Synthesis of D-Alanine Oligopeptides Catalyzed by D-Aminopeptidase in Non-Aqueous Media

Synthesis of D-alanine oligopeptides from D-alanine methylester hydrochloride has been demonstrated by use of immobilized D-aminopeptidase from Ochrobactrum anthropi (Achromobacter sp.) in non-aqueous media. D-Alanine dimer and trimer were obtained in 56% and 6% yield, respectively, when 250 mM of the substrate was incubated for 3 hours with urethane-prepolymer immobilized D-aminopeptidase (1.5 U/ml) and 3 equivalents of triethylamine in water-saturated toluene. The k_cat of this reaction was calculated to be 19,500 (min^?1), which is several ten thousand times greater than that of the known enzymatic syntheses of amino acid oligomers.     

Optimization of cultural conditions for tannase production by Pseudomonas aeruginosa IIIB 8914 under submerged fermentation

A tannase yielding bacterial strain was isolated from soil sample collected from the area situated nearby small-scale tannery. It was identified as Pseudomonas aeruginosa IIIB 8914. The bacterial strain produced extra-cellular tannase under sub-merged fermentation (Smf) using amla (Phyllanthus emblica), keekar (Acacia nilotica), jamoa (Eugenia cuspidate) and jamun (Syzygium cumini) leaves. Among different substrates, amla and keekar leaves resulted in maximal extra-cellular production of tannase. Various process parameters were studied to optimize the extra-cellular yield of tannase under Smf. Maximum yield of tannase i.e., 13.65 and 12.90 U/ml was obtained when Smf was carried out using amla and keekar leaves (2% w/v) respectively in minimal media supplemented with MgSO₄·7H₂O (amla)/HgCl₂ (keekar), NH₄NO₃ and 0.2% Tween 80; inoculated with 2% cell suspension, and incubated at 37°C for 24 h. The bacterial strain produced about 2 times (13.65 U/ml) higher yield of tannase than the highest reported yield of tannase (6 U/ml). Our finding suggests that agro residues in the form of amla and keekar leaves can be one of the best and cost effective alternatives to the costly pure tannic acid for industrial production of microbial tannase.     

Chromium (D-phenylalanine)3 alleviates high fat-induced insulin resistance and lipid abnormalities

High-fat diet has been implicated as a major cause of insulin resistance and dyslipidemia. The objective of this study was to evaluate the impact of dietary-supplementation of chromium (d-phenylalanine)₃ [Cr(d-Phe)₃] on glucose and insulin tolerance in high-fat diet fed mice. C57BL/6-mice were randomly assigned to orally receive vehicle or Cr(d-Phe)₃ (45 μg of elemental chromium/kg/day) for 8-weeks. High-fat-fed mice exhibited impaired whole-body-glucose and -insulin tolerance and elevated serum triglyceride levels compared to normal chow-fed mice. Insulin-stimulated glucose up-take in the gastrocnemius muscles, assessed as 2-[³H-deoxyglucose] incorporation was markedly diminished in high-fat fed mice compared to control mice. Treatment with chromium reconciled the high-fat diet-induced alterations in carbohydrate and lipid metabolism. Treatment of cultured, differentiated myotubes with palmitic acid evoked insulin resistance as evidenced by lower levels of insulin-stimulated Akt-phosphorylation, elevated JNK-phosphorylation, (assessed by Western blotting), attenuation of phosphoinositol-3-kinase activity (determined in the insulin-receptor substrate-1-immunoprecipitates by measuring the extent of phosphorylation of phosphatidylinositol by γ-³²P-ATP), and impairment in cellular glucose up-take, all of which were inhibited by Cr(d-Phe)₃. These results suggest a beneficial effect of chromium-supplementation in insulin resistant conditions. It is likely that these effects of chromium may be mediated by augmenting downstream insulin signaling.     

Statistical optimization of glucose oxidase production from Aspergillus niger NRC9 under submerged fermentation using response surface methodology

Response surface methodology (RSM), employing the fractional factorial design (FFD) was used to optimize the fermentation medium for the production of glucose oxidase (GOD) from a marine isolate (NRC9) of Aspergillus niger under submerged fermentation. The design was employed by selecting glucose, CaCO_3, ammonium phosphate and MgSO₄ concentrations as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum concentrations (g/l) glucose, 100; CaCO₃, 25; (NH₄)₂HPO₄, 1.8 and 0.4 of MgSO₄, resulted in an improvement of GOD production (170?±?0.88 U/ml) as compared to the initial level (109.81?±?1.38 U/ml) after four days of incubation at 200 rpm and 30 °C, whereas its predicted value obtained by the quadratic model was 164.36 U/ml. Analysis of variance (ANOVA) showed a high coefficient of determination value (R ²) of 0.967, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is the first report on production of glucose oxidase from a marine fungal isolate, Aspergillus niger NRC9, using statistical experimental design and response surface methodology in optimization of its production under submerged fermentation.     

Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system

A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na₂, 0.5 mM ATP, 5 mM Mg²⁺, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.     

Production and characterization of a solvent-tolerant protease from a novel marine isolate Bacillus tequilensis P15

Thirty-six proteolytic bacteria were isolated from the Jakhau coast, Kutch, India, amongst which isolate P15 identified as Bacillus tequilensis (JQ904626) was found to produce an extracellular solvent-- and detergent-tolerant protease (116.69?±?0.48 U/ml) and was selected for further investigation. Deoiled Jatropha seedcake (JSC) was found to be a suitable substrate for protease production under submerged condition. Upon optimization of process parameters following one-factor-at-a-time approach, an overall 6.4-fold (860.27?±?18.48 U/ml) increase in protease production was achieved. The maximum protease yield was obtained using a medium containing 2 % (w/v) deoiled JSC as substrate (pH of 8.0) upon 36 h of fermentation at 30 °C. The optimum temperature and pH for activity of B. tequilensis P15 protease was found to be 50 °C and 8.0, respectively. The enzyme exhibited a half-life of 190 min at 50 °C, which was enhanced to 270 min in presence of 5 mM Ca²⁺. The enzyme exhibited significant stability in almost all the solvents tested in the range of log P _ow varying from 8.8 to ?0.76. The enzyme activity was strongly inhibited by PMSF at 5 mM concentration, whereas the presence of EDTA (5 mM) and pCMB (5 mM) enhanced enzyme activity by 20.9 and 13.7 %, respectively. The enzyme was also found to be stable in the presence of surfactants, commercial detergents and bleach-oxidant (H₂O₂). This protease was demonstrated to be effective in removal of blood stains from fabrics, dehairing of hide, and stripping off the gelatin from used photographic films.     

Efficient synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose by using new 2-deoxy-d-ribose-5-phosphate aldolase from Streptococcus suis with moderate activity and aldehyde tolerance

The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg⁻¹, K_M of 0.8 mM, and V_max of 32.9 μmol min⁻¹ mg⁻¹ toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.     

Optimal conditions for enhanced β-mannanase production by recombinant Aspergillus sojae

Optimization of the growth conditions for maximum β-mannanase production in shake flasks by using recombinant Aspergillus sojae ATCC11906 (AsT1) was carried out by Box–Behnken design of response surface methodology. The highest β-mannanase activity on the fourth day of cultivation at 30 °C was obtained as 363 U/ml in the optimized medium consisting of 7% sugar beet molasses, 0.43% NH₄NO₃, 0.1% K₂HPO₄ and 0.05% MgSO₄ (by weight per volume) at 207 rpm. On the sixth day of cultivation under the optimized conditions, the highest β-mannanase activity was achieved as 482 U/ml which is 1.4-fold of 352 U/ml activity found on glucose medium previously.     

Effect of Urea Concentration on Growth of Ureaplasma urealyticum (T-Strain Mycoplasma)

The effect of urea on growth of Ureaplasma urealyticum type VIII was studied by cultivating the organisms in a dialysate broth, prepared from soy peptone and autoclaved yeast, supplemented with 5% dialyzed horse serum, 100 mM 2-(N-morpholino)ethane sulfonic acid buffer (pH 5.75), and defined amounts of urea. Without urea, growth did not occur. Total growth was directly related to urea concentration. The least amount of urea that supported growth was 0.032 mM, which resulted in 3 × 10⁴ colony-forming units per ml. The maximum yield of organisms, 8.0 × 10⁷ colony-forming units per ml, was observed at 32 mM urea. Growth was limited not only by urea concentration, but also by the buffer capacity of the medium. The maximum amount of 2-(N-morpholino)ethane sulfonic acid buffer that could be employed was 100 mM; at higher concentrations, growth was inhibited. The yield of U. urealyticum was small even in medium with 32 mM urea and 100 mM 2-(N-morpholino)ethane sulfonic acid buffer: 0.63 mg of protein per liter of culture containing 5 × 10¹⁰ total colony-forming units. The molar growth yield was 20 mg of protein per mol of urea. The growth rate was also a function of urea concentration. Generation times ranged from 8 h at 0.032 mM urea to 1.6 h at 3.2 mM urea, where the substrate level was saturating. The K_s value for growth was 2.0 × 10⁻⁴ M urea. Thus, urea is a growth-limiting factor for U. urealyticum, but remarkably large amounts of this substrate are required.     

