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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #8)

Agrobacterium-Mediated Genetic Transformation of Pogostemon cablin (Blanco) Benth. Using Leaf Explants: Bactericidal Effect of Leaf Extracts and Counteracting Strategies by Anamika Paul; Souvika Bakshi; Debee Prasad Sahoo; Mohan Chandra Kalita; Lingaraj Sahoo (pp. 1871-1895).

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 μM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait.

Keywords: Agrobacterium ; Leaf disk; l-glutamine; Patchouli; Physiological age; Tween 20

Butanol Production from Cane Molasses by Clostridium saccharobutylicum DSM 13864: Batch and Semicontinuous Fermentation by Ye Ni; Yun Wang; Zhihao Sun (pp. 1896-1907).

Clostridium acetobutylicum strains used in most Chinese ABE (acetone–butanol–ethanol) plants favorably ferment starchy materials like corn, cassava, etc., rather than sugar materials. This is one major problem of ABE industry in China and significantly limits the exploitation of cheap waste sugar materials. In this work, cane molasses were utilized as substrate in ABE production by Clostridium saccharobutylicum DSM 13864. Under optimum conditions, total solvent of 19.80 g/L (13.40 g/L butanol) was reached after 72 h of fermentation in an Erlenmeyer flask. In a 5-L bioreactor, total solvent of 17.88 g/L was attained after 36 h of fermentation, and the productivity and yield were 0.50 g/L/h and 0.33 g ABE/g sugar consumption, respectively. To further enhance the productivity, a two-stage semicontinuous fermentation process was steadily operated for over 8 days (205 h, 26 cycles) with average productivity (stage II) of 1.05 g/L/h and cell concentration (stage I) of 7.43 OD₆₆₀, respectively. The average batch fermentation time (stage I and II) was reduced to 21−25 h with average solvent of 15.27 g/L. This study provides valuable process data for the development of industrial ABE fermentation process using cane molasses as substrate.

Keywords: Acetone–butanol–ethanol; Fermentation; Cane molasses; Clostridium saccharobutylicum ; Semicontinuous

Advances and Developments in Strategies to Improve Strains of Saccharomyces cerevisiae and Processes to Obtain the Lignocellulosic Ethanol−A Review by C. Laluce; A. C. G. Schenberg; J. C. M. Gallardo; L. F. C. Coradello; S. R. Pombeiro-Sponchiado (pp. 1908-1926).

The conversion of biomass into ethanol using fast, cheap, and efficient methodologies to disintegrate and hydrolyse the lignocellulosic biomass is the major challenge of the production of the second-generation ethanol. This revision describes the most relevant advances on the conversion process of lignocellulose materials into ethanol, development of new xylose-fermenting strains of Saccharomyces cerevisiae using classical and modern genetic tools and strategies, elucidation of the expression of some complex industrial phenotypes, tolerance mechanisms of S. cerevisiae to lignocellulosic inhibitors, monitoring and strategies to improve fermentation processes. In the last decade, numerous engineered pentose-fermenting yeasts have been developed using molecular biology tools. The increase in the tolerance of S. cerevisiae to inhibitors is still an important issue to be exploited. As the industrial systems of ethanol production operate under non-sterile conditions, microbial subpopulations are generated, depending on the operational conditions and the levels of contaminants. Among the most critical requirements for production of the second-generation ethanol is the reduction in the levels of toxic by-products of the lignocellulosic hydrolysates and the production of low-cost and efficient cellulosic enzymes. A number of procedures have been established for the conversion of lignocellulosic materials into ethanol, but none of them are completely satisfactory when process time, costs, and efficiency are considered.

Keywords: Biomass pretreatments and hydrolysis; Pentose-fermenting yeasts; Pentose metabolism in yeast; Yeast tolerance to biomass hydrolysates; Genes related to yeast stresses; Yeast diversity and dynamics; Saccharomyces cerevisiae

Enhancement of the Activity and Enantioselectivity of Lipase by Sol–Gel Encapsulation Immobilization onto β-cyclodextrin-Based Polymer by Elif Yilmaz; Mehmet Sezgin (pp. 1927-1940).

