Research Journal of Biotechnology

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Process Optimization for the Production of Rainbow Trout Growth Hormone (rtGH) Fusion Protein

Beshay Usama, Moreira Antonio and Chen Thomas

Recombinant Escherichia coli pRE1-rtGH 121/MZ1 strain harboring a plasmid coexpressing a fusion protein of rainbow trout growth hormone (rtGH) was used in fermentations to optimize levels of growth hormone production. Medium compositions, inducer level, and growth stage at inductions were optimized in shake-flasks and in stirred tank bioreactors with respect to volumetric productivity of the recombinant growth hormone. A complex medium, with glycerol (45 g/l) as a carbon source and yeast extract (25 g/l) as a nitrogen source, was chosen. Isopropyl-b-D-thiogalacto-pyranoside (IPTG) concentration of 5.0 mM was found to give the best rtGH production level. The volumetric yield of rtGH was maximal when the culture was induced at the early-logarithmic growth phase. The maximum concentration of produced rtGH obtained from batch fermentation in stirred tank bioreactor was 0.34 g/l as compared to 0.033 g/l in shake-flasks after 24 h.

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Immobilization of Invertase on Functionalized Mesoporous Silica and properties of the immobilized biocatalyst

Prabhune Asmita, Karandikar Sulabha, Pundle Archana, Deshpande Shilpa and Pardhy Sanjeevani

Invertase, which catalyses the hydrolysis of sucrose, is one of the simplest commercial carbohydrases and is used for the production of invert syrup (mixture of glucose and fructose). Fructose is the sweetest natural sugar having beneficial medicinal effects and is emerging as an alternative sweetener to sucrose in the pharma-ceutical industry. b fructofuranosidase (E C purified from Kluyveromyces marxianus NCYC 2675, a thermotolerant yeast, was immobilized on functionalized mesoporous silica SBA- 15. Attempts were made to immobilize invertase on functionalized silicalite and cross linked with glutaraldehyde so as to obtain a highly active and stable immobilized enzyme preparation suitable for commercial exploitation. Cross linked enzyme silica complex as well as enzyme silica complex showed very good temperature and pH stability. Extrudates of the mesoporous silica also showed similar results. Bound enzyme showed excellent stability to repeated use and retained 90% of its initial activity after ten cycles of reuse in batch mode.

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Physico-Chemical and Biochemical Characteristics of the Fermentation of Boganda, a Brandy of Congo

Diakabana P., Louembe D. and Kobawila S.C.

Boganda, a brandy made from retted and dried cassava (Manihot esculenta sp.) and malted corn (Zea mays sp.), is a spirit beverage appreciated in Congo-Brazzaville. The porridge (pasted dense wort) obtained by mixing the thick wort of the corn malt and the foufou starch (floor of retted and dried cassava root) makes a mixed fermentation that is compared to the fermentation in the brewery. The addition of yeast in the wort improves the rate of the saccharification with 4.5 % and of starch conversion into ethanol with 6 %. It allows the diminution of the acetic acid content four times less and the increase of iso amylic content with a factor of 1.3 into the boganda. Ethanol represents 96% of volatile organic substances in the boganda and the other metabolites are in minor quatities, notably 3.5 % for the acetic acid and 0.114 % for isoamylic alcohol.However, they participate all to the sensory quality. The boganda is an ethanolic fermentation.

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Role of Oxygen Scavengers in Improving the Stability and Viability of Pediococcus pentosaceus

Rao T. Surya Chandra, Rachappa S.B. and Agrawal Renu

Studies were done to improve the stability and viability of probiotic lactic acid bacteria Pediococcus pentosaceus by supplementing cysteine hydrochloride (Cys HCl) and ascorbic acid (AA) as oxygen scavengers at different concentrations. The adjuvants were supplemented to the culture in MRS broth stored at 280C and 180C for 12 days keeping in mind the milk products like biocurd (patent no: NF482005) prepared with the same culture in the laboratory to be used for a period of 10 days. It was found that Cys HCl and AA at a concentration of 100mg/L were the best to improve the stability and viability of probiotic lactic acid bacteria. During the study, the decrease in the pH and an increase in the acidity in control with lower growth were attributed to autolysis as visualized by scanning electron microscopy. Release of proteins was also observed. The increase in growth in the supplemented samples can be correlated to the decrease in the O2 content thus improving the stability and viability by four logs as compared to the control.

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Production of Citric Acid from Aspergillus niger using Sugar Beet as Substrate

Tiwari K.L., Jadhav S.K. and Fatima A.

Citric Acid, one of the most important industrial organic acids, is found in all citrus fruits. Aspergillus niger produces citric acid from various substrates. Aspergillus niger was isolated from orange peel and UV treatment was given for strain improvement. The amount of citric acid produced by A. niger was estimated using different substrates. Sugar beet was chosen for the study and effects of different nitrogen and carbon sources were tested on the production of citric acid. At pH 6 there was optimum production. The study was also done to determine effects of different metals on production of citric acid by A.niger. Day-wise production for citric acid was tested with 4th day giving optimum production

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A Biolistic Approach for Transformation and Expression of Cry 1Ac Gene in Shoot Tips of Cotton (Gossypium hirsutum)

Dangat S.S., Rajput S.G., Wable K.J., Jaybhaye A.A. and Patil V.U.

