Indian Journal of Biotechnology


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VOLUME 5

CODEN:IJBNAR 5(3) 257-426 (2006)

NUMBER 3

JULY 2006

ISSN:0972-5849

 

 CONTENTS

 

Review

 

 

Marine Biotechnology: An overview

263

 

        IPC Code: Int. Cl.8 A01N63/00; A61K31/00

 

 

        Narsinh L Thakur & Archana N Thakur

 

 

 

 

 

Papers

 

 

Somatic embryogenesis and Agrobacterium mediated genetic transformation in Indian accessions of lucerne (Medicago sativa L.)

269

 

        IPC Code: Int. Cl.8 A01H4/00 & 5/10; C12N15/09

 

 

        Sanjay Gupta, Shweta Gupta, Vishnu Bhat & M G Gupta

 

 

 

 

 

Synthesis and characterization of poly-β-hydroxybutyrate from Bacillus thuringiensis R1

276

 

IPC Code: Int. Cl.8 A01N63/00; C08G63/00; C12R1/07

 

 

D Rohini, S Phadnis & S K Rawal

 

 

        

 

 

Purification and characterization of the pectin lyase secreted within the macerating fluid of Rhizopus oryzae (Went, Prinsen Geerligs) grown on orange peel

284

 

IPC Code: Int. Cl.8 C12N19/88; C12R1/845

 

 

Hossam S Hamdy

 

 

       

 

 

Properties of hydrogel-entrapped lipase of thermophilic Pseudomonas aeruginosa BTS-2

292

 

IPC Code: Int. Cl.8 C12N9/20; C12N11/04; C12R1/385

 

 

S S Kanwar, M Gupta, R Gupta, R K Kaushal & S S Chimni

 

 

 

 

 

Genetic variability in pea wilt pathogen Fusarium oxysporum f. sp. pisi in north-western Himalaya

298

 

IPC Code: Int. Cl.8 C12N15/10

 

 

Pooja Sharma, Kamal Dev Sharma, Rajan Sharma & P Plaha

 

 

 

 

 

Thidiazuron induced high frequency shoot bud formation and plant regeneration from cotyledonary node explants of Capsicum annuum L.

303

 

        IPC Code: Int. Cl.8 A01H4/00, 5/04

 

 

Iram Siddique & Mohammad Anis

 

 

 

 

 

Short Communication

 

 

Micropropagation of Azadirachta indica A. Juss. via cotyledonary nodes

309

 

        IPC Code: Int. Cl.8 A01H4/00

 

 

        A Rajasekhar Reddy, M Bavaji & J V S Rao

 

 

 

 

 

Supplement Papers

 

 

 

 

 

Isolation and characterization of recombinant Brugian parasitic transglutaminase

317

 

        IPC Code: Int. Cl.8 C12N9/64

  

 

        V Uma, M Geetha, V Murugan & P Kaliraj

 

 

 

 

 

Bacterial lipid modification in vitro: Synthetic peptide substrate for phosphatidyl-glycerol—Prolipoprotein diacylglyceryl transferase

327

 

        IPC Code: Int. Cl.8 C12N1/38

 

 

        A Tamil Selvan & K Sankaran

 

 

          

 

 

Solid-state fermentation of lignocellulosic substrates for cellulase production by Trichoderma reesei NRRL 11460

332

 

        IPC Code: Int. Cl.8 C12N9/42; C12R1/885

 

 

        Reeta Rani Singhania, Rajeev K Sukumaran, Anu Pillai, P Prema, George Szakacs & Ashok Pandey

 

 

          

 

 

Production of a high maltose-forming, hyperthermostable and Ca2+-independent amylopullulanase by an extreme thermophile Geobacillus thermoleovorans in submerged fermentation

337

 

        IPC Code: Int. Cl.8 C12N9/44; C12R1/01

 

 

        S M Noorwez, M Ezhilvannan & T Satyanarayana

 

 

 

 

 

Production of tannase under mSSF and its application in fruit juice debittering

346

 

        IPC Code: Int. Cl.8 C12N9/16

 

 

        S Rout & R Banerjee

 

 

 

 

 

Purification and characterization of an endoxylanase from solid-state culture of alkalitolerant Aspergillus fumigatus MKU1

351

 

        IPC Code: Int. Cl.8 C12N9/42; C12R1/68

 

 

        S Thiagarajan, M Jeya & P Gunasekaran

 

 

 

 

 

Characterization of chitinases from microorganisms isolated from Lonar lake

357

  

        IPC Code: Int. Cl.8 A01N63/04

 

 

        Vijay B Bansode & Shyam S Bajekal

         

 

 

 

 

Esterification reactions catalysed by surfactant-coated Rhizopus arrhizus lipase

364

 

        IPC Code: Int. Cl.8 C12N9/20; C12N11/08; C12R1/845

 

 

        Preetha Sasi, Rohit R Mehrotra & Mira Debnath (Das)

 

 

 

 

 

Entrapment of lipase in polymer of polyvinyl alcohol-boric acid for esterification in organic media

368

 

IPC Code: Int. Cl.8 C12N9/20; C12N11/08; C12R1/72

 

 

        Rachna Dave & Datta Madamwar

 

 

 

 

 

Selection of optimal growth medium for the synthesis of a-galactosidase from mangrove actinomycetes

373

 

        IPC Code: Int. Cl.8 C12N9/40; C12R1/01

 

 

        G S Anisha & P Prema

 

 

 

  

 

Purification and characterization of laundry detergent compatible alkaline protease from Bacillus cereus

380

 

        IPC Code: Int. Cl.8 C12N9/52; C12R1/085

 

 

        R M Banik & Monika Prakash

 

 

Biopulping studies using an effluent isolate Curvularia lunata LW6

385

 

