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Double Displacement Reaction
Studies on thermophilic protease production by bacillus sps
INTRODUCTION
The gastrointestinal microbiota has a role in the nutrition, growth and disease susceptibility of the fish. Symbiotic bacteria in an animal's digestive tract often produce complement enzymes for digestion of plant foods as well as synthesize compounds that are assimilated by the host. The bacterial diversity of marine and estuarine provides a wide array of enzymes with unique properties. In recent years, proteases from the gut of fishes received much attention (4). Substrates that have been traditionally fermented by solid state include a variety of agricultural products such as rice, wheat, millet, barley, grains, beans, corns and soy beans. However, non-traditional substrate which may also be of interest in industrial process development includes an abundant supply of agricultural, forest and food processing wastes. Bacteria have been used for the enzyme production, ensiling, composting and some food processes e.g. sausages, Japanese natto, fermented soybean paste and Chinese vinegar (10). Thermostable proteases are advantageous where higher processing temperatures employed and also results in faster reaction rate, increase in the solubility of nongaseous reactants and reduced the incidence of microbial contamination by mesophilic organisms. Proteases secreted from thermophilic bacteria are thus of particular interest and have become increasingly useful in a range of commercial applications (14). A variety of thermostable proteases is produced by the heterogeneous species Bacillus stearothermophilus (5). Hence the present work was undertaken to investigate the thermophilic protease production by Bacillus aquimaris AP.MSU7 isolated from the gut of Etroplus surratensis.
MATERIALS AND METHODS
The rice bran was obtained from the local market in Nagercoil,Tamilnadu,India. . Five grams of wet rice bran was taken in 250ml flasks, sterilized at 121.5°C for 15 min, cooled. The solid substrate was inoculated with 2 ml of bacterial suspension and incubated at 35°C for 48 h.
Extraction of crude enzyme. 20 ml of distilled water was added to the fermented substrate on a rotary shaker at 120 rpm for 1 h. The enzymes were removed from the homogenate by ultra centrifugation at 4˚C and the resultant clear supernatant was used for further analytical studies.
Assay for protease. The assay system consists of following ingredients such as 1.25 ml Tris buffer (pH 7.2), 0.5 ml of 1% aqueous casein solution and 0.25 ml culture supernatant. Approximate controls were also made. The mixture was incubated for 30 min at 300C. Then 3 ml of 5% TCA was added to this mixture and placed at 40C for 10 min to form precipitate. Then it was centrifuged at 5000 rpm for 15 min. From this, 0.5 ml of supernatant was taken, to this 2.5 ml of 0.5M sodium carbonate was added, mixed well and incubated for 20 min. Then it was added with 0.5 ml of folin phenol reagent and the absorbance was read at 660 nm using UV-Vis Spectrophotometer (TECOMP 8500). The amount of protease produced was estimated and expressed in microgram of tyrosine released under standard assay conditions.
Screening of thermophilic protease producers. Screenings of thermostable protease producing strains were made from nine different bacterial strains.
Strain identification
Genomic DNA extraction, Cloning and sequencing of 16S rRNA gene. The isolated bacterial strain was grown in 2ml Zobell Marine Broth (HiMedia cat#) overnight at 27o C. The culture was centrifuged at 7000 rpm for 3 min. The pellet was resuspended in 400 µl of sucrose TE. Lysozyme was added to a final concentration of 8 mg/ml and incubated for 1h at 37°C. To this tube, 100 µl of 0.5M EDTA (pH 8.0), 60 µl of 10% SDS and 3 µl of proteinase K from 20 mg/ml were added and incubated at 550C overnight. The supernatant was extracted twice with phenol: chloroform (1:1) and once with chloroform: isoamylalcohol (24:1) and ethanol precipitated. The DNA pellet was resuspended in sterile distilled water. The amplifiedproduct (1,500-bp) was purified using GFX ™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences) according to manufacturer's instruction. The 16S rDNA amplicon was cloned in pTZ57R/T vector according to the manufacturer's instruction (InsT/AcloneTM PCR Product Cloning Kit #K1214,MBI Fermentas). Full length sequencing of the rRNA gene (about 1500 bp) for the isolated bacteria was carried out in Macrogen (Seoul,Korea). The full-length sequences obtained were matched with previously published sequences available in NCBI using BLAST.
