Research Journal of Biotechnology

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Structural and Functional analysis of Glutamate Decarboxylase System in Bacillus aryabhattai

Bora Sudipta Sankar, Sarma Kishore, Das Saurav and Barooah Madhumita

An isolate designated as AM was isolated from acidic soil of Golaghat District of Assam, India. Based on the phenotypic, biochemical, 16S rRNA sequencing and chemotaxonomic characteristics, the isolate was identified to be Bacillus aryabhattai. Bacillus aryabhattai is a known neutralophile. However, its ability to grow and survive at low pH (at 4.5) was intriguing. Here, we report the activity of glutamate decarboxylase (GAD) a reported enzyme with ability to confer tolerance to various stress condition including soil acidity. This pyridoxal phosphate (Vitamin B6) based enzyme plays an important role in pH homeostasis by catalysing the decarboxylation of glutamate to ɣ-aminobutyrate. A rapid pH indicator method showed the presence of this enzyme activity at low pH. Significant activity was observed with substrate L-glutamic acid but no activity with D-glutamic acid and α-methyl-DL-glutamic acid. The purified protein showed a single band of ~53.0 kDa on SDS-PAGE. A set of gad specific primers were designed to amplify the gene. A partial sequence of the gad (780 bp) was submitted to the GenBank. It showed significant homology with the gadB of Bacillus megaterium WSH-002 and DSM319.

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Breeding and fermentation study of a high-yield spinosad producing Saccharopolyspora spinosa strain

Guo Weiqun, Zhang Xiaolin , Zou Qiulong, Chen Yuan, Hartke Axel, Ashraf Muhammad Aqeel and Song Yuan

Saccharopolyspora spinosa parental strain CG-73 was exposed to cosmic radiation during a flight aboard a Shenzhou 9 spacecraft. 12,480 recovered colonies potentially harboring mutations were screened for increased spinosad production. Of these strains, 172 exhibited improved yields. The maximal spinosad yield of one strain in a shaking flask reached 573.8 µg/mL which correspond to an improvement of 80.3% compared to that of the parental strain. The effects of S. spinosa biomass on the spinosad yield were also studied by shaking flask fermentation and in a 30-L fermentation tank. Using central composition design (CCD), the optimal medium formula for enhancing biomass was obtained with five important factors: glucose, peptonized milk, dextrin, cottonseed protein and soybean oil. Finally, the tank-based fermentation yield of spinosad reached 1156.8 µg/mL by the fermentation process regulation.

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Screening and Characterization of Auricularia delicata Strain for Mushroom Production under Tropical Temperature Conditions to make use of Rubberwood Sawdust

Wang Xing-Hong, Zhang Chaobin, Fevereiro Pedro and Zhang Changhe

The renewal of rubber trees produces a huge amount of wood waste yearly. Auricularia delicata is an edible wild mushroom species occurring naturally in the tropics. This work aimed at screening an A. delicata strain suitable for cultivation under the tropical temperature conditions to make use of the rubberwood waste. Chemical analysis showed that rubberwood was suitable for mushroom cultivation. Two new A. delicata strains were isolated. Optimal carbon source, inorganic and organic nitrogen sources, culture temperature and medium pH for strain A. delicata were determined for mycelial growth. Optimal temperature range for fruiting body differentiation was 20-280C. 5 tested substrate formulations with rubberwood sawdust as main composition significantly affected fruiting body differentiation time; the best formulation contained 68% rubberwood sawdust. At an industrial scale culture, the average mushroom yield was 750±60 g/kg substrate. The micronutrient contents of the cultured mushrooms were higher than those of the wild mushrooms. The spent substrate was found to be a good organic fertilizer. Results show a novel utilization of rubberwood waste in producing A. delicata mushroom and simultaneously producing organic fertilizer in tropical regions. A. delicata was proposed as a novel species for mushroom cultivation especially in the tropical and sub-tropical regions.