        IPC Code: Int. Cl.8 C12S9/00; C12R1/645

 

 

        K P Narkhede & N N Vidhale

 

 

 

 

 

Cholesterol biotransformation in monophasic systems by solvent tolerant Bacillus subtilis AF 333249

389

 

        IPC Code: Int. Cl.8 C07J9/00; C12R1/125

    

 

        M S Andhale & S A Sambrani

 

 

 

 

 

Optimization of secondary hardening process of banana plantlets (Musa paradisiaca L. var. grand nain)

394

 

        IPC Code: Int. Cl.8 A01H4/00, 5/00

 

 

        Shailesh R Vasane & R M Kothari

 

 

 

 

 

Integrated biological approach for the enhanced degradation of lindane

400

 

        IPC Code: Int. Cl.8 B09C1/10

 

 

        Varima Nagpal & K M Paknikar

 

 

 

 

 

Decolorization of textile dyes by Aspergillus ochraceus (NCIM-1146)

407

 

        IPC Code: Int. Cl.8 C02F3/02; C02F103/30

 

 

        G D Saratale, S D Kalme & S P Govindwar

 

 

 

 

 

Efficient Pseudomonas putida for degradation of p-nitrophenol

411

 

        IPC Code: Int. Cl.8 B09C1/10

 

 

        Meenal Kulkarni & Ambalal Chaudhari

 

 

 

 

 

Microbial degradation of melanoidins in distillery spent wash by an indigenous isolate

416

 

        IPC Code: Int. Cl.8 C02F3/34; C02F101/30

 

 

        M N Chavan, M V Kulkarni , V P Zope & P P Mahulikar

 

 

 

 

 

Decolorisation of reactive red 11 and 152 azo dyes under aerobic conditions

422

 

        IPC Code: Int. Cl.8 C02F3/02; C02F103/30

 

 

        K M Kodam & K R Gawai

 

 

 

 

 

Instructions to Contributors

425

 


 

 

 

AUTHOR INDEX

 

 


Andhale M S

389

Anis M

303

Anisha G S

373

Bajekal S S

357

Banerjee R

346

Banik R M

380

Bansode V B

357

Bavaji M

309

Bhat V

269

Chaudhari A

411

Chavan M N

416

Chimni S S

292

Dave R

368

Debnath (Das) M

364

Ezhilvannan M

337

Gawai K R

422

Geetha M

317

Govindwar S P

407

Gunasekaran P

351

Gupta M G

269

Gupta M

292

Gupta R

292

Gupta S

269

Gupta Sh

269

Hamdy H S

284

Jeya M

351

Kaliraj P

317

Kalme S D

407

Kanwar S S

292

Kaushal R K

292

Kodam K M

422

Kothari R M

394

Kulkarni M V

416

Kulkarni M

411

Madamwar D

368

Mahulikar P P

416

Mehrotra R R

364

Murugan V

317

Nagpal V

400

Narkhede K P

385

Noorwez S M

337

Paknikar K M

400

Pandey A

332

Phadnis S

276

Pillai A

332

Plaha P

298

Prakash M

380

Prema P

332

Prema P

373

Rao J V S

309

Rawal S K

276

Reddy A R

309

Rohini D

276

Rout S

346

Sambrani S A

389

Sankaran K

327

Saratale G D

407

Sasi P

364

Satyanarayana T

337

Selvan A T

327

Sharma K D

298

Sharma P

298

Sharma R

298

Siddique I

303

Singhania R R

332

Sukumaran R K

332

Szakacs G

332

Thakur A N

263

Thakur N L

263

Thiagarajan S

351

Uma V

317

Vasane S R

394

Vidhale N N

385

Zope V P

416


 


Indian Journal of Biotechnology
Vol 5, July 2006, pp 263-268

 

 

Marine biotechnology: An overview

Narsinh L Thakur* and Archana N Thakur

Marine biotechnology is the creation of products and processes from marine organisms through the application of the techniques of biotechnology, molecular and cellular biology, and bioinformatics. This is a scientifically fascinating and economically expanding field of science. No ecosystems provide greater genetic diversity or possibilities for new products and processes than the world's marine environments. Marine organisms, from bacteria to eukaryotes are certainly a source of molecules of great interest in biotechnology. Today, marine biotechnology has numerous applications from the production of lifesaving drugs to better food and conservation of organic waste. This article provides a selective overview of past achievements, present scenario and future directions of marine biotechnology

Key words: aquaculture, bioactive compounds, biodiversity, biotechnology, marine

IPC Code: Int. Cl.8 A01N63/00; A61K31/00

Indian Journal of Biotechnology
Vol 5, July 2006, pp 269-275

 

 

Somatic embryogenesis and Agrobacterium mediated genetic transformation in Indian accessions of lucerne (Medicago sativa L.)

Sanjay Gupta*, Shweta Gupta, Vishnu Bhat and M G Gupta

Plant regeneration in lucerne (Medicago sativa) is highly genotype dependent and inspite of considerable information on in vitro regeneration in this species the Indian genotypes have not been investigated so far. A system of recurrent somatic embryogenesis (RSE) was established for the first time in Indian accessions of lucerne and utilized for Agrobacterium mediated genetic transformation. Seeds, hypocotyls and cotyledons of LLC-3, C-10, A-3 and IL-75 accessions and ovary explants of 5 somaclones of LLC-3 were used and globular shaped somatic embryos were observed in all of the cultures. All the developmental stages of somatic embryogenesis were observed in ovary culture of one somaclone only. These somatic embryos have been undergoing cycles of RSE and the system has perpetuated for 24 months. For Agrobacterium mediated genetic transformation, this system of RSE required creation of injury in individual somatic embryos for enhanced transformation efficiency that limited its utilization for large-scale transformation experiments. An efficient system to overcome the requirement of creation of injury was developed in which pre-culture of somatic embryos on 2,4-D supplemented medium prior to their co-cultivation with Agrobacterium and supplementation of 2,4-D in co-cultivation medium could significantly improve the transformation efficiency.

