Research Journal of Biotechnology

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Micropropagation of Papilionanthe teres (Roxb.) Schltr. by seed and shoot tip culture

Neupane Pradeep, Pandey Bikram, Tripathi Salina and Pant Bijaya

Page No: 1-8

Abstract: Papilionanthe teres (Roxb.) Schltr. is an epiphytic orchid having ornamental and medicinal value. The main aim of this study is to develop a reliable protocol for seed germination and micropropagation of P. teres. In vitro seed germination was carried out from immature seeds on different strength (full, half and quarter) of Murashige and Skoog (MS) medium and MS medium supplemented with 0.5mg/l NAA and 0.5 mg/l BAP. MS medium supplemented with NAA(0.5mg/l) and BAP (0.5mg/l) was found to be the best condition for germination. The germination was also favored in half strength of MS.

Shoot multiplication and rooting were carried out in different concentrations and combinations of Gibberellic acid (GA3) and auxins (IAA, IBA and NAA) in half-strength MS medium. Among them, half-strength MS medium supplemented with 2 mg/l GA3 was found to be effective condition for shoot development while half-strength MS medium supplemented with 0.5 mg/l GA3 favored root multiplication. The present study shows that this species needs gibberellic acid rather than auxins for in vitro growth. This protocol might be useful to select the best condition for asymbiotic germination, micropropagation and ex situ conservation of this valuable orchid.

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In vivo, in vitro and in silico screening of a potent Angiotensin Converting Enzyme (ACE) inhibitor from Trigonella foenum-graecum extract using Zebrafish as a model organism to reduce hypertension

Teena Suresh and Suma Sarojini

Page No: 9-16

Abstract:The number of patients suffering from hypertension is on the rise worldwide and there is a need to explore natural products which can supplement current drugs to treat this disease. RAAS (Renin Angiotensin Aldosterone System) is one of the factors maintaining blood pressure. In the present investigation, we explored the potential of methanolic extract of fenugreek seeds in inhibiting Angiotensin Converting Enzyme (ACE), a key enzyme in the RAAS system, thereby reducing hypertension. In addition to in vivo studies conducted on zebrafish, in vitro and in silico studies were also performed to assess the inhibitory effect of the fenugreek extract on ACE. The bioactive components in Trigonella foenum-graecum revealed by GC-MS were further subjected to docking and binding studies with the receptor protein ACE. Of the various phytochemicals studied, arachidonic acid exhibited the maximum inhibitory effect on ACE.

Thus, the present investigation was able to favorably screen a potent ACE inhibitor in Trigonella foenum-graecum extract which shows a potential to be used alone or supplemented with synthetic ACE inhibitors to treat high blood pressure. Further investigations are required to quantify the phytochemical for its inhibitory activity and also to understand the mechanism of inhibition of the enzyme.

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Enhanced Production of polygalacturonase from mangrove soil isolate Aspergillus terreus RR105

Ketipally Ravi and Raghu Ram Mukkavilly

Page No: 17-24

Abstract:In the present study, fungal strains were isolated from mangrove habitats of Krishna district, A.P., India. The isolated strains were screened for pectinolytic activity and the potent fungus was identified based on cultural, morphological and molecular characterization as Aspergillus terreus RR 105. Various physical parameters and nutritional substrates were analyzed to enhance the polygalacturonase production.

Highest enzyme production was achieved when the strain was grown on modified pectin broth (pH 6.5) amended with sucrose and malt extract at 35°C after 168 h of incubation. Production of polygalacturonase by Aspergillus terreus RR 105 is considered suitable for industrial scale production as it has high productivity.

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Genetic Relationships and Population Structure of Central Asian Apple Species collected from Different Parts of Kyrgyzstan

Aydin Uzun, Kubanichbek Turgunbaev, Abdykerim Abdullaev and Hasan Pinar

Page No: 25-32

Abstract: Apple is among the most widely grown fruit species worldwide. It is included in the genus Malus and there are several species of apple. Although a consensus is not available about the origin of domesticated apples, it is widely accepted that Malus sieversii, known as Central Asia wild apple, was the primary progenitor of apples. Central Asia is a quite significant origin center of apple and harbors different species and a great diversity. In this study, genetic relations among 79 apple materials belonging to 4 different species (M. domestica, M. sieversii, M. kirghisorum and M. niedzwetzkyana) collected from different regions of Kyrgyzstan, Central Asia and their population structures were put forth. A total of 15 ISSR markers were used and 79 bands were obtained with a polymorphism ratio of 75.2%.

