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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #8)

Purification, Characterization and Kinetic Studies of a Novel Poly(β) Hydroxybutyrate (PHB) Depolymerase PhaZ Pen from Penicillium citrinum S2 by Srividya Shivakumar; Sneha Jani Jagadish; Hardik Zatakia; Jayasmita Dutta (pp. 1225-1236).

A fungal isolate, identified as Penicillium citrinum S2, produced ≈1 U/mL of PHB depolymerase by 72 h when grown in BHM containing 0.2%, w/v PHB, pH 6.0 at 30 °C. Partial purification of an extracellular poly(-β-)hydroxybutyrate (PHB) depolymerase PhaZ Pen from P. citrinum S2 by two steps using ammonium sulphate (80% saturation) and affinity chromatography using concanavalin A yielded 16.18-fold purity and 21.53% recovery of protein. The enzyme was composed of three polypeptide chains of 66, 43 and 20 kDa, respectively, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All the three bands stained positive for glycoprotein by PAS staining. Optimum enzyme activity was detected at pH 6.0 and 50 °C. The enzyme was stable between pH 4.0 and 7.0 at 50 °C, 2 h. β-hydroxybutyrate monomer was detected as the major end product of PHB hydrolysis. The enzyme also showed distinct behaviour towards different inhibitors tested, which suggests the role of serine, serine residue, carboxyl group, tyrosine and sulfhydryl groups in its active site.

Keywords: PHB depolymerase; P. citrinum ; Purification; Characterization; Kinetic studies

Improvement of Ethanol Production Using Saccharomyces cerevisiae by Enhancement of Biomass and Nutrient Supplementation by Santosh N. Sankh; Preetee S. Deshpande; Akalpita U. Arvindekar (pp. 1237-1245).

Optimization of ethanol production through addition of substratum and protein-lipid additives was studied. Oilseed meal extract was used as protein lipid supplement, while rice husk was used as substratum. The effect of oil seed meal extract and rice husk was observed at varying concentration of medium sugar from 8% to 20%. Of the three oil seed meal extracts used, viz. groundnut, safflower, and sunflower, safflower was found to be most efficient. The use of oilseed meal extract at 4% was found to enhance ethanol production by almost 50% and enhanced sugar tolerance from 8% to 16%. A further increase of almost 48% ethanol was observed on addition of 2 g of rice husk per 100 ml of medium. An increase in cell mass with better sugar attenuation was observed. Further optimization was sought through use of sugarcane juice as the sugar source. While 8.9% ethanol yield with 75% sugar attenuation was observed at 20% sucrose concentration, it was found to increase to 12% (v/v) with almost complete utilization of medium sugar when sugarcane juice was used. Cell weight was also observed to increase by 26%.

Keywords: Ethanol; Oilseed meal extract; Rice husk; Sugarcane juice; Saccharomyces cerevisiae

In Vivo Evalution of Hypoglycemic Activity of Aloe spp. and Identification of Its Mode of Action on GLUT-4 Gene Expression In Vitro by Rajiv Kumar; Bhavna Sharma; Neha R. Tomar; Partha Roy; Atul K. Gupta; Anil Kumar (pp. 1246-1256).

The present study evaluated the hypoglycemic activity of Aloe extract on streptozotocin-induced diabetic mice and focuses its effect on GLUT-4 gene expression under in vitro cell-culture system. Administration of extract at the dosage of 130 mg/kg body weight per day for 4 weeks resulted in significant decrease in blood glucose and total cholesterol in streptozotocin (60 mg/kg body weight) induced diabetic mice. The hypoglycemic effect was compared with metformin. The activities of carbohydrate metabolizing enzymes were brought back to near normal level after the treatment and glucose homeostasis was maintained. Lyophilized aqueous Aloe extract (1 mg/ml) upregulated the GLUT-4 mRNA synthesis in mouse embryonic NIH/3T3 cells.

Keywords: Diabetes mellitus; Aloe extract; In vivo; Glucose tolerance test; Lipid profile; Hexokinase assay; In vitro; GLUT-4 gene; NIH/3T3 cells

Bioremediation of Single and Mixture of Pesticide-Contaminated Soils by Mixed Pesticide-Enriched Cultures by K. Rama Krishna; Ligy Philip (pp. 1257-1277).

