Evolutionary operation (EVOP) to optimize protease biosynthesis by Rhizopus oryzae

To optimize the operating conditions of any microbial process, evolutionary operation (EVOP) technique can be applied as a tool. This is very efficient to optimize the combined effect of two or three variables and their interaction on a biological process. The application of EVOP methodology to optimize the environmental conditions for any biological system has not yet been reported in the literature. In this paper firstly EVOP methodology for optimizing two-variable experiments has been outlined and then applied to optimize protease biosynthesis by Rhizopus oryzae (RO, IIT KGP).     

Experimental optimization of a step aeration waste treatment process

Evolutionary operation (EVOP) was used to experimentally investigate the optimum steady state operating conditions for a step aeration activated sludge waste treatment process. A laboratory scale two tank step aeration activated sludge unit with fixed total volume, total influent flow rate, recycle flow rate, and sludge wasting rate was employed. The volume ratio and flow rate ratio which minimized effluent chemical oxygen demand were determined. The results indicate that EVOP is a useful technique for improving the performance of biological processes.     

Optimization of physicochemical parameters for gallic acid production by evolutionary operation-factorial design technique

The interaction effects of five variable parameters on gallic acid yield by the application of Evolutionary Operation (EVOP)-factorial design technique is presented in this paper. The methodology combines the advantage of factorial techniques for designing experiments with five parameters and that of EVOP methodology for the systematic analysis of experimental results. It facilitates the selection of optimum conditions or directs the changes desired for individual parameters for the designing of subsequent experiments. Decision were taken according to the decision-making procedure available in the literature. Standard deviation and error limits based on 95% confidence were also calculated according to the relationship given in literature. The best combinations of the physicochemical parameters at optimal levels of 32 °C, 93% relative humidity, 4.5 pH, 72 h incubation period, 20 g substrate weight and 0.4:1 solid–liquid ratio resulted in gallic acid yield of 90.9% under modified solid-state fermentation (MSSF).     

Facile and statistical optimization of transfection conditions for secretion of foreign proteins from insect Drosophila S2 cells using green fluorescent protein reporter

Insect Drosophila melanogaster S2 cells were developed as a plasmid-based and therefore nonlytic expression system for functional foreign proteins. Transfection is an important step to introduce foreign target DNA into cells and should be properly optimized to obtain maximum production yield. Single factor search (SFS) methodology is still generally used to determine optimal condition in a biological system. Although this method is relatively simple to perform, it has many disadvantages such as not considering interactions between several factors and not covering the entire region of the solution pool. Therefore, we approached this optimization problem statistically with response surface (RSM) and evolutionary operation (EVOP) methodologies and compared the transfection efficiencies with the traditional SFS method. We employed secreted green fluorescent protein (GFP) as a reporter for determination of optimal transfection condition and secreted human erythropoietin (hEPO) as a confirming foreign model protein. Consequently, we arrived at the best optimal transient transfection condition (1 microg of plasmid DNA, 5 microg of lipofectin, 2 x 10(6) cells of initial cell number, and 18 h of transfection duration time) through a systematic access in a series of SFS, RSM, and EVOP. The secreted hEPO yield using optimal transient transfection condition by EVOP methodology was enhanced by about 1.8-fold compared to that of traditional SFS. This optimized transient transfection condition can be used as a basis for optimal stable transfections. A linear relationship between secreted GFP fluorescence intensity and secreted hEPO concentration indicated that facile and noninvasive determination of optimal transfection conditions for expression and secretion of foreign proteins in S2 cell cultures was made possible by simple measurement of GFP fluorescence.     

