Research Journal of Biotechnology

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Efficient Bacillus subtilis Promoters for Graded Expression of Heterologous Genes in Escherichia coli

Wang Y.Y. , Ng K.L. , Lam C.C. , Chan K.N. , Sze K.F. , Fu Z.B. and Wong W.K.R.

A variant of the veg I promoter (abbreviated vegA P in this work), designated vegG P, was isolated from its natural host, Bacillus subtilis and characterized to contain a spontaneous A to G transition mutation at the -11 position of the -10 region of vegA P. The mutant promoter functioned well to provide efficient transcription of foreign genes in B. subtilis. Moreover, despite its origin from a gram-positive bacterium, vegG P was found to facilitate efficient gene expression in E. coli and was shown to mediate thus far the highest level of expression of the reporter protein, an endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi. Surprisingly, vegG P was also shown to function more efficiently than one of the strongest E. coli promoters, tac (tac P) in E. coli, as judged by Northern blot analysis and excretory expression of Eng. Two derivatives of vegG P, designated vegC P and vegT P, together with the wild-type promoter, vegA P, were also found to be more efficient than tac P to express excretory Eng in E. coli, despite at lower efficiencies than that of vegG P. These four B. subtilis promoters, which were shown to exhibit different transcriptional efficiencies, may be employed for stepwise or graded gene expression in E. coli, thereby facilitating the task of fine tuning to achieve “optimal” expression of the target gene, which appears to be a crucial milestone for the maximum production of the desired protein in the culture medium.

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Copper Bioremediation from Aqueous Solution and Pigment Industries Waste using Dried Aquatic Plant Biomasses

Dave S. R., Damani M. S., Tipre D.R. and Dutt P.S.

Copper biosorption was studied with dried biomass of Ceratophyllum, Eichhornia, Vallisneria and Salvinia in the batch system. The optimum conditions were analysed in terms of pH, contact time, sorbate and biomass concentration, adsorption isotherm and desorption. For all four biomass pH 5 was found to be optimum for the copper sorption. Contact time of 2 h was found to be optimum for copper sorption by Ceratophyllum and Salvinia biomass, where as 3 h was optimum for Eichhornia and Vallisneria biomass. The copper sorption was directly proportional to the taken biomass concentration. Under the experimental conditions, optimum copper concentration for all the four biomass was 100 ppm. HNO3 was better desorbing agent for Salvinia, Vallisneria and Eichhornia biomass compared to HCl, whereas HCl was better for Ceratophyllum. Langmuir adsorption isotherm was better fitted with all for biomass than Freundlich adsorption isotherm. Treatment of actual waste also showed 92, 80, 73 and 46% copper removal with Ceratophyllum, Vallisneria, Salvinia and Eichhornia biomass respectively.

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Antiparasitic Effects of Some Essential Oils on the Scuticociliate, Uronema Sp.

Al-Yaqout A. and Azad I.S.

The essential oils (Eos) investigated for their antiparasitic effect were; tea tree oil (TTO), thyme oil (TO), clove oil (CO), sage oil (SO), garlic oil (GO), cinnamon oil (CnO), eucalyptus oil (EO) and oregano oil (OO). Response of the live parasite to the EOs was recorded on a time scale until lysis or death of cells. Cessation of ciliary motility which was an end point indicator for the calculation of lethal dose (LD100) showed that many of the tested oils, except CO and CnO, were lethal to the parasite at 20 ppm concentration within 10 min of exposure. Faster effects were noticed with TTO, OO, GO and EO producing 90% or higher lethality within 3 min of exposure. Complete lethality was achieved with GO at 4 min followed by OO, TO and EO at 5 min. Scuticociliate infected Pampus argenteus was treated with 20 ppm of TTO for 30 min daily for 5 days resulting in complete healing of the skin erosions. The study while indicating the potentials of EOs in controlling the scuticociliatosis also emphasized on the need for future research in the field of EOs as environ-friendly disease control agents.

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Mutational analysis of MLH1 protein in Hereditary Nonpolyposis Colorectal Cancer-2

Chellasamy Selvaakumar, Kirolikar Pravin Saurabh, Borgaonkar Swaranshu Pramod and Adi Kumara Veena

The MLH1 gene product plays an essential role in DNA repair during the process of cell cycle. MutL alpha is a heterodimer between Mismatch repair (MMR) protein MLH1 and PMS2. Mutational analysis was carried out for crystal structure of MLH1 using What if server. The site directed mutation was done at position 112. This residue is substituted by Arginine and Methonine respectively. After the mutation the structura­l stability of the mutant proteins of MLH1 was calculated using Prosa software. It reveals that with the mutation of Ser to Arg (1st mutant) the z score value is -7.18. With the mutation of Ser to Met (2nd mutant) the value is -7.15. But for the wild type MLH1 the z score is -7.31. This clearly signifies that with the mutation of Arginine and Methonine there is a decrease in the stability of the protein. Further docking studies were carried using HEX software to study the interaction of these three structures viz. wild MLH1 (S112), first mutant MLH1 (R112) and second mutant MLH1 (M112) protein with PMS2. The binding energy was calculated separately for each docked pose. For the wild MLH1 the energy was -578.70 kcal/mol. For the first mutant the energy was -563.18 kcal/mol and for the second mutant it was -341.72 kcal/mol. The structural stability of the wild type, mutant 1 and mutant 2 were comparatively analyzed. It reveals that the stability of the MLH1 protein decreases with the mutation of Arginine and Methonine. Docking studies also confirms this with a reduced energy value for mutant 1 and mutant 2. But the structural stability and docking energy is low for Methonine compared to Arginine. Position 112 withstands conservative substitution viz. Ser but not with Arg and Met. This is very much evident from the bioinformatics analysis.

