Research Journal of Biotechnology

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Research Journal of Biotechnology





Expression, purification and characterization of a NAD+- malic enzyme from Oryza sativa L. in Escherichia coli

Zhou Hao1 , Liu Shenkui2 and Chuanping Yang1*

The cDNA fragment of NAD+-malic enzyme was cloned from Oryza sativa L. (OsNAD-ME1) and constructed into expression vector (pGEX-6p-3). OsNAD-ME1 was successfully expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21. The optimal concentration of IPTG for induction was 1mmol/L and the optimal growth temperature was 30°C. The fusion protein was purified by using affinity chromatography with a Glutathione Sepharose 4B column. After enzymatic cleavage of the GST tag, the OsNAD-ME1 recombinant protein was collected for studying its kinetic properties. The optimum pH and temperature for OsNAD-ME1 were pH 6.4 and 35°C respectively. The kcat value determined at pH 6.4 was 36.38 s-1 and the Km values for NAD+ and malate were 0.10 and 15.98mmol/L respectively. The maximum activity of OsNAD-ME1 using NADP+ as coenzyme is 64.47% of the maximum activity with NAD+ as coenzyme.

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Modeling and Maximizing AFLP Pre-Amplification Yield using Response Surface Methodology with Covariate

Muhanad Walid Akash

Abstract: This study was performed to model annealing temperature and [Mg+2] changes during AFLP preamplification step in order to maximize their levels. Response surface methodology (RSM) with covariate was employed for DNA samples obtained from barley, crocus and faba bean plants. Applying a second order regression model showed that annealing temperature and [Mg+2] had a significant influence on band concentration obtained from the pre-amplification process. The canonical analysis results showed that the highest band concentration was obtained with annealing temperature equal to 56.2 °C and [Mg+2] equal to 1.76 mM. The contour plot of the predicted response surface confirmed this conclusion.

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Identification of Genotype and Allelic Frequencies of Vitamin D Receptor Gene (Taq1) Polymorphisms in Type 1 Diabetes Mellitus Patients from Turkey

Hasibe Cingilli Vural

Abstract: Changes in the DNA or polymorphisms of the VDR cause the protein to bind more or less tightly to 1,25OH. The tighter the vitamin D binds, the stronger and longer lasting the metabolic changes are. Some of the different polymorphisms of the VDR have been associated with an increased risk for Diabetes Mellitus. Recent studies suggest that allelic variations of the vitamin D receptor (VDR) gene can influence Diabetes Mellitus. In brief, the aim of this study was to assess the contribution of these VDR polymorphisms to the susceptibility to Type 1 Diabetes Mellitus in the Turk population. There are several polymorphisms for the VDR gene, but only the three most commonly studied polymorphisms are Taq1, Bsm1 and Fok1 examined. This study suggests that while Taq1 polymorphisms may be functionally different, it may also play a role in serum levels. TT allele of VDR gene has been associated with higher Diabetes Mellitus risk for study on young adults or 100 patients with Type 1 Diabetes Mellitus (DM) (50 women, 50 men) and 120 healthy subjects. The Polymerase chain reaction (PCR) was used for amplification of a 200 bp fragment of the Vitamin D Receptor gene. One study found that TT genotype are overrepresented in Type 1 Diabetes Mellitus patients and those with the TT allele had a 3 fold increase in Type 1 Diabetes Mellitus risk. In addition, the aim of the present study was to adapt PCR amplification, the PCR-RFLP and the most effective DNA isolation method. Taq1 polymorphism indicates susceptibility to Type 1 Diabetes Mellitus in the Turk population. The results indicate that the Taq 1 polymorphism in the VDR gene plays a significant role in protection against T1DM.

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Silver Recovery from Waste X-Ray films using Alkaline Protease from Aspergillus Wentii and its Antibacterial Studies < /p>

Lee Kulkarni Prema and Rathod Vandana*

Abstract: Kulkarni Prema and Rathod Vandana*

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Aspergillus niger- A potential enzyme producer on cost effective agro industrial wastes

Suganthi R., Anjana Hari, Arumugam B., Arungopal M., Ramesh Kumar V. and Fathima Benazir J.*

Solid state fermentation (SSF) techniques are gaining foothold in commercial processes to produce a wide variety of enzymes mainly from fungal origin. Production of amylase and protease by solid state fermentation employing two strains of Aspergillus niger isolated from bread was evaluated. Various substrates used were cheap agro industrial wastes such as wheat bran, rice bran, black gram bran, coconut oil cake, gingely oil cake and groundnut oil cake. The process parameters tested were incubation time, incubation temperature and pH, carbon and nitrogen sources. The strain Aspergillus niger BAN3E exhibited the highest amylase production. The maximum amylase activity was recorded in groundnut oil cake supplemented with starch as carbon source and ammonium sulphate as nitrogen source after an incubation period of 6 days at temperature 37 ºC and pH 7. The best protease producing strain was Aspergillus niger BAN1E which gave the maximum yield of the enzyme in wheat bran supplemented with 0.2 % maltose and ammonium sulphate as carbon and nitrogen sources respectively on the sixth day. The optimum pH and temperature for enzyme production was found to be 7 and 40°C respectively. The optimum parameters were utilized to formulate production media for the two fermentation processes. The proteins were separated on SDSPAGE and compared with markers. Zymogram analyses were conducted with starch and gelatin as the respective substrates for amylase and protease respectively. This study could be instrumental in the large scale industrial production of amylase and protease for commercial utilization.