Optimization of media components
Optimization of incubation period. The production profile of protease was studied by conducting fermentation for different time intervals (24, 48, 72, 96, 120 and 140 h).
Effect of carbon sources and nitrogen on protease production. To asses the effect of carbon sources on protease production by using various carbon sources such as glucose, sucrose, dextrin, sorbitol, mannitol, fructose, maltose, lactose, galactose and control were supplemented individually at 0.2% concentration in the optimized basal medium inoculated with a 1% inoculums of isolated bacterial strain. The effect of nitrogen sources was studied by using various nitrogen sources such as skim milk powder, peptone, beef extract, urea, yeast extract, casein, soy meal, ammonium chloride, ammonium nitrate, potassium nitrate and sodium nitrate. They were tested individually at 0.2% concentration in the optimized carbon sources in basal medium inoculated with 1% inoculums of isolated bacterial strain.
Effect of sodium chloride and metal ions on protease production. The effect of NaCl on protease production was studied by supplying various concentrations of NaCl to the production media. The experiment was carried out individually at 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% and 4% NaCl in the optimized carbon and nitrogen sources. The effect of metal ions on protease production was determined by using various kinds of metal ions such as magnesium sulphate, ferric chloride, magnesium chloride, EDTA, copper sulphate, barium chloride, zinc sulphate, calcium chloride and control. They were incorporated individually into optimized carbon and nitrogen sources.
Effect of pH and Temperature on protease activity. Optimum pH for protease activity by the experimental microorganism was determined by using different pH in the production medium. The assay was carried out individually at various pH such as 3, 4, 5, 6, 7, 8 and 9. The effect of temperature on protease activity was studied by incubating the enzyme and substrate solution at various temperatures such as 10, 20, 30, 40, 50, 60, 70, 80 and 900C at 48h of incubation.
Partial purification of protease and molecular weight determination.
Partial purification was carried out at 4°C. Enzymes (supernatant of fermented broth obtained after centrifugation at 10 000_/g for 10 min) were precipitated by adding two volumes of acetone and kept for 1 h at 0 - 4°C to allow complete precipitation. The resulting precipitate was collected by centrifugation (10 000 rpm, 30 min) and the pellet was air dried and resuspended in a minimal volume of 20 mM Tris HCl buffer, pH 7.2. SDS-PAGE (12%) was performed according to the method of Laemmli (9) under reducing conditions. The molecular weight was determined by interpolation from a linear semi-logarithmic plot of relative molecular weight versus the Rf value (relative mobility) using a standard molecular weight marker
RESULTS
Screening of protease producing microbes. Based on the morphological, physiological and biochemical characteristics the suspected colony was identified as Bacillus sps by the following standard keys of Bergey's Manual of Determinative Bacteriology and the isolated bacterium was screened for thermophilic protease producing ability on skim milk agar. The zone formation around the bacterial colony indicated the protease positive strain which may be due to hydrolysis of casein. Hence the strain was identified as a protease producer and it was taken for further experimental studies. Phylogenetic studies revealed that the 16S rRNA gene sequencing of the strain Bacillus aquimaris has 97% (Accession number FJ 52234) similarity with the nearest match in the Genbank (Fig 
.
Time course on protease production
Under optimal condition, protease activity was reached in the culture of the bacterial isolate at 48h of the fermentation when the cell growth reached the late log phase (Fig. 1).
Effect of carbon, nitrogen sources, NaCl, metal ions and surfactants
Among these tested carbon sources, the protease production was maximum in sucrose supplemented medium (135.85 U/ml) when compared with other supplied sources (Fig.2) after 48 h of incubation period. In the case of nitrogen sources, the maximum amount of protease production was recorded in skim milk powder (340.79 U/ml) supplemented medium (Fig. 3). Among the tested NaCl concentrations, (Fig. 4) the protease production was high in the 2.5% added medium (244.88 U/ml). Fig-5 shows the effect of metal ions on protease production after 48 h of incubation period. Among the tested metal ions, the maximum amount of enzyme production was recorded in magnesium sulphate(177.98 U/ml) added medium.