Candida rugosa lipase was encapsulated within a chemically inert sol–gel support prepared by polycondensation with tetraethoxysilane and octyltriethoxysilane in the presence of β-cyclodextrin-based polymer. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenylpalmitate and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yield of the encapsulated lipase was 65 U/g, which is 7.5 times higher than that of the covalently immobilized lipase. The β-cyclodextrin-based encapsulated lipases had higher conversion and enantioselectivity compared with covalently immobilized lipase. The study confirms an excellent enantioselectivity (E >300) for the encapsulated lipase with an enantiomeric excess value of 98% for S-naproxen.

Keywords: Candida rugosa lipase; β-cyclodextrin; Sol–gel encapsulation; Enantioselective hydrolysis; S-naproxen

In Vitro Cultures of Schisandra chinensis (Turcz.) Baill. (Chinese Magnolia Vine)—a Potential Biotechnological Rich Source of Therapeutically Important Phenolic Acids by Agnieszka Szopa; Halina Ekiert (pp. 1941-1948).

The contents of free phenolic acids and cinnamic acid were determined using an HPLC method in methanolic extracts from biomass of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) at different stages of organogenesis, cultured in vitro on a few variants of Murashige and Skoog (MS) medium, containing different concentrations of plant growth regulators 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (from 0.1 to 3.0 mg/l) and in extracts from overground parts of plants growing in vivo. Six of 12 analysed compounds were detected in all extracts: chlorogenic, p-coumaric, p-hydroxybenzoic, protocatechuic, salicylic and syringic acids. Total contents of the examined metabolites in biomass of shoot-differentiating callus culture cultivated on six MS medium variants were dependent on concentrations of growth regulators in the media and ranged from 14.90 to 60.05 mg/100 g d.w. Total contents of the compounds in biomass extracts from undifferentiating callus culture maintained only on two of six MS medium variants were higher and amounted to 74.54 and 78.24 mg/100 g d.w. Maximum total contents of phenolic acids in both types of in vitro cultures were greater than in fruits (55.73 mg/100 g d.w.) and leaves (4.55 mg/100 g d.w.) of plants gowning in vivo. Chlorogenic acid and salicylic acid were the main compounds identified in biomass extracts of shoot-differentiating callus cultures (max 22.60 and 21.17 mg/100 g d.w., respectively), while chlorogenic acid (max 38.43 mg/100 g d.w.) and protocatechuic acid (max 20.95 mg/100 g d.w.) prevailed in the extracts from undifferentiating callus cultures. Other compounds dominated in fruits, namely p-coumaric acid (23.36 mg/100 g d.w.) and syringic acid (14.96 mg/100 g d.w.). This is the first report on biochemical potential of cells from S. chinensis in vitro cultures to produce the biologically active phenolic acids. These are the first results on the analysis of this group of metabolites in overground parts of plants growing in vivo, too.

Keywords: Schisandraceae; Shoot-differentiating callus culture; Undifferentiating callus culture; Chlorogenic acid; Protocatechuic acid; Salicylic acid

Topoisomerase Inhibition, Nucleolytic and Electrolytic Contribution on DNA Binding Activity Exerted by Biological Active Analogue of Coordination Compounds by Mohan N. Patel; Bhupesh S. Bhatt; Promise A. Dosi (pp. 1949-1968).

The neutral mononuclear copper complexes with the quinolone antibacterial drug ciprofloxacin and bipyridine derivatives have been synthesized and characterized. Complexes were screened for their antibacterial activity against three Gram⁽⁻⁾ and two Gram⁽⁺⁾ bacteria, and study suggests inhibition of gyrase activity by metal complexes as the possible mechanism. The nucleolytic activity of adducts was carried out on double stranded pUC19 DNA using gel electrophoresis in the presence of radical scavenging agents that suggest hydrolytic cleavage mechanism for plasmid DNA.

Keywords: Gyrase inhibition; DNA binding free energy; Radical scavenging agents; Thermal denaturation study; Hydrolytic cleavage

Sugar Ester Synthesis by Thermostable Lipase from Streptomyces thermocarboxydus ME168 by Aran H-Kittikun; Poonsuk Prasertsan; Wolfgang Zimmermann; Phisit Seesuriyachan; Thanongsak Chaiyaso (pp. 1969-1982).

