichia coli was purified through a two-step chromatography process. The effect of temperature and pH on purified rGALNS stability was evaluated, as well as the stability in human serum. Finally, the uptake of rGALNS by HEK 293 cells and MPS IVA fibroblasts was evaluated. The use of a semi-continuous process allowed the production of an active extracellular rGALNS, which was used for protein purification. The purified rGALNS showed a specific activity of 0.29 U mg^?1 and a production yield of 0.78 mg L^?1. The rGALNS presented an optimal pH of 5.5 and was stable for 8 days at 4 °C. In human serum it was stable for up to 6 h. rGALNS was not taken up by the cultured cells, suggesting that N-linked oligosaccharides are not necessary for the production of an active enzyme or enzyme stability but for the cell uptake of protein. This study shows the first characterization of rGALNS produced by E. coli, and provides important information about purification, stability, and glycosylations effect for this type of enzymes.