Almost all of the apple materials used in this study were separated from each other. Genetic similarity values varied between 0.76-1.00. Present materials were gathered under two main groups and 4 sub-groups of the dendrogram. Sub-structuring analysis indicated that there were four sub-populations among the accessions studied. It was observed that there were significant admixtures and transitions among the populations. With this study, apple genetic diversity of Kyrgyzstan was put forth and significant data were presented for the preservation and use of these materials in further studies.

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3D perfusion culture of Huh-7 cell line for expression of drug-metabolizing markers

Suma M.S., Jamuna K.S., Ramesh C.K. and Riaz Mahmood

Page No: 33-39

Abstract:Development of ex vivo cell culture models that adequately recreate in vivo conditions is useful in improved drug screening. In this regard, we developed a scaffold incorporated perfusion culture system which is shown to have improved gene expression of drug metabolizing enzymes in comparison with the static culture in the multi-well plates. The purpose of this work is to provide a simple, easy-to-fabricate 3D hexagonal, hexa-channel micromachined scaffolds for studying liver cell line (Huh-7). Scaffolds are further incorporated into custom-made perfusion-enabled bioreactors that emulate flow as seen in vivo. Cells are cultured in the scaffolds and then incorporated into a perfusion culture system. RNA isolated on day 3 and day 7 of culture were subjected to gene expression on these two different time points by qPCR. Cytoskeletal assessment is done by rhodamine-phalloidin staining.

This work shows the effects of using Polydimethylsiloxane (PDMS) hexagonal scaffolds of 50µm height as a three-dimensional model to study Huh-7 cells. PDMS scaffolds are biocompatible, reusable and can be easily incorporated into custom made perfusion system as they are simple to fabricate and scalable due to the mould based design.

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Multiplex PCR assay for detection of major bacterial blight resistance genes Xa21 and xa13 in Basmati (Oryza sativa L.)

Raina Meenakshi and Salgotra R.K.

Page No: 40-46

Abstract:Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastating diseases of rice that limits the crop productivity worldwide. Resistance genes for bacterial blight disease are known and routinely used in marker-assisted breeding either singly or in combination. In this study, a simple multiplex PCR-based functional marker system was developed to simultaneously identify genotypes of wide compatibility conferring resistance against BB pathogens. Two major BB resistance target genes, viz. xa13 and Xa21employed for rice yield improvement were amplified using gene specific primers in a single tube.

A total of 124 backcross population, 125 F2 population and 14 F1 lines were evaluated by multiplex PCR to identify plants that are either homozygous or heterozygous for xa13 and Xa21 genes. The resistance R alleles were amplified using specific functional primers. This method proved to be less time consuming and cost effective as compared to marker by marker selection.

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Comparative evaluation of biochemical changes in the leaves of resistant and susceptible mungbean plants infected by Mungbean Yellow Mosaic Virus

Balasubramaniam Madhumitha, Adhimoolam Karthikeyan, Gurusamy Poornima Devi, Karuppiah Eraivan Arutkani Aiyanathan and Manickam Sudha

Page No: 47-54

Abstract:Mungbean yellow mosaic virus (MYMV) is a wide spread, destructive and economically important disease in mungbean growing regions of Southern India. However, information about mungbean and MYMV interaction is still limited. In this study, the amounts of chlorophyll, total soluble proteins, carbohydrates (starch and total sugars), phenolic compounds, polyphenol oxidase (PPO) and peroxidase (POX) were examined in leaves of resistant (VGGRU1) and susceptible (VRM (Gg) 1) mungbean genotypes. VGGRU1 plants agroinoculated with the MYMV infectious clone VA 239 (KA30 DNA A + KA27 DNA B) showed no visible symptoms while agroinoculated VRM (Gg) 1 plants produced mosaic symptoms in leaves.

The chlorophyll, protein, starch, phenolic compounds, POX and PPO activities were increased in the inoculated plants of VGGRU1, but decreased in VRM (Gg) 1 compared to their respective control. On the other hand, total sugars were recorded to be high on both VGGRU1 and VRM (Gg) 1 plants comparing their uninoculated controls and the total sugars were slightly increased in inoculated plants of VRM (Gg) 1 over the VGGRU1. Taken together, the findings from the present study shed light on the biochemical defense mechanisms involved in mungbean against MYMV.