In the present study, degradation efficiencies for individual as well as mixed pesticide in different Indian soils, by mixed pesticide-enriched cultures, were evaluated under submerged and unsaturated conditions, Lindane (L), methyl parathion (MP), carbofuran (C), and a mixture of L, MP, and C were used in the study. For all the various conditions considered, methyl parathion degradation was the maximum and lindane degradation was the minimum. The degradation kinetics of the pesticides in sandy, clayey, compost, and red soils by various microbial isolates were studied. It was observed that adsorption was maximum and degradation of pesticides was minimum in compost soil. The degradation efficiencies of pesticides in liquid phase associated with soil sediment were less than those under the normal liquid phase conditions as leaching of pesticides from soil phase was continuous. Pesticide degradation was more in submerged soils compared to that in unsaturated soils. The degradation by-products of individual and mixed pesticides in liquid, unsaturated, and submerged soils were identified. Different metabolites were produced under submerged and unsaturated conditions.

Keywords: Lindane; Methyl parathion; Carbofuran; Mixed pesticides; Soil and liquid phase

Global Gene Response in Saccharomyces cerevisiae Exposed to Silver Nanoparticles by Javed H. Niazi; Byoung-In Sang; Yeon Seok Kim; Man Bock Gu (pp. 1278-1291).

Silver nanoparticles (AgNPs), exhibiting a broad size range and morphologies with highly reactive facets, which are widely applicable in real-life but not fully verified for biosafety and ecotoxicity, were subjected to report transcriptome profile in yeast Saccharomyces cerevisiae. A large number of genes accounted for ∼3% and ∼5% of the genome affected by AgNPs and Ag-ions, respectively. Principal component and cluster analysis suggest that the different physical forms of Ag were the major cause in differential expression profile. Among 90 genes affected by both AgNPs and Ag-ions, metalloprotein mediating high resistance to copper (CUP1-1 and CUP1-2) were strongly induced by AgNPs (∼45-folds) and Ag-ions (∼22-folds), respectively. A total of 17 genes, responsive to chemical stimuli, stress, and transport processes, were differentially induced by AgNPs. The differential expression was also seen with Ag-ions that affected 73 up- and 161 down-regulating genes, and most of these were involved in ion transport and homeostasis. This study provides new information on the knowledge for impact of nanoparticles on living microorganisms that can be extended to other nanoparticles.

Keywords: Silver nanoparticles; Nanotoxicity; Microarray; Transcriptome analysis; Yeast genome analysis

Simulating the Degradation of Odor Precursors in Primary and Waste-Activated Sludge During Anaerobic Digestion by Saad Aldin; Fuzhou Tu; George Nakhla; Madhumita B. Ray (pp. 1292-1304).

Degradation of known odor precursors in sludge during anaerobic digestion was systematically studied and simulated using the Anaerobic Digestion Model Number 1 (ADM1). The degradation of various protein fractions (particulate, soluble, and bound), volatile fatty acids (VFAs), lipids, and amino acids of primary sludge (PS) and waste-activated sludge (WAS) were monitored during anaerobic digestion. The degradation kinetic constants of the odor precursors namely, protein, lipid, and VFAs were determined. Relationships between degradations of protein fractions and volatile suspended solid were established; a strong relationship between bound protein, a major odor precursor, and volatile suspended solid degradation was found. No statistically significant difference in bound protein reduction was observed between PS and WAS. ADM1 was successfully used to simulate the lab scale continuous anaerobic digestion; model results with optimized parameters showed good agreement with the experimental data for methane production and several other sludge parameters including odor precursors such as lipids, VFAs, and proteins.

Keywords: Anaerobic digestion; Odors precursors; Cell protein; Bound protein; ADM1 model

In Vivo Regulation of Alcohol Dehydrogenase and Lactate Dehydrogenase in Rhizopus Oryzae to Improve l-Lactic Acid Fermentation by Sitanan Thitiprasert; Sarintip Sooksai; Nuttha Thongchul (pp. 1305-1322).

Rhizopus oryzae is becoming more important due to its ability to produce an optically pure l-lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4′-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.

Keywords: Fermentation; Lactic acid; Ethanol; In vivo inhibition; Rhizopus oryzae ; Alcohol dehydrogenase; Lactate dehydrogenase; Sulfhydryl reagent; Substrate/product analog

Identification and Characterization of a Novel Thermostable gh-57 Gene from Metagenomic Fosmid Library of the Juan De Fuca Ridge Hydrothemal Vent by Hui Wang; Yingxue Gong; Wei Xie; Wenjuan Xiao; Junmei Wang; Yangyang Zheng; Jia Hu; Zehuan Liu (pp. 1323-1338).

