Research Journal of Biotechnology

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Research Journal of Biotechnology

A Molecular Study on Thermostable Carboxyl Esterase and Esterase/ Acylaminoacyl-peptidase enzymes of Geobacillus thermoleovorans YN

Soliman Nadia A.1* and Gaballa Ahmed A.2

Thermostability is considered a promising characteristic for industrial enzyme applications. Screening of the genomic library of thermophilic G. thermoleovorans YN (ac: AF385083) for esterase/lipase activity has led to the isolation of two positive clones. Sub-cloning and sequence analyses identified two potential genes estB (560 bps, partial sequence) and estC (2022 bps), encoding two different esterases. The complete ORF of estB (1500 bps) was amplified using degenerate primers based on multiple sequence alignment. EstB showed 95% identity to putative carboxyl esterases of G. kaustophilus HTA426 (ac: BAD77334) G. stearothermophilus (ac: AAN81910) and 84% similarity to G. thermodenitrificans NG80 (ac: ABO68347). On the other hand, ORF of estC (2022 bps), encoded a protein with typical esterase/lipase motif GXSXG with 37% identity to acylpeptide hydrolase/esterase of hyperthermophilic Aeropyrum pernix K1 (ac: BAA80835). In addition, EstC showed high similarity to proteins with esterase and peptidase dual activity. Both, estB and estC were subcloned and efficiently expressed in E. coli DH5α under control of temperature-inducible λp L-promoter using the vector pCYTEX-P1. SDS-PAGE of the recombinant E. coli crude cell lysates showed induction of 54.7 kDa and 75.4 kDa proteins corresponding to EstB and EstC respectively. The temperature optima of Est B and EstC were 65oC and 55oC respectively; however, they showed almost quite similar pH optima ranging from 8 to 9, The recombinant proteins (EstB or C) showed a complete stability at 65oC after one hr and pH 10 after 24 hr exposure, moreover detergents addition to the EstB enhanced its activity.

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Screening of a high yield polysaccharide strain from ten edible and medicinal fungi and optimization of its culture conditions

Duan Yan-Qing2, Xing Zhan-Chang3 and Xu Jun-Wei1*

Ten edible and medicinal fungi were studied for their abilities to produce polysaccharide in submerged culture. Strain specific variation in intracellular and extracellular polysaccharide production was quantified under laboratory conditions. Among the fungi examined, Agrocybe aegirita Mo-Aa was found to produce high amount (3.77 g/L) of total polysaccharide. Furthermore, optimization of production conditions for A. aegirita Mo-Aa polysaccharide was conducted in submerged culture. Production of A. aegirita Mo-Aa polysaccharide was optimal at pH 5.0, an inoculation size of 480 mg/L, culture period of 9 days and mushroom fermentation medium. Under these optimal conditions, the maximum production of A. aegirita Mo-Aa polysaccharide reached 5.46 g/L. The results obtained will be useful for enhanced production of A. aegirita Mo-Aa polysaccharide on a large scale.

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Diversity for Cell Morphology, Nitrogenase Activity and DNA Profile of Azospirillum species present in Rhizosphere Soils of Six Different Physiographic Regions of Maharashtra

Murumkar D.R.,* Borkar S.G. and Chimote V.P.

A total of 93 Azospirillum isolates were recovered during the year 2010 from the rhizosphere soils of six different physiographic regions of Maharashtra. The distinct variation was observed in cell morphology among the different Azospirillum isolates. The cells of 83 Azospirillum isolates were non-motile whereas 10 isolates had motile cells. A wide variation was observed in cell size varying from 1.0 to 2.8 × 3.3 to 24.8 µm among the Azospirillum isolates. Out of 93 Azospirillum isolates, 83 isolates utilized glucose and malic acid as a sole carbon source and were positive for biotin requirement, whereas 10 isolates utilized only malic acid as a sole carbon source and showed negative results for glucose and biotin requirement. Based on the morphological, cultural, biochemical and physiological characteristics, 83 isolates were identified as Azospirillum lipoferum and 10 isolates as Azospirillum brasilense. A wide variation was observed in nitrogenase activity (from 8.5 to 394.7 nmol among the 93 Azospirillum isolates. The A. lipoferum isolates exhibited a higher average nitrogenase activity compared to A. brasilense isolates (105.9 vs. 20.8 nmol protein-1. hr-1, respectively). Based on the nitrogenase activity, seven highly efficient Azospirillum isolates alongwith MPKV strain Asp-BNF were studied for their genetic variability by employing RAPD-PCR technique. On the basis of UPGMA clustering analysis, the A. lipoferum isolates were classified into four broad clusters and the genetic variation observed among A. lipoferum isolates was due to domestication of the isolates in different agro-ecological regions as well as their nitrogenase activity. Based on nitrogen-fixing ability, highly efficient Azospirillum isolates may be further exploited in biofertilizer production

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Genetic characterization of Majaheem camel population in Saudi Arabia based on microsatellite markers

