Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.142, #3)

Production of Raw Starch-Saccharifying Thermostable and Neutral Glucoamylase by the Thermophilic Mold Thermomucor indicae-seudaticae in Submerged Fermentation by Sanjeev Kumar; Pardeep Kumar; T. Satyanarayana (pp. 221-230).

Among physical and nutritional parameters optimized by “one variable at a time” approach, four cultural variables (sucrose, MgSO₄ ^.7H₂O, inoculum size, and incubation period) significantly affected glucoamylase production. These variables were, therefore, selected for optimization using response surface methodology. The p-values of the coefficients for linear effect of sucrose and inoculum size were less than 0.0001, suggesting them to be the key experimental variables in glucoamylase production. The enzyme production (34 U/ml) attained under optimized conditions (sucrose, 2%; MgSO₄ ^.7H₂O, 0.13%; yeast extract, 0.1%; inoculum size, 5 × 10⁶ spores per 50 ml production medium; incubation time, 48 h; temperature, 40°C; and pH 7.0) was comparable with the value predicted by polynomial model (34.2 U/ml). An over all 3.1-fold higher enzyme titers were attained due to response surface optimization. The experimental model was validated by carrying out glucoamylase production in shake flasks of increasing capacity (0.25–2.0 l) and 22-l laboratory bioreactors (stirred tank and airlift), where the enzyme production was sustainable. Furthermore, the fermentation time was reduced from 48 h in shake flasks to 32 h in bioreactors.

Keywords: Glucoamylase; Thermomucor indicae-seudaticae ; Thermophilic mold; Response surface methodology; Shake flasks; Stirred tank fermenter; Airlift fermenter; Bioreactor

Mesophilic Digestion Kinetics of Manure Slurry by Khursheed Karim; K. Thomas Klasson; Sadie R. Drescher; Whitney Ridenour; Abhijeet P. Borole; Muthanna H. Al-Dahhan (pp. 231-242).

Anaerobic digestion kinetics study of cow manure was performed at 35°C in bench-scale gas-lift digesters (3.78 l working volume) at eight different volatile solids (VS) loading rates in the range of 1.11–5.87 g l⁻¹ day⁻¹. The digesters produced methane at the rates of 0.44–1.18 l l⁻¹ day⁻¹, and the methane content of the biogas was found to increase with longer hydraulic retention time (HRT). Based on the experimental observations, the ultimate methane yield and the specific methane productivity were estimated to be 0.42 l CH₄ (g VS loaded)^–1 and 0.45 l CH₄ (g VS consumed)^–1, respectively. Total and dissolved chemical oxygen demand (COD) consumptions were calculated to be 59–17% and 78–43% at 24.4–4.6 days HRTs, respectively. Maximum concentration of volatile fatty acids in the effluent was observed as 0.7 g l^–1 at 4.6 days HRT, while it was below detection limit at HRTs longer than 11 days. The observed methane production rate did not compare well with the predictions of Chen and Hashimoto’s [1] and Hill’s [2] models using their recommended kinetic parameters. However, under the studied experimental conditions, the predictions of Chen and Hashimoto’s [1] model compared better to the observed data than that of Hill’s [2] model. The nonlinear regression analysis of the experimental data was performed using a derived methane production rate model, for a completely mixed anaerobic digester, involving Contois kinetics [3] with endogenous decay. The best fit values for the maximum specific growth rate (μ _m) and dimensionless kinetic parameter (K) were estimated as 0.43 day^–1 and 0.89, respectively. The experimental data were found to be within 95% confidence interval of the prediction of the derived methane production rate model with the sum of residual squared error as 0.02.

Keywords: Anaerobic; Digestion; Kinetics; Manure; Mesophilic; Mathematical model; Methane production

Statistical Determination of Optimal Baculovirus Infection Condition for Recombinant Protein Production in Drosophila S2 Cells by Hye Sook Cho; Kyoung Ro Kim; Yeon Kyu Kim; Hyung Joon Cha (pp. 243-252).

Insect Drosophila melanogaster S2 cell was developed as plasmid-based and, therefore, a nonlytic expression system for functional foreign proteins. To achieve multiple protein expressions, it was suggested that baculovirus be used on S2 cell system because baculovirus can infect S2 cells but cannot replicate inside the cells. Therefore, establishment of baculovirus infection conditions is the first important step and this should be properly optimized for production yield. We used statistical methodology to optimize the baculovirus infection conditions using green fluorescent protein (GFP) as a reporter protein. Consequently, we arrived at optimal infection conditions through a statistical regression method. The secreted GFP yield from vMT-GFP baculovirus-infected wild-type S2 cells under optimal infection conditions was >15-fold higher than that under nonoptimal conditions and comparable to that from stably transfected recombinant S2 cells.