The extracellular lipase from Streptomyces thermocarboxydus ME168 was purified to 9.5-fold with 20% yield, following concentration by acetone precipitation, ion exchange chromatography (Resource Q) and gel filtration chromatography (Superdex 200), respectively. The purified enzyme had an apparent molecular mass of 21 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of the lipase was ASDFDDQILG and was different from most other reported lipase. The enzyme showed maximum activity at 50 °C with the half-life of 180 min at 65 °C. It showed high stability at a broad pH range of 5.5–9.5 and was thermostable at the temperature range of 25–60 °C. The K m and V _max were 0.28 mM and 1,428 U/mg, respectively, using p-nitrophenyl palmitate as substrate. It was active toward p-nitrophenyl ester with medium to long acyl chain (C₈–C₁₆). Lipase activity was inhibited by Zn²⁺, dithiothreitol (DTT), EDTA and some organic solvents, e.g., ethanol, acetone, dioxane, acetronitrile, tert-butanol and pyridine. Immobilized crude lipase of S. thermocarboxydus ME168 on celite could be used to synthesize sugar esters from glucose and vinyl acetate, vinyl butyrate or vinyl caproate in tert-butanol:pyridine (55:45 v/v) at 45 °C with conversion yields of 93, 67 and 55%, respectively.

Keywords: Streptomyces thermocarboxydus ; Lipase; Sugar esters; Vinyl acetate; Vinyl butyrate

Effect of Penicillin on Nitrite-Oxidizing Bacteria in Activated Sludge by Shao-Hui Zhang; Francis L. de los Reyes III (pp. 1983-1990).

The effects of penicillin G on the number and activity of nitrite-oxidizing bacteria were investigated in laboratory-scale reactors and batch tests. At a concentration of 100 mg L⁻¹, addition of penicillin G for short periods did not significantly affect nitrite oxidation, while addition for more than 2 months suppressed nitrite oxidation. Fluorescence in situ hybridization with 16S ribosomal RNA-targeted probes revealed a slight decrease in the abundance of Nitrospira, while Nitrobacter was not affected by addition of penicillin G for more than 39 days. The resistance of nitrite-oxidizing bacteria to penicillin appeared to be positively affected by intermittent aeration only when accompanied by denitrification; otherwise, the aeration mode (continuous or intermittent aeration) did not significantly affect the abundance of Nitrobacter and Nitrospira.

Keywords: Nitrite oxidation; Penicillin; Resistance

Induced Biofilm Cultivation Enhances Riboflavin Production by an Intertidally Derived Candida famata by Sayani Mitra; Dheeraj Thawrani; Priyam Banerjee; Ratan Gachhui; Joydeep Mukherjee (pp. 1991-2006).

The aim of the investigation was to ascertain if surface attachment of Candida famata and aeration enhanced riboflavin production. A newly designed polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) holding eight equidistantly spaced rectangular strips mounted radially on a circular disk allowed comparison of riboflavin production between CCFs with hydrophobic surface (PMMA-CCF), hydrophilic glass surface (GS-CCF), and 500-ml Erlenmeyer flask (EF). Riboflavin production (mg/l) increased from 12.79 to 289.96, from 54.44 to 238.14, and from 36.98 to 158.71 in the GS-CCF, EF, and PMMA-CCF, respectively, when C. famata was grown as biofilm-induced cultures in contrast to traditional planktonic culture. Production was correlated with biofilm formation and planktonic growth was suppressed in cultivations that allowed higher biofilm formation. Enhanced aeration increased riboflavin production in hydrophilic vessels. Temporal pattern of biofilm progression based on two-channel fluorescence detection of extracellular polymeric substances and whole cells in a confocal laser scanning microscope followed by application of PHLIP and ImageJ volume viewer software demonstrated early maturity of a well-developed, stable biofilm on glass in contrast to PMMA surface. A strong correlation between hydrophilic reactor surface, aeration, biofilm formation, and riboflavin production was established in C. famata. Biofilm culture is a new-found means to improve riboflavin production by C. famata.

Keywords: Candida famata ; Riboflavin; Biofilm; Confocal laser scanning microscopy

Effect of Feed Strategy on Methane Production and Performance of an AnSBBR Treating Effluent from Biodiesel Production by Giovanna Lovato; Roberto A. Bezerra; José A. D. Rodrigues; Suzana M. Ratusznei; Marcelo Zaiat (pp. 2007-2029).