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Response Surface Methodology for the Optimized Production of Polylactide from Lactic Acid produced by Lactobacillus Delbrueckii

Srivastava Gaurava and Srivastava Pradeep

Response surface methodology involving central composite design was successfully applied to evaluate the optimized conditions for the polymerization of lactic acid to produce polylactide. Effect of different parameters such as temperature, time and catalyst concentration for polymerization of lactic acid was optimized. Statistical analysis of the results showed that the quadratic terms of these three variables had significant effect. The interaction between the three variables was found to contribute to the response at significant level. A quadratic model was developed through RSM in terms of independent variables to describe experimental yield as the response. Based on contour, 3D-plots and variance analysis, optimum operational conditions for maximizing initial experimental yield were determined. Maximum polylactide production achieved was at time 15 hrs, temperature 165 ºC and catalyst concentration 0.06%. Experimental findings were in dose agreement with the model predictions.

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Isolation and Characterization of a Novel Protease Enzyme from Bacillus Cereus

Kavitha B. and Thankamani V.

Proteases producing bacteria were isolated locally from soil. Seven Bacillus sps, were isolated, which were grown in selective media for screening. Microscopic and cultural characters were studied. The different isolates were coded and identified as per Bergeys Manual of Systematic Bacteriology. KB5 (Bacillus cereus) that showed highest zone of hydrolysis was studied in detail. Species was confirmed by FAF. The growth curve of the best isolate Bacillus cereus was studied. The organism showed maximum enzyme production at pH 9.0, temperature 370C, incubation over 72 hours and in shaking conditions. The best carbon and nitrogen sources and minerals which enhanced enzyme production were glucose, casein and calcium respectively. An optimized medium was formulated for maximum enzyme production. The optimum assay conditions for proteases activity were determined. The enzyme showed best activity at pH 9.0, temperature 500C, duration of incubation for one hour, substrate concentration of 20 mg of casein. The apparent Km value of the crude enzyme produced by Bacillus cereus for casein was found to be 10 mg/ml from the growth curve. Maximum pH and temperature stability of Bacillus cereus were pH 9.0 and 500C respectively.

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Molecular Diversity of CMS, Restorer Lines and Selective Hybrids in Pearl Millet [pennisetum glaucum (l.) Br. R.] based on Randomly Amplified Polymorphic DNA (RAPD) markers

Patil Harshal E and Jadeja G. C.

Molecular studies revealed the high genetic diversity in CMS, Restorer lines and selective hybrids by using RAPD analysis. The genotype specific amplification profile was observed with minimum of 12 polymorphic amplicons by ICMA 95222 genotype and a maximum of 18 amplification with ICMA 92777 genotype across seven primers for CMS lines. The polymorphism shown by seven CMS varied from 44.45 to 72.00 per cent. Among the restores, IPC 1657, IPC 1658 and IPC 1964 showed great divergence from others. Seven RAPD primers of OP-D series (OP-D 6, OP-D 9, OP-D 10, OP-D 12, OP-D 13, OP-D 14 and OP-D 15) were able to distinguish all the seven male sterile and thirteen restorer genotypes. The genotype specific amplification profile was observed with minimum of 12 polymorphic amplicons by ICMA 95222 x J 2340 and a maximum of 18 amplification with ICMA 94555 x J 2340 across four primers and among the five selective hybrids. The polymorphism shown by hybrids varied from 76.47 to 87.50 per cent. A high genetic diversity is observed in pearl millet genotypes and discriminated all from each other. Molecular investigation has considerable importance in crop improvement programme and can help in identifying parents for the best hybrid combinations and cataloging the genetic diversity in the breeding material without any limitations of environmental influences associated with the conventional breeding programme. The importance of these results is useful not only in making preliminary predication of yield performance on the basis of molecular diversity but also useful as a tool in identifying gene pools having broad genetic base for further use in pearl millet.

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Biomineralisation of Organophosphorus Pesticide (Malathion) using Fungal Species Aspergillus Niger from Cotton Soils of Andhra Pradesh, India

Udaya Sri P., Sambasiva Rao K.R.S. and Subbu Rathinam K.M.