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Skin Anti-Inflammatory Activities of the Ginseng Polysaccharides from Homogenization Extraction Process

Lee Hyeon Yong

856, 1137 and 2100 kiloDaltons (kD) of three acidic polysaccharides were obtained from the Ginsengs through a simple one-step homogenization process at 30000 psi with two cycles for 30 min. Of these three polysaccharides, the 1137-kD polysaccharide showed the highest skin anti-inflammatory activities, increasing both human B and T cell growth up to 1.5x105 viable cells/mL. These polysaccharides also showed the ability of regulating iNOS gene expression down to 65% as compared to the case of treating lipopolysaccharides (LPS). This down-regulation resulted in good suppression of NO production down to 8.22 and 10.23 µM in adding 856 and 2100 k D of the polysaccharides respectively while 12.5 µM of NO production from human macrophage was observed in treating LPS only. In addition, 26.7 pg/mL and 839.1 pg/mL of IL-6 and TNF-α production from human T cells were estimated in treating 1137kD polysaccharide. This polysaccharide was also found to effectively inhibit both hyaluronidase enzyme activities and Prostaglandin E2 (PGE2) production compared to the other two polysaccharides (856 and 2100 kD).These results strongly indicate that 1137kD polysaccharide obtained from the fresh ginseng has better skin anti-inflammatory activities than those of 856 and 2100-kD polysaccharides. Selective polysaccharides from a relatively simple pretreatment process could have high skin anti-inflammatory activities possibly due to the efficient destruction of the crystal structures of the intact large polysaccharides of ginseng with small amounts of the polysaccharide sulfates.

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Comparative study of GCMS, antimicrobial, antioxidant activity of callus and leaf extracts from Baccharoides anthelmintica (L.)Moench

Kalimuthu K., Chinnadurai V., Prabakaran R. and Saraswathy M.

Baccharoides anthelmintica (L.) Moench. (Syn. Vernonia anthelmintica) belonging to the family Asteraceae is an annual herb distributed throughout India, commonly known as purple fleabane. The species contain 40% seed oil with 70%-80% vernolic acid which is extensively used in manufacture of adhesives, varnishes, paints and industrial coatings and many medicinal properties. The present work is comparative study of therapeutic compound present in in vivo leaf and leaf callus extracts of economically and medicinally important plant B. anthelmintica through preliminary phytochemical and GCMS studies and its effect on antimicrobial and antioxidant activity. Ethanolic leaf and leaf callus powder was used for the analysis of preliminary phytochemical, GCMS, antimicrobial and antioxidant potential. Preliminary phytochemical analysis revealed the presence of various secondary metabolites. GCMS analysis of ethanolic extracts of leaf and leaf derived callus showed the presence of 31 bioactive compounds with many known bioactive principles. The ethanolic leaf extract showed good antibacterial activity against Escherichia coli (20 mm), Streptococcus pyogenes (20 mm) and Klebsiella pneumoniae (18 mm) at 60 µl concentrations. The ethanolic leaf callus extract in the concentration of 60 µl shows higher antibacterial activity against Escherichia coli (18 mm) and Staphylococcus aureus (22 mm). These extracts showed moderate antifungal activity at 60 µl concentration against Candida albicans and Trichoderma viridae. Highest 2, 2- diphenyl picrylhydrazyl (DPPH) Scavenging activity was obtained using leaf and callus extracts (IC50= 1.94 mg/ml and 2.4 mg/ml respectively). The leaf and callus extracts exhibit better reducing power activity. There was a direct relationship between dose and absorbance. There is no major difference between in vivo leaf and leaf derived callus.

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Bacillus thuringiensis regulates Chinese cabbage growth beyond growth promotion

Huang Tianpei, Jiang Jie and Guan Xiong

Seventy-seven Bacillus thuringiensis (Bt) strains, isolated from diverse resources had been investigated for their regulation on plant growth. Chinese cabbage was selected as tested plant. Height, fresh weight and number of leaves of the seedlings were measured after 30 days cultivation. Of the 77 Bt strains, 32 strains promoted the growth while 28 strains inhibited Chinese cabbage production, indicating that Bt regulates Chinese cabbage growth beyond growth promotion Interestingly, Bt plant-growth promoting strains BRC-CWS5, BRC-SFR2, BRC-SFR6 and HQ20 produced indole-3-acetic acid (IAA) in LB liquid media supplementing with tryptophan but they were not able to soluble phosphate. The results suggested that IAA production and phosphate-solubilizing ability may not be universal mechanisms for plant-growth-promoting Bt strains. Our data implied that both good effects (insecticidal spectrum, plant growth promotion etc.) and potential adverse effects (plant growth inhibition etc.) should be considered when choosing a new Bt strain for production.