Optimization of Process Variables for Lipase Biosynthesis from <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> NRRL 5905 Using Evolutionary Operation Factorial Design Technique

Extracellular lipase was produced from Rhizopus oligosporus NRRL 5905 through solid state fermentation (SSF). To provide an optimum fermentation conditions for maximum lipase yield, five process variables (temperature, liquid–solid ratio, pH, incubation period and spore concentration) were optimized using evolutionary operation (EVOP) factorial design technique taking into account the interaction between the process variables. Optimization through EVOP resulted in around 3 fold increase in lipase activity (77 U gds⁻¹) at a liquid–solid ratio of 1.5:1, fermentation temperature of 35°C, initial fermentation pH 6, incubation period 5 days and a spore concentration of 10⁸ spores ml⁻¹.     

Production of proteolytic enzyme by Rhizopus oryzae in a bubble column bioreactor

Production of proteolytic enzyme in a bubble column bioreactor was carried out using the microorganism Rhizopus oryzae. Optimization of the production conditions were done adopting the EVOP (evolutionary operation technique). Studies on two variable and three variable parameters were carried out. The variable parameters were air flow rate, aeration time and incubation period. 8 hours aeration time per day, 1.66 vvm air flow rate and 2 days incubation was found to be optimum.     

Use of evolutionary operation (EVOP) factorial design technique to develop a bioprocess using grease waste as a substrate for lipase production

The aim of the present work was to develop a bioprocess using EVOP-factorial design technique employing grease waste as a substrate for the production of lipase. A newly isolated fungal strain of Penicillium chrysogenum was explored for the fermentation process. Solid-state fermentation (SSF) was carried out using grease waste and Czapek-dox medium, supplemented with wheat bran. The yield of lipase was 38 U/ml when SSF was carried out at 32 °C for 8 days and grease:wheat bran:Czapek-dox media in 1:1:2 (w/w/v). Different physicochemical parameters affecting the production of lipase were optimized through evolutionary operation (EVOP) factorial design technique and after optimization yield was enhanced up to 46 U/ml at 30 °C, pH 7.0 with 1:1:2 (w/w/v) grease waste:wheat bran:Czapek-dox media. Industrial grease waste has never been reported before for the production of industrially important lipase enzyme.     

Alkaline elution of rat testicular DNA: detection of DNA cross-links after in vivo treatment with chemical mutagens

The alkaline elution technique was used to measure DNA damage in the rat testis after intraperitoneal injection of 3 chemicals known to cause heritable mutations in rodents. These 3 chemicals are triethylenemelamine (TEM), mitomycin C, and cyclophosphamide. All three of these chemicals produced DNA damage which was readily detectable by alkaline elution. Both TEM and mitomycin C produced DNA interstrand cross-links, although TEM was a more potent cross-linker on an equimolar basis than mitomycin C. Cyclophosphamide produced both DNA cross-links and DNA strand breaks. Alkaline elution in the absence of proteinase K indicated that some of the strand breaks appeared to be closely associated with protein. These studied indicate that the alkaline elution technique is capable of detecting DNA damage in mammalian germ cells produced by chemical mutagens. This technique may prove useful as a screening tool for identifying chemicals which cause heritable mutations in mammals.     

Effect of reducing the top concentration used in the in vitro chromosomal aberration test in CHL cells on the evaluation of industrial chemical genotoxicity

A current concern with in vitro mammalian cell genotoxicity testing is the high frequency of false or misleading positive results caused in part by the past use of excessively high test concentrations. A dataset of 249 industrial chemicals used in Japan and tested for genotoxicity was analyzed. Of these, 116 (46.6%) were positive in the in vitro chromosomal aberration (CA) test, including 6 that were positive only at test concentrations >10mM. There were 59 CA-positive chemicals at test concentrations ≤ 1mM. At >1mM, 51 chemicals were CA-positive, including 13 Ames-positive chemicals, which were therefore not "missed" by the test battery. Thus, 38 potentially positive chemicals would not have been detected in the test battery if the top test concentration was limited to 1mM in CA test. Analysis of the relevance of CA results on the 38 missed chemicals was conducted based on a weight of evidence approach, including evaluations of effects of extreme culture conditions (low pH, high toxicity, or precipitation), in silico structural alert analysis, in vivo genotoxicity and carcinogenicity test data (where available), mode of action, or information from closely related chemicals. After an exhaustive review, there were four chemicals with some concern for human health risk assessment, nine with minimal concern, and the remaining 25 with negligible concern. We apply different top concentrations to the 38 missed chemicals to identify the most accurate approach for predicting the genotoxicity of industrial chemicals. Of these 2mM or 1mg/mL, whichever is higher, was the most effective in detecting these chemicals, i.e., relatively higher (8/13) or lower (17/25) detection among 13 chemicals with some or minimal concern, or 25 with negligible concern, respectively. Lower top concentration limits, 1mM or 0.5mg/mL, whichever is higher, are not as effective (2/13) for detecting these chemicals with concern. Therefore, we conclude 2mM or 1mg/mL, whichever is higher, would be an appropriate top concentration limit for testing industrial chemicals for chromosome damage.     