Cotton is the world’s most important non-food agricultural commodity easily damaged by various sucking pests like jassids, aphids, thrips, wh­itefly, red spider, mites, mealy bug and bollworms. Pesticides and their residues pose a major problem by their accumulation in different parts of the world in general and in different biological organisms of the food chain in particular. In the light of the importance and essentiality to overcome the deleterious effects of pesticides, the present study has made an attempt to isolate the fungal species capable of degrading orga­no­phosphorus pesticides (chlorpyriphos, malathion and triazophos) that are widely sprayed on cotton crop in Andhra Pradesh. The isolated species were identified basing on microscopic structural and growth characterization studies together with scann­ing electron microscopy. Molecular characteri­zation was also done basing on the sequence analysis of the DNA coding for 18s rRNA of the test fungal organism. It was elucida­ted that the selected fungal species is an effective biodegrading agent and by optimizing the different cultural conditions and by using large scale culture techniques, this fungal species, Aspergillus niger, can be very effectively used as biodegrading agent in cotton fields as effective biocleaner and will be an economically effective microbial process which is an alternative for control of environmental pollution.

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Rapid and Comparatively Cheaper Protocol for DNA Extraction in Molecular Approaches

Patel Naimesh J. and Jasrai Yogesh T.

To improve the variety of crops, usage of molecular markers is very essential and for making it possible, proper method needs to be established for extraction of DNA. Moreover, such DNA extracts need to be free from protein, RNA and other interfering impurities. Usually, DNA extraction methods are very tedious, time consuming, labor intensive and hence escalate the cost. Though isolation protocols are available, all have lengthy proceedings. DNA extraction from cotton seed becomes difficult as seed material is dry with hard seed coat. The proposed method involves efficient extraction of DNA with few steps. It is inexpensive, requires no special kit, column or liquid nitrogen. The method can efficiently extract DNA from 30-40 samples in just few hours. The extracted DNA from seed is suitable even for further studies like RFLP, RAPD, AFLP and PCR techniques.

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Citric Catalysis on the Microbiological Leaching of Chalcopyrite Fine Grained Tailings in Shake Flask

Li Dou and Li Dongwei

The effect of citric catalysis on the microbiological leaching of chalcopyrite fine grained tailings using Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans in shake flask under mesophilic cnditions (30±1 oC) was studied. The test results suggested that citric catalysis and suitable pulp density could extract Cu remarkably better than in absence of catalyst. Once the tolerance is exceeded, the microbial activity would be inhibited. The citric catalyzed the bacteria to accelerate the leaching bioreaction of microbiological leaching but also extracted the Cu (II) directly as a organic acid. Scanning electron microscopy (SEM) was used to study microscopic changes occurring in chalcopyrite fine grained tailings when the microbiologica leaching medium contained citric catalysis.

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Cloning and Expression of Heat Shock Protein 90 Gene from the Diapausing Larvae of the Rice Stem Borer, Chilo Suppressalis (Lepidoptera: Pyralidae) exposed to Temperature Stress

Qiang Cheng-kui, Du Yu-zhou, Yu Lin-ya , Cui Ya-dong and Zhou Bao-ya

The rice stem borer, Chilo suppressalis Walker, is an economically important pest distributed in rice producing regions of China. To understand its molecular adaptation, the authors cloned the cDNA sequence of CcsHsp90 which is essential for dealing with many environmental stresses in all organisms and measured the mRNA expression in the diapausing larvae exposed to temperature stress. The results showed that CcsHsp90 (GenBank accession no. FJ866608) is potentially encoded to 312 amino acids with calculated molecular weight of 35.59KDa and the theoretical isoeletric point of 4.72. The deduced amino acid sequence of CcsHsp90 is displayed over 80% of the degree of conservation to other Lepidopteran insects. The relative mRNA expression levels of CcsHsp90 in the diapausing larvae which were exposed to different temperatures from 28 oC (CK) to -14 oC with a gradient of 7 oC for 4h increased first and decreased later and peaked at 0 oC. Then the larvae were exposed to 0 oC for 0~8h, the change of the levels was the same as the above and peaked at 4h treatment. Therefore, it could be concluded that 0 oC for 4h treatment might induce the maximal expression of CcsHsp90 in the diapausing larvae of the rice stem borer.

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CEffect of Cyanobacterial Biofertilizer on Growth and Biochemical Characteristics of Vinca rosea Linn

Das Pradipa Kumar

The biofertilizer effect of the aqueous extract of four cyanobacteria viz: Anabaena circinalis (H), Scytonema coactail (H), Lyngbya mucicola (NH) and Oscillatoria princeps (NH) on the growth and biochemical characteristics of an antileukemic plant Vinca rosea Linn. was investigated. Cyanobacterial extracts were added to soil bed in three different concentrations (1%, 5% and 10% w/v). All the treated extracts were found to be stimulatory in action. Vinca rosea showed best response at 10% extracts of Anabaena circinalis in all the growth parameters studied. Similarly a significant increase in the content of photosynthetic pigments, leaf soluble protein, anti-oxidant enzyme activity of catalase and peroxidase was observed.

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