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α-amylase from Bacillus aquimaris VITP4 by solid state fermentation exhibits broad operational range

Anupama A. and Jayaraman G.*

Abstract In order to fulfill the increasing demand of α- amylases at the level of industries, cost effective procedures should be applied in α-amylase production. The present study investigates the production of α-amylase in the presence of potato starch as the main carbon source for the halotolerant bacterium Bacillus aquimaris VITP4. This halotolerant bacterium exhibited enhanced enzyme production in 1 M NaCl concentration (14.48 U/mg) and in the presence of 5 g substrate. At optimal conditions, pH 7.5 and temperature 50oC, the hydrolytic activity of the enzyme was found to be 1,090 U/ml (specific activity 947 U/mg). The activity was enhanced by the presence of calcium and ferrous ions whereas other metal ions had an inhibitory activity. Partial purification of the enzyme was carried out using 80 % (w/v) ammonium sulphate precipitation that resulted in 2 fold purity and the molecular mass as determined from SDS-PAGE was 49 kDa. The ease of production and partial characterization reveals application of α-amylase in industries like paper, bread, bakery, starch saccharification and food industries.

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Purification of Protein from Probiotic Leuconostoc Mesenteroides Active against Vibrio Cholerae

Rajendra Vanitha and Agrawal Renu*

Abstract: Aim of the research is to purify the protein fraction active against V. cholerae from probiotic Leuconostoc mesenteroides and optimization of physical parameters for maximum production. Physical parameters like pH, time and temperature were optimized for maximum production of antimicrobial compound which was found to be at pH 5.5 and 300oC when the culture was grown for a period of 20 h. The active fraction was separated after HPLC and molecular mass was determined using LCMS which was found to be 22,000. The activity was destroyed with protease and trypsin. It was found to be thermostable and could resist 100 0C and also 1210C. The antimicrobial active fraction with a molecular mass of 22,000 from probiotic lactic acid bacteria Leuconostoc mesenteroides significantly reduced the colonies of V. cholerae.The probiotic lactic acid bacteria could be used in various food formulations which would inhibit pathogenic bacteria V. cholerae.

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Lipid Lowering and Antioxidant Activities of Methanolic Extract of Andrographis paniculata Leaves in Normal and Streptozotocin-induced Diabetic Rats

Radhika P1*., Annapurna A.2 and Nageswara Rao S.2

Methanolic extract of Andrographis paniculata leaves was evaluated for its blood glucose reduction in normal, type I and type II diabetic rats. A single oral dose 100 mg/kg was used. The extract was found to produce significant hypoglycemic effect in normal rats and antihyperglycemic effect in type II diabetic rats. Dose dependent percent glucose lowering effect of the extract was evaluated in type II diabetic rats. As 100 mg/kg of the extract was producing a significant glucose lowering effect, the effect of chronic administration (7 days) of the extract on various biochemical parameters was evaluated. Blood glucose, hepatic glycogen content, serum lipid profile and oxidative stress were measured. The extract was found to have glucose lowering, lipid lowering and antioxidant activities in normal and type II diabetic rats.

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Enhancement of Lipase Production from Pseudomonas sp. by Induced Mutations

Arunasri R., Anilkumar S., Reddy Praveen kumar, Soumya and Sulochana M. B.*

Abstract: Strain improvement program was developed to increase extracellular lipase production from Pseudomonas sp. The use of any scientific techniques that allows the isolation of cultures exhibiting a desired phenotype is the process of strain improvement. The utilization of improved microbes for industrial processes is not new. Today, the large scale production of health care products, amino acids, food additives, enzymes and antibiotics serves testimony to the important role of strain improvement in shaping the pharmaceutical and fermentation industries. The improvement of microbial strains offers the greatest opportunity for the cost reduction without significant capital investment. The purpose of the present investigation is to enhance the production of lipase by subjecting the indigenous lipase producing strain Pseudomonas aeruginosa RAS-4 to strain improvement by using UV light as a physical mutagen.

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Production of Xyloglucanases from Three Species of Filamentous Fungi - Fusarium equiseti, Aspergillus terreus and Cephalosporium sp.

Rashmi R.1 and Siddalingamurthy K. R.2*

Abstract: Agro-industrial residues containing lingocellulosic material have attracted wide attention in recent years as raw material for the production of many industrial enzymes. Three filamentous fungi viz. Fusarium equiseti, Aspergillus terreus and Cephalosporium sp. capable of producing xyloglucanase, using tamarind seed powder as the sole organic carbon source for growth, were isolated from soil samples collected near Bangalore. The present work aims at production of xyloglucanases from these organisms and alteration of production conditions for enhancement of activity. Czapek Dox medium produced higher activity of 97.2, 67.2 and 65.2 U/hr/ml culture filtrate from Aspergillus terreus, Fusarium equiseti and Cephalosporium sp. respectively. The enzyme activity of Cephalosporium sp. and A. terreus XG increased by 3.45 and 1.5 folds in the presence of 40 mM Mg2+ while in F. equiseti, XG activity increased 3.35 fold in the presence of 60 mM Mg2+. All three enzymes were secretory while the xyloglucanase from A. terreus was identified to be membrane bound.