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Management of blight disease of litchi (Litchi chinensis) caused by Alternaria alternata

Anal Ajit Kumar Dubedi, Kumar Vinod and Varma Ajit

Page No: 55-62

Abstract: Litchi or Lychee (Litchi chinensis Sonn.) is a member of the Sapindaceae family and is regarded as one of the most popular and nutritious fruit. Blight, in the form of leaf, panicle and fruit, was observed in litchi plants cultivated at the farm in ICAR-National Research Centre on litchi, Muzaffarpur, Bihar (India). Based on disease severity, an attempted was taken to control this newly emerged disease caused by Alternaria alternata using different fungicides and bio-control agents in laboratory, glass house and field conditions. Among different fungicides, Difenoconazole at 1000 ppm, Copper Oxy chloride at 1500 ppm, Azoxystrobin at 2000 ppm completely inhibited (100%) mycelial growth of A. alternata under in vitro condition. Two indigenous bio-control agents viz. Trichoderma viride (NRCL T01) and Bacillus subtilis (NRCL BS01) showed 63.3% and 53.8% of Percent Inhibition of Radial Growth (PIRG) of Alternaria alternata in dual culture plate respectively. In glass house condition, Azoxystrobin (PDI=16.7% and PDC=80.2%), Difenoconazole (PDI=18.5% and PDC=78.0%), Thiophanate methyl (PDI=19.3% and PDC=77.1%) and Copper oxy chloride (PDI=24.4% and PDC=70.9%) were found to be highly effective in reduction of leaf blight.

In addition to this, for management of panicle and fruit blight, experiments were also conducted in field condition at season 2017 and 2018 where almost same trends of effectiveness of fungicides were observed. Besides, bio-control agents viz. Trichoderma viride (NRCL T01) and Bacillus subtilis (NRCL BS01) also reduce the incidence of both panicle and fruit blight. Present study is not only giving a promising solution to minimizing the loss caused by Alternaria alternata in litchi by using available commercial fungicides but is also providing an alternate option of using eco-friendly bio-control agents.

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Plackette-Burman Design in optimization of Medium Components for Keratinase Production by a Marine Fungus and Determination of Biological Activities

Siragam Satyalakshmi, Guntaku Girijasankar and Gatte Geetha

Page No: 63-70

Abstract: A marine fungus was isolated from marine sponge Clathria procera which was collected from south-east coast of India and optimized for keratinase production using Plackette-Burman design. Eleven variables in 12 run experiment were conducted to evaluate the effect of different carbon and nitrogen sources. From the ANOVA results, significant variables (P<0.05), improved keratinase production. Soyabean meal and glucose at negative level or low concentration have the highest effect on the keratinase production. Purified enzyme with 28 kDa of molecular weight was used for determination of biological activities like deharing and nail degrading activity.

Antimicrobial activity of fungus was determined by well diffusion method against different bacterial and fungal organisms. The fungal culture was identified as Scopulariopsis brevicaulis and provided with a reference number of MTCC11794 by Microbial Type Culture Collection, Chandigarh, India. Marine fungus in this present work was isolated from a novel marine sponge which was exhibiting significant antimicrobial, keratin degrading and dehairing ability. Keratin digestion property of the purified enzyme was further useful in ungual drug delivery to enhance permeation of applied drug through nails to treat nail infections.

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Gene expression analysis of EGFR and PI3K genes in A549 lung cancer cell line treated with Withania somnifera root extract

Krishna Battula Vamsi, Theboral J. Glory, Tilton Florida, Nair Aneesh, Loganathan Rekha and Pratheep Thangaraj

Page No: 71-75

Abstract:The present study investigated the gene expression of EGFR and PI3K genes in Human Non-Small Cell Lung Cancer cell line (NSCLC) when treated with root extract of Withania somnifera. NSCLC (A549) was treated with ethanolic root extract of W. somnifera and gene expression levels of Epidermal Growth Factor Receptor (EGFR) and phosphatidyl inositol-3-kinase (PI3K) measured by RT PCR. EGFR gene was constantly expressed in both control and treated cell line. But PI3K gene expression was less in treated cells than control.

This study revealed that the root extract of W. somnifera can down regulate the expression of PI3K gene and its downstream molecules in pathway up to some extent. Further research is required to know about whether this drug can prevent metastasis of tumor cells or not.

