Biochemical Characterization of Yeast Isolates from Murcha

Kiran Babu Tiwari1,2*, Manindra Lal Shrestha1, and Vishwanath Prasad Agrawal1,2

1Universal Science College, Pokhara University, Maitidevi, Kathmandu, Nepal
2Research Laboratory for Agricultural Biotechnology and Biochemistry, Maitidevi,
Kathmandu, Nepal

*Corresponding author: Kiran Babu Tiwari, Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal. Email: kiranbabu.babukiran@gmail.com

Abstract

Eight Murcha samples were collected from different localities of Nepal. Altogether 31 yeasts (unicellular fungi) were isolated in Potato-Dextrose Agar (PDA). Compared to Saccharomyces cerevisiae (white/round/raised, Gluose/Fructose/Galactose/Sucrose/Maltose - positive, Mannose/Arabinose/Lactose/Citrate/Urea - negative), the yeasts were characterized morphologically and biochemically. Of the total isolates, eight (25.8%) were similar to S cerevisiae for their colonial properties. All isolates could assimilate Glucose, Maltose and Starch; and none of them could utilize citrate as carbon source. Higher proportions of the strains were able to assimilate Lactose (7, 22.6%), Mannose (6, 19.4%) and Arabinose (5, 16.2%) compared to S. cerevisiae. Further, nine (29.0%), five (16.2%) and one (3.2%) were unable to assimilate Galactose, Fructose and Sucrose respectively. Out of seven (22.6%) urea positive strains, two were able to hydrolyze urea strongly as nitrogen source.

A Novel Class of Protease from Choerospondias axillaris (Lapsi) Leaves

Sudeep Karki1, Rupendra Shakya1 and Vishwanath P.Agrawal1,2

1Universal Science College and 2Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal.

Abstract

A novel protease from leaves of Choreospndias axillaris has been reported. C. axillaris , locally called Lapsi is dioceous, deciduous fruit – bearing large tree, having multiple daily uses. In an attempt to find method for determining sex of Lapsi at seedling stage , we stumbled upon a unique protease that has thwarted our effort to find sex - related protein. Thus protease is highly thermo – stable and acid resistant. Its preparation can be autoclaved without significant loss in activity. Its activity can be repeatedly precipitated by trichloroacetic acid. It possesses a Km value of 29 µM and Vmax 52.63 pmoles/min for bovine serum albumin as the substrate. It is catalytically so powerful that the level of soluble protein in leaf is below 20 µg per g dry weight. Lapsi leaf protease is an specific endopeptidase attacking peptide bonds that have phenylalanine, tyrosine, alanine and threonine / aspartic acid residues.

Introduction

Choerospondias axillaries:(Locally called “lapsi”), is a large, deciduous fruit-bearing tree of the family Anacardiaceae.A native of the Nepal hills (850–1900m) Lapsi wood is used as light construction timber and fuel wood; seed stones are used as fuel in brick kilns and the bark has medicinal value. Nepal is unique in processing and utilizing lapsi fruits. The fruits are rich in vitamin C content.

Agrawal and Kesari (1992) were first to observe strong proteotytic activity in Lapsi leaves. Lapsi Protease was found to be active even under autoclave condition. Dekhang and Sharma (2006) reported optimium pH of 7 for the protease. The protease is not inhibited at all by phenylmethanesulfonylfluoride ( PMSF ) and 20-30% inhibited by sodium iodoacetic acid, thus revealing that protease is not a serine protease. No smaller proteolytic products of BSA could be seen in SDS-PAGE using silver staining indicating that the protease is not exopeptidase, Protease activity can be repeatedly precipitated by 0.2 M tricloroacetic acid TCA (Singh and Giri 2007). In order to find an insight into the mechanism of protease action, the present research was carried out

______________________________

The research was done for the fulfilment of degree requirement for B. Sc. Biochemistry (SK and RS)

Materials and Methods

Preparation of Lapsi Leaf Powder : Dried leaves of lapsi were blended in a glass blender to get fine powder.

Partial purification and concentration of protease : It involved following steps.

1. Washing of the lapsi powder (5gm) with 50 ml acetone
2. Extraction of acetone washed powder with 10 ml phosphate buffer (0.1M of pH 7).
3. Heat treatment of the extract at 70. ºC for 30min
` 4. TCA precipitation of protein in the extract by adding TCA (2.45M) to heat treated solution to a final concentration of 0.2M followed by keeping in freeze for 10min, centrifuging at 10000rpm/10min and dissolving the pellet dissolve in 200μl of phosphate buffer.

Determination of proteolytic activity :

A typical reaction mixture cntaining 28.75μg of protein and 50 μg BSA in the total volume of 200μl of 0.1M pH 7 phosphate buffer was incubated for 30 min . The proteolytic activity is measured by 3 following methods.

1. Direct Method : The reaction was stopped by adding Bradford reagent for determination of BSA ( Bradford 1976, Saleemudin 1980 ).