Keywords: Drosophila S2 cells; Baculovirus; Infection condition; Statistical regression

Effect of the Pretreatment of Lipase with Organic Solvents on its Conformation and Activity in Reverse Micelles by Muhammad Moniruzzaman; Mahabubur Rahman Talukder; Yoshishige Hayashi; Takuya Kawanishi (pp. 253-262).

The activity and conformation of Chromobacterium viscosum lipase-pretreated with various organic solvents were investigated. The pretreatment of lipase led to a substantial increase of enzyme activity in AOT (sodium bis [2-ethyl -1-hexyl] sulfosuccinate)/isooctane/water reverse micelles. Among the organic solvents used, n-hexane was found to be most effective. It was observed that higher hexane content with shorter agitation time and vice versa had almost the same effect on the initial activity of lipase. The kinetic study showed that the Michaelis constant (K _m) and the substrate adsorption equilibrium constant (K _ad) were reduced by the pretreatment of lipase with hexane, whereas the change in the maximum reaction rate (V _max) was insignificant. The two spectroscopic techniques (Fluorescence spectra of lipase encapsulated in RMs and Fourier transform infrared [FTIR] spectra of lipase powders) were performed to detect possible conformational changes in the enzyme caused by the pretreatment. A correlation between the maximum fluorescence intensity and the activity of treated lipase was found as a function of agitation time. The FTIR spectrum of lipase showed a new shape peak corresponding to 1,500 cm⁻¹ as a result of pretreatment with organic solvents.

Keywords: Lipases; Pretreatment; Hexane; Hydrolysis; FTIR; Fluorescence

Optimization of Chlorophyllase-catalyzed Hydrolysis of Chlorophyll in Monophasic Organic Solvent Media by Paula Arriagada-Strodthoff; Salwa Karboune; Ronald J. Neufeld; Selim Kermasha (pp. 263-275).

The effects of selected reaction parameters, including solvent hydrophobicity, initial water activity, agitation speed, temperature and enzyme concentration, on the biocatalytic efficiency of a chlorophyllase enzymatic extract from Phaeodactylum tricornutum in neat organic solvent media were investigated. The highest chlorophyllase specific activity of 322 nmol hydrolyzed chlorophyll per gram of protein per minute and bioconversion yield of 91% were obtained in the reaction mixture of hexane/2-octanone (98.3:1.7, v/v), at a controlled initial water activity of 0.90. R _O/A value, which is the ratio of the specific activity in the organic solvent to that in the aqueous/miscible organic solvent medium, was 1.5 × 10⁻³. To reduce the substrate diffusional limitations, the appropriate agitation speed and enzyme concentration were determined. The optimum temperature for maximal enzymatic activity and activation energy were 35°C and 105.0 kJ/mol, respectively. Although the catalytic efficiency of chlorphyllase in the neat organic solvent mixture was lower than that in the aqueous medium, its half-life time in the first environment at temperature ranging from 35 to 50°C was increased by 5.0 to 15.0 times.

Keywords: Chlorophyllase; Biocatalysis; Neat organic solvent media; Kinetics

Potential of Agricultural Residues and Hay for Bioethanol Production by Ye Chen; Ratna R. Sharma-Shivappa; Deepak Keshwani; Chengci Chen (pp. 276-290).

Production of bioethanol from agricultural residues and hays (wheat, barley, and triticale straws, and barley, triticale, pearl millet, and sweet sorghum hays) through a series of chemical pretreatment, enzymatic hydrolysis, and fermentation processes was investigated in this study. Composition analysis suggested that the agricultural straws and hays studied contained approximately 28.62–38.58% glucan, 11.19–20.78% xylan, and 22.01–27.57% lignin, making them good candidates for bioethanol production. Chemical pretreatment with sulfuric acid or sodium hydroxide at concentrations of 0.5, 1.0, and 2.0% indicated that concentration and treatment agent play a significant role during pretreatment. After 2.0% sulfuric acid pretreatment at 121°C/15 psi for 60 min, 78.10–81.27% of the xylan in untreated feedstocks was solubilized, while 75.09–84.52% of the lignin was reduced after 2.0% sodium hydroxide pretreatment under similar conditions. Enzymatic hydrolysis of chemically pretreated (2.0% NaOH or H₂SO₄) solids with Celluclast 1.5 L–Novozym 188 (cellobiase) enzyme combination resulted in equal or higher glucan and xylan conversion than with Spezyme® CP- xylanase combination. The glucan and xylan conversions during hydrolysis with Celluclast 1.5 L–cellobiase at 40 FPU/g glucan were 78.09 to 100.36% and 74.03 to 84.89%, respectively. Increasing the enzyme loading from 40 to 60 FPU/g glucan did not significantly increase sugar yield. The ethanol yield after fermentation of the hydrolyzate from different feedstocks with Saccharomyces cerevisiae ranged from 0.27 to 0.34 g/g glucose or 52.00–65.82% of the theoretical maximum ethanol yield.