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Impact of Heavy Metals on Genotoxic by ISSR, Growth and Biosorption by Some Fungi

Abd-El-Aziz Abeer R.M. and Sayed Shaban R.M.

Both essential and nonessential metals form the basis of biological activities. However, all metals induce toxicity at certain levels depending on the tolerance of organisms to these metals. Therefore, it is important to identify novel microbial strains that can tolerate increasing concentrations of heavy metals. Microorganisms including fungi, remove heavy metals from soil through bioaccumulation and biosorption in an eco-friendly and cost-effective manner. In this study, we used fungi Aspergillus niger, A. flavus, Fusarium solani, and Penicillium notatum to remove heavy metals. Effects of 4 different concentrations (125, 250, 500, and 1000 mg/l) of MnCl2, AlCl3, HgCl2 and ZnSO4 on the radial growth of the 4 tested fungi were examined. Toxicity of the metals to A. niger was HgCl2 > ZnSO4 > MnCl2 > AlCl3, A. flavus was HgCl2 > ZnSO4 > MnCl2 > AlCl3, F. solani was HgCl2 > ZnSO4 > MnCl2 > AlCl3 and P. notatum was HgCl2 > ZnSO4> MnCl2 > AlCl3. Both living (bioaccumulation) and non-living (biosorption, alkali treated) fungal biomass effectively removed heavy metals. Our results showed that the living biomass of F. solani and P. notatum removed 2.2–62.1 mg·g-1 of AlCl3 and HgCl2 respectively. Treatment with alkali biomass can significantly consolidate the heavy metal adsorption well described in this study. Maximum adsorption of HgCl2, ZnSO4, MnCl2 and AlCl3 by the alkali biomass of P. notatum was 45.2–72.1, 67.3–75, 36.1–66.1, and 29.1–54.7 mg·g-1 respectively. Thus, our results suggested that A. niger, A. flavus, F. solani, and P. notatum can be used for removing high concentrations of heavy metals from contaminated soil and wastewater. In our study all four heavy metals HgCl2, ZnSO4, MnCl2 and AlCl3 showed a serious damage on DNA especially with high concentration of metal. Total of 3 primers were screened with treated and untreated fungi.

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Characterization of Mitochondrial D-loop Sequences from Three Chinese Theraponidae

Wenyan Fan, Junna Jiao and Zhenping Xu

Previous morphological classification within Theraponidae and some related families was problematic, and no comprehensive molecular evaluation was conducted on these groups. In order to protect the genetic resource of Chinese Theraponidae, the genetic polymorphisms of mitochondrial DNA (mtDNA) D-loop of three Theraponidae (T. jarbua, T.theraps and P. quadrilineatus) in southeast China was investigated. A total of 381 polymorphic sites were found in 2567 sequences of the 44 samples by PCR. The average nucleotide divergence in the three populations was 28% (at T.jarbua), 14.1% (at T. theraps), 44.8% (at P. quadrilineatus) respectively. Complete sequences of the mitochondrial DNA control region from three species of Theraponidae were gained by software splicing. The results of this study showed 3 blocks (CSB-F, CSB-E and CSB-D) for central domain and 3 blocks (CSB1, CSB2, CSB3) for conserved sequence domain. The findings showed that a high level of genetic polymorphisms in D-loop region and T.theraps is closer in molecular resolution with P.quadrilineatus which is different from the traditional phenotype cluster analysis.

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The genetic determinism and the feeding influence on the Oryctolagus Cuniculus Domesticus male genital system