Keywords: Medicago sativa, lucerne, somatic embryogenesis, recurrent somatic embryogenesis, plant regeneration, genetic transformation

IPC Code: Int. Cl.8 A01H4/00, 5/10; C12N15/09

Indian Journal of Biotechnology
Vol 5, July 2006, pp 276-283

 

 

Synthesis and characterization of poly-β-hydroxybutyrate from
Bacillus thuringiensis R1

D Rohini, S Phadnis and S K Rawal*

A poly-β-hydroxybutyrate (PHB) accumulating, Gram positive Bacillus thuringiensis R1 was isolated from the soil samples. The growth of the organism and PHB accumulation in the presence of different carbon sources was studied. While maximum dry cell mass accumulation (3.90 to 4.11g/L) resulted in the presence of glycerol (1% v/v), least biomass formation (1.28g/L) was in the presence of 1%(v/v) lactic acid. Glycerol also supported maximum accumulation of PHB (34.18 to 34.23%) on cell dry mass basis. PHB accumulation in the presence of table sugar (sucrose) and molasses was 28.23 % and 23.06%, respectively. No PHB accumulation was observed in the presence of acetic acid or ethanol. B. thuringiensis R1 cells grown to log phase in the basal medium in the absence of a additional carbon source and then reinoculated into the same medium supplemented with 1% (v/v) glycerol or 1% (w/v) table sugar accumulated 64.10% and 31.36% PHB of the dry cell mass, respectively. The recovered polymer was characterized by NMR, FTIR, GPC, DSC and TGA.

Keywords: Bacillus thuringiensis, β-hydroxybutyric acid, carbon sources, biphasic studies, physical characterization

IPC Code: Int. Cl.8 A01N63/00; C08G63/00; C12R1/07

Indian Journal of Biotechnology
Vol 5, July 2006, pp 284-291

 

 

Purification and characterization of the pectin lyase secreted within the macerating fluid of Rhizopus oryzae (Went & Prinsen Geerligs) grown on orange peel

Hossam S Hamdy*

Potentiality of Rhizopus oryzae to utilize orange peel, an inexpensive and low-cost substrate, under solid state fermentation (SSF) conditions to produce macerating fluid with high cellulolytic and pectinolytic activities was confirmed in the present work. Addition of NH4NO3 and NH4Cl to the fermentation medium improved the macerating potentiality due to an increase in cellulase and pectinase levels. The pectin lyase (PL) secreted by R. oryzae was also purified to electrophoretic homogeneity, using ammonium sulfate fractionation and 2-step-column chromatography. The PL was purified 22-folds and its specific activity was 2313 U/mg protein. The purified PL expressed its maximum activity at 50°C and pH 7.5, showed good stability in the pH range of 7 to 9.5 and its midpoint of thermal inactivation (Tm) was recorded at 70°C after 45 min of exposure. Presence of Ca2+ enhanced the activity and thermal stability of the purified PL. Ions of Mg, Na and K showed stimulatory effects on the enzyme activity, while ions of Zn, Co, Mn and Hg were inhibitory. The results suggest that possibly SH group in PL structure participated in the activity of the enzyme. Values of Km, Vmax, Kcat and molecular mass of the purified enzyme were 3.87 mg/mL, 297 U/mL, 5.94 mg U-1 min-1 and 31 kDa, respectively.

Keywords: cellulase, orange peel, pectin lyase, Rhizopus oryzae, solid state fermentation

IPC Code: Int Cl.8 C12N19/88; C12R1/845

Indian Journal of Biotechnology
Vol 5, July 2006, pp 292-297

 

 

Properties of hydrogel-entrapped lipase of thermophilic
Pseudomonas aeruginosa BTS-2

 

S S Kanwar*, M Gupta, R Gupta, R K Kaushal and S S Chimni

Pseudomonas aeruginosa BTS-2 produced lipase (total yield, 55.4 U/L) optimally on a mineral based (MB) broth, supplemented with 1% (v/v) cottonseed oil as sole carbon source, after incubation at 56°C for 48 h. The presence of 4% (w/v) yeast extract in the MB broth further increased the lipase production by ~ 3-folds. The purified lipase was efficiently (~80%) immobilized in alginate than carrageenan beads. The alginate-immobilized lipase (beads) showed optima at pH 7.5 and 55°C temperature. An increase in C-chain of alcohols increased the activity of entraped lipase. Exposure of alginate beads to decyl (C:10) and dodecyl (C:12) alcohol enhanced the lipase activity by 47 and 64%, respectively. Whereas, most alkanes decreased the activity of immobilized lipase but exposure to nonane enhanced the activity by 52%. The lipase efficiently esterified ethanol and acetic acid to ethyl acetate.

Keywords: esterification, immobilization, Pseudomonas aeruginosa BTS-2, stability in alcohols/alkanes

IPC Code: Int. Cl.8 C12N9/20; C12N11/04; C12R1/385

Indian Journal of Biotechnology
Vol 5, July 2006, pp 298-302

 

 

Genetic variability in pea wilt pathogen Fusarium oxysporum f. sp. pisi

in north-western Himalayas

 

Pooja Sharma, Kamal Dev Sharma*, Rajan Sharma and P Plaha

Genetic diversity in Fusarium oxysporum f. sp. pisi isolates from three agroclimatically distinct regions, sub-tropical, sub-humid and wet-temperate, of north-western Himalayas was studied using cultural characteristics, DNA (RAPD) and protein (native-proteins and esterase isozyme) markers. Variability in growth pattern, colour of mycelium and pigments was observed. Based on cultural characters, 24 isolates from three regions could be assigned to six groups. Amplification of genomic DNA of 24 isolates with ten 10-mer primers generated 134 polymorphic markers, whereas native-protein and esterase profiles revealed 27 markers. Based on NTSYS analysis of RAPD and protein data, the isolates were delineated into four region specific groups. Group PRI, PRIII and PRIV represented isolates from sub-tropical and sub-humid regions, whereas group PRII consisted of isolates from dry-temperate region, indicating that pathogen populations from sub tropical and sub humid regions evolved from three distinct lineages and those from temperate regions from the fourth lineage.