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Long term effect of Di (2-ethylhexyl) phthalate on the liver of female Swiss albino mice Mus musculus

Singh Anjali 1*, Kumar Ravish 1, Singh J.K.1 and Tanuja2

Abstract: Investigations on female Swiss albino mice in order to evaluate the long term toxicity of orally administered DEHP at a dose of 20 mg/kg/ bodyweight/ day for a period of six weeks on biochemical parameters of liver showed a decrease in body weight that was statistically not significant (P>0.05) in two weeks while it was statistically very significant (P<0.01) in four and six weeks DEHP treated groups of mice. The relative weight of liver increased in treated groups and the changes in liver weight were statistically not quite significant (P>0.05) in two weeks treated mice whereas it was statistically extremely significant (P<0.001) in four and six weeks treated mice. The increased SGPT level was statistically significant (P<0.01) in two weeks treated mice while it was statistically extremely significant (P<0.001) in four weeks and six weeks treated mice. Increase in serum ALP level in DEHP treated groups was statistically significant (P<0.05) in two weeks, statistically very significant (P<0.01) in four weeks and statistically extremely significant (P<0.001) in six weeks treated groups. The serum level of albumin and total protein decreased progressively from second week to sixth week and the decreased value was statistically not significant (P>0.05) in two weeks while statistically very significant (P<0.01) in four weeks and found statistically extremely significant (P<0.001) in six weeks treated group.

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CFTR Gene mutations and Clinical correlation in Indian patients with Congenital Bilateral Absence of the Vas Deferens

Jain Manish Kumar1* and Saraf D. K.2

Abstract: Cystic fibrosis (CF) is the most common potentially lethal autosomal recessive disorder. Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. Here we identify different mutations of CFTR and the poly-T variant of intron 8 (IVS8) in Indian patients (Sagar district of MP) and analyze sweat test values and clinical characteristic related to Cystic Fibrosis (CF). For counseling purposes the most frequent possible mutation in Indian population: deltaF508 was screened in wives. Four patients (23%) showed abnormal chloride values (> 60 mmol/l). A second group of 3 patients (18%) had borderline values of sweat chloride (40-59 mmol/l). We defined another group with 3 patients (18%), with normal sweat chloride levels (30-39 mmo/l) and a fourth group of 8 (41%) patients with sweat chloride below 30 mmol/l, delta F508 muation was found in 3 of the 18 patients (16%). On a sample of 14 patients, IVS8 analysis showed a frequency of 6/56 chromosomes (11%) of 5T allele.

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Fed-batch Fermentation for Pleuromutilin Production by Pleurotus mutilis

Xiang Zoua,1,2* Bing Li1 and Chang Hua Hu1

Abstract: The effects of carbon source (i.e. glucose concentration) on cell growth and production and productivity of pleuromutilin were investigated. A relatively higher initial glucose concentration could improve the production of pleuromutilin but will decrease the productivity of pleuromutilin in shake flask. Different fed-batch strategies including constant feed rate fed-batch, exponential fed-batch, constant glucose concentration fed-batch and pH control fedbatch were further compared with batch fermentation in 7 l fermentor. Constant glucose concentration fedbatch was an effective method for simultaneously enhancing the production and productivity of pleuromutilin that reached 4.99 g/l and 0.026 g/l per hour at 192 h which was 55.9 % and 100 % higher than that in batch fermentation. The fed-batch method was successfully scaled up in 50 l fermentor which the production and productivity of pleuromutilin were 10.15 g/l and 0.053 g/l per hour at 192 h. This work is helpful for the large-scale production of pleuramutilin

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Molecular Identification of root nodule bacteria from Cicer arietinum

Rao Agrawal Pooja

Abstract: A total of 85 samples of Cicer arietinum, collected from different fields of district Sagar (M.P.) were processed for the isolation of root nodule bacteria. A total of 94 bacteria were identified as Rhizobium on the basis of Lactose agar test, nitrate reduction test, citrate utilization test, motility, oxidase, catalase, different staining techniques like Gram’s staining and carbol fuschin staining. All the isolates were tested for phosphatase activity on solid plate assay. Maximum phosphatase production was noted in the test strains and were selected for quantitative estimation of phosphatase and indole acetic acid production where as siderophore production was tested for qualitative assay. Selected rhizobia were subjected to RAPD and ARDRA analysis to identify the species. On the basis of RAPD and ARDAR all the 10 isolates were identified as Rhizobium meliloti. Higher phosphatase activity was noted in ten Rhizobium isolates i.e. Rhizobium G04, G16, G20, G29, G30, G45, G75, G77, G88 and G98.

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