A novel glycoside hydrolases family 57 gene (gh-57) was found from a metagenomic fosmid library constructed from a black smoker chimney sample 4143-1 from the Mothra hydrothermal vent at the Juan de Fuca Ridge. Sequence and homology analysis using BLAST revealed that it had high similarity to gh-57 family. Conserved domain research revealed that the novel gh-57 contained a Glyco-hydro-57 domain and five conserved regions, including two putative catalytic residues Glu¹⁵⁴ and Asp²⁶³. The three-dimensional features of the protein and its homologue from Pyrococcus horikoshii OT3 known as α-amylase were generated by homology modeling. The gh-57 gene was cloned, expressed, and purified in Escherichia coli using pQE system. Enzyme activity revealed that the recombinant protein could hydrolyze soluble starch and demonstrated amylase activity. It showed an optimal pH of 7.5, an optimal temperature of 90°C, and its thermostability at 90°C could remain over 50% enzyme activity for 4 h. The enzyme activity could be increased by DTT and Mg²⁺ while an inhibitory effect was observed with EDTA, ATP, and Ca²⁺. These results showed that the gh-57 gene was a novel thermostable amylase from oceanic microorganisms.

Keywords: Metagenomics; Thermophile; Glycosidase; Cloning; Gene expression; Enzyme activity; Amylase

System Establishment of ATPS for One-Step Purification of Glutamate Decarboxylase from E. coli After Cell Disruption by Wanying Yao; Xiao Wu; Jun Zhu; Bo Sun; Curtis Miller (pp. 1339-1349).

The partition of glutamate decarboxylase (GAD) from Escherichia coli in polyethylene glycol (PEG) and sodium sulfate aqueous two-phase systems (ATPS) has been explored with the purpose of establishing a phase system for the purification of GAD after cell disruption. The results showed that the partitioning of GAD was slightly influenced by PEG molecular weight (MW) but depended on the tie line length (TLL) and NaCl and loading sample concentrations. The optimum system obtained for GAD purification was composed of a PEG MW of 4,000, TLL of 63.5%, a volume ratio of 2.31, a loading sample concentration of 0.4 g/mL, which produced a GAD recovery of 90% with the purification fold of 73. Furthermore, the feasibility of directly purifying GAD from the cell disrupts using ATPS was evaluated. The established ATPS for GAD purification exhibited an efficient integrated purification process compared to the reported purification process in terms of purification efficiency and recovery.

Keywords: Aqueous two-phase system; One-step purification of GAD; Molecular weight; Tie line length; Sample concentration

The Effect of Naphthalene-Acetic Acid on Biomass Productivity and Chlorophyll Content of Green Algae, Coccolithophore, Diatom, and Cyanobacterium Cultures by Ryan W. Hunt; Senthil Chinnasamy; K. C. Das (pp. 1350-1365).

The application of biochemical stimulants to enhance biomass and metabolite productivity is being investigated here and may be a simpler approach to achieve our goals of higher productivity and lower costs than methods such as genetic modification. The research builds on prior work of screening various biochemical stimulants representing different types of plant growth regulators with the green alga, Chlorella sorokiniana. Here, we report the impact on biomass and chlorophyll productivity by comparing the delivery method of a previously identified superior stimulant, the synthetic auxin naphthalene-acetic acid (NAA), solubilized in ethanol or methanol. Algae evaluated included the green alga, C. sorokiniana, as well as a mixed consortium that includes C. sorokiniana along with two other wild-isolated green algae, Scenedesmus bijuga and Chlorella minutissima. It was found that NAA dissolved in ethanol was more effective in enhancing biomass productivity of C. sorokiniana. However, no differences were observed with the mixed consortia. The most effective treatment from this step, EtOH_500ppm + NAA_5ppm, along with two other NAA concentrations (EtOH_500ppm + NAA_2.5ppm and EtOH_500ppm + NAA_10ppm), was then applied to six diverse species of microalgae to determine if the treatment dosage was effective for other freshwater and marine green algae, cyanobacteria, coccolithophore, and diatoms. It was found that three of the species bioassayed, Pleurochrysis carterae, C. sorokiniana, and Haematococcus pluvialis exhibited a substantial boost in biomass productivity over the 10-day growth period. The use of ethanol and NAA at a combined dosage of EtOH_500ppm + NAA_5ppm was found to generate the highest biomass productivity for each of the species that responded positively to the treatments. If scalable, NAA and ethanol may have the potential to lower production costs by increasing biomass yields for commercial microalgae cultivation.

