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Open Access
Characterization of psychrotolerant rhizobacteria for Zn biofortification in lentil (Lens culinaris L. Medikus)
(Punjab Agricultural University, Ludhiana, 2018) Jaskiran Kaur; Khanna, Veena
Seventy psychrotolerant rhizobacterial isolates were evaluated for their PGP traits at 10°, 20° and 30°C. Highest IAA production was recorded with isolates PRh-9, PRh-14, PRh- 15, PRh-60, PRh-61 and that was confirmed by HPLC (8.98, 4.91, 5.66, 2.73 and 1.62 μg/ml respectively). Isolate PRh-14 recorded maximum Zn-solubilization potential of 354, 304.8 and 284.5 ppm at 10°, 20° and 30°C. It also recorded the maximum P-solubilization efficiency, gibberellins, EPS production and biofilm formation at 10°, 20° and 30°C. Eleven isolates amplified acdS gene which confirmed the presence of ACCD activity. Maximum naringin and quercetin production was shown by isolate PRh-61 and PRh-9, further confirmed by TLC studies. Highest antagonistic activity was recorded by PRh-9 against Rhizoctonia bataticola and Fusarium oxysporum (33.3% and 48.1% respectively) and was also established by SEM analysis. Potent siderophore, salicyclic acid and HCN producers were PRh-9, PRh- 61 and PRh-60. The SEM analysis of root surface revealed that coinoculation with Rhiobium and PRh-14 helped in maintaining the root integrity owing to biofilm formation on the root surface. Under field conditions coinoculation of Rhizobium with PRh-15, PRh-14, PRh-9, PRh-61, PRh-60 and PRh-30 further increased by grain yield by 1.7, 4.4, 3.7, 2.5, 2.09, 1.5% respectively, over Rhizobium alone. A significant increase in the grain and straw zinc content was recorded with the treatment R+PRh-14 (105.9 and 141.0 ppm respectively). The DGGE analysis of soil DNA depicted a composite banding pattern that reflected a high microbial diversity. The promising rhizobacterial isolates were identified as PRh-9 (Pseudomonas flourescens), PRh-14 (Aeromonas hydrophila), PRh-60 (Lysinibacillus fusiformis) and PRh-61 (Pseudomonas korensis) based on 16S rDNA sequencing.
Open Access
Impact of long term organic and inorganic fertilization on microbial community and screening for cellulose degrading bacteria
(Punjab Agricultural University, Ludhiana, 2018) Bhagat, Poonam
Impact of long term incorporation of rice straw and inorganic nitrogen was studied on microbial communities, soil enzymatic activities and on soil parameters at different time intervals (0, 45, 90 and 120 DAS) for two consecutive years. The different doses of rice straw (0, 5, 7.5 and 10 t/ha) along with different doses of inorganic nitrogen (0, 90, 120 and 150 kg/ha) were incorporated in the wheat field. The first year results revealed maximum total bacterial count (8.31 log 10 cfu/g of soil), fungal count (4.89 log 10 cfu/g of soil), celllulse degrading bacteria count (7.11 log 10 cfu/g of soil) with 7.5 t/ha RS + 120 kg N/ha while actinomycetes count (7.14 log 10 cfu/g of soil), diazotrophic count (6.07 log 10 cfu/g of soil) with 10 t/ha RS alone at 45 DAS. The maximum dehydrogenase activity (23.96 µg TPF/g soil/hr), alkaline phosphatase activity (14.77 µg pNP/g soil/hr) and urease activity (330.0 µg urea/hr/g soil) observed at 45 DAS with 7.5 t/ha RS plus 120 kg N/ha. The microbial population and enzymatic activities found higher in second year over first year. The soil pH was found to be altered from 7.82 to 6.99 and EC from 0.219 to 0.201 dSm-1 with treatment 10 t/ha RS +120 kg N/ha whereas OC from 0.26% to 0.49% with 10 t/ha RS + 150 kg N/ha in two years at 120 DAS. The maximum soil available nutrients viz. nitrogen (136.7 kg/ha), phosphorous (29.94 kg/ha) observed with treatment10 t/ha RS + 150 kg N/ha, potassium (119.78 kg/ha) with treatment 7.5 t/ha RS + 120 kg N/ha at 45 DAS in the first year. The maximum straw and grain nitrogen (0.46%) and (1.37%), phosphorus (0.15%) and (0.38%), potassium (1.72%) and (0.53%) contents respectively in first year found with 10 t/ha RS + 150 kg N/ha. The maximum straw and grain yield (9.65 t/ha) and (6.28 t/ha) respectively observed in first year with 7.5 t/ha RS + 120 kg N/ha. The available nutrients and yield found higher in second year than first year. The cellulose degrading bacteria were isolated and halozone formation confirmed their cellulolytic activity. Seventeen isolates were characterized biochemically and most were positive for oxidase and catalase while negative for H2S test, indole test and MR-VP test and few were positive for citrate utilization, gelatin solubilization and starch hydrolysis. The functional characterization of isolates showed highest cellulolytic activity (1.44µg/ml), IAA production (34.85µg/ml), ammonia excretion (2.048µg/ml) and phosphate solubility (28.56 µg/ml) was exhibited by isolate SKPB3. Isolates were analysed phylogenetically by 16S rDNA sequencing. Isolates clustered in phylogentic trees indicated high similarity and the abundance of particular cellulolytic strains. Identification of representative cultures using parial sequencing of 16S rDNA revealed presence of Acinetobacter sp, Pseudomonas sp, Stenotrophomonas sp, Bacillus sp.
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Synergistic effect of Mesorhizobium and non rhizobial endophytes on plant growth promotion in chickpea (Cicer arietinum L.)
(Punjab Agricultural University, Ludhiana, 2018) Chhabra, Deepika; Sharma, Poonam
The present investigation was carried out to exploit non rhizobial endophytic bacteria from root and nodules of chickpea. Of 263 non rhizobial endophytic bacteria 74.4 %, 87.4%, 12.6%, 36.6%, 37.4% and 55.7% were found to be positive for catalase, oxidase, citrate utilization, nitrate reduction, methyl red and Voges Proskauer‟s, respectively. 71 were found to be good for phosphate solubilization and IAA production. Significantly high P solubilization was observed in root isolate RBR20 (20.60 mg100ml-1) whereas, among nodule‟s isolates with RBN17 (21.0 mg100ml-1). In the presence of tryptophan, the isolate LCRE 9 produced the maximum amount of IAA (39.60 μgml-1) whereas in the absence of tryptophan the isolate RBR164 produced the maximum amount of IAA (19.93 μgml-1). RBN16 isolate showed highest growth in DF medium with ACC (OD 1.04) followed by RBR164 (OD 0.9985). High amount of GA production was observed in RBR19, RBR127, RBR136, RBR164 and LCNE6 (112.15 μgml-1). Highest amount of EPS production was observed LCRE8 (105.01 μgml-1). Out of 75 isolates 38.7%, 82.7%, 9.3% and 21.3% isolates were able to produce cellulase, protease, HCN and fluorescent pigment, respectively. On the basis of overall PGP traits and compatibility studies 3 potential non rhizobial endophytic bacteria (RBR164, LCRE9 and RBN17) were selected for colonization study in different combinations with ampicillin (6μgml-1) an antibiotic marker. Colonization was maximum in RB1+LGR33+ RBN164 treatment (1.16 and 1.60) at 15th and 50th respectively. Triple inoculant treatment LGR33+RB1+RBR164 resulted into maximum increase in plant height, dry weight of shoot and total bacterial count at 90 DAS as compared to dual, single and uninoculated control under field conditions. Maximum increase in dry weight of root, root shoot ratio, nodule dry weight and leghaemoglobin was observed with same treatment at 60 and 90 DAS. Similar trend was observed for total NPK content of shoot and seed. Grain yield was increased with triple inoculants treatment LGR33+RB1+RBR164 by 8.9% and 3.6% over uninoculated control and recommended consortium of chickpea, respectively. It seems from the foregoing study that consortium of Mesorhizobium with multifarious plant growth promoting endophytic bacteria can be developed and used as biofertilizer for chickpea.