Characterization of a robust glucose 1-dehydrogenase,SyGDH, and its application in NADPH regeneration for the asymmetric reduction of haloketone by a carbonyl reductase in organic solvent/buffer system

To realize coenzyme regeneration in the reduction of haloketones, a codon-optimized gene Sygdh encoding glucose 1-dehydrogenase (SyGDH) was synthesized based on the putative GDH gene sequence (Ta0897) in Thermoplasma acidophilum genomic DNA, and expressed in E. coli BL21(DE3). Recombinant SyGDH was purified to homogeneity by affinity chromatography with the specific activity of 86.3 U/mg protein towards D-glucose at the optimum pH and temperature of 7.5 and 40 °C. It was highly stable in a pH range of 4.5–8.0 and at 60 °C or below, and resistant to various organic solvents. The K_m and catalytic efficiency (k_cat/K_m) of SyGDH towards NADP+ were 0.67 mM and 104.0 mM⁻¹ s⁻¹, respectively, while those towards NAD+ were 157.9 mM and 0.64 mM⁻¹ s⁻¹, suggesting that it preferred NADP+ as coenzyme to NAD+. Additionally, using whole cells of E. coli/Sygdh-Sys1, coexpressing SyGDH and carbonyl reductase (SyS1), as the biocatalyst, the asymmetric reduction of 60 mM m-chlorophenacyl chloride coupled with the regeneration of NADPH in situ was conducted in DMSO/phosphate buffer (2:8, v/v) system, producing (R)-2-chloro-1-(3-chlorophenyl)ethanol with over 99.9% ee_p and 99.2% yield. Similarly, the reduction of 40 mM α-bromoacetophenone in n-hexane/buffer (6:4, v/v) biphasic system produced (S)-2-bromo-1-phenylethanol with over 99.9% ee_p and 98.3% yield.     

A 31P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Synthesis of D-Alanine Oligopeptides Catalyzed by D-Aminopeptidase in Non-Aqueous Media

Synthesis of D-alanine oligopeptides from D-alanine methylester hydrochloride has been demonstrated by use of immobilized D-aminopeptidase from Ochrobactrum anthropi (Achromobacter sp.) in non-aqueous media. D-Alanine dimer and trimer were obtained in 56% and 6% yield, respectively, when 250 mM of the substrate was incubated for 3 hours with urethane-prepolymer immobilized D-aminopeptidase (1.5 U/ml) and 3 equivalents of triethylamine in water-saturated toluene. The k_cat of this reaction was calculated to be 19,500 (min^-1), which is several ten thousand times greater than that of the known enzymatic syntheses of amino acid oligomers.     

Insulin-sensitizing and cholesterol-lowering effects of chromium (D-Phenylalanine)3

Low-molecular weight organic chromium complexes are thought to play a key role in carbohydrate and lipid metabolism and therefore have been gaining popularity as nutritional supplement for patients with diabetes and concomitant lipid disorders. The aim of the present study was to evaluate the effects of a novel synthetic chromium (d-phenylalanine)(3) complex on insulin-sensitivity, plasma lipid-profile and oxidant stress in a mouse model of type II diabetes. Plasma glucose levels following intraperitoneal insulin-challenge (1U/kg) to obese ob/ob(+/+) mice treated with Cr(d-Phe)(3) (150 microg/kg/day for 6 weeks) were significantly lower compared to vehicle-control (control: 175.8+/-43.2mg/dL versus Cr(d-Phe)(3) 115.3+/-18.0mg/dL, p<0.01, n=12). Total serum cholesterol to high-density lipoprotein ratio was significantly reduced following Cr(d-Phe)(3)-treatment (control: 2.19+/-0.08 versus Cr(d-Phe)(3) 1.63+/-0.05; p<0.05). Hepatic oxidant stress, assessed as malondialdehyde equivalents and protein-carbonyl content were significantly attenuated following Cr(d-Phe)(3) treatment. The complex also inhibited lipid-peroxidation in vitro, in a concentration dependent manner. Taken together, these data suggest that Cr(d-Phe)(3) may be of potential value in the therapy or prophylaxis of insulin-resistance and dyslipidemia associated with obesity.     