Keywords: Chemical pretreatment; Enzymatic hydrolysis; Feedstocks; Fermentation; Glucan; Lignin; Xylan

Immobilization of Urease from Pigeonpea (Cajanus cajan) on Agar Tablets and Its Application in Urea Assay by Swati Mulagalapalli; Sandeep Kumar; Ravi Charan Reddy Kalathur; Arvind M. Kayastha (pp. 291-297).

The pigeonpea urease was immobilized on agar, a common gelling substance. The tablet strips were used as moulds to cast agar tablets of uniform shape and size. The time and temperature of solidification of agar was 6 min and 44 °C, respectively. The 5 % agar (w/v) and 0.019 mg protein/agar tablet yielded an optimum immobilization of 51.7%. The optimum pH was shifted through 0.2 U (from 7.3 to 7.5) towards basic side upon immobilization. The optimum temperature of soluble and immobilized urease was 30 °C and 60 °C, respectively, showing the improvement in thermal stability of urease. There was an increase in K _m from 3.23 to 5.07 mM after immobilization. The half-lives of soluble and immobilized urease were 21 and 53 days, respectively, at pH 7.3 and 4 °C. The urea was estimated in different blood samples with the help of immobilized urease and the results were consistent with those from clinical pathology laboratory through an autoanalyzer® (Zydus Co., Rome, Italy).

Keywords: Immobilization; Urease; Cajanus cajan ; Agar; Urea

Ultrastructural and Intracellular Chemical Changes of a Novel Halophilic Strain V430 of Staphylococcus saprophyticus under CaCl₂ Stress by Xin Xin; Yanxin Wang (pp. 298-306).

Observation of the ultrastructural and intracellular chemical changes of CaCl₂-tolerant strains is important both for understanding their adaptation mechanism under high salt stress and for providing theoretical basis of their application in treating high-CaCl₂ wastewaters. A novel strain V 430 isolated by us has been successfully used to decrease the chemical oxygen demand (COD) concentration of diosgenin wastewater from 20 g l⁻¹ to less than 0.5 g l⁻¹. For this study, strain V430 was incubated in culture media of different CaCl₂ concentrations (up to 9.0%). Strain V430 cells incubated in media of high CaCl₂ concentration excreted extracellular substances and accumulated intracellular Ca²⁺ and K⁺ and free amino acids. The levels of intracellular cations and free amino acids increased with increase in CaCl₂ concentration of the medium. The increase in total free amino acids was mostly due to accumulation of glutamic acid. The strain cells under 9.0% CaCl₂ stress took up K⁺ in a short time, while accumulation of Ca²⁺ proceeded over the whole growth process.

Keywords: Staphylococcus saprophyticus ; CaCl₂-tolerant; Ultrastructure; Cations; Free amino acid

Decolorization of Some Azo Dyes by Immobilized Geotrichum sp. Biomass in Fluidized Bed Bioreactor by Youssef Zeroual; Beom Su Kim; Myoung Won Yang; Mohamed Blaghen; Kang Min Lee (pp. 307-316).

Geotrichum sp. strain, which is able to decolorize azo dyes enzymatically, was used in this study for decolorization of synthetics solutions contaminated by toxic azo dyes orange G, trypan blue, azorubine, and methyl red. The biomass of Geotrichum sp. was immobilized in calcium alginate and polyacrylamide gels and used for the decolorization of tested azo dyes in fluidized bed bioreactor. The highest specific decolorization rate was obtained when the fungal biomass was entrapped in calcium alginate beads. Immobilized biomass in calcium alginate continuously decolorized azo dyes after eight repeated batch decolorization experiments without significant loss of activity whereas polyacrylamide immobilized biomass retained only 10% of its activity after 4 days of incubation. The effects of some physicochemical parameters such as temperature, pH, and dyes concentration on decolorization performance of isolated fungal strain were also investigated.

Keywords: Decolorization; Azo dyes; Geotrichum sp.; Immobilization

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.142, #3)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.142, #3)