Open Access
Development of microbial consortia for pretreatment of paddy straw and its utilization for ethanol production
(Punjab Agricultural University, Ludhiana, 2017) Lota, Pardeep; Kocher, Gurvinder Singh
The present study was conducted with the objective of standardization of a fungal consortium from selected ligninolytic cultures for biological pretreatment of paddy straw and its subsequent conversion to bioethnaol. The proximate analysis of paddy straw revealed that 30 mesh straw had highest proportion of cellulose (39.5%) besides, 23.3% hemicellulose and 11.5% lignin. Among different chemical pretreatment methods, acid (1.0% H2SO4) –autoclave (15 psi for 90 min) followed by alkali (2.0% NaOH) pretreatment resulted in 82.61% and 59.23% decrease in lignin and hemicellulose, respectively and 36.60% increase in relative proportion of cellulose. Four fungal strains (viz., LS1, LS2, LCu1 and LCu2) from a total of fifty nine strains isolated from diverse natural habitats, tested positive for the production of laccase (Lacc), lignin peroxidase (LiP) and manganese peroxidase (MnP). Out of three consortia developed on the basis of ligninolytic activities, consortium III comprising Pleurotus ostreatus and Phanerochaete chrysosporium, resulted in Lacc, LiP and MnP activities of 2.40, 37.92 and 62.50 nkat/gds, respectively, significantly higher than the activities of individual fungal strains. Biological pretreatment of paddy straw under Response surface methodology optimized conditions (moisture, 121.0 %; temperature, 31.3°C and log spore count, 8.0 spores/ml) resulted in cellulose, 43.0%; hemicellulose, 12.7%; lignin, 7.0%; total sugars,13.65 mg/gds and reducing sugars, 4.00 mg/gds. The pretreatment of paddy straw with concentrated (10x) ligninolytic enzyme under shake flask conditions using optimized physico-chemical parameters viz. paddy straw concentration, 2.5 g; enzyme volume, 8.0 ml; Ca2+ ions (10 mM) and incubation temperature, 45°C in 72 h of enzymatic action resulted in 80.87% and 47.64% decrease in lignin and hemicellulose, respectively and 29.96% increase in relative proportion of cellulose. For saccharification studies, from a total of forty seven fungal strains isolated from diverse natural sources, two strains (Aspergillus sp. CTS1 and Aspergillus sp. CTS2) tested positive for thermophilic and cellulolytic nature. The two strains were used for the development of fungal consortium which revealed filter paper, carboxymethyl cellulase and cellobiase activities of 10.2, 30.0 and 7.9 U/gds, respectively, significantly higher than the activities of individual fungal strains. Under the RSM optimized conditions [temperature, 55°C; pH,4.8 and substrate concentration, 5% (w/v)] and 30FPU/g dry substrate Arrowzyme yielded 476.0 mg/gds reducing sugars from biological pretreated paddy straw which were further increased to 492.0 mg/gds by the addition of Tween 20 at 0.2% (v/v). Enzymatic saccharification of biological pretreated straw by in-house concentrated cellulase (CC1) from thermophilic fungal consortium resulted in 229.0 mg/gds reducing sugars. The saccharification of chemical pretreated straw with Arrowzyme and CC1 resulted in 566.0 mg/gds and 250.0 mg/gds reducing sugar, respectively. Optimization of fermentation under separate hydrolysis and fermentation (SHF) conditions using RSM [S.cerevisiae inoculum size 10.0 % (v/v) and DAHP concentration, 0.2% (w/v)] resulted in 0.172 g/g and 0.193g/g ethanol from biological and chemical pretreated hydrolysate, respectively. Similarly, the CC1 saccharified hydrolysate resulted in an ethanol content of 0.076 g/g (biological pretreated) and 0.085 g/g (chemical pretreated). Fermentation of non-enzymatically saccharified biological and chemical hydrolysate by S.cerevisiae produced 0.013 g/g and 0.041 g/g ethanol, respectively, whereas sequential fermentation of chemical hydrolysate with S.cerevisiae and Pachysolen tannophilus produced 0.047g/g ethanol. The combined ethanol production from enzymatically and non-enzymatically saccharified biological and chemical pretreated paddy straw hydrolyaste was observed to be 0.185 g/g and 0.240 g/g, respectively.