盐析萃取生物基化学品的研究进展

廉价生物质的生物炼制研究主要集中在菌种和发酵方面,对下游分离研究较少。廉价生物质资源的利用导致发酵液中引入更多杂质,成分较单糖发酵更复杂,致使生物基化学品的下游分离过程成为其工业化生产亟需解决的关键问题。文中介绍了一种基于两相分配差异分离亲水性生物基化学品的盐析萃取技术及其在生物基化学品分离方面的应用,重点阐述了短链醇和盐对双水相形成的影响,并对1,3-丙二醇、2,3-丁二醇、乙偶姻、乳酸等的盐析萃取研究进展进行了总结和展望。盐析萃取技术可有效地回收发酵液中的小分子亲水性产品,同时除去大多数的杂质 (细胞和蛋白质等),在生物基化学品的分离过程中将是一种有前景的分离技术。     

Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals tested for carcinogenicity by the U.S. National Toxicology Program

This paper is an extension of compilations published previously in this journal. (Ashby and Tennant, 1988; Ashby et al., 1989). A summary of the rodent carcinogenicity bioassay data on a further 39 chemicals tested by the U.S. National Toxicology Program (NTP) is presented. An evaluation of each chemical for structural alerts to DNA-reactivity is also provided, together with a summary of its mutagenicity to Salmonella. Chemicals with an aliphatic nitro group (-C-NO2) have been added to the composite structure of DNA-reactive sub-groups. The 39 chemicals were numbered and evaluated as an extension of the earlier analysis of 264 NTP chemicals. The activity patterns and conclusions derived from the earlier studies are followed by these 39 chemicals, albeit a detailed analysis of the total database of 301 chemicals is reserved for the succeeding paper.     

The effect of acetylsalicylic acid and oxalic acid on Myzus persicae and Aphidius colemani

Plants can respond to damage by pests with both induced direct defences and indirect defences by the attraction of their natural enemies. Foliar application of several plant-derived chemicals, such as salicylic acid and oxalic acid, can induce these defence mechanisms. The effect of acetylsalicylic acid and oxalic acid on the aphid Myzus persicae Sulzer (Homoptera: Aphididae) and its parasitoid Aphidius colemani Viereck (Hymenoptera: Aphidiidae) was investigated. Experiments were carried out with direct application of acetylsalicylic and oxalic acids on these insects, as well as choice and no-choice tests using foliar application of both chemicals on Brussels sprouts plants, Brassica oleracea var. gemmifera L. (Brassicaceae). Parasitoids were given a choice between treated and untreated plants for oviposition, and the effects of the chemicals on aphid and parasitoid development were determined. Although direct application of both chemicals increased aphid mortality, their foliar application did not induce resistance against aphids. The foliar application of such compounds, even in low concentration as shown in the choice tests, has the potential to induce indirect plant defences against aphids by encouraging aphid parasitisation. Although the direct application of both chemicals reduced parasitoid emergence from their hosts, the foliar application of acetylsalicylic acid and low concentrations of oxalic acid did not have a negative effect on parasitoid emergence ability. However, 10 m m oxalic acid reduced the number of emerged parasitoids in no-choice experiments. This study shows that foliar application of acetylsalicylic and oxalic acids has the potential to encourage aphid parasitisation, but care is needed as high concentrations of oxalic acid can have a negative effect on these beneficial organisms.     