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Improvement of growth and development of olive tree by mycorrhizal autochthonous inoculum

Boutaj Hanane, Meddich Abdelilah, Wahbi Said, Moukhli Abdelmajid, El Alaoui-Talibi Zainab, Douira Allal, Filali-Maltouf Abdelkarim and El Modafar Cherkaoui

Page No: 76-84

Abstract:The aim of the present work is to study the effect of arbuscular mycorrhizal fungi (AMF) “autochthonous inoculum” on olive tree growth and development (Picholine Marocaine cultivar). Inoculated olive plants with mycorrhizal autochthonous inoculum namely Rhizolive consortium or with commercial pure strain Glomus irregulare were grown for nine months under greenhouse conditions. Our results indicated that inoculation with Rhizolive consortium and G. irregulare caused a significant (p < 0.05) improvement in vegetative growth and plant biomass. Indeed, root colonization as well as mycorrhizal frequency and intensity were greatly improved in Rhizolive consortium (100% and 58.91% respectively) and G. irregulare (97.77 and 57.77% respectively) treatments.

Moreover, the highest values of vesicular and arbuscular content were recorded in AMF treatments as compared to control plants. Significant difference (p < 0.05) in nutrient concentrations such as sodium, calcium and phosphorus was shown in olive shoot in Rhizolive consortium treatment (3.24, 3.14 and 0.119 mg g-1 respectively) whereas, in olive root similar significance was recorded in Rhizolive consortium and G. irregulare treatments. Rhizolive consortium was slightly better that G. irregulare regarding nutrient accumulation. In general, AMF increased significantly the growth and development of olive plants “Picholine Marocaine” as compared to control plants.

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Isolation and screening of tannase producing bacterial isolates from a hyper saline environment in Tamil Nadu, India

Lekshmi R., Arif Nisha S. and Kaleeswaran B.

Page No: 85-90

Abstract:Halophilic or halotolerant microorganisms survive at wide range of salinity which offers great potential uses in the field of biotechnology. These microorganisms are playing very important role in nutrient recycling in hypersaline environment and soil management. Hypersaline environment is a niche between marine and terrestrial environment that supports colonization of diverse group of microorganisms.

In the present study, an attempt was made to isolate and screen tannase producing bacteria from the hypersaline environment in South India. Tannin acyl hydrolase which is generally referred as tannase, hydrolyse tannins and produces gallic acid, galloyl esters and glucose. In order to screen tannase producing bacteria in salt pans, a total of 243 bacterial strains were isolated and only 20.9% bacteria exhibited tannase activity. Most of the screened bacterial isolates were grown between 5 and 15% NaCl and pH between 7.0 and 8.0. Tannase producing ability of the bacterial isolate was analyzed using various methods including substrate containing minimal medium, enriched medium and assay of cell free supernatant using well diffusion method. The morphological and biochemical features of the potent isolates were studied. A potent bacterial strain B48 was identified as a gram-positive Bacillus sp. which is rod shaped, motile and spore forming. Tannase from halotolerant environment has potent application in the treatment of tannery effluents.

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Evaluation of secretory Galectin9 in uncontrolled type 2 diabetes mellitus

Mudaraddi Rakesh and Perumalreddy Hrudya

Page No: 91-95

Abstract:Homeostasis of glucose is essentially done by glucose transporter (GLUT). GLUT-2 is expressed by pancreatic β cells and Galectin-9 is associated with GLUT-2 in pancreatic β cells and is involved in glucose transport activity. The main aim of this study is to analyze and compare levels of urinary Galectin-9 in healthy controls and diabetics with or without complication. Study subjects (n=82) were divided into 3 groups as normal healthy controls, type 2 diabetics without nephropathy and type 2 diabetics with nephropathy. We have estimated blood sugar, glycosylated haemoglobin (HbA1C), serum urea, serum creatinine, urinary micro albumin and urinary galectin9.

Statistically increased urinary albumin (p<0.05) was observed in patients with micro albuminuria and nephropathy. Urinary galectin9 levels among groups were 5.63 pg/ml, 3.0 pg/ml and 41.8 pg/ml respectively. Cell cycle is dysregulated in diabetic state resulting in high glucose induced cellular hypertrophy. Progression of diabetes with renal system involvement may increase urinary excretion of albumin as well as filtration of Galectin9 through glomerular basement membrane. To conclude this study, increased urinary Galectin9 and albumin excretion in type 2 diabetic nephropathy patients could be related to the micro and macro vascular complications leading to chronic kidney disease.

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Expression of heat shock genes in Egyptian wheat (Triticum aestivum) varieties in response to high temperature

Elseehy Mona M. and Alotaibi Saqer S.