Effect of pH and temperature
Fig- 6 shows the effect of pH on protease production. Among the tested pH, the maximum protease production was obtained at pH 7.0 (187.5 U/ml) when compared with control (74.64 U/ml). Similarly in the experimentation on the effect of temperature (Fig-7), the maximum protease production was obtained at 600C (287.64 U/ml).
Partial purifications
Purified protease from Bacillus licheniformis migrated as a single band of 63 kDa in SDS-PAGE under reducing conditions, suggesting that the purified protein was homogeneous (Plate. 1).
DISCUSSION
In recent years, solid state fermentation (SSF) has shown much promise in the development of bioprocesses and products of industry. Several reports on SSF were made on the production of fine chemicals (17). Screening of organic nitrogen source on protease production resulted that, skim milk powder was the best organic nitrogen sources in Bacillus aquimaris. This was supported by the earlier findings of Ferraro et al. (6). They stated that the production of protease was induced by the medium supplemented with casein and skim milk powder by Bacillus licheniformis. The effect of various carbon sources on protease production indicated that sucrose gave higher production when compared to other carbon sources supplied. This is in accordance with the earlier study of Sumantha et al. (15) and Sonleither et al. (16). The present study on the effect of sodium chloride showed the protease production was remarkably high in 2.5% sodium chloride supplied medium. Shanmuga Priya et al. (13) reported that the protease production by marine bacterium Roseobacter sp. absolutely require 3% NaCl. The present study on the effect of metal ions and trace elements on protease production revealed that the magnesium sulphate had influence on the protease production. Sumantha et al. (15) reported that, some bacteria and fungi showed maximum protease production in media supplemented with metal ions such as Ca2+, Mg2+ and inhibited by EDTA. The present study was revealed that the maximum enzyme production was found to be at pH 7.0 and suitable temperature at 60ºC. Gupta et al. (7) reported that the protease from Virgibacillus pantothenticus and Aspergillus oryzae U1521 has its maximum activity with the temperature range between "300C – 500C". In this study, protease production was high in the culture of the bacterial isolate at 48h of the fermentation when the cell growth reached the late log phase. The result obtained was in accordance with other halophilic bacterium Salinivibrio sp. strain AF-2004 (1). Earlier workers have reported the molecular weight determination of various bacterial proteases. He estimated the molecular weight of the particular proteinase was 63 kDa by using SDS-PAGE. In the present study also, the molecular weight of the purified protease of Bacillus sp. was determined and it was 45 kDa. Similar observation were reported by Kobayashi et al. (8) and Singh et al. (14) on SDS-PAGE analysis of the serine alkaline proteases of Bacillus KSM-K16 and B. sphaericus, respectively. Generally the molecular weight of the alkaline proteases of various Bacillus species lay within the range from 15 to 36 kDa (2).
REFERENCES
- Amoozegar MA, FatemiAZ, Karbalaei-Heidari HR, Razavi MR. Production of extracellular alkaline metallo protease from newly isolated, moderately halophile, Salinivibrio sp. Strain AF-2004. Microbial Res 2006;162 (4):369-377,
- Beg QK and Gupta R. Purification and characterization of an oxidation stable thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme microbial Technol 2003; 32: 294-304.
- Cappuccino JG. Sherman, N. Microbiology: A Laboratory Manual. Pearson Educaiton, Singapore 2004; pp – 491,.
- Chi Z, Ma C, Wang P, Li HF. Optimization of medium and cultivation for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresource Technology 2007; 98: 534-538.
- ChopraAKand Mathur DK. Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products. J. Appl. Bacteriol 1984; 57(2): 263 – 271..
- Ferrero MA, Cartro GR, Abate CM, Baigori MD, Sineriz F. Thermostable alkaline protease producing bacteria from Visakhapatnamsoil. Ind. J. Microbiol 1996; 42: 173 – 175.