The possibility of regenerating the tetraploid cotton Gossypium hirsutum cv NH 545 plants using shoot tips excised from 3-5 day old seedlings was investigated. MS basal medium along with 0.1 mg/l BAP and 0.1 mg/l NAA was found better for shooting and the root initiation was obtained using 0.3 mg/l IBA. We also tried to transfer these with Cry 1Ac gene using the particle bombardment process. Shoot tips were used as explants in transformation experiment with Cry 1Ac gene and npt II as selection marker gene driven by a CaMV 35S promoter in pBIN Bt3 plasmid vector. All the conditions for optimal delivery of DNA to explants were provided. Transformed putative plantlets were selected on the MS media containing growth regulators along with 75 mg/l kanamycin. The integration of the Cry 1Ac into the genomic DNA of plant was confirmed by PCR method using both npt II gene specifics. Transient expression of Cry 1Ac protein was confirmed by dipstick method

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Study of Callus and Shoot Tip Culture for Micropropagation in Chilli (Capsicum annum L.)

Patil Dharmendra P. and Patil Virupakshagouda U.

Red pepper (Capsicum annum L.) is an economically important crop and used as spice and vegetable through out the world. Besides being an important food crops, Chilli peppers are used in pharmaceutical industries. Capsaicin, the main alkaloid responsible for pungency in chilies is used as a counter irritant balm form external application and it is also used in creamy for to provide enhanced pain relief for arthritis patients. Present investi-gation was carried out to see the effect of different growth regulators on the callus induction and plantlet proliferation from the Chilli (C. annum L.) cv. Fof. 2.

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Optimization of Media for Expression and Economic Production of Recombinant Proteins of Therapeutic and Diagnostic Interest in a Novel Osmotically Inducible E.coli GJ1158

Janardhan S., Srinivasa Babu K., Kaliraj P. and Meenakshisundaram S.

Human granulocyte-macrophage colony stimulating factor (hGM-CSF), an important therapeutic cytokine and Wuchereria bancrofti protein Wb-SXP1 a potential candidate for immunodiagnosis of Human Lymphatic filariasis, were cloned in our laboratory considering future industrial applications. For economic production of recombinant proteins, a novel osmotically (salt) inducible Escherichia coli GJ1158 host was prefered. The recombinant protein expression in E.coli GJ1158 during batch operation with LB/ON and basal M9 media supporting low cell densities was identical, whereas glucose based M9 media showed better recombinant protein expression. Continuous cultivation was carried to understand GJ1158 growth kinetics. Components were tested during steady states for media optimization. However, with E.coli GJ1158, basal level expression was observed in modified M9 medium supporting higher cell density which was unreported in LB/ON, making media design very critical. The performance of GJ1158 in varied glucose and yeast extract levels was studied for further improvisation. A final inducer (NaCl) concentration at 200mM and biomass concentration at ½ OD600max for any media was found to be optimal. In GJ1158, proteins were expressed more in soluble form and purification was easily done under native condition. A short fed batch using recombinant GJ1158 was designed and studied. Leaky expression, cell lysis, foaming and plasmid mediated burden interfered with biomass maximization in fed batch. The maximum cell concentration successfully increased several fold in fed batch compared to batch with basal M9 or LB/ON medium. The cell growth and recombinant protein expression in GJ1158 are discussed. This study demonstrates a successful media optimization for over-expression of recombinant protein in E.coli GJ1158

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In vitro rapid multiple shoots proliferation and plant regeneration of herbal spice Patchouli - Pogostemon cablin (Blanco) Benth

Gopi C. and Polenin Sripal

Efficient protocol for rapid multiplication and plant regeneration of the herbal spice Pogostemon cablin (Lamiaceae) through node explant culture was established using Murashige and Skoog (MS) medium. MS medium supplemented individually with 0.5 mgl-1 N6-benzyladenine (BA) and 1.0 mgl-1 kinetin (KN) showed the higher rate of shoots proliferation; induced a mean of 38.8±0.721 shoots/culture for the periods of four weeks. These shoot clumps were separated and sub cultured MS media containing BA (0.5mgl-1) and NAA (1.0mgl-1) combination for three weeks. Four to five shoots were maintained in each clump. Each shoot attained a height of more than nine cm bearing more than five to seven nodes within 35 days. Well developed plantlets were transferred for acclimatization

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Enhanced Activity of L-asparaginase Produced by Genetically Engineered Recombinant E.coli cells

Verma Neelam, Kumar Kuldeep, Kaur Gurnoor and Anand Sneh

An experimental system has been developed for the production of L-asparaginase by recombinant E.coli cells. mRNA from the strain E.coli K-12 was isolated and cDNA generated from it using RT-PCR. This cDNA was later modified and ligated to pARC 035 vector that was then used for the transformation of E.coliGJ1158 cells. The transformed colonies were selected on IPTG X-gal medium and later tested for L-asparaginase production whose enzyme activity was calculated as well. The enzyme was produced intracellularly. There was a marked improvement in the activity of L-asparaginase (5.5 ± 0.23U/ml/min) obtained from recombinant cells, which rose to five-fold than the native enzyme

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