Open Access
Bioprospects of microalgal isolates from water logged area of Punjab for biogas production
(Punjab Agricultural University, Ludhiana, 2017) Dar, Rouf Ahmad; Phutela, Urmila Gupta
The present study was aimed at isolation, identification, screening and characterization of microalgae from the waterlogged area of south-west Punjab, India. Optimization of cultural conditions of potential microalgal strains, their biogas production potential and anaerobic co-digestion studies were also conducted. Nineteen microalgal cultures (BGLR1-BGLR19) were isolated and were screened using different culture media for their growth kinetics. Isolate BGLR6 followed by BGLR5 showed the highest growth and biomass production in Algae culture medium and BG-11 medium respectively. These isolates upon molecular identification were found to be Asterarcys quadricellulare BGLR5 (MF661929) and Spirulina subsalsa BGLR6 (MF191711). The cultivation conditions for the enhanced production of biomass and other functional components (chlorophyll, carbohydrate, lipid and protein) of BGLR5 and BGLR6 were first screened by Plackett–Burman design and then the significant factors were optimized by Central Composite design (CCD). The optimal cultural conditions for BGLR5 and BGLR6 as per the model were pH of 9.92 & 11.5; temperature of 21.84 & 20°C; light intensity of 80.99 & 81 μmol m-2 s-1; growth period of 25 days (both); 15.00 mM NH4Cl/ CaCl2; 12.28 & 5.00 mM NaNO3 and 7.09 & 2.00 mM K2HPO4 respectively. Under these conditions, the response variables generated a desirability index of 84.10 and 94.91% for BGLR5 and BGLR6 respectively. A 0.42 and 1.60-fold increase in dry cell biomass yield was achieved in BGLR5 and BGLR6, compared to the basal condition (0.8858 and 1.0890 g biomass L-1) respectively. The biogas potential of the microalgal biomass under controlled temperature (35 ± 2°C) conditions revealed that in case of BGLR5, the highest biogas yield (51.712 Lkg-1algal biomass) was obtained in the hydrothermally (100°C 30min) pretreated biomass whilst in BGLR6, the highest biogas yield (42.73 Lkg-1algal biomass) was obtained in enzymatically (10% 24hr) pretreated biomass. Further, co-digestion of 50% paddy straw and 50% BGLR5 biomass (replacement of paddy straw by equal amount of microalgal biomass) produced 168.32 L biogas kg-1 feedstock with the ultimate biogas yield potential of 361.81 mLg-1 VS, reduced lag phase (λ:2.81 d) and increased rate of biogas production (Rm: 8.19 mLg-1d -1) which was higher not only in comparison to the anaerobic digestion of paddy straw and algal biomass individually but also to that of co-digestion of BGLR6 biomass with paddy straw. Likewise, 50 and 100% supplementation of BGLR5 biomass led to enhanced biogas production. Utilization of algal biomass cultivated in the open pond for co-digestion study with paddy straw in field scale digesters resulted in an increase of 17.27% biogas compared to control. The enhancement of methane content from 46.4% (control) to 66.5% (digester containing algal biomass) was also achieved. Hence, the present study signifies that the Asterarcys quadricellulare BGLR5 and Spirulina subsalsa BGLR6 biomass could be utilized as a cofeedstock with paddy straw for biogas production.
