Current Biotechnology (v.2, #2)

Reality Check: Cancer Stem Cell Route to Cancer by Meera Nair, Richa Jain, Pooja Saxena (89-105).
Cancer stem cells originate from normal cells and gain proliferation to grow clonally into metastatic tumors.They stay hidden as seeds, hence their identification and prognosis are necessary for eradication of cancer from root level.Different models are put forth to prove cancer stem cells subsistence via cancer epidemiology, carcinogenesis,biochemistry and molecular biology. This review provides an update on fundamental role of cancer stem cells in cancerprogression, with a brief overview on its biochemistry and molecular biology along with its in vitro and in vivo analysisusing modern assays with promising therapeutic regimens.

The most serious pre-harvest disease of avocado in South Africa is still Avocado Black spot (Cercospora spot).Two methods were compared to assess Cercospora infection. The conventional assessment method (CA) relies on adisease intensity rating system that classifies fruit as local, export or rejects market quality. The disease incidence andseverity assessment method (ISA) combines spore release intensity (through a weather based prediction formula) withdisease intensity on fruit (as measured for specific lesion sizes representing different infection periods). In the currentstudy, Fuerte avocado fruit from the Limpopo Province in South Africa was subjected to both CA and ISA. Data obtainedthrough CA proved to be very limited and only reflected total disease control at the end of the season. ISA allowed a moredetailed assessment of the spray program and it was possible to evaluate the efficiency of individual spray applications.Both the effect of environmental influences on disease severity and the resulted disease intensity can also be evaluatedthrough ISA visualization.

Butanol Production by Submerged Fermentation of White Grape Pomace by Laurent Law, Noemi Gutierrez (114-116).
White grape pomace, containing residual fermentable sugar, has been evaluated as a substrate for production ofbutanol by submerged fermentation using Clostridium saccharobutylicum P262. Using an initial value of pH 5.5, aninoculum size of 10% (v/v) and addition of supplementary yeast extract (10 g/l), butanol concentrations of up to 6 g/l wereobtained, at a yield, based on sugar utilized, of 0.2 g/g. Significantly, the requirement for supplementary yeast extractcould be replaced by selected salts, indicating that white grape pomace is not lacking in any organic nutrients for growthof, and butanol production by, C. saccharobutylicum, and hence has potential commercial application.

The effects of operational parameters (pH, contact time, initial Cd(II) concentration, and temperature), kinetics,equilibrium, and thermodynamics of Cd(II) biosorption by compound bioflocculant (CBF), produced by mixed culture ofRhizobium radiobacter F2 and Bacillus sphaeicus F6, were investigated. Desorption-reuse study and Fourier transforminfrared (FTIR) spectra were also examined towards a better standing of the biosorption mechanism. The maximumbiosorption capacity was obtained at pH of 7.0 and contact time of 60 min. The biosorption data obeyed pseudo-first-ordermodel better than pseudo-second-order equation and Elovich equation. The equilibrium data were analyzed using theLangmuir, Freundlich and Redlich-Peterson isotherm models, and Langmuir isotherm gave the best fit. Thethermodynamic parameters indicated that the biosorption process was endothermic and spontaneous. The adsorbed Cd(II)on CBF could be effectively desorbed by HCl and NaOH, and about 65% of the initial biosorption capacity was regainedafter 2 cycles of biosorption-desorption-elution using HCl as the desorbing agent. The FTIR characterization indicatedthat -OH, -NH, -C=O, and ?COOH and C-N groups may be involved in the interaction. It was concluded that CBF in thisstudy can be used as an effective and environmental friendly biosorbent for the removal of Cd(II) ions from aqueoussolution.

Statistical Modeling and Optimization of Chitosan Production from Absidia coerulea Using Response Surface Methodology by Ahmed ElMekawy, Ashraf F. El-Baz, Emad A. Soliman, Samuel Hudson (125-133).
Optimization of chitosan production from Absidia coerulea mycelia was presented through a three-level fullfactorial design in terms of nitrogen source, glucose concentration, incubation time and temperature. The four factors,individually or interacted, had significant effects (95%) on chitosan production. The highest chitosan content (0.1829 g/l)was obtained after 72 h at 32°C using a medium containing ammonium sulfate (14 g/l) and glucose (20 g/l). Chitosanproduction was further optimized to improve yield using the response surface methodology. Various second orderquadratic models were generated for each nitrogen source with respect to the other studied growth conditions.Subsequently, the optimum chitosan production factors were scaled up in a bioreactor. The maximum dry cell weight wasnearly doubled on comparing the propagation in the shake flask level (7.841 g/l) against the bioreactor level (13.87 g/l), aswell the incubation time was reduced from 72 h to 48 h.