Page No: 96-102

Abstract:Abiotic stresses are determinant factors of plant growth, development and production. Heat is a biotic stress that impact plants in many regions of the world. Wheat is affected by heat stress during its life cycle especially in arid and semiarid regions of the world. Nine commercial Egyptian bread wheat varieties with different biotic and abiotic stress tolerance were used in this study. Expression of two Heat Shock Protein (HSP17, HSP90) and two abiotic stress-induced transcription factors (DREB2, NAC6D) was investigated using semiquantitative PCR of cDNA generated from total RNA under normal and heat stress (40ºC). Results showed substantial differences in the response of wheat varieties in HSP17, HSP90, DREB2 genes expression in response to high temperature whereas NAC6D showed up-regulation in all varieties compared to normal condition.

Giza168 and Sids1 showed high induction of the four investigated genes. They represented 70.6, 55.2 (HSP17), 4.4, 2.7 (HSP90), 7.5, 4.8 (DREB2) and 2.2, 2 (NAC6D) folds of expression level relative to expression of minimal variety. High induction of gene expression in Giza168 and Sids1 seems to be linked to their tolerance to abiotic and biotic stresses. Giza168 is tolerant to high temperature, drought and rusts whereas Sids1 is tolerant to high temperature, salinity and yellow rust. Results of this study will contribute in evaluation of commercial new wheat varieties and their possible integration in wheat breeding programs.

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In Vitro Plant Regeneration of Momordica cymbalaria Fenzl. and Assessment of Genetic Fidelity using ISSR primers

Gopu Chaitanya, Vankudoth Suvarchala, Dasari Ramakrishna, Dharavath Sunitha and Taduri Shasthree

Page No: 103-110

Abstract:Present investigation successfully established the plant regeneration from different explants of Momordica cymbalaria Fenzl., an important medicinal Cucurbit. Leaf, node and shoot tip explants were successfully callused on Murashige and Skoog’s medium (MS) supplemented with various concentrations of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), IBA (indole-3-butyric acid) and cytokinin BAP (6-benzylaminopurine) alone. Maximum percentage of callus (95%) was obtained from leaf explants at 1.0 mg/l 2,4-D. Leaf derived callus highly responded for the shoot regeneration (13.33±0.667shoots) on half strength MS medium containing 1.5 mg/l BAP in combination with 1.5 mg/l IBA. The elongated shoots were rooted on half strength MS media supplemented with 0.2-2.0 mg/l NAA (naphthalene acetic acid) and IBA (indole-3-butyric acid).

The highest number of roots were obtained (5.33±0.667) in 0.5 mg/l NAA. The rooted plants were successfully hardened and transferred to the green house with 90% of survival. Inter Simple Sequence Repeat (ISSR) analysis revealed the genetic stability of in vitro raised plants with mother plant. The present investigation showed that regeneration of Momordica cymbalaria offers a good opportunity to raise micropropagation from leaf, node and shoot tip explants.

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Effect of CuSO4.5H2O on regenerative capacity of mature foliar explants of Papilionanthe teres (Roxb.) Schltr: A study in vitro

Sharma Vishal

Page No: 111-116

Abstract:The regenerative competence of in vivo derived mature leaves of Papilionanthe teres depends significantly on physiological age of donor tissue, copper sulphate treatment and nutritional regime. The juvenility of the tissues emerged as the important factor in inducing regeneration as mature foliar explants(>3cm long), excuded phenolics profusely and remained recalcitrant to embryogenesis, whereas, those from the younger folded ones(<3cm long) released little exudates and regenerated depending upon their position on the source tissue and the regeneration pathway is markedly influenced with exogenous application of cytokinins and auxins which manipulate their endogenous level favouring morphogenetic response. In present studies, the quality of the cytokinin determined the regeneration pathway; however, their efficacy is obligatory to the NAA and modified BM18 medium containing Cu++ ions (2.2 mg.l-1 ), 44x the normal concentration. A combination containing BAP in dose double than that of NAA (10:5) favoured direct somatic embryogenesis producing 10.25±0.25 PLBs in 51.56 ±1.56 responded explants, however, replacing them with KN, the regeneration and embrogenetic response is impaired.

The meristimatic loci are randomly spread in juvenile leaves (<2.5cm long) but is basal gradient in the mature leaves (2.5-3cm). The neo-formations show basipetal polarity. Histological studies revealed that the proliferation is manifested only on adaxial side and prominently restricted to dermal cells. The regenerated plantlets are accilimatized and transferred to pots filled with moss, pinebark, brick and charcoal pieces mixture (1:1:1:1) with 90% survival.