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Development of diagnostic protocol for assessment of microbiological quality of fresh vegetables
(Punjab Agricultural University, Ludhiana, 2017) Sood, Bhavish; Param Pal
The epidemiological surveillance for microbiological quality of seven fresh vegetables (N=725) sourced from Village fields along Buddha Nallah, Supermarkets and Street vendors was conducted and concluded the high incidence of 11 important food borne pathogens with highest positive percentage of K. pneumonia (69%) in village fields, Y. enterocolitica in supermarkets (63.54%) and vendor samples (78.98%). The highest mean total plate count was observed in Spinach (>6 log cfu g-1) collected from village fields and vendors. Most probable number (MPN) analysis reflected the deteriorated quality of irrigation water with index range upto >1100/100ml (above desirable limit 1000/100ml). Pathogenic bacterial load was dominated in internal part of tomato (3.98 log cfu g-1) and on the surface region of cucumber (4.12 log cfu g-1). Phenotying of isolates of 11 pathogens was carried out to study the diversification of pathogens on basis of biochemical, virulence and antibiogram profiling. Clustering analysis of isolates (Unweighted Pair Group Method with Arithmetic Mean) revealed their significant diversity among themselves which provides insight of the indigenous pathogens inhabiting the vegetable commodity. Antibiotic profiling using 25 antibiotics belonging to 10 classes was established for all the isolates and complete tolerance to five antibiotics [Clindamycin (2mcg), Tetracycline (30mcg), Cloxacillin (5mcg), Metronidazole (5mcg) and Imipenem (10mcg)] was observed in Salmonella enterica. The bacterial isolates showed high MAR index range of 0.08-0.84. The species-specific virulence markers (enterotoxin, effector proteins encoding genes) used in PCR assay to detect the gene distribution pattern among the pathogens and their presence prove the potentiality of causing illness. A Bacteriological Food testing Kit (BFTK) (12h detection time) was devised based on the biochemical characteristics of indicator (Escherichia coli, Staphylococcus aureus, Shigella spp., Salmonella spp., Listeria monocytogenes) and emerging (Yersinia enterocolitica, Aeromonas hydrophila, Campylobacter jejuni, Bacillus cereus and Klebsiella pneumoniae) and validated with food samples (N=1000) against BIS Standard Methods at 5% level of significance. Another rapid yet inexpensive detection technique based on the Multiplex PCR was devised in which hblD gene (430bp fragment) of Bacillus cereus; ystA gene (79bp fragment) of Yersinia enterocolitica, invA gene (280bp fragment) of Salmonella enterica and iap gene in Listeria monocytogenes (225bp fragment) were targeted. Furthermore, a combined culturing (BFTK) and molecular tool (multiplex PCR) was found promising to detect pathogens in time slot of 15 h using specific markers with detection limit of 106 cells per target bacteria. A simulation study on concentration method stepped with vacuum filtration, low speed centrifugation and high speed centrifugation has shown high rate of recovery of all the 11 pathogens with sensitivity limit not less than 104 cfu/10g by reducing the sample to final volume of 2ml. A useful intervention was made for decontamination of vegetables by effectively using 1000ppm of citric acid in wash water.