Keywords: cultural characteristics, Fusarium oxysporum f. sp. pisi, protein markers, RAPD, variability

IPC Code: Int. Cl.8 C12N15/10

Indian Journal of Biotechnology
Vol 5, July 2006, pp 303-308

 

 

Thidiazuron induced high frequency shoot bud formation and plant regeneration from cotyledonary node explants of Capsicum annuum L.

 

Iram Siddique and Mohammad Anis*

An efficient protocol for rapid in vitro propagation of Capsicum annuum L. cv. Pusa jwala through multiple shoot bud formation from cotyledonary node explants of 15-day-old aseptic seedlings has been developed. Multiple shoot bud formation and highest rate of multiplication was standardized on Murashige and Skoog (MS) medium supplemented with 1.5 µM thidiazuron (TDZ) and 0.5 µM indole 3-acetic acid (IAA). The regenerated shoot buds when subcultured on hormone free MS medium gave the highest rate of shoot proliferation and shoot length by the end of third subculture. Individual elongated shoots were rooted best on MS media containing 1.0 µM α-naphthalene acetic acid (NAA). Ex vitro rooting was also achieved when the basal cut ends of the in vitro regenerated shoots were dipped in 200 µM indole-3-butyric acid (IBA) for half an hour followed by transplantation in plastic pots containing sterile garden soil. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to field transfer. Of all the micropropagated plants, about 95% survived following transfer to soil. Growth performance of 2-month-old in vitro raised plants was compared with in vivo seedlings of the same age on the basis of selected morphological and physiological parameters. Carotenoids and relative water content were significantly higher in in vitro regenerated plants as compared to in vivo control plants of same age.

Keywords: Capsicum annuum, cotyledonary node explants, plant regeneration, ex vitro rooting, relative water content

IPC Code: Int. Cl.8 A01H4/00, 5/04

Indian Journal of Biotechnology
Vol 5, July 2006, pp 309-311

 

 

Micropropagation of Azadirachta indica A. Juss. via cotyledonary nodes

A Rajasekhar Reddy*, M Bavaji and J V S Rao

An efficient protocol for micropropagation of Azadirachta indica A. Juss., a medicinally important plant, has been standardized. Cotyledonary nodes (1 cm long) excised from 15-20 d old in vitro-germinated seedlings were used as explants. The seeds were germinated on half strength MS medium devoid of phytohormones. Cotyledonary nodes were cultured on MS medium supplemented with different concentrations of cytokinins (BA/TDZ/2-ip) and auxins (2,4-D and NAA). Maximum shoot proliferation from single explant was obtained on MS media incorporated with TDZ (1.5 mM), 2,4-D (0.5 mM), adenine sulphate (40 mg/L), glutamine (100 mg/L) and thiamine HC1 (10 mg/L). In vitro-produced shoots were induced roots on half strength MS medium supplemented with a range of IBA concentrations (0.5-5.0 mM). However, the highest frequency of root proliferation was observed on half strength MS medium supplemented with 2.0 mM IBA. The regenerants were transferred to field conditions after acclimatization with a success rate of 80%.

Keywords: Azadirachta indica, cotyledonary node, micropropagation

IPC Code: Int. Cl.8 A01H4/00

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 317-326

 

 

Isolation and characterization of recombinant Brugian parasitic transglutaminase

V Uma, M Geetha, V Murugan and P Kaliraj*

An attempt has been made to identify and characterize a putative chemotherapeutic target antigen for human lymphatic filariasis by recombinant DNA techniques. Identification of transglutaminase (TGA), an enzyme vital for the growth and survival of the parasites, has opened a new drug target for developing potent therapeutic agents against the parasites. TGA gene from Brugia malayi adult female parasite (BmTGA) has been subcloned, expressed and purified as a 47 kDa fusion protein with N-terminal histidine tag. Since BmTGA lacks homology to mammalian TGAs, it is a promising drug target for developing chemotherapeutic agent and candidate vaccine against human lymphatic filariasis.

Keywords: adjuvants, filaricidals, immunoprophylaxis, molting, nematodes, protein disulphide isomerase, transgluta-minase, vaccine

IPC Code: Int. Cl.8 C12N9/64

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 327-331

 

 

Bacterial lipid modification in vitro: Synthetic peptide substrate for phosphatidylglycerol—Prolipoprotein diacylglyceryl transferase

 

A Tamil Selvan and K Sankaran*

Lipid modification is an emerging protein-engineering tool for providing hydrophobic anchor to hydrophilic proteins for immobilizing them onto a variety of man-made and biological surfaces. In this respect, N-acyl S-diacylglyceryl modification of N-terminal cysteine, called bacterial lipid modification has many advantages including the fact that it can be achieved through popular prokaryotic expression of proteins. The modification proceeds in a three-step cascade reaction in which three inner membrane enzymes, Phosphatidylglycerol:Prolipoprotein diacylglyceryl transferase, signal peptidase II and apolipoprotein N-acyl transferase, participate. Bacterial expression has limitations including the necessity of expensive downstream processing. However, in vitro modification has not been possible so far because the enzymology of this pathway is not well studied due to difficulty in assaying these enzymes. As a first step to overcome this problem we have designed the peptide substrate, MKATKSAVGSTLAGCSSHHHHHH, for in vitro lipid modification specifically the first enzyme, Phosphatidylglycerol:Prolipoprotein diacylglyceryl transferase, which catalyzes the committed step. The design was based on bioinformatics analysis of more than 1000 bacterial lipoprotein precursors. This synthetic peptide substrate was soluble and contained other built-in features useful in easier handling and purification. The designed substrate exhibited expected properties and in vitro diacylglyceryl modification was confirmed by Tricine SDS PAGE based mobility shift assay.