2. Indirect Method : The reaction was stopped by adding 2.45M TCA to a final concentration is 0.2M, centrifuged at 10,000rpm/10min and pellet dissolved in 200μl of phosphate buffer (0.1M of pH-7) and protein determined by Bradford method.

3. Determination of amino acid produced in reaction .by Ninhydrin method :
To the reaction mixture (100μl), 0.9ml distilled water and 4ml Ninhydrin (0.5% in ethanol) reagent were added and heated at 80ºC for 10 min, cooled and absorbance measured at 570nm. Alanine was used as standard.

Detection of amino acids by paper chromatography :
Ascending paper chromatography of the reaction mixture (60μl 0.1% isopropanol) was done on Whatman paper 1 using Butanol: Glacial acetic acid: water (40:10:50 V/V) as the solvent system. Chromatogram was dried in air for 6 hrs.Amino acid spots were made visible by spraying the chromatogram with 0.2% Ninhydrin reagent. Then the purple coloured zones were marked and Rf value calculated.


Determined RF value of Standard Amino acid used.

Amino-Acids Rf-value Amino-Acids Rf-value
Alanine 0.35 Threonine 0.27
Tryptophan 0.63 Valine 0.8
Leucine 0.89 Aspartic acid 0.25
Iso-leucine 0.86 Serine 0.15
Phenylalanine 0.85 Cystine 0.05
Glycine 0.1 Arginine 0.07
Histidine 0.08 Glutamic acid 0.2
Tryrosine 0.45 Lysine 0.06

Results and Discussion

In order to measure the effect of enzyme concentration on protease activity, the double TCA precipitated enzyme (50μl) preparation (5.75ug/10μl) was used. It was noticed that enzyme activity was linear up to 28.75μg of protein. In order to determine the effect of time of incubation on protease activity, 50μl of double TCA precipitation enzyme (28.75μg) of protein and 50μg of the substrate (BSA) were used and incubated for various periods. Result obtained showed that the proteolytic activity was linear only up to 30min. Therefore, further experiments were carried out using 28.75μgm of protein and 30mins of incubation.In order to determine the Km and Vmax by Lineweaver-bulk plot, the effect of BSA concentration on enzyme activity was measured.. It was found that protease has Km of 29.1μM for BSA and Vmax of 52.63pmoles/min.A low value of Km indicates that the substrate is tightly bound to enzyme.

Since BSA has 607 amino acids , 607nmoles of amino acids should be produced per nmoles of BSA degraded. In order to check this equivalency, the amino aid contents of reaction mixture were analysed ( Table 1 ).

Table 1 : Comparison of experimental and the theoretical values of amino acid content of reaction mixture

BSA (μg)
a BSA degraded(μg)
b Theoretical value of amino acids produced
( nmoles)
c Experimental value of amino acids produced ( nmoles)
D c/d Calculated no. of free amino aacids
10 5.5 48.207 13.6 0282 171
20 12 105.180 28 0.266 161
50 26.8 234.904 65 0.277 168
100 60.4 529.410 145 0.273 166

*For BSA degradation, reaction mixture containing 28.75μg of enzyme and equired amount of BSA in total volume of 200μl of 0.1 M pH 7 phosphate buffer .After 30 min of incubation 100 μl of the mixture was used for protein determination using direct method.
*molecular weight of BSA 69323.4 Da, 1 mole of BSA contains 607 moles of amino acids
( Hilger et al. ) ; 50μg of BSA contains 0 .722 nmoles of BSA.
*For amino acid determination, to the reaction mixture (100 μl ) 0. 8.ml water was added and ninhydrin method used.

It was observed that upon proteolysis one molecule of BSA yielded only 161 -171 molecules of amino acids ( Table 1 ) suggesting that in addition to free amino acids 70 – 75 % BSA is degraded to smaller peptides which cannot be observed in SDS-PAGE as well as not amenable to the Ninhydrin method.

In order to determine the identity of released amino acids during proteolysis of BSA ,we embarked upon the paper chromatography of the reaction mixture . In paper chromatogram, only 4 spots could be seen. Comparing with the Rf-values of standard amino acids ( Fig. 1 ) we concluded that amino acids produced by degradation of BSA are phenylalanine, tyrosine, alanine and threonine / aspartic acid.

Table 2. Rf valuue of amino acids

Sample(enzyme) Rf-value Amino acid
Spot A 0.26 Thr , Asp
Spot B 0.35 Ala
Spot C 0.44 Tyr
Spot D 0.85 Phe

So, we conclude that lapsi protease is very special type of protease. It’s not a exopeptidase, it is an endopeptidase with very specific activity attacking peptide bonds containing phenylalanine, tyrosine, alanine, threonine / aspartic acid residues. A serious literature search shows that such type of protease has not been reported in the literature.

References

Agrawal VP, Keshari and Singh D, (1992) Study of Lapsi (Choreospondias axillaris) Protease Unpublished result.

Bradford MM (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72: 248-254.