Open Access
STRAIN IMPROVEMENT OF Calocybe indica THROUGH MUTAGENESIS AND ITS AGRONOMIC OPTIMIZATION
(PAU Ludhiana, 2011) JATINDER KAUR; H. S. Sodhi
Tremendous revolution in the mushroom technology in recent years with respect to types and strains of cultivated mushrooms has made Calocybe indica, a specialty mushroom, the third most popular and commercially grown mushroom in India. A lot of work has been done as far its cultivation technology is concerned but no systematic work has yet been undertaken for its genetic improvement. During present investigation nine strains of C. indica were cultivated on wheat straw using recommended technology. Strain Ci- 3 gave maximum biological efficiency of 81.28%. Basidiospores from all the nine strains were collected and subjected to germination trial on different media but no germination was observed. Improvement of high yielding strain, Ci-3 using mutagenic treatment on protoplasts was conducted. Four mutagenic treatments (one physical and three chemical) yielded 30 putative mutants. Growth studies of putative mutants indicated maximum growth rate of CMB-4 on wheat straw while CMN-11 gave maximum biomass on complete yeast extract medium. Mutant CMN-9 gave maximum endoglucanase (0.345 μg/min/ml) and xylanase (0.540 μg/min/ml) activity. Seven mutants were identified on the basis of growth and enzymes studies. A set of 14 primers were used for molecular characterization of mutants along with the parent using RAPD-PCR. Out of these only 6 primers resulted gave distinct amplification products. Six primers yielded 68 scorable bands ranging from 40bp to 280bp. Similarity coefficient of Ci-3 ranged from 0.706 for CMN-3 to 0.794 for CMU-2. Three mutants (CMU-2, CMU- 5 and CME-2) showed 4 bands each having similar relative mobility values (0.2, 0.35, 0.5, 0.65) during protein profiling using SDS-PAGE. Three bands were obtained for CMN-11, CMB-4 and Ci-3 and 2 bands for CMN-9. Only one band was obtained in case of CMN-3 with relative mobility of 0.2. Cultivation of the mutants was carried out on wheat straw following hot water treatment. Four mutants (CMN-3, CMN-9, CMN-11 and CMB-4) were found to give significantly higher yield than the parent while only three mutants (CMN-3, CMN-9 and CMN-11) gave better results on mixture of wheat straw and paddy straw and on steam sterilized wheat straw. During nutritional studies mutant CMN-9 was found to be nutritionally better with high protein, tocopherol, β- carotene and lycopene content. Postharvest treatment of mutants indicated cabinet drying as a better option in terms of colour and texture retention.
Open Access
STUDY ON ERGOSTEROL CONTENT AND PROTEIN PROFILE OF MEDICINAL MUSHROOM, Ganoderma lucidum
(PAU Ludhiana, 2013) Anna Goyal; H. S., SODHI
Ganoderma lucidum, a specie belonging to the class basidiomycetes, family polyporaceae of the order aphyllophorales has been widely used as a source of potent nutracuetical products. Present study was planned to identify and characterize the role of proteins and ergosterol in the developmental process of Ganoderma lucidum. Four strains of Ganoderma lucidum (GL I - IV) showed a gradual increase in biomass to give 25.52g to 31.72g of biomass after three weeks of growth in mushroom complete medium broth with maximum in strain GL-III. Ganoderma lucidum strains were grown on wheat straw supplemented with 5% wheat bran with maximum biological efficiency for GL-I strain (31.27%) followed by GL-II (26.76%) and number of fruit bodies were 927 and 693 each weighing 33.7g and 38.6g, respectively. Ganoderma lucidum strain GL-I showed maximum ergosterol content (4601μg/g) whereas strain GL-III showed only 32μg/g. From spawn run, maximum ergosterol was obtained from GL-IV strain followed by strain GL-II while at pin head formation and fruit body formation ergosterol content was better for GL-II. Ergosterol content of fruit body of GL-1 was observed maximum (7009μg/g). Overall observation indicated that the ergosterol content increased with each stage of cultivation process i.e. from spawn run to pinhead and finally to fruit body formation. The intracellular and extracellular enzymatic studies have indicated enhanced activity during spawn run on solid substrate in comparison to that grown in the broth. The esterase and peroxidase activity significantly increased during the pinning of the cultures thus, indicating a positive role of these enzymes in fructification process. The FTIR analysis of proteins made during different stages of cultivation namely spawn run, pin head formation and fruiting