Keywords: bacterial lipid modification, signal sequence, peptide substrate, assay

IPC Code: Int. Cl.8 C12N1/38

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 332-336

 

 

Solid-state fermentation of lignocellulosic substrates for cellulase production by Trichoderma reesei NRRL 11460

Reeta Rani Singhania, Rajeev K Sukumaran, Anu Pillai, P Prema, George Szakacs and Ashok Pandey*

Cellulase production studies were carried out using the fungal culture Trichoderma reesei NRRL 11460 using four different lignocellulosic residues (both raw and pre-treated) by solid-state fermentation. The effect of basic fermentation parameters on enzyme production was studied. Maximal cellulase production obtained was 154.58 U/gds when pre-treated sugarcane bagasse (PSCB) was used as substrate. The optimal conditions for cellulase production using PSCB were found to be initial moisture content - 66%, initial medium pH-7.0, incubation temperature -28°C, NH4NO3 at 0.075 M, and 0.005 M cellobiose. The optimal incubation time for production was 72 h. Results indicate the scope for further optimization of the production conditions to obtain higher cellulase titres using the strain under SSF.

Keywords: cellulase, endoglucanse, Trichoderma, solid-state fermentation, lignocellulosic residues, sugarcane bagasse

IPC Code: Int. Cl.8 C12N9/42; C12R1/885

Indian Journal of Biotechnology
Vol. 5 (Suppl), July 2006, pp. 337-345

 

 

Production of a high maltose-forming, hyperthermostable and Ca2+-
independent amylopullulanase by an extreme thermophile Geobacillus thermoleovorans in submerged fermentation

S M Noorwez, M Ezhilvannan and T Satyanarayana*

Production of a hyperthermostable, high maltose-forming and Ca2+-independent amylopullulanase by an extreme thermophile Geobacillus thermoleovorans was studied in submerged fermentation. Parametric optimization led to secretion of amylopullulanase (2850 U L-1 of a-amylase, 840 U L-1 of pullulanase) in a starch-yeast extract medium (2% starch, 0.1% ammonium sulphate, 0.3% yeast extract, 0.1% K2HPO4, 0.1% NaCl, 0.01% MgSO4.7H2O and 0.1% Maltose) using 2% inoculum (12 h) at 70°C and 200 rpm in 20 h. The addition of trace elements, detergents, surfactants and additives did not stimulate growth or enzyme production. The hydrolysis of pullulan and starch yielded maltotriose, and maltose, maltotriose and maltotetraose as the major end products, respectively. The cultivation of the organism in a laboratory fermenter showed (i) abolition of the lag phase, (ii) a 2-fold increase in enzyme production (5880 U L-1 of a-amylase, 1900 U L-1 of pullulanase) and (iii) a reduction in fermentation time to 7-8 h.

Keywords: Geobacillus thermoleovorans, amylopullulanase, submerged fermentation, starch hydrolysis

IPC Code: Int. Cl.8 C12N9/44; C12R1/01

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 346-350

 

 

Production of tannase under mSSF and its application in fruit juice debittering

 

S Rout and R Banerjee*

Enzymatic treatment of fruit juices to reduce the bitterness has got advantages such as the higher quality of juice due to the lower haze and non-deterioration of juice quality. The pomegranate has recently been acclaimed for its health benefits, in particular, for its disease-fighting antioxidant potential. Presence of high tannin content in the pomegranate is responsible for haze and sediment formation as well as for colour, bitterness and astringency of the juice upon storage. Due to the inability of conventional fruit juice debittering processes for removing the bitterness effectively, enzymatic debittering should be preferred. This present work is aimed at the debittering of pomegranate juice using tannase, which has potential applications in the degradation of tannins. The juice was treated with tannase (10% v/v) of 35.6 U/mL at 37şC for two h and then the enzyme was inactivated at 50°C for 30 min. Tannase treatment resulted in 25% degradation of tannin while a combination of tannase and gelatin (1:1) resulted in 49% of tannin degradation. Ascorbic acid content, pH, total sugar, protein, and viscosity of both the treated and non-treated fruit juices were studied.

Keywords: pomegranate, tannase, debittering, tannin degradation and ascorbic acid content

IPC Code: Int. Cl.8 C12N9/16

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 351-356

 

 

Purification and characterization of an endoxylanase from solid-state
culture of alkalitolerant Aspergillus fumigatus MKU1

S Thiagarajan, M Jeya and P Gunasekaran*

A novel high molecular weight endoxylanase (XylF1) from the solid state culture of Aspergillus fumigatus MKU1 strain was purified to homogeneity by a combination of tube gel electrophoresis and electroelution methods. The purity of XylF1 was demonstrated by SDS-PAGE, which has the molecular mass of 43 kDa. The optimal pH and temperature for enzyme activity were 7.0 and 70° C, respectively. Further, the apparent Km and Vmax values of XylF1 with oat spelt xylan as substrate were 6.25 mg/mL and 0.05 mmol/mL/min (2.5 mmol/min/mg protein), respectively. The enzyme showed high activity on oat spelt xylan, while only trace of activity was observed on carboxy methylcellulose. The activity of XylF1 was strongly inhibited by Hg 2+, Cu 2+, Fe3+ and NBS, whereas stimulated by DTT, DTNB and L-Cysteine. The hydrolysis of oat spelt xylan released only xylooligosaccharides.