Keywords: Plant growth regulators; Phytohormones; Auxins; Naphthalene-acetic acid; Bioenergy; Biofuels; Biomass; Biostimulants; Microalgae; Phytohormones; Chlorella sorokiniana ; Haematococcus pluvialis ; Phaeodactylum tricornutum ; Pleurochrysis carterae ; Dunaliella bardawil ; Nostoc

Expression and Purification of Bioactive High-Purity Recombinant Mouse CXCL14 in Escherichia coli by Jingjing Li; Jin Gao; Sunita Sah; Uttam Satyal; Ruliang Zhang; Wei Han; Yan Yu (pp. 1366-1375).

Mouse CXCL14/BRAK is a monocyte-selective chemokine which is expressed in almost all normal tissues. A flood of reports on its new functions of tumor suppression and fat metabolism modulation has left CXCL14 a potential therapeutic candidate for these diseases. Therefore, a simple accessible method is on demand for large-scale production of recombinant mouse CXCL14 protein for in vivo animal studies. Here, we introduce an efficient method for large-scale production of recombinant mouse CXCL14, by which an 18-mg protein is produced from 2-L Escherichia coli culture with good bioactivity and low level of endotoxin.

Keywords: Mouse CXCL14; Nickel affinity; Large-scale preparation; Chemokines

glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II by Hiten J. Gutka; Kamolchanok Rukseree; Paul R. Wheeler; Scott G. Franzblau; Farahnaz Movahedzadeh (pp. 1376-1389).

The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6-bisphosphatase II (FBPase II; EC 3.1.3.11); a key gluconeogenic enzyme. Mtb possesses glpX homologue as the major known FBPase. This study explored the expression, purification and enzymatic characterization of functionally active FBPase II from Mtb. The glpX gene was cloned, expressed and purified using a two step purification strategy including affinity and size exclusion chromatography. The specific activity of Mtb FBPase II is 1.3 U/mg. The enzyme is oligomeric, followed Michaelis–Menten kinetics with an apparent km = 44 μM. Enzyme activity is dependent on bivalent metal ions and is inhibited by lithium and inorganic phosphate. The pH optimum and thermostability of the enzyme have been determined. The robust expression, purification and assay protocols ensure sufficient production of this protein for structural biology and screening of inhibitors against this enzyme.

Keywords: glpX ; Fructose 1,6–bisphosphatase; Mycobacterium tuberculosis ; Gluconeogenesis

Fractionation of Corn Fiber Treated by Soaking in Aqueous Ammonia (SAA) for Isolation of Hemicellulose B and Production of C5 Sugars by Enzyme Hydrolysis by Nhuan P. Nghiem; Justin Montanti; David B. Johnston; Caye Drapcho (pp. 1390-1404).

A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cleavage into xylo-oligosaccharides and subsequently recovered by precipitation with ethanol. The pretreatment step resulted in high retention of major sugars and improvement of subsequent enzymatic hydrolysis. The recovered hemicellulose B was hydrolyzed by a cocktail of enzymes that consisted of β-glucosidase, pectinase, xylanase, and ferulic acid esterase (FAE). Xylanase alone was ineffective, demonstrating yields of less than 2% of xylose and arabinose. The greatest xylose and arabinose yields, 44% and 53%, respectively, were obtained by the combination of pectinase and FAE. A mass balance accounted for 87% of the initially present glucan, 91% of the xylan, and 90% of the arabinan. The developed process offered a means for production of corn fiber gum as a value-added co-product and C5 sugars, which could be converted to other valuable co-products through fermentation in a corn wet-milling biorefinery.

Keywords: Lignocellulosic biomass; Pretreatment; Hemicellulose; Arabinoxylan; Soaking in aqueous ammonia; Corn fiber; Ethanol

Influence of Feedstock Particle Size on Lignocellulose Conversion—A Review by Bernardo C. Vidal Jr.; Bruce S. Dien; K. C. Ting; Vijay Singh (pp. 1405-1421).