High Podophyllotoxin Producing Hairy Root Line of Linum Album: Influence of Carbohydrates, Temperature and Photoperiod by Sunita Farkya, Gaurav Rajouria, Vinod Kumar, Virendra S. Bisaria (134-141).
High producing hairy root line of Linum album LYR2i was established following transformation of segmentsof aseptically grown seedlings with A. rhizogens LBA9402. The transformed cultures showed rapid growth andaccumulation of comparatively higher content of podophyllotoxin (PT) and 6-methoxy podophyllotoxin (6-MPT). Hairyroot line LYR2i was subjected to various media, carbon sources, temperatures, and photoperiodic conditions to assesstheir effect for optimum growth and production in shake flask. Only B5 gamborg medium was conducive for growth ofhairy root line LYR2i. Sucrose was preferentially consumed by the culture for highest growth and volumetricproductivity. Dark conditions supported hairy root growth and continuous light enhanced formation of PT and 6-MPT ingrowing hairy root line LYR2i. On the basis of above results detailed batch kinetics was established which featured 11.62g/l of biomass with a volumetric productivity of 11.30 mg/l.d of lignan in Gamborg B5 medium supplemented with 30 g/Lsucrose under 16/8 light/dark regime at 25°C ?1°C.

Aim of the present study was to verify the utility of industrial waste material in the production of an enzymepenicillin G acylase (PGA). Different industrial waste material such as black liquor, molasses, and distillery spent washwere used as a complex organic source (COS) for the production of PGA. Comparatively, black liquor showed higheractivity (24±0.8%) and microbial growth (54±1.7%) than the cornsteep liquor a conventional COS. Interestingly, theoptimum pH (7.2), temperature (23 °C), and growth time (23 h) obtained for black liquor was similar as obtained forcornsteep liquor. In the presence of peptone, yeast extract and optimized black liquor concentration, 39±1.3% increase inPGA activity (3.97±0.12 U/mg) was observed. The black liquor was stored at 8±2°C and used for the production of PGAfor 12 months. The average PGA activity obtained over a period of 12 months was 3.48±0.85 U/mg. The black liquorobtained from different source showed similar PGA activity (3.82±0.15 U/mg). In comparison with uninoculated growthmedium, inoculated medium showed 52±1.5% decrease in chemical oxygen demand and 55±1.6%, 59±1.9%, and91±3.3% decrease in total protein, amino acid and total sugar respectively after 24 h of growth. Comparatively, sevendifferent Escherichia coli strains showed 32±1.5% higher average PGA activity in the presence of black liquor. Theutilization of our findings may results in the reduction of overall production cost of PGA and a high value byproduct fromwaste material to the black liquor generating industries.

Cholesterol oxidase (COX, E.C., catalyses the oxidation of cholesterol to 4-cholesten-3-one with thereduction of oxygen to hydrogen peroxide. Qualitative analysis of bacterial strain (Bacillus sp. BT COX-T3) gave Grampositive rod-shaped colonies with high cholesterol degradation. Organic solvent tolerant microorganisms are novel groupof extremophilic microorganisms that have developed resistance to withstand solvent toxicity. These organisms play animportant role in biotransansformation and bioconversion of organic compounds. An organic solvent-tolerant Bacillus sp.BT COX-T3 obtained after primary screening for production of extracellular COX transformed cholesterol to 4-cholesten-3-one in toluene, and resulted in a higher COX production in a biphasic medium due to an increase in solubility ofcholesterol in the medium making it easily available to be metabolized by the organism. A simple screening method for6 β-hydroperoxycholest-4-en-3-one formed by Bacillus sp. COX-T3 was devised in this study and bioconversion wasmonitored spectrophotometerically. Presence of cholesterol in 20% (v/v) toluene was observed to be most effectivebiotransformation system for bacterial COX production. A maximum COX activity of 0.892 U/ml was obtained at 45°Cby using 8% (v/v) inoculum, 0.1% (w/v) cholesterol as the main carbon source and 0.5% (w/v) yeast extract. Amongstvarious salt ions only Co2+ ions improved the COX production by Bacillus sp. COX-T3. Moreover, the cholesterolprovided in the bioconversion system was completely transformed by Bacillus sp. COX-T3. Bacillus sp COX-T3effectively degraded cholesterol in the presence of toluene and the extracellular COX produced by it was highly stable atthe end of 144 h.