Keywords: Aspergillus fumigatus, biochemical characterization, purification, solid state culture, xylanase,

ICC Code: Int. Cl.8 C12N9/42; C12R1/68

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 357-363

 

 

Characterization of chitinases from microorganisms isolated from Lonar lake

 

Vijay B Bansode and Shyam S Bajekal*

Chitinases, the enzymes that breakdown chitin—the second most abundant polysaccharide in nature—are known to have numerous uses. Alkaline chitinases, in particular are considered to have a greater potential in this respect. Fifteen chitinolytic microbial strains were isolated from the alkaline soil of Lonar lake in Buldhana district of Maharashtra. In this paper, the characters of chitinases produced by the five most potent isolates are presented. All the enzymes exhibited maximum activity in the neutral to alkaline range of pH (from 7.0 to 9.6) with a wide stability range from pH 4 to 11. The temperature optima for all the enzymes were slightly on the higher side, between 35 and 60°C, with a stability range from 25 to 60°C. All the enzymes showed a typical response to substrate concentration. None of the enzymes had any specific requirement for any particular metal ion, though a considerable stimulatory effect of Ca2+ was observed on all the enzymes. Minor effects of Cu2+, Mn2+ and Na1+ were also observed on some of the enzymes.

Keywords: alkaline, biocontrol, chitinases, fungal protoplasting, seafood waste

IPC Code: Int. Cl.8 A01N63/04

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 364-367

 

 

Esterification reactions catalysed by surfactant-coated Rhizopus arrhizus lipase

Preetha Sasi, Rohit R Mehrotra and Mira Debnath (Das)*

The esterification activity of microbial lipase (triacyl glycerol hydrolase, E.C. 3.1.1.3) in water-organic biphasic solvent system has been investigated. The lipase produced from Rhizopus arrhizus NCM997 strain by solid-state fermentation, using rice bran as substrate, exhibited novel capability of catalyzing esterification reaction. The partially purified lipase was coated with nonionic surfactant (Span 60). The esterification activity of the enzyme was found to be optimum at 30°C and pH 6.5, and isooctane was the best solvent for esterification. Surfactant-coated lipase showed enhanced esterification activity compared to native enzyme. Among the substrates tested, palmitic acid and glycerol gave maximum conversion of 74.02%. Effect of initial water activity has been investigated using different salts of varying water activity. The maximum conversion (85.75 %) of ester was obtained with salts having less water activity (LiCl; aw. 0.12). Thus, this surfactant-coated microbial lipase in organic solvent (isooctane) has a good potential of biocatalysis.

Keywords: esterification, lipase, solvent, surfactant coating, water activity

IPC Code: Int. Cl.8 C12N9/20; C12N11/08; C12R1/845

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 368-372

 

 

Entrapment of lipase in polymer of polyvinyl alcohol-boric acid for esterification in organic media

Rachna Dave and Datta Madamwar*

Polyvinyl alcohol (PVA)-boric acid method has been utilized for entrapment of Candida rugosa lipase. Immobilized lipase was used to produce ethyl butyrate, a flavour ester showing 80.2% conversion in 72 h. Lipase in PVA-boric acid beads possessed the ability to synthesize variety of esters and was stable in various organic solvents with varying log P value from 2 to 8 under incubation at 50°C for 1 h.The immobilized lipase showed nearly full retention of activity even after 8 cycles of use, the activity then gradually decreased reaching to 56% conversion efficiency after 20 cycles and possesses a shelf-life of 10 months. The thermostability of the lipase increased three times upon immobilization. The immobilized enzyme possessed 40% higher activity compared to its free counterpart.

Keywords: boric acid, entrapment, ethyl butyrate, lipase, polyvinyl alcohol

IPC Code: Int. Cl.8 C12N9/20; C12N11/08; C12R1/72

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 373-379

 

 

Selection of optimal growth medium for the synthesis of a-galactosidase from mangrove actinomycetes

G S Anisha and P Prema*

a-Galactosidases (a-galactoside galactohydrolase, EC.3.2.1.22) are exoglycosidases that cleave terminal a-1→6-linked galactose residues from glycoconjugates. To select best medium for the enhanced production of this enzyme by the mangrove actinomycete cultures (AGP 42 and AGP 47), four different media reported in literature were tried. The medium containing locust bean gum as the carbon source (Medium I) gave the highest level of a-galactosidase activity and was selected for further studies. The actinomycete culture AGP 42 was found to give higher enzyme titers than AGP 47. A comparison of the characteristics of crude a-galactosidase from these cultures showed that the enzyme from AGP 42 was active over a wider range of pH (4.0 to 8.5) compared to that from AGP 47 (pH 5.5 to 8.5). The optimum pH for the maximum a-galactosidase activity from AGP 42 and AGP 47 was 7.0 and 6.4, respectively. Similarly, the optimum temperatures for both the cultures were 55 and 40°C. The enzyme from AGP 42 was found to be stable at pH range 6.0 to 7.5 and that from AGP 47 at pH range 6.0 to 7.0. Similarly, the temperature stability of a-galactosidase from both the cultures was from 30 to 45°C and 30 to 40°C.