indicated that the fruiting strains (GL-I and GL-II) have an ordered protein structure with hydrophobic amino acids. In case of GL-IV, unordered structure was obtained that could be related to the role of hydrophobin proteins in mushroom fructification process. Another observation on GL-IV indicated the presence of basic amino acids and aromatic amino acids with very low amount of acidic amino acids like aspartic acid and glutamic acid. The observation recorded during present study indicated a positive role of hydrophobic amino acids and hydrophobin proteins in mushroom fructification process. Ganoderma lucidum strain GL-II was also grown on selenium fortified mushroom minimal medium at different concentrations of 5ppm to 25ppm of sodium selenate. Scanning electron micrographs exhibited gradual decrease in hyphal diameter, spore number and spore diameter with increase in selenium concentration and the spore structures were altered. A significant decrease in spore diameter is observed in concentration of 20ppm and 25ppm (5.60 and 1.26 μm, respectively) as compared to control (10.04 μm). The SEM-EDS studies showed no selenium traces on the hyphal surface, however, on the contrary, SEM-EDS studies of crushate samples revealed selenium traces indicating selenium absorption as the cytosolic moieties as selenoproteins. Atomic absorption spectroscopy indicated an increasing trend in the uptake by the hyphal biomass as the concentration of sodium selenate increased with maximum absorption at concentrations of 15 ppm and 25 ppm (9.9%). It was concluded that fortification till 15 ppm can be used as stress was not that prominent and culture could grow rapidly without significant alteration in structure and morphology to enhance its biomedicinal properties. Present study has indicated that during the mushroom development process, ergosterol content increases with a positive role of proteins like peroxidases and hydrophobins at each stage of morphogenesis.
Open Access
“BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ALKALINE PROTEASE OF BACILLUS CIRCULANS MTCC 7906”
(PAU Ludhiana, 2011) Inderjeet Kaur; Gurvinder Singh, Kocher
The present study was conducted to characterize alkaline protease of Bacillus circulans MTCC 7906. Cotton deoiled meal (2.1%) supported optimum enzyme production in 144 hours of incubation. Among the agricultural byproducts used to improve alkaline protease production, the combination of Soybean meal and Glucose supported maximum enzyme production in 96 hours of incubation which was comparable with the control Reese medium (Casein + Glucose) in terms of alkaline protease activity as well as incubation time required for enzyme production. The alkaline protease was purified (16.2 fold) to homogeneity from the culture supernatant by the combination of ammonium sulphate precipitation (30-60%) and DEAE-cellulose anion exchange chromatography. The biochemical characterization of partially purified alkaline protease displayed maximum activity at a pH of 9.0, temperature of 60°C, an enzyme concentration of 0.1 ml/3.0 ml and substrate concentration of 12 mg/ml. The Km and Vmax of partially purified alkaline protease were found to be 4.5 mg/ml and 5555 nmole tyrosine min-1 ml-1, respectively. The enzyme activity was activated by divalent ions whereas a metal chelator EDTA and ammonium hydroxide reduced the activity. The molecular weight of the enzyme was estimated to be 46 kDa on SDS-PAGE. For, molecular analysis, full length alkaline protease gene (BcAP, 1329 bp) was PCR amplified from B. circulans DNA and cloned into pTrcHisA vector. Sequencing and alignment of nucleotide and predicted amino acid sequences of alkaline protease region identified a number of substitutions (mutation) which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The multiple alignment of nucleotide and amino acid sequences of alkaline protease gene of B. circulans established major matches with three closely related subspecies of B. subtilis (B. subtilis subspecies subtilis strain 168, B. subtilis BSn5 and B. subtilis subspecies spizizenii strain W23). Molecular evolution of B. circulans strain was determined by phylogenetic analysis of alkaline protease gene and predicted amino acid sequences. This analysis also suggested that alkaline protease gene is novel gene, which has been accessioned in GenBank with accession number JN645176.1. Recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the protein with estimated molecular size of 46 kDa as observed earlier.

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