The aim of this work was to investigate the effect of different feeding times (2, 4 and 6 h) and applied volumetric organic loads (4.5, 6.0 and 7.5 gCOD L⁻¹ day⁻¹) on the performance of an anaerobic sequencing batch biofilm reactor (AnSBBR) treating effluent from biodiesel production. Polyurethane foam cubes were used as inert support in the reactor, and mixing was accomplished by recirculating the liquid phase. The effect of feeding time on reactor performance showed to be more pronounced at higher values of applied volumetric organic loads (AVOLs). Highest organic material removal efficiencies achieved at AVOL of 4.5 gCOD L⁻¹ day⁻¹ were 87 % at 4-h feeding against 84 % at 2-h and 6-h feeding. At AVOL of 6.0 gCOD L⁻¹ day⁻¹, highest organic material removal efficiencies achieved with 4-h and 6-h feeding were 84 %, against 71 % at 2-h feeding. At AVOL of 7.5 gCOD L⁻¹ day⁻¹, organic material removal efficiency achieved with 4-h feeding was 77 %. Hence, longer feeding times favored minimization of total volatile acids concentration during the cycle as well as in the effluent, guaranteeing process stability and safety.

Keywords: AnSBBR; Methane production; Feeding time; Applied volumetric organic load; Biodiesel effluent

Effects of Selenium and Light Wavelengths on Liquid Culture of Cordyceps militaris Link by J. Z. Dong; M. R. Liu; C. Lei; X. J. Zheng; Y. Wang (pp. 2030-2036).

To investigate the effects of selenium and light wavelengths on the growth of liquid-cultured Cordyceps militaris and the main active components’ accumulation, culture conditions as selenium selenite concentrations and light of different wavelengths were studied. The results are: adenosine accumulation proved to be significantly selenium dependent (R ² = 0.9403) and cordycepin contents were determined to be not significantly selenium dependent (R ² = 0.3845) but significantly enhanced by selenium except for 20 ppm; there were significant differences in cordycepin contents, adenosine contents, and mycelium growth caused by light wavelengths: cordycepin, blue light > pink light > daylight, darkness, red light; adenosine, red light > pink light, darkness, daylight, blue light; and mycelium growth, red light > pink light, darkness, daylight > blue light. In conclusion, light wavelength had a significant influence on production of mycelia, adenosine, and cordycepin, so lightening wavelength should be changed according to target products in the liquid culture of C. militaris.

Keywords: Cordyceps militaris ; Cordycepin; Adenosine; Light wavelength; Selenium; Mycelium growth

Angiotensin I-Converting Enzyme Inhibitory Proteins and Peptides from the Rhizomes of Zingiberaceae Plants by Maneerat Yodjun; Aphichart Karnchanatat; Polkit Sangvanich (pp. 2037-2050).

Ammonium sulphate cut protein extracts, and their pepsin hydrolysates, from the rhizomes of 15 plants in the Zingiberaceae family were screened for their in vitro angiotensin I-converting enzyme inhibitory (ACEI) activity. The protein extract from Zingiber ottensii had the highest ACEI activity (IC₅₀ of 7.30 × 10⁻⁷ mg protein/mL) and was enriched for by SP Sepharose chromatography with five NaCl step gradients 0, 0.25, 0.50, 0.75 and 1 M NaCl collecting the corresponding five fractions. The highest ACEI activity was found in the F75 fraction, which appeared to contain a single 20.7-kDa protein, suggesting enrichment to or near to homogeneity. The ACEI activity of the F75 fraction was moderately thermostable (−20–60 °C), showed >80% activity across a broad pH range of 4–12 (optimal at pH 4–5) and appeared as a competitive inhibitor of ACE (K _i of 9.1 × 10⁻⁵ mg protein/mL). For the pepsin hydrolysates, that from Zingiber cassumunar revealed the highest ACEI activity (IC₅₀ of 0.38 ± 0.012 mg/mL), was enriched to a single active hexapeptide by RP-HPLC with a strong ACEI activity (IC₅₀ of 0.011 ± 0.012 mg/mL) and acted as a competitive inhibitor of ACE (K _i of 1.25 × 10⁻⁶ mg protein/mL).

Keywords: Angiotensin I-converting enzyme; Proteins; Peptides; Zingiberaceae plant

Examining the Potential of Plasma-Assisted Pretreated Wheat Straw for Enzyme Production by Trichoderma reesei by Divanery Rodriguez-Gomez; Linda Lehmann; Nadja Schultz-Jensen; Anne Belinda Bjerre; Timothy John Hobley (pp. 2051-2063).

Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw (which contained antibiotics) was best suited for the production of xylanases (110 IU ml⁻¹) and cellulases (0.5 filter paper units (FPU) ml⁻¹). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml⁻¹) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml⁻¹) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising and suitable substrate for cellulase and hemicellulase production.

Keywords: Pretreatment; Ozonisation; Avicel; Enzyme production; Fungal growth

Initial Proteome Analysis of Caffeine-Induced Proteins in Aspergillus tamarii Using Two-Dimensional Fluorescence Difference Gel Electrophoresis by Gerardo Gutiérrez-Sánchez; James Atwood; V. S. Kumar Kolli; Sévastianos Roussos; Christopher Augur (pp. 2064-2077).

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.

Keywords: Two-dimensional electrophoresis; Difference gel electrophoresis; Proteomics; Filamentous fungi; Caffeine degradation; Mass spectrometry

Enhanced Biodecolorization of Reactive Dyes by Basidiomycetes Under Static Conditions by Ismat Bibi; Haq Nawaz Bhatti (pp. 2078-2090).

This study presents the biodecolorization potential of basidiomycete fungi Trametes hirsuta, Pycnoporus sp., and Irpex sp. for different reactive dyes viz. Reactive Red 120, Remazol Brilliant Blue R (RBBR), Reactive Orange G, and Reactive Orange 16 under static and shaking conditions. The screening trials revealed that T. hirsuta exhibited maximum potential (83.75 %) for biodecolorization of RBBR dye under static conditions after the fifth day of incubation. However, the rate of biodecolorization of RBBR dye by Pycnoporus sp. was much slow and reached maximum (81.25 %) after 15 days of incubation under shaking conditions. By process optimization, enhanced decolorization (91.2 %) of RBBR by T. hirsuta was achieved at pH 5.5 within 24 h using a defined salt medium amended with p-coumaric acid under static conditions. pH was found to be an important parameter for the enzymatic system involved in RBBR dye decolorization by T. hirsuta and Pycnoporus sp. Biodecolorization of RBBR dye was determined by a reduction in optical density at the wavelength of maximum absorbance (λ, 578 nm) by UV–vis spectrophotometer. The shift in maximum wavelength toward shorter/longer wavelength in UV–vis scanning spectrum revealed the degradation of RBBR dye into different transformation products.

Keywords: White rot fungi; Reactive dyes; Biodegradation; Peroxidases

Effects of Different Amino Acids in Culture Media on Surfactin Variants Produced by Bacillus subtilis TD7 by Jin-Feng Liu; Juan Yang; Shi-Zhong Yang; Ru-Qiang Ye; Bo-Zhong Mu (pp. 2091-2100).

Surfactin produced by Bacillus subtilis has different variants, which are affected by the composition of substrate available. To demonstrate the effects of amino acids on surfactin variants, B. subtilis TD7 was cultivated under the same conditions but with different amino acids supplied in media, respectively, and the type as well as the proportion of surfactin variants produced was analyzed with electrospray ionization mass spectrometry and gas chromatography–mass spectrometry. The result shows that the addition of different amino acids significantly influences the proportion of surfactin variants with different fatty acids. When Arg, Gln, or Val was added to the culture medium of B. subtilis TD7, the proportion of produced surfactin variants with even β-hydroxy fatty acids significantly increased, while the addition of Cys, His, Ile, Leu, Met, Ser, or Thr enhanced the proportion of surfactin variants with odd β-hydroxy fatty acids markedly. This result may be of some reference value in enhancing the production of specific surfactin variants as well as in the research on the relationship between culture media and the corresponding products of a certain bacterium.

Keywords: Bacillus subtilis ; Surfactin; Amino acid; Hydroxy fatty acid; ESI-MS; GC/MS

The Impact of Long-Term In Vitro Expansion on the Senescence-Associated Markers of Human Adipose-Derived Stem Cells by Wan Kamarul Zaman Wan Safwani; Suzana Makpol; Somasundaram Sathapan; Kien Hui Chua (pp. 2101-2113).

Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P < 0.05). The data showed that growth of ASCs reduced in long-term culture and the ß-galactosidase activity was significantly increased at later passage (P20). The morphology of ASCs in long-term culture showed the manifestation of senescent feature at P15 and P20. Significant alteration in the senescence-associated genes expression levels was observed in MMP1, p21, Rb and Cyclin D1 at P15 and P20. Significant increase in CD45 and HLA DR DQ DP surface marker was observed at P20. While cell cycle analysis showed significant decrease in percentage of ASCs at S and G2/M phase at later passage (P15). Our data showed ASCs cultured beyond P10 favours the senescence pathway and its clinical usage in cell-based therapy may be limited.

Keywords: Adipose-derived stem cells; Long-term culture; Senescence; Cell-based therapy; Regenerative medicine

Granulation of Nitrifying Bacteria in a Sequencing Batch Reactor for Biological Stabilisation of Source-Separated Urine by Fei-Yun Sun; Ya-Jing Yang; Wen-Yi Dong; Ji Li (pp. 2114-2126).

Biological stabilisation of human urine highly depends on the abundance and activities of nitrifying bacteria. However, it is quite difficult to enrich nitrifiers as bio-aggregation by self-immobilized biomass. In this study, granulation of nitrifying bacteria involving inoculation strategy was developed. Two sequencing batch reactors, the one inoculated with nitrifying bacteria and the other inoculated with aerobic granules, were operated in laboratory side by side with a feeding of urine solution. Aerobic nitrifying granules (ANG), with compact morphological structure and good nitrifying activity, were achieved in the reactor inoculated with aerobic granules. Enrichment of nitrifying bacteria favors the nutrient uptake, and hence, to obtain a high ammonia oxidation efficiency. Nonetheless, nitrite accumulation gradually dominated in reactor, partly attributes to a high concentration of free nitrous acid and free ammonia in bulk. The matured ANG had a rather stable microbial profile, as that a number of activated bacteria occupied the surface of granule. It was also found that ANG were much more impermeable than aerobic granules and activated sludge, which was demonstrated as smaller porosities, and therewith an excellent settleability. The results herein reveal that granulation of nitrifying bacteria could enrich the biomass to implement stabilisation of urine in biological way.

Keywords: Aerobic nitrifying granulation; Nitrification; Sequencing batch reactor (SBR); Source-separated urine

Effects of Light Intensity on the Growth and Lipid Accumulation of Microalga Scenedesmus sp. 11-1 Under Nitrogen Limitation by Junhan Liu; Cheng Yuan; Guangrong Hu; Fuli Li (pp. 2127-2137).

Scenedesmus spp. have been reported as potential microalgal species used for the lipid production. This study investigated the effects of light intensity (at three levels: 50, 250, and 400 μmol photons m⁻² s⁻¹) on the growth and lipid production of Scenedesmus sp. 11-1 under N-limited condition. Carotenoid to chlorophyll ratio was higher when algae 11-1 grew under 250 and 400 μmol photons m⁻² s⁻¹ than that under 50 μmol photons m⁻² s⁻¹, while protein contents was lower. Highest biomass yield (3.88 g L⁻¹), lipid content (41.1 %), and neutral lipid content (32.9 %) were achieved when algae 11-1 grew at 400 μmol photons m⁻² s⁻¹. Lipid production was slight lower at 250 μmol photons m⁻² s⁻¹ level compared to 400 μmol photons m⁻² s⁻¹. The major fatty acids in the neutral lipid of 11-1 were oleic acid (43–52 %), palmitic acid (24–27 %), and linoleic acid (7–11 %). In addition, polyunsaturated fatty acids had a positive correlation with total lipid production, and monounsaturated fatty acids had a negative one.

Keywords: Light intensity; Microalgae; Biodiesel; Neutral lipid; Fatty acid composition

A Superoxide Dismutase Purified from the Rhizome of Curcuma aeruginosa Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line by Wanwisa Moon-ai; Ploypat Niyomploy; Ruethairat Boonsombat; Polkit Sangvanich; Aphichart Karnchanatat (pp. 2138-2155).

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K _m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V _max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min⁻¹ mg protein⁻¹. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn²⁺and Fe²⁺ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC₅₀ = 14.36 ± 0.15 μg protein/ml).

Keywords: Superoxide dismutase; Nitric oxide; Macrophage RAW 264.7; Zingiberaceae plants

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #8)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.166, #8)