Keywords: a-galactosidase, mangrove actinomycetes, locust bean gum, crude enzyme, characterization

ICC Code: Int. Cl.8 C12N9/40; C12R1/01

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 380-384

 

 

Purification and characterization of laundry detergent compatible
alkaline protease from Bacillus cereus

R M Banik* and Monika Prakash

An alkalophilic bacterium, Bacillus cereus produced an extracellular alkaline protease, which was found to be active at high temperature and pH range, suitable for commercial laundry detergents. B. cereus protease was partitioned in different aqueous two-phase systems such as PEG/dextran, PEG/potassium phosphate, PEG/sodium citrate, PEG/magnesium sulphate and PEG/sodium dihydrogen phosphate and best separation was found in PEG/potassium phosphate system. Therefore, production of protease was performed by the method of extractive fermentation in aqueous two-phase system composed of PEG 4000/potassium phosphate. Enhanced production was obtained in aqueous two-phase system as it overcomes the limitation of catabolic repression and product inhibition. The enzyme was purified to homogeneity by procedures including ammonium sulphate precipitation, concentration by ultrafiltration, anion exchange chromatography and gel filtration. The purified enzyme had specific activity 3256.05 U/mg and found to be a monomeric protein with a molecular weight of 28 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Its maximum protease activity against casein was found to be at pH 10.5 and at 50°C. Proteolytic activity of the enzyme was detected by casein zymography, which gave a very clear protease activity zone on gel that correspond to the band obtained on SDS-PAGE with a molecular weight nearly 31 kDa. Protease was inhibited by specific serine protease inhibitors, suggesting the presence of serine residues at the active site of the enzyme.

Keywords: alkaline protease, Bacillus cereus, detergent compatible, ATPS, purification, characterization

IPC Code: Int. Cl.8 C12N9/52; C12R1/085

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2005, pp 385-388

 

 

Biopulping studies using an effluent isolate Curvularia lunata LW6

K P Narkhede* and N N Vidhale

The lignin is unwanted constituent for a pulp and paper manufacturer, which must be removed for getting good quality paper. The aim of bio-delignification of raw material is to make pulping process easier, to save energy requirements, to improve pulp properties and to minimize pollution. The present study aims to find suitability of isolate Curvularia lunata LW6 for biopulping of sugarcane bagasse. The organism was screened from effluent of pulp and paper mill and tested for lignolytic ability. The isolate produced the enzyme lignin peroxidase (3.31 U/mg of protein) and laccase (6.12 U/mL). It showed good saprophytic ability (38.14 cm2) on sterile bagasse when supplemented with glucose and yeast extract as co-substrate. The solid-state fermentation of bagasse for biopulping resulted in loss of weight by 21.66%, while lignin content and kappa number decreased by 44.05% and 40.78%, respectively. The chemical pulping showed 55.25% reduction in kappa number; however, it required high temperature, pressure and alkaline medium. The mutagenesis studies showed slight decrease in cellulase activity of the isolate.

Keywords: biodelignification, Curvularia lunata LW6, laccase, lignin peroxidase, solid-state fermentation

IPC Code: Int. Cl.8 C12S9/00; C12R/645

IndianJournal of Biotechnology
Vol. 5 (Suppl), July 2006, pp 389-393

 

 

Cholesterol biotransformation in monophasic systems by solvent tolerant Bacillus subtilis AF 333249

M S Andhale and S A Sambrani*

New developments in biotechnology are focused on new products or the introduction of new processes as a substitute to the conventional way, or improvements in performance of existing biotechnological applications. The examples of application of non-conventional media are organic solvents, supercritical solvents or aqueous two phases systems. Steroid biotransformation is a multi million-dollar industry and the pharmaceutical uses of steroids are numerous. Steroids like cholesterol are completely soluble in some organic solvents like benzene, toluene and butanol. Biphasic systems wherein the cells are present in the aqueous phase and steroids dissolved in the organic phase is considered an ideal set-up. The major problem here is the fact that most bacteria and their enzymes are inactivated or destroyed in the presence of these toxic organic solvents, also it is necessary that the microbes contain relevant enzymes in sufficient amounts and in high specific activities, even in the presence of the generally destructive apolar phase. This can be very well accomplished by using organic solvent tolerant bacteria having desired enzyme activities. One such application is being discussed.

Keywords: cholesterol, organic solvents, tolerance, solubility, bio-transformation, monophasic systems

IPC Code: Int. Cl.8 C07J9/00; C12R1/125

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 394-399

 

 

Optimization of secondary hardening process of banana plantlets
(Musa paradisiaca L. var. grand nain)

 

Shailesh R Vasane and R M Kothari*

In view of significance of well hardened planting material for ensuring highest percentage survival upon transplantation of tissue culture derived plants from shade house (secondary hardening) to farm, studies were undertaken to optimize (i) material for plantlet container, (ii) container size, (iii) potting medium, (iv) use of Azotobacter (for nitrogen fixation), Aspergillus (for phosphate solubilization) and Vesicular Arbuscular Mycorrhizae (VAM) like G. intraradices (for growth promotion), (v) spray of organic extracts and (vi) spacing of plantlets in shade house. These studies have revealed that (a) for the time being, there is no substitute to polybags due to susceptibility of jute bags for decay, (b) optimum size of polybag is 20´16 cm (H´W), (c) soil:press mud cake (3:1) is cost-effective and eco-friendly potting medium, (d) incorporation of Azotobacter, Aspergillus and VAMs during media preparation helps in better establishment and growth of plants, (e) sprays of organic extract have insignificant effect on the vitality of plantlets and (f) 1000 cm2 provided optimal spacing for 3.8 plants. Financial implications of these findings are enormous.