Simeanu D., Creangă Ş., Pop I.M., Gâlcă Valerica and Simeanu Cristina

The purpose of our study was to evaluate the effect of feeding on the male gonads development and functionality. Our research was carried using four animal batches (a control group – CGand three experimental ones - EG), each one of five males belonging to the commercial hybrid "Cunirom-PF-310". All of the subjects were bred from the weaning age up to the age of 120 days when they reach a medium weight of 2800 grams. The feed administrated to the investigated rabbits was represented by complete mixed fodders including more types of ingredients providing 2500-3000 Kcal energetic content /kg dry matter, 183-188 g gross protein /kg dry matter and a different content of gross fibre. All of the males were slaughtered at the age of sexual maturity (120 days)and the testicles (including epididymis) were harvested in order to make histological slides. For the males which were fed with 12% or 16% gross fibres content in mixed fodder, the testicles and the related epididymis were normally developed with many seminiferous tubules; the seminal epithelium have characteristic cellular elements of seminal line such as spermatogonia, spermatocyte I and II and spermatids. There were also observed many Sertoli cells with large, fringe and granulated nuclei, numerous mitoses indicating a very active spermatogenesis process in these animals. The statistical interpretation of the data related to the structure of the testes showed insignificant differences. The feeding of the rabbit males with this kind of mixed fodder did not negatively influence the male gonadal development and function and the installation time of the puberty status. For the males which were fed with a mixed fodder containing 20% or 24% gross fibre, there were found large differences in negative terms for testicles and epididymis development and functionality and in regard to the installation of puberty status; the statistical interpretation of the data related to the structure of the testes showed with few exceptions significant differences; therefore, it is possible to conclude that the feeding with a mixed fodder containing 20% or 24%, gross fibre had a negative influence on the male gonadal development and function and the installation time of the puberty status.

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Ribosomal DNA sequence based characterization of Trichoderma spp. against Sclerotium rolfsii causing collar rot of brinjal in Bay Islands

Bhagat Someshwar, Ahmad Israr and Tripathi Arvind Kumar

The biocontrol fungi, Trichoderma are the most important biocontrol agent and have been used extensively worldwide for the management of various plant pathogens. Trichoderma isolates were isolated from different crops rhizosphere of Bay Islands and established antagonistic potential against Sclerotium rolfsii based on phylogenetic analysis of nucleotide sequences. All isolates of Trichoderma spp significantly inhibited the S. rolfsii by dual culture and production of non-volatile inhibitors at 5 and 10% concentrations of culture filtrate. DNA sequences of the isolates included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1-ITS2). The 5.8S region was found conserved and much of the sequence variability was due to indels or transition/transversion mutations in the ITS1 and 2 regions. Both the isolates belonged to the T. harzianum clade. These isolates are distinct yet have close genetic similarity with several established strains/isolates of T. harzianum reported worldwide. Correct identification of these bioagents would augment the effective utilization of these fungi of immense agricultural importance in managing the collar rot disease of brinjal.

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The Ripening Characterization of Low- and Full-fat Cheese fermented by Lactobacillus acidophilus

Zhao Ruixiang, Ran Junjian, Zhao Lili, Liang Xinhong and Li Gang

Change of soluble nitrogen content at pH 4.6 (pH 4.6 SN), change of 12% trichloracetic acid soluble nitrogen (12% TCA-SN), amino acid, texture, micro structure of low- and full-fat cheese fermented by L. acidophilus have been investigated. The results showed that there were more than 28 main flavor components in full-fat cheese and at least 19 in low-fat cheese. The protein had been proteolysed in varying degrees after ripening for 90d at 400C. The pH 4.6 SN was 4.97% and 3.88% and that of 12% TCA-SN was 5.17% and 3.74% in low- and full-fat cheese respectively. The total amino acids contents in low- and full-fat cheese were 29.17 mg/100g and 16.02 mg/100g. The hardness, gumminess and chewiness of low-fat cheese were higher than that of full-fat cheese while the springiness, cohesiveness and adhesiveness were less than that of full-fat cheese. The structure of low-fat cheese after ripening was more tightening and compact while that of full-fat cheese had a lot of cavity.

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Anticancer Mechanism of Unexplored Plant Compounds – A Review

Subramanyam Deepika and Immanuel Selvaraj C.

The elucidation of structures and functions of plant derived natural products having various biological properties plays a vital role in treating several lethal diseases. This review mainly focuses on the study of rarely explored plants and their compounds which have been scientifically proved to be prospective anticancer agents. Existing cytotoxic drugs affects rapidly dividing cells without adequate differentiation of normal and cancerous cells, it also damages the normal tissues. So the strategies for discovering cancer drug mainly reflect on targeting specific proteins implicated in tumour growth and progression. As the use of phytochemicals are promising and escalating rapidly, the appropriate scientific study to extract bioactive chemicals, detail assessment of its role in anticancer treatment and clinical studies might be the appealing issue of upcoming cancer research. This review summarizes proof of benefits by presenting the list of non-toxic and prevailing natural sources of drugs for patients who are in need. Thus, it is worthwhile to overview the traditional and current status on the usage of bioactive compounds in anticancer activity.

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