Current Biotechnology - Volume 2, Issue 2, 2013

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 cancer progression, with a brief overview on its biochemistry and molecular biology along with its in vitro and in vivo analysis using 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 a disease intensity rating system that classifies fruit as local, export or rejects market quality. The disease incidence and severity assessment method (ISA) combines spore release intensity (through a weather based prediction formula) with disease intensity on fruit (as measured for specific lesion sizes representing different infection periods). In the current study, Fuerte avocado fruit from the Limpopo Province in South Africa was subjected to both CA and ISA. Data obtained through CA proved to be very limited and only reflected total disease control at the end of the season. ISA allowed a more detailed 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 evaluated through ISA visualization.

White grape pomace, containing residual fermentable sugar, has been evaluated as a substrate for production of butanol by submerged fermentation using Clostridium saccharobutylicum P262. Using an initial value of pH 5.5, an inoculum size of 10% (v/v) and addition of supplementary yeast extract (10 g/l), butanol concentrations of up to 6 g/l were obtained, at a yield, based on sugar utilized, of 0.2 g/g. Significantly, the requirement for supplementary yeast extract could be replaced by selected salts, indicating that white grape pomace is not lacking in any organic nutrients for growth of, 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 of Rhizobium radiobacter F2 and Bacillus sphaeicus F6, were investigated. Desorption-reuse study and Fourier transform infrared (FTIR) spectra were also examined towards a better standing of the biosorption mechanism. The maximum biosorption capacity was obtained at pH of 7.0 and contact time of 60 min. The biosorption data obeyed pseudo-first-order model better than pseudo-second-order equation and Elovich equation. The equilibrium data were analyzed using the Langmuir, Freundlich and Redlich-Peterson isotherm models, and Langmuir isotherm gave the best fit. The thermodynamic 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 regained after 2 cycles of biosorption-desorption-elution using HCl as the desorbing agent. The FTIR characterization indicated that -OH, -NH, -C=O, and –COOH and C-N groups may be involved in the interaction. It was concluded that CBF in this study can be used as an effective and environmental friendly biosorbent for the removal of Cd(II) ions from aqueous solution.

High producing hairy root line of Linum album LYR2i was established following transformation of segments of aseptically grown seedlings with A. rhizogens LBA9402. The transformed cultures showed rapid growth and accumulation of comparatively higher content of podophyllotoxin (PT) and 6-methoxy podophyllotoxin (6-MPT). Hairy root line LYR2i was subjected to various media, carbon sources, temperatures, and photoperiodic conditions to assess their effect for optimum growth and production in shake flask. Only B5 gamborg medium was conducive for growth of hairy root line LYR2i. Sucrose was preferentially consumed by the culture for highest growth and volumetric productivity. Dark conditions supported hairy root growth and continuous light enhanced formation of PT and 6-MPT in growing hairy root line LYR2i. On the basis of above results detailed batch kinetics was established which featured 11.62 g/l of biomass with a volumetric productivity of 11.30 mg/l.d of lignan in Gamborg B5 medium supplemented with 30 g/L sucrose 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 enzyme penicillin G acylase (PGA). Different industrial waste material such as black liquor, molasses, and distillery spent wash were used as a complex organic source (COS) for the production of PGA. Comparatively, black liquor showed higher activity (24±0.8%) and microbial growth (54±1.7%) than the cornsteep liquor a conventional COS. Interestingly, the optimum pH (7.2), temperature (23 °C), and growth time (23 h) obtained for black liquor was similar as obtained for cornsteep liquor. In the presence of peptone, yeast extract and optimized black liquor concentration, 39±1.3% increase in PGA activity (3.97±0.12 U/mg) was observed. The black liquor was stored at 8±2°C and used for the production of PGA for 12 months. The average PGA activity obtained over a period of 12 months was 3.48±0.85 U/mg. The black liquor obtained from different source showed similar PGA activity (3.82±0.15 U/mg). In comparison with uninoculated growth medium, inoculated medium showed 52±1.5% decrease in chemical oxygen demand and 55±1.6%, 59±1.9%, and 91±3.3% decrease in total protein, amino acid and total sugar respectively after 24 h of growth. Comparatively, seven different Escherichia coli strains showed 32±1.5% higher average PGA activity in the presence of black liquor. The utilization of our findings may results in the reduction of overall production cost of PGA and a high value byproduct from waste material to the black liquor generating industries.

Cholesterol oxidase (COX, E.C.1.1.3.6), catalyses the oxidation of cholesterol to 4-cholesten-3-one with the reduction of oxygen to hydrogen peroxide. Qualitative analysis of bacterial strain (Bacillus sp. BT COX-T3) gave Gram positive rod-shaped colonies with high cholesterol degradation. Organic solvent tolerant microorganisms are novel group of extremophilic microorganisms that have developed resistance to withstand solvent toxicity. These organisms play an important 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 of cholesterol in the medium making it easily available to be metabolized by the organism. A simple screening method for 6β-hydroperoxycholest-4-en-3-one formed by Bacillus sp. COX-T3 was devised in this study and bioconversion was monitored spectrophotometerically. Presence of cholesterol in 20% (v/v) toluene was observed to be most effective biotransformation system for bacterial COX production. A maximum COX activity of 0.892 U/ml was obtained at 45°C by using 8% (v/v) inoculum, 0.1% (w/v) cholesterol as the main carbon source and 0.5% (w/v) yeast extract. Amongst various salt ions only Co2+ ions improved the COX production by Bacillus sp. COX-T3. Moreover, the cholesterol provided in the bioconversion system was completely transformed by Bacillus sp. COX-T3. Bacillus sp COX-T3 effectively degraded cholesterol in the presence of toluene and the extracellular COX produced by it was highly stable at the 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 optimum temperature and pH for purified peroxidase were 37°C and 5.5, respectively. However, the bacterial peroxidase was active at an alkaline pH too, unlike previously reported microbial peroxidases. The Km(H2O2) and Vmax(H2O2) of the bacterial peroxidase by using H2O2 as a substrate were found to be 0.133 mM and 33.3 U/mg/min, respectively representing a Kcat(H2O2) of 20.53/sec. The Bacillus sp. F31 peroxidase exhibited good affinity towards OPD as observed from measured Km(OPD) and Vmax(OPD) of 5.138 mM and 29.41 U/mg/min, respectively (Fig. 11). The half-life of the purified peroxidase was 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 and Tween 20) also had an inhibitory effect on the enzyme activity.

Agriculture industrial waste or byproducts could be a valuable biological material for synthesis of metal nanoparticles. 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 and characterized by using UV-Vis spectrophotometer, X-ray diffraction (XRD), scanning electron microscope (SEM) and fourier transform infrared spectrum (FT-IR). The antimicrobial activity of silver nanoparticles was evaluated against gram-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 having particle size 9.22 nm. FT-IR analysis showed phenolic components of flaxseed hydroalcoholic extract could be responsible for the synthesis of silver nanoparticles. The resultant potent antimicrobial activity of synthesized silver nanoparticles could corroborate its usefulness in food and health product industry including flaxseed oil.

Microbial degradation of cellulose has economic potential in enzyme industry as well as for the production of biofuels from plant biomass. In this study bacteria were isolated from soil sample of Leh region and screened for different hydrolases. The strain Bacillus SL4 was found as potent producer of cellulase. The enzyme from Bacillus SL4 was purified by ion exchange chromatography and characterized. The molecular weight of enzyme was 82kDa. Enzyme showed 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 in this paper was able to hydrolyze both -β-1, 4 and β-1,3 glycosidic linkages and thermostable at high temperature.