The genetic toxicology of Gene-Tox non-carcinogens

The Gene-Tox Program has identified 61 chemicals that have been tested in chronic rodent carcinogenesis bioassays and found to be inactive. The genetic toxicology data of these 61 non-carcinogens is reviewed and summarized. A large proportion of these chemicals have been tested to a limited extent in genetic toxicity bioassays: 32 in 2 tests or less. Of the remaining 29 chemicals, 28% have been tested in 9 or more tests which encompass a range of genetic endpoints: gene mutation, chromosomal effects, other genetic endpoints, and cell transformation. The genetic toxicity of 12 chemicals with sufficient data is discussed in detail: benzoin, caffeine caprolactam, ethanol, halothane, hycanthone methanesulfonate, malathion, maleic hydrazide, methotrexate, 1-naphthylamine, 4-nitro-o-phenylenediamine, and p-phenylenediamine. A new technique for the evaluation of multiple test data, the "genetic activity profile", has been applied to 6 of these chemicals, allowing the qualitative and quantitative information to be compared collectively. In the evaluation of the genotoxicity effects of these non-carcinogens, a number of discrepancies between the results from genetic toxicity bioassays and chronic rodent bioassays have been uncovered. These discrepancies are discussed in light of current knowledge on the strengths and weaknesses of both genetic toxicity bioassays and chronic rodent bioassays.     

The nongenotoxic carcinogens naphthalene and para-dichlorobenzene suppress apoptosis in Caenorhabditis elegans

Naphthalene (1) and para-dichlorobenzene (PDCB, 2), which are widely used as moth repellents and air fresheners, cause cancer in rodents and are potential human carcinogens. However, their mechanisms of action remain unclear. Here we describe a novel method for delivering and screening hydrophobic chemicals in C. elegans and apply this technique to investigate the ways in which naphthalene and PDCB may promote tumorigenesis in mammals. We show that naphthalene and PDCB inhibit apoptosis in C. elegans, a result that suggests a cellular mechanism by which these chemicals may promote the survival and proliferation of latent tumor cells. In addition, we find that a naphthalene metabolite directly inactivates caspases by oxidizing the active site cysteine residue; this suggests a molecular mechanism by which these chemicals suppress apoptosis. Naphthalene and PDCB are the first small-molecule apoptosis inhibitors identified in C. elegans. The power of C. elegans molecular genetics, in combination with the possibility of carrying out large-scale chemical screens in this organism, makes C. elegans an attractive and economic animal model for both toxicological studies and drug screens.     

Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate

A silver stain technique using treatment with potassium permanganate for visualisation of proteins and DNA separated by gel electrophoresis was developed and applied both to the very thin (thickness 0.1-0.2 mm) and to the usual 1-2 mm thick gels. The technique is reproducible, only stable chemicals are used and it is shortened to about 1 h even for the 1 mm gel (30 min for the 0.2 mm gel). It is applicable to gels thicker than 2 mm with somewhat longer washes. Amounts as low as 2 X 10(-10) g of protein per band have been detected. Protein bands of different colours are obtained. The technique has been used with success in continuous, discontinuous and 2D-gel systems. Bands have been detected which were not observed when the other silver staining techniques were used.     

Microbial production of diols as platform chemicals: recent progresses

Diols are chemicals with two hydroxyl groups which have a wide range of appealing applications as chemicals and fuels. In particular, four diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO) and 1,4-butanediol (1,4-BDO) can be biotechnologically produced by direct microbial bioconversion of renewable materials. These diols are considered as platform green chemicals. We review and discuss here the recent development in the microbial production of these diols, especially regarding the engineering of production strains and optimization of the fermentation processes.     