- Gupta A, Joseph B, Mani A, Thomas G. Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus panthenticus. World. J. Microbiol Biotechnol 2007; 36: 417 – 426.
- Kobayashi T, Hakamada Y, Hifomi J. Koike K, Ito S. Purification of alkaline proteases from a Bacillus strain and their possible interrelationship. Appl. Microbiol. Biotechnol 1996; 45: 63 – 71.
- Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage, Nature (London) 1970; 227: 680-685.
- Mitchell DA. Definition, characterization and potential. In: Solid state cultivation Ed. Doelle, H. W., D. A. Mitchel and C. R. Rolz, New York, Elsevier, Applied Sciences 1992; 1 – 13,.
- Moriarty DJW. Control of luminous Vibrio species in penaeid aquaculture ponds. Aquaculture 1998; 164: 351 – 358.
- Ochoa S J L, Olmos S J. The functional property of Bacillus for shrimp feeds. Food Microbiology 2006; 23: 519 – 525.
- Shanmuga Priya S. Krishnareni J, Joseph Selvin, Gandhimathi,. Arun Kumar R, Thangavelu M, Seghal Kiran, TG, Natarajaseenivasan K. Optimization of extracellular thermotolerant alkaline protease produced by marine Roseobacter sp (MMD040). Bioprocess Biosyst. Eng 2008; 31: 427-433.
Singh J, Batra N, Sobti RC. Serine alkaline protease from a newly isolated
About the Author
M. SELVENDRAN
Ph.D ,in Microbiology
51,Deiva kanni street ,
Anuppanadi,Madurai-9
TamilNadu,India
Tel : + 91- 9944932360
Email : selvendran008@gmail.com
mselvendran@ymail.com
OBJECTIVE
To carve my niche in Bioscience research by updating my knowledge in recent technologies for the sustainable development of Microbiology and Biotechnology
current status TECHNIQUES KNOWN
UGC Research fellow &
Pursuing Ph.D, in Microbiology
Submitted a report on the "isolation and characterization lignin degradating micro organism from termite gut" Leading to the award of M.Sc degree.
Submitted a report on the "isolation and partial characterization of active compounds from microalgae which function as antimicrobials against aquatic pathogens" Leading to the award of M.Phil degree.
PhD ) Doing research in the field of Marine Micro biology "isolation and characterization of antimicrobial peptides from marine micro algal symbionts as a probiotic approach to aquatic industry"
Personal Details
:
FATHER'S NAME : P. Mahalingam
DATE OF BIRTH & AGE : 11.05.1982 & 29
NATIONALITY : Indian
MARITAL STATUS : UN Married
Address for communication :
M.Selvendran,
s/o P.Mahalingam (Rtd Forester)
51, Deivakanni Street,
Anuppanadi,
Madurai-9
Education and training
2008-2011: PhD,. MICROBIOLOGY
ManonmaniamSundarnarUniversity, Tamilnadu
2006-2007: M.Phil., MARINE BIOTECHNOLOGY
ManonmaniamSundarnarUniversity, Tamilnadu
2004-2006 : M.Sc., MICROBIOLOGY
Gandhigram Rural University, Tamilnadu
2000-2003: B.Sc.,MICROBIOLOGY
Yadava collage ,Madurai Kamaraj University, Tamilnadu
- 10 days summer program in Quality Control Madurai Aavin.
- 15 days Training program on Oyster Mushroom Cultivation organized by Mushroom Project by CAPART, New Delhi. organized by Gandhigram Rural Institute (Deemed University)
ü 3 year experience in teaching for PG and M.phil ( Micro biology ) in M.S university Centre for Marine Science and Technology Rajakkamangalam, Nagercoil. During the period of PhD, and also 4 years Research experience in the field of Marine micro biology and biotechnology, pharmaceutical biology and Aquaculture.
- Currently working as a Research Assistant in the project of UGC - CMST,M.S.University,Rajakkamangalam
Languages
- English
- Tamil
Computer skills
- Basic computer knowledge , MS office -Word, Excel, PowerPoint, etc., Scientific databases
Hobbies
- Reading books, Travel ,Driving
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