Keywords: Banana grand nain, secondary hardening, vesicular arbuscular mycorrhizae, container size, potting media

IPC Code: Int. Cl.8 A01H4/00, 5/00

Indian Journal of Biotechnology
Vol 5 (Suppl), July 2006, pp 400-406

 

 

Integrated biological approach for the enhanced degradation of lindane

Varima Nagpal and K M Paknikar*

Lindane is a persistent organic pollutant (POP) and its removal from various environmental compartments is a global priority. In the present study, three lindane degrading bacterial cultures, viz. Pseudoarthrobacter sp., Pseudomonas sp. and Klebsiella sp., were selected from among ten isolates obtained from lindane-exposed soils by enrichment culture technique. The cultures exhibited a maximum lindane degradation efficiency of ~50%, having little significance in bioremediation. However, in the presence of protozoa (ciliates) obtained from the same enrichment culture, lindane degradation efficiency of bacterial cultures increased to the tune of 90-92% with concomitant formation of 1,2,4-trichlorobenzene, a key degradation product. Lindane degradation by the bacterial cultures was also found to get enhanced by 10-15% in the presence of root exudates of lindane-tolerant plants, viz. corn, chili and coriander, even though these plants were unable to uptake any lindane and thus were unsuitable for phytoremediation per se. These data highlight the need for an intensive investigation on (a) bacteria-protozoa interactions for bioremediation of POPs and (b) use of POP-degrading bioinoculants in the rhizospheric soil of resistant plants as a possible alternative, in the absence of a viable conventional remedial option.

Keywords: lindane, bioremediation, phytoremediation, root exudates, protozoa

IPC Code: Int. Cl.8 B09C1/10

Indian Journal of Biotechnology

Vol 5 (Suppl), July 2006, pp 407-410

 

 

Decolorisation of textile dyes by Aspergillus ochraceus (NCIM-1146)

G D Saratale, S D Kalme And S P Govindwar*

Aspergillus ochraceus (NCIM-1146) has ability to decolorize various xenobiotic dyes. Biodegradation of dyes was demonstrated by their decolorisation  in the culture medium. The extent of biodegradation was determined by monitoring the decrease in absorbance of each dye. Malachite green decolorisation  activity is affected by various conditions such as composition of media, concentration of dye, amount of mycelia and agitation. The durability of decolorisation  activity under optimum conditions was investigated in repeated batch mode. An increase in the amount of mycelia positively affected the durability of decolorisation  activity. The decrease in dye decolorisation  capability of mycelia occurred with increasing dye concentration in repeated batch mode. Spectrophotometric data revealed that the process involved in decolorisation  is through microbial metabolism but not biosorption. This study showed that fungal mycelia (A. ochraceus) could effectively be used as an alternative to the traditional physico-chemical process.

Keywords: decolorisation; Aspergillus ochraceus; malachite green; textile dyes, repeated batch mode

IPC Code: Int. Cl.8 C02F3/02; C02F103/30

Indian Journal of Biotechnology

Vol 5 (Suppl), July 2006, pp 411-415

 

 

Efficient Pseudomonas putida for degradation of p-nitrophenol

Meenal Kulkarni and Ambalal Chaudhari*

Exploration of Pseudomonas putida for controlling environmental (p-nitrophenol) PNP pollution revealed that 0.5 OD inoculum at 600 nm degraded 100 ppm of PNP completely under the ambient conditions within 24 h as established by UV-visible spectrophotometric and TLC analysis of treated broth and confirmed the same by HPLC analysis. An attempt to elucidate the mechanism of PNP degradation indicated that (i) it was oxidative with a stoichiometric release of nitrite, (ii) PNP was converted to hydroquinone, which accumulated in the presence of 2,2˘-dipyridyl, (iii) hydroquinone was the first product of ring oxygenation and nitrite removal, as confirmed by TLC and HPLC analysis and (iv) concomitant formation of a keto acid by ring fission, which was confirmed by Rothera's test.

Keywords: p-nitrophenol; biodegradation; Pseudomonas putida; hydroquinone; ring fission

IPC Code: Int. Cl.7 B09C1/10

Indian Journal of Biotechnology

Vol 5 (Suppl), July 2006, pp 416-421

 

 

Microbial degradation of melanoidins in distillery spent wash by an indigenous isolate

M N Chavan, M V Kulkarni*, V P Zope and P P Mahulikar

Spent wash is pollution intensive wastewater generated by distilleries. Its dark brown colour is due to recalcitrant melanoidin pigments. Bacterial cultures were screened for the ability to degrade these pigments. One isolate, identified as Pseudomonas sp., was selected for degradation studies. Spent wash dilution up to 10% and addition of externally added carbon source was essential for decolorisation. The optimum conditions for decolorisation were pH 6.8-7.2, temperature 30-35°C and glucose 0.4 g%, (W/V) of the spent wash medium. The maximum decolorisation up to 56% and 63% reduction in COD of the spent wash could be achieved after 72 h by the microbial treatment. Spectrophotometric and HPLC analysis of treated effluent confirmed biodegradation of melanoidin pigments by the isolate. This approach could be used to develop a cost-effective, eco-friendly biotechnology package for the bioremediation of spent wash before its disposal.

Keywords: biodegradation, decolorisation, melanoidins, spent wash

IPC Code: Int. Cl.8 CO2F3/34; CO2F101/30

Indian Journal of Biotechnology

Vol. 5 (Suppl), July 2006, pp. 422-424

 

 

Decolorisation of reactive red 11 and 152 azo dyes under aerobic conditions

K M Kodam* and K R Gawai

A microorganism isolated from a textile-dyeing effluent disposal site was found to decolorise textile azo dyes viz. reactive red 11 and reactive red 152 (200 mg L-1) within 24 h of incubation at room temperature and neutral pH. Fast decolorisation (within 42 h) was observed with higher concentration of azo dyes (1000 mg L-1) and the decolorisation took place strictly under aerobic conditions. Thus, this microorganism seems to have potential for effective bioremediation of textile-dyeing industry effluents.

Keywords: Aerobic, bioremediation, decolorisation, sulfonated azo dyes

IPC Code: Int. Cl.8 C02F3/02; C02F103/30