Feedstock particle sizing can impact the economics of cellulosic ethanol commercialization through its effects on conversion yield and energy cost. Past studies demonstrated that particle size influences biomass enzyme digestibility to a limited extent. Physical size reduction was able to increase conversion rates to maximum of ≈50%, whereas chemical modification achieved conversions of >70% regardless of biomass particle size. This suggests that (1) mechanical pretreatment by itself is insufficient to attain economically feasible biomass conversion, and, therefore, (2) necessary particle sizing needs to be determined in the context of thermochemical pretreatment employed for lignocellulose conversion. Studies of thermochemical pretreatments that have taken into account particle size as a factor have exhibited a wide range of maximal sizes (i.e., particle sizes below which no increase in pretreatment effectiveness, measured in terms of the enzymatic conversion resulting from the pretreatment, were observed) from <0.15 to 50 mm. Maximal sizes as defined above were dependent on the pretreatment employed, with maximal size range decreasing as follows: steam explosion > liquid hot water > dilute acid and base pretreatments. Maximal sizes also appeared dependent on feedstock, with herbaceous or grassy biomass exhibiting lower maximal size range (<3 mm) than woody biomass (>3 mm). Such trends, considered alongside the intensive energy requirement of size reduction processes, warrant a more systematic study of particle size effects across different pretreatment technologies and feedstock, as a requisite for optimizing the feedstock supply system.

Keywords: Cellulosic ethanol; Lignocellulose conversion; Biomass particle size; Biomass recalcitrance

Biochemical Studies on Hemoglobin Modified with Reactive Oxygen Species (ROS) by Tejinder Pal Khaket; Rizwan Ahmad (pp. 1422-1430).

Hemoglobin is the iron-containing oxygen transporting metalloprotein in the red cells of blood in mammals and other animals. Hemoprotein-mediated oxidative stress is thought to play a major role in pathophysiology of cerebral hemorrhage, blast pressure injury, crush injury, myocardial ischemia reperfusion injury. Hemoglobin undergoes oxidation–reduction reactions that lead to both generation and consumption of highly reactive oxygen and nitrogen species. In the present study, hemoglobin molecule was treated with hydrogen peroxide and the modification so incurred was analyzed by UV spectra, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detection of carbonyl content. Our observations suggest that carbonyl content increases with increase in concentration of hydrogen peroxide. Production of hydroxyl radical was assessed by using benzoate degradation analysis. Our results was in tandem with the fact that hemoglobin on treatment with hydrogen peroxide rapidly generates free-radical species that can degrade benzoate to thiobarbituric acid reactive material which on reacting with thiobarbituric acid gives color. The increase in absorbance of ROS-modified hemoglobin at 532 nm shows the increase in benzoate degradation, which is a parameter of hydroxyl radical formation with increase in concentration of hydrogen peroxide. Modified hemoglobin was treated with catalase, mannitol, thiourea, glutathion, sodium benzoate and their effect were detected by spectroscopy and SDS-PAGE (12%). Substantial scavenging effect of aforementioned antioxidants reiterates the formation of hydroxyl radical. Catalase shows the maximum scavenging effect followed by thiourea and mannitol.

Keywords: Hemoglobin; Reactive oxygen species; Hydrogen peroxide

A Novel Glutamate Transport System in Poly(γ-Glutamic Acid)-Producing Strain Bacillus subtilis CGMCC 0833 by Qun Wu; Hong Xu; Dan Zhang; Pingkai Ouyang (pp. 1431-1443).

Bacillus subtilis CGMCC 0833 is a poly(γ-glutamic acid) (γ-PGA)-producing strain. It has the capacity to tolerate high concentration of extracellular glutamate and to utilize glutamate actively. Such a high uptake capacity was owing to an active transport system for glutamate. Therefore, a specific transport system for l-glutamate has been observed in this strain. It was a novel transport process in which glutamate was symported with at least two protons, and an inward-directed sodium gradient had no stimulatory effect on it. K _m and V _m for glutamate transport were estimated to be 67 μM and 152 nmol⁻¹ min⁻¹ mg⁻¹ of protein, respectively. The transport system showed structural specificity and stereospecificity and was strongly dependent on extracellular pH. Moreover, it could be stimulated by Mg²⁺, NH ₄ ⁺ , and Ca²⁺. In addition, the glutamate transporter in this strain was studied at the molecular level. As there was no important mutation of the transporter protein, it appeared that the differences of glutamate transporter properties between this strain and other B. subtilis strains were not due to the differences of the amino acid sequence and the structure of transporter protein. This is the first extensive report on the properties of glutamate transport system in γ-PGA-producing strain.

Keywords: Bacillus subtilis CGMCC 0833; Glutamate transport; Poly(γ-glutamic acid); Secondary transport system

Production and Physico-chemical Characterization of a Biosurfactant Produced by Pseudomonas aeruginosa OBP1 Isolated from Petroleum Sludge by Pranjal Bharali; Bolin Kumar Konwar (pp. 1444-1460).