Lycopersicon assays of chemical/radiation genotoxicity for the study of environmental mutagens

From a literature survey, 21 chemicals are tabulated that have been evaluated in 39 assays for their clastogenic effects in Lycopersicon. Nineteen of the 21 chemicals are reported as giving a positive reaction (i.e. causing chromosome aberrations). Of these, five are reported positive with a dose response. In addition, 23 assays have been recorded for six types of radiation, all of which reacted positively. The results of 102 assays with 32 chemicals and seven types of radiation tested for the induction of gene mutations are tabulated, as well as 20 chemicals and/or radiation in combined treatments. The Lycopersicon esculentum (2n=24) assay is a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis and for somatic mutations induced by chemicals and radiations. The Lycopersicon bioassay has been shown to be as sensitive and as specific an assay as other plant genotoxicity assays, such as Hordeum vulgare, Vicia faba, Crepis capillaris, Pisum sativum and Allium cepa and should be considered in further studies in assessing clastogenicity. Tests using L. esculentum can be made for a spectrum of mutant phenotypes of which many are identifiable in young seedlings.     

Biochemical strategies for the detection and detoxification of toxic chemicals in the environment

Addressing the problems related to the widespread presence of an increasing number of chemicals released into the environment by human activities represents one of the most important challenges of this century. In the last few years, to replace the high cost, in terms of time and money, of conventional technologies, the scientific community has directed considerable research towards the development both of new detection systems for the measurement of the contamination levels of chemicals in people's body fluids and tissue, as well as in the environment, and of new remediation strategies for the removal of such chemicals from the environment, as a means of the prevention of human diseases. New emerging biosensors for the analysis of environmental chemicals have been proposed, including VHH antibodies, that combine the antibody performance with the affinity for small molecules, genetically engineered microorganisms, aptamers and new highly stable enzymes. However, the advances in the field of chemicals monitoring are still far from producing a continuous realtime and on-line system for their detection. Better results have been obtained in the development of strategies which use organisms(microorganisms, plants and animals) or metabolic pathway-based approaches(single enzymes or more complex enzymatic solutions) for the fixation, degradation and detoxification of chemicals in the environment. Systems for enzymatic detoxification and degradation of toxic agents in wastewater from chemical and manufacturing industries, such as ligninolytic enzymes for the treatment of wastewater from the textile industry, have been proposed. Considering the high value of these research studies, in terms of the protection of human health and of the ecosystem, science must play a major role in guiding policy changes in this field.     

Chromosome aberration assays in Pisum for the study of environmental mutagens

From a literature survey, 117 chemicals are tabulated that have been assayed in 179 assays for their clastogenic effects in Pisum. Of the 117 chemicals that have been assayed, 65 are reported at giving a positive reaction (i.e. causing chromosome aberrations), 30 positive with a dose response, five borderline positive. Seventeen chemicals gave a negative response. Eighty-one percent of the chemicals gave a definite positive response. A c-mitotic effect was detected from treatment with 17 chemicals. In addition to the above tabulation of chemicals, 39 chemicals have been reported with an antimitotic effect. Thirteen assays have been recorded for five types of radiation, which with the exception of ultrasound reacted positively. The results of assays with 38 chemicals and/or radiations in combined treatments, as well as 15 chemicals and three types of radiations that induce somatic mutations are tabulated. The Pisum sativum (2n=14) bioassay has been shown to be a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis for somatic mutations induced by chemicals, radiations, and environmental pollutants. For some chemicals, the Pisum assay is not as sensitive in assessing clastogenicity as the Allium assay, although this should be considered in relative terms. Pisum fulvum (2n=14) has been used in clastogenic studies also, but to a much lesser extent.     

Tryptophan oxidation catabolite,N‐formylkynurenine,in photo degraded cell culture medium results in reduced cell culture performance

Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC‐MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N‐formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:74–82, 2016