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Hyperproduction of laccase by an indigenous species of Pleurotus pulmonarius under solid state fermentation and in vitro potential applications

Rahi Deepak Kumar, Sidhu Arshdeep and Kaur Maninderjeet

Page No: 117-128

Abstract:Production of laccase by an indigenous species of Pleurotus pulmonarius was evaluated using solid state fermentation with one-factor at a time method at different physical and biochemical conditions. Optimization conditions of production yielded an enzyme with activity 8.56 x 104 U/gds which was 2.3 folds higher than the initial. Laccase was partially purified using ammonium sulphate precipitation and characterization of the enzyme was also performed. The crude laccase was further used for evaluating in vitro applications in decolorization of different classes of dyes, apple juice clarification and deinking of various waste papers.

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Evaluation of antibacterial and antioxidant activities of crude Polysaccharide extracted from medicinal Mushroom Ganoderma lucidum

Jandaik Savita, Jarial R.S. and Jarial Kumud

Page No: 129-135

Abstract:Polysaccharide contents of Ganoderma lucidum extracts are often quoted as an index of the active components. This study was undertaken to reveal the amount of polysaccharides in Ganoderma lucidum fruit bodies. To examine the sugar composition of Ganoderma lucidum, its polysaccharides were hydrolyzed by chemical methods to release the monosaccharide/oligosaccharide components for paper chromatographic analysis. A great variation was found in polysaccharide content of different Ganoderma isolates. Five sugars of medical relevance were identified i.e. rhamnose, fucose, galactose, mannose and glucose. Polysaccharide extract was found very active against all tested bacterial pathogens.

The results show that the G. lucidum polysaccharide has remarkably good antimicrobial activities than mycelial and fruit body extracts. With regard to reducing power, polysaccharide extract (PE) showed the best antioxidant property because of its lowest efficient concentration or EC50 value (0.96 mg/ml) indicating that PE was more effective as an antioxidant.

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Genotypic and Phenotypic Characterization of Endophytic Microbispora SUK 14 from Labisia pumila

Noraziah Mohamad Zin, Noridayu Zakaria, Aishah Ismail, Siti Junaidah Ahmad and Nurul Izzah Mohd Sarmin

Page No: 136-143

Abstract:This study characterized a novel endophytic species designated as Strain Universiti Kebangsaan (SUK) 14 isolated from Labisia pumila plant. The isolate forms closely arranged paired spores along the aerial hyphae and produced dark olive-green aerial mycelium on International Streptomyces Project (ISP) 2 agar. Phylogenetic analysis of SUK 14 using 16S rRNA gene sequence with closely related species revealed a similarity between SUK 14 and Microbispora corallina NBCR 16416T (98.86%), Microbispora rosea subsp. rosea NBCR 14044T (98.64%) and Microbispora siamensis DMKUA-245T (97.72%). The G + C content of SUK 14 is 72.89% and contains -LL DAP isomer in the cell wall component.

Endophytic Microbispora SUK 14 contains only MK-9 (H0) in its plasma membrane as compared to M. corallina having MK-9 (H4), MK-9(H2) and MK-9(H0). Lipid analysis showed that SUK 14 contains phospholipids of L-a-phosphatidylethanolamine (PE) and L-a-phosphatidyl-DL-glycerol (PG). The morphological and chemotaxonomic properties clearly demonstrated that this isolate belongs to the genus Microbispora. Based on these genotypic and phenotypic data, strain SUK 14 might represent a novel species in the genus Microbispora.

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Production, partial purification and characterization of laccase from rhizospheric bacteria Pseudomonas putida strain LUA15.1

Dhiman Karuna and Shirkot Poonam

Page No: 144-152

Abstract:A new laccase producing rhizospheric bacteria Pseudomonas putida strain LUA15.1, was isolated from the rice rhizospheric soil samples of Himachal Pradesh. Optimization of culture conditions was done for bacterial growth and laccase production. The bacterial laccase was purified to homogeneity by ammonium sulphate precipitation (50-90 %) followed by gel filtration and ion exchange chromatography. The procedure yielded 1.11 mg protein with 59.06 fold purification with a percent yield of 19.04.

The purified enzyme had optimal activity at pH 8.0 and 400C temperature. The molecular weight of laccase was found to be 42.5 kDa whereas Km value was found to be 0.028. Enzyme activity was inhibited by DTT and sodium azide. The enzyme exhibited novel characteristics such as activity and stability at alkaline pH and a low Km value. These two characters are quite significant from the industrial point of view making the enzyme an ideal candidate for industrial applications.

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