Pseudomonas aeruginosa strain OBP1, isolated from petroleum sludge, was used to produce biosurfactant from a modified mineral salt medium with 2% n-hexadecane as sole source of carbon. The crude biosurfactant was fractionated using TLC and HPLC. Using FTIR spectroscopy, ¹H NMR, and LC-MS analyses, the chemical structure of the purified fraction of crude biosurfactant was identified as rhamnolipid species. The LC-MS spectra show that monorhamnolipid (l-rhamnopyranosyl-β-hydroxydecanoyl-β- hydroxydecanoate, Rha-C₁₀-C₁₀) was produced in abundance with the predominant congener [M–H]⁻ ions for l-rhamnopyranosyl-l-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-Rha-C₁₀-C₁₀). Seven different carbon substrates and five nitrogen sources were examined for their effect on rhamnolipid production. Using n-hexadecane (20 g/l) as carbon substrate and urea along with (NH₄)₂SO₄ (2 g/l each) as nitrogen source was found to be the best, with a maximum yield of 4.8 g/l. The biosurfactant reduced the surface tension of water to 31.1 mNm⁻¹ with a critical micelle concentration of 45 mg/l. The biosurfactant showed a better emulsifying activity against a variety of hydrocarbon and achieved a maximum emulsion index of 82% for diesel. The purified biosurfactant showed a significant antibacterial activity against Staphylococcus aureus at a minimum inhibitory concentration of 8 μg/ml.

Keywords: Biosurfactant; Rhamnolipid; n-Hexadecane; Pseudomonas aeruginosa

Production of Fumaric Acid by Rhizopus oryzae: Role of Carbon–Nitrogen Ratio by Yueyue Ding; Shuang Li; Chang Dou; Yang Yu; He Huang (pp. 1461-1467).

Cytosolic fumarase, a key enzyme for the accumulation of fumaric acid in Rhizopus oryzae, catalyzes the dehydration of l-malic acid to fumaric acid. The effects of carbon–nitrogen ratio on the acid production and activity of cytosolic fumarase were investigated. Under nitrogen limitation stress, the cytosolic fumarase could keep high activity. With the urea concentration decreased from 2.0 to 0.1 g l⁻¹, the cytosolic fumarase activity increased by 300% and the production of fumaric acid increased from 14.4 to 40.3 g l⁻¹ and l-malic acid decreased from 2.1 to 0.3 g l⁻¹. Cytosolic fumarase could be inhibited by substrate analog 3-hydroxybutyric acid. With the addition of 3-hydroxybutyric acid (50 mM) in the fermentation culture, fumaric acid production decreased from 40.3 to 14.1 g l⁻¹ and l-malic acid increased from 0.3 to 5.4 g l⁻¹.

Keywords: Fumaric acid; Cytosolic fumarase; Substrate analog; Rhizopus oryzae

Production of Lipids Containing High Levels of Docosahexaenoic Acid by a Newly Isolated Microalga, Aurantiochytrium sp. KRS101 by Won-Kyung Hong; Dina Rairakhwada; Pil-Soo Seo; Sung-Yong Park; Byung-Ki Hur; Chul Ho Kim; Jeong-Woo Seo (pp. 1468-1480).

In the present study, a novel oleaginous Thraustochytrid containing a high content of docosahexaenoic acid (DHA) was isolated from a mangrove ecosystem in Malaysia. The strain identified as an Aurantiochytrium sp. by 18S rRNA sequencing and named KRS101 used various carbon and nitrogen sources, indicating metabolic versatility. Optimal culture conditions, thus maximizing cell growth, and high levels of lipid and DHA production, were attained using glucose (60 g l⁻¹) as carbon source, corn steep solid (10 g l⁻¹) as nitrogen source, and sea salt (15 g l⁻¹). The highest biomass, lipid, and DHA production of KRS101 upon fed-batch fermentation were 50.2 g l⁻¹ (16.7 g l⁻¹ day⁻¹), 21.8 g l⁻¹ (44% DCW), and 8.8 g l⁻¹ (40% TFA), respectively. Similar values were obtained when a cheap substrate like molasses, rather than glucose, was used as the carbon source (DCW of 52.44 g l⁻¹, lipid and DHA levels of 20.2 and 8.83 g l⁻¹, respectively), indicating that production of microbial oils containing high levels of DHA can be produced economically when the novel strain is used.

Keywords: Aurantiochytrium sp.; Heterotrophic microalga; Lipid; Docosahexaenoic acid

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #8)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.164, #8)