An extracellular peroxidase produced by a bacterial isolate Bacillus sp. F31 was purified by successive(NH4)2SO4 fractionation, dialysis and DEAE-cellulose anion-exchange chromatography to 14.5-fold with a yield of~7.0%. The molecular mass of purified peroxidase was determined to be ~37 kDa by SDS-PAGE. The optimumtemperature and pH for purified peroxidase were 37°C and 5.5, respectively. However, the bacterial peroxidase was activeat an alkaline pH too, unlike previously reported microbial peroxidases. The Km(H2O2) and Vmax(H2O2) of the bacterialperoxidase by using H2O2 as a substrate were found to be 0.133 mM and 33.3 U/mg/min, respectively representing aKcat(H2O2) of 20.53/sec. The Bacillus sp. F31 peroxidase exhibited good affinity towards OPD as observed from measuredKm(OPD) and Vmax(OPD) of 5.138 mM and 29.41 U/mg/min, respectively (Fig. 11). The half-life of the purified peroxidasewas approximately 155 min at 37°C. The peroxidase activity was found to be stimulated only in the presence of Mg+2 (1,3 and 5 mM) ions added to the reaction cocktail whereas other salt-ions (Li2CO3, ZnSO4, KH2PO4, NaCl, HgCl2, CaCl2,CuSO4, MnSO4 and FeSO4) inhibited the enzyme activity. All the selected detergents (SDS, Triton X-100, Tween 80 andTween 20) also had an inhibitory effect on the enzyme activity.

Agriculture industrial waste or byproducts could be a valuable biological material for synthesis of metalnanoparticles. Deoiled flaxseed meal is a byproduct of flaxseed oil industry. In present study its hydroalcoholic extract(70%) was assessed for the synthesis of silver nanoparticles. Synthesis of silver nanoparticles was confirmed andcharacterized by using UV-Vis spectrophotometer, X-ray diffraction (XRD), scanning electron microscope (SEM) andfourier transform infrared spectrum (FT-IR). The antimicrobial activity of silver nanoparticles was evaluated againstgram-negative (E. coli), gram-positive (S. aureus) bacteria, and mycotoxin producing fungi A. flavus and A. parasiticus.The results of XRD, SEM analysis confirmed the face centered cubic structure of colloidal silver nanoparticles havingparticle size 9.22 nm. FT-IR analysis showed phenolic components of flaxseed hydroalcoholic extract could beresponsible for the synthesis of silver nanoparticles. The resultant potent antimicrobial activity of synthesized silvernanoparticles could corroborate its usefulness in food and health product industry including flaxseed oil.

Purification and Characterization of Thermostable Cellulase from Soil Bacteria of Northwestern Himalayas by Sarika Sharma, Kuldeep Kaur, Vikas Sharma, Sandeep Shama (167-171).
Microbial degradation of cellulose has economic potential in enzyme industry as well as for the productionof biofuels from plant biomass. In this study bacteria were isolated from soil sample of Leh region and screened fordifferent hydrolases. The strain Bacillus SL4 was found as potent producer of cellulase. The enzyme from Bacillus SL4was purified by ion exchange chromatography and characterized. The molecular weight of enzyme was 82kDa. Enzymeshowed an optimum activity at pH 6.0 and highly stable between 55-65 °C. The crude cellulase had activity toward CMC,avicell, β - glucan and cellobiose, but there was no detectable activity on xylan and p-nitrophenyl- β-d-glucopyranoside(PNPG). The rate of CMC degradation was higher than any other substrates used in this study. The enzyme reported inthis paper was able to hydrolyze both - β-1, 4 and β -1,3 glycosidic linkages and thermostable at high temperature.