Mahmoud A.H. 1*, AlShaikh M.A.2, Aljummah R.S.2 and Mohammed O.B.3

Sixteen microsatellite markers were used to investigate the genetic polymorphism in Majaheem population of camels in the Kingdom of Saudi Arabia. Genomic DNA was extracted from hair roots from 40 unrelated Majaheem camels. Out of these 16 markers, only microsatellite VOLP67 did not produce any PCR amplification from all individuals studied. Results showed that 102 alleles were generated by the 15 microsatellites loci with a mean of 6.8 and a range of 3 to 14 alleles per locus. The mean expected heterozygosity (He) was 0.652, ranged from 0.422 to 0.807 and the mean observed heterozygosity (Ho) was 0.665 with a range of 0.275 to 0.900. The Polymorphic Information Content (PIC) values ranged from 0.340 to 0.768 with a mean of 0.590. No significant heterozygote excess or bottleneck in nearest past was detected in the studied camel population as indicated by sign, standardized differences and Wilcoxon tests, along with the normal L shaped distribution of mode-shift test. The results indicated the utility of these 15 microsatellite loci for studying genetic polymorphism in the dromedary camel (Camelus dromedarius).

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Fermentative production of extracellular pigment from Streptomyces coelicolor MSIS1

Mohanasrinivasan V.*, SriramKalyan P., Ipsita Nandi, Subathradevi C., Selvarajan E., Suganthi V. and Jemimah Naine S.

In the present research work, the pigment producing actinomycetes was isolated from a rhizosphere soil of ornamental plants and identified as Streptomyces coelicolor MSIS1 (FR856603).The pigment was produced in shake flask as well as in bioreactor. The results were evident that there was threefold increase in the pigment production in bioreactor compared with shake flask.The extracted pigment was characterized based on TLC, HPLC and FT-IR. The HPLC data showed that the compound may be one of actinorhodinic acid but on the other side FT-IR data infers that there were no presence of aromatic ring but prominent aliphatic stretch has been found.

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Evaluation of Antibacterial Potential of Various Seed Extracts of Caesalpinia bonducella F.

Shukla Shruti1*, Mehta Archana 2 and Ayyadurai N. 3

In this study, Caesalpinia bonducella F. (Leguminosae) seed powder was defatted with petroleum ether. The non-defatted seed powder as well as the defatted plant material was then individually extracted using different solvents with increasing polarity viz. acetone, chloroform, ethanol and water. The percentage yield values of C. bonducella for non-defatted and defatted seed extracts ranged from 4.36 - 17.5% and 3.6 - 13.84%, respectively. All the extracts (non-defatted and defatted) of C. bonducella seeds were evaluated for their antibacterial activity against B. subtilis (ATCC 15563), S. aureus (ATCC 25973), K. pneumoniae (NCIM 2729), E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) by agar well diffusion method, MIC and MBC determination and cell viability assay. At the used concentrations (10, 20 and 50 mg/ml), the defatted extracts showed better antibacterial activity against the tested pathogens as compared to non-defatted extracts and the maximum activity was observed at the highest concentration (50 mg/ml). The defatted acetone and ethanol extracts of C. bonducella were found highly active against all the tested bacterial pathogens as compared to other extracts with diameter of zones of inhibition ranging from 5 to 20 and 9 to 23 mm respectively. The MIC and MBC values of non-defatted and defatted extracts ranged from 3.2 to 13 mg/ml and 1.5 to 11 mg/ml and 3.5 to 13.5 mg/ml and 1.5 to 11 mg/ml respectively. Further, based on the bioactivity, exposure of 240 min of defatted acetone and ethanol extracts of C. bonducella exerted potential antibacterial effect on the viable counts of the tested bacterial pathogens with 100% inhibitory effect. These findings indicate that C. bonducella has potential value to control various pathogenic microbes.

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Arsenic-induced Oxidative Stress and Antioxidant Defense System of Pisum sativum and Pennisetum typhoides: A Comparative Study

Sharma Iti

A study of arsenic–induced oxidative damage and response of antioxidant enzymes in the aerial parts of Pennisetum typhoides and Pisum sativum was carried out. Seedlings of test plants were grown hydroponically in Hoagland nutrient solution and enriched with different concentrations of arsenic (0, 10, 25, 50, 100 and 200 µM) for 10 days. Time course study was carried out in test plants exposed to sublethal concentration of Arsenic (50 µM). This work aims to carry out a comparative study between the two test plants based on the above parameters. Arsenic accumulation increased with an increase in arsenic concentration in the growth medium, the most being found in P. typhoides showing less toxicity symptoms than P. sativum. In addition, plant growth inhibition was higher in P. sativum than P. typhoides exposed to increasing concentration of arsenic. Lipid peroxidation and H2O2 were found to be lowest in P. typhoides which correspond with its observed tolerance to arsenic. Also, the amount of thiol content and photosynthetic pigments was decreased with increasing concentration of Arsenic. Both test plants responded differentially to arsenic exposure in terms of anti-oxidative defence. Higher levels of superoxide dismutase, catalase and ascorbate peroxidase were observed in P. typhoides than in P. sativum, showing their active involvement in the arsenic detoxification mechanism. Result shows that due to strong enzymatic antioxidant defense system P. typhoides is comparatively arsenic tolerant than P. sativum.