Production of steviol from steviol glucosides using β-glycosidase from Sulfolobus solfataricus

Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, was cloned and expressed in E. coli Rossetta BL21(DE3)pLyS using lactose as an inducer. Through fermentation, SSbgly was expressed as a 61 kDa protein with activity of 24.3 U/mg and the OD₆₀₀ of 23 was reached after 18 h induction with 10 mM lactose. Purified protein was obtained by Ni-Sepharose chromatography with a yield of 92.3%. SSbgly hydrolyzed steviol glycosides to produce steviol with a yield of 99.2%. The optimum conditions for steviol production were 50 U/ml SSbgly and 90 mg/ml Ste at 75 °C as determined by the response surface method.     

Purification and properties of pyruvate kinase type M2 from rat lung

(1) Pyruvate kinase type M₂ from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of M_r = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M₂, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K_0.5 value for phosphoenolpyruvate is 0.26 mM (n_H = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (n_H = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The K_m value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The K_m value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M₂ from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M₂ from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M₂ may represent a separate form of the pyruvate kinase type M₂ family.     

Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H₂, CO₂, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N₂-15% CO versus N₂ alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield.     

Photosynthetic characteristics and effects of exogenous glycine of Chorispora bungeana under drought stress

We investigated the photosynthetic characteristics of Chorispora bungeana under conditions of drought stress caused by different concentrations of polyethylene glycol-6000 (PEG; 0, 5, 20, and 40%) and various concentrations of exogenous glycine (0, 5, 10, and 20 mM) with 20% PEG. We showed that moderate and severe drought stress of PEG reduced the chlorophyll (Chl) content (both Chl a and b), maximal quantum yield of PSII photochemistry (F_v/F_m), actual photochemical efficiency of PSII in light (Y_II), and quantum yield of regulated energy dissipation (Y_NPQ), while Chl a/b and quantum yield of nonregulated energy dissipation (Y_NO) increased. The low and moderate drought stress increased Mg²⁺ and Fe³⁺ contents, while a decrease in Mg²⁺ and Fe³⁺ was found under severe drought stress. Compared to sole PEG stress, the addition of exogenous 10 mM glycine increased Chl, Mg²⁺ and Fe³⁺ contents, F_v/F_m, Y_II, and Y_NPQ, and reduced Y_NO. On the contrary, 20 mM glycine showed an opposite effect, except for Y_NO. Our results proved that Chl contents and fluorescence parameters are reliable indicators for drought tolerance of C. bungeana. We suggest that a proper glycine content can relieve the effect of drought stress on C. bungeana.     

Ethanol production from seaweed (Undaria pinnatifida) using yeast acclimated to specific sugars

Ethanol production from Undaria pinnatifida (Sea mustard, Miyuk) was performed using yeast acclimated to specific sugars. Pretreatment conditions were optimized by thermal acid hydrolysis and enzyme treatment to increase the monosaccharide yield. Pretreatment by thermal acid hydrolysis was carried out using seaweed powder at 8 ～ 17% (w/v) solid content with a treatment time of 30 ～ 60 min. Enzyme treatment was carried out with 1% (v/v) Viscozyme L (1.2 FGU/mL), 1% (v/v) Celluclast 1.5 L (8.5 EGU/mL), 1% (v/v) AMG 300 L (3.0 AGU/mL), and 1% (v/v) Termamyl 120 L (0.72 KNU/mL). All enzymes except Termamyl 120 L, which was applied during pretreatment, were treated at 45°C for 24 h following pretreatment. Optimal pretreatment and enzyme conditions were determined to be 75 mM H₂SO₄, 13% (w/v) slurry, and 2.88 KNU/mL Termamyl 120 L at 121°C for 60 min. A maximum monosaccharide concentration of 33.1 g/L with 50.1% theoretical yield was obtained. To increase the ethanol yield, Pichia angophorae KCTC 17574 was acclimated to a high concentration (120 g/L) of galactose and mannitol at 30oC for 24 h. Ethanol production of 12.98 g/L with 40.12% theoretical yield was obtained from U. pinnatifida through fermentation with 0.35 g dry cell weight/L P. angophorae KCTC 17574 acclimated to mannitol and galactose.     

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

Abstract

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2?mM CaCO_3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55?U/ml, 79.22?U/ml, 12.43?U/ml, 64.66?U/ml, and 21.12?U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.