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Isolation and Characterization of Cold active lipase producing Pseudomonas sp. 4 from Marine samples of Tamilnadu Coast

Kavitha M. and Shanthi C.

Twenty lipase producing bacterial isolates were screened from marine samples collected from Northern coast of Tamilnadu, India. Isolate-4, identified as Pseudomonas sp. was found to be the potential cold active lipase producer with enzyme activity of 172 U/ml/min at 250C and 133 U/ml/min at 150C. The isolate was identified as halotolerant capable of tolerating up to 1.5 M NaCl and grew well at both neutral and alkaline pH. The isolate produced lipase optimally at pH 5.5 in presence of 0.5 M NaCl. Optimum incubation period for lipase production was recognized as 8 days. The crude enzyme extract from the isolate showed highest activity at 250C and retained almost 80% of activity at 15 and 200C, strongly suggesting its high catalytic activity at low temperatures. Optimum pH for lipase activity was found at pH 8. The enzyme displayed maximum thermal stability at 400C by retaining 80% of initial activity for about 4 h, making it a promising candidate for industrial applications after due purification and characterization to find out its suitability for various applications.

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A method to suppress the browning in banana (Musa, AAA) embryogenic callus induced

Chang S.1,2,3 and Shu H.1,2*

Establishing a stable and homogeneous embryogenic cell suspension (ECS) is the prerequisite for biotechnological breeding of banana. Inducing embryogenic callus is the key step for establishing an ECS. But explants were often browning and necrotic when they were induced that decreased the possibility for getting ECS and much more labor has to be paid. In this paper, we found that browning rate of flowers cultured on 1/2 MS or 1/3 MS was lower than that of those cultured on MS basal medium. Neither activated charcoal nor pH had significant effects on decreasing flowers’ browning. Flowers cultured on medium containing DTT or Na2S2O3 did not show clear superiority to those cultured on control medium. Ascorbic acid combined with 1/2 MS or 1/3 MS had much better effects than other media in controlling flowers’ browning. No flowers cultured on these two media were browning and necrotic after being cultured one month. But if the dose of ascorbic acid was higher than 50 mg/L, the flowers developed slower than control. 30 mg/L of ascorbic acid was the descent concentration.

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Biofunctionalized nanoparticle for drug delivery

Sathish kumar K.1*, Madhusudhanan J.2, Thanigaivel2, Robin Abraham2 and Veni V. A2.

Doxorubicin (DOX), a common cancer chemotherapeutics, was conjugated to folate-modified polyethyleneimine-functionalized gold nanoparticles. The controlled release behaviour of DOX-loaded gold nanoparticles was analysed using Tris HCl and PBS. The TEM and FTIR characterization says that there is a strong association of DOX over folate modified gold nanoparticle. DOX-loaded gold nanoparticles had higher cytotoxicity for folate-receptor-positive cells (KB cells) compared to folate-receptor-negative cells (A549 cells) which show lower cells viability at higher concentration of folate modified gold nanoparticle. The viability of KB cells is 7% and A549 cells are 17% at high concentration AuNP-PEI-FOL-DOX. The DOX effects on cancer cell DNA were determined by DNA cleavage pattern.

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Response surface optimization for the production of marine eubacterial protease and its application

Richa K., Bose H., Singh K., Karthik L., Kumar G. and Bhaskara Rao K.V.*

Marine Bacillus VITRKHB showed substantial amount of proteolytic activity. The impact of different carbon and nitrogen source on protease yield and activity was investigated following its production. The maximum enzyme activity was observed in case of fructose as carbon and nutrient broth as nitrogen supplement. Statistical experimental designs using Response surface methodology (RSM) were employed to study the mutual interactive effect of major nutrients and physicochemical parameters influencing the enzyme production. Optimal level of different variables for maximum protease activity was determined as fructose 2.50%, nutrient broth 0.70%, pH 6.75, NaCl 4.75% and temperature 40oC after 24h of incubation. Determination of co-efficient (R2=0.8528) close to 1.0 indicated significant precision and reliability of the response data. Extracellular protease was isolated, purified to 1.19 fold by DEAE ion – exchange chromatography and its specific activity was determined to be 1.59 IU/mg. Industrial efficacy of the marine eubacterial protease was ascertained.

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Recent Trends in Stem Cell Research on Cancer Therapeutics

Shanju S.,1* Sangeetha K.,2 Chandran K.Nair.3 and Satheesan. B 4

Stem cells are unique cells, having the property of self renewal characteristics and differentiation. Protein antigen markers in surface of cells are generally used to identify stem cell population. Cancer stem cells or tumor initiating cells generally occur within tumor cells causing cancer. Cell surface antigen markers on the stem cells are not able to identify the specific type of cancer. Further research on tumor suppressor genes, signaling pathways during stem cell differentiation would be able to combat the disease.

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