Dekhang RN and Sharma G (2006) Study of Protease from the leaves of Choreospondias axillaries (Lapsi). Submitted to Universal Science College, Biochemistry Department for the fulfilment of degree requirement of . B. Sc. Biochemistry.

Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Hentges F ( 2001 ) Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. J. Clin. Exp. Immupol. 123 (3), 387-394. http://www.ncbi.nlm.nih.gov/protein/3336842?report=genpep

Saleemudin M, Ahmad H and Hussain A. (1980) A Simple, Rpapid and Sensitive Procedure for the Assay of endoprotease using Coomassie Brilliant Blue G-250. Analytical Biochemistry 105: 202-205.

Singh R and Giri S (2007) Characterization and purification of protease from leaves Choerospondias Axillaris (Lapsi).Submitted to Universal Science College, Biochemistry Department for the fulfilment of degree requirement of B.Sc. Biochemistry.

Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

Shrestha KO¹, Adhikari S¹, Tiwari KB^1,2 and Agrawal VP^1,2

¹Universal Science College, Maitidevi, Kathmandu, Nepal

²Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal

Abstract

Lactose utilizing yeasts have a potential application in the lactose pollution management in brewery and dairy industries as the organisms can grow at environmental conditions well. Eleven strains of lactose positive yeast were isolated from murcha samples collected from local markets in Kathmandu. Because of highest lactose positive activity among the isolated strains, L4 was selected for production, partially purification and characterization of β-galactosidase. Mass culture of the strain was treated with 2% (v/v) Chloroform to disrupt the cells. The enzyme was purified with Acetone precipitation method to 7.7 fold activity with 77.6% yield; specific activity was found to be 0.054 nmol/min/mg of protein. Optimum pH and temperature were determined to be 6.6 and 37°C respectively with the reaction time period 180 min. Michaelis-Menten constant (Km) value of the enzyme was found to be 8.798 mM of O-nitrophenyl-β-D-galactopyranoside.

Introduction

Murcha is a mixed starter inoculum, used in production of local alcoholic beverages in India, Tibet, Nepal, Bhutan etc. (Tsuyoshi et al., 2005). Murcha is a round cake, which is mildly acidic and has a pH around 5.2 containing 13% w/w moisture and 0.7% w/w ash (dry weight basis). The Murcha cakes contain mixed microbial population viz. molds, yeasts and bacteria (Tamang and Sarkar, 1995).

Yeasts are the world's premier industrial microorganisms, which have wide exploitation in the production of foods, beverages and pharmaceuticals. Alcoholic beverages are one of the major products in the world’s market. Yeasts can contaminate different dairy products because they have relatively low water activity (a_w, 0.88) (Frazier and Westhoff, 1995), can easily grow at room temperature and can utilize (assimilate and/or ferment) a variety of carbohydrates (Nahvi and Moeini, 2004), eg. pentoses, hexoses, disaccharides and, rarely polysaccharides (Barnett et al., 1990). The capacities of the organisms, thus, can be exploited to manage the biodegradeable wastes of the food, dairy and beverage industries. Most of the yeasts, except Saccharomyces spp., can grow on cellulosic materials, however; only few genera are able to degrade starch. Among disaccharides, lactose is one of the most refractory carbon substrate to most of the yeasts. Among hundreds of genera, only few are lactose positive, viz. many Cryptococcus spp. and Trichosporon spp.; some Debaryomyces spp., Kluyveromyces spp. and Myxozyma spp.; and occasionally Bullera spp., Candida spp., Rhodotorula spp. and Tremella spp. (Barnett et al., 1990). Lactose is one of the major components in whey in cheese industry that is non-friendly in the environment (Nahvi and Moeini, 2004). As they can grow at 25-30⁰C, yeasts can be exploited to manage lactose pollution (Sarova and Nikolova, 2002).

Yeast has been considered the predominant microbial enzyme source for food applications. Lactose utilizing yeast are good source of β-galactosidase. With rigorous search, Tiwari et al (2008) elucidated the presence of abundant lactose positive yeasts in murcha samples collected from various parts of Nepal. Hence, the study was designed to collect various yeasts capable to utilize lactose efficiently, and production, purification and characterization of β-galactosidase.

Materials and Methods

Isolation: Murcha samples were collected from local markets in Kathmandu, Nepal. The pure culture of yeast strains were isolated by serial dilution methods in Potato Dextrose Agar (PDA) (20% potato extract, 2% dextrose, and 1.5% agar, pH 4.5) incubating for 48 hr at 27⁰C. The purified isolates were stored on PDA slant at 4⁰C. The pure culture of the isolates was Gram stained for microscopic morphology.

Sugar assimilation test: The sugars used were glucose, fructose, sucrose, maltose, mannitol, galactose, lactose, and arabinose using a basal medium (4.5gm yeast extract, 7.5gm peptone and 20gm sugar in 1-lit distilled water) with phenol red (1mg/ml) as indicator. The pH was adjusted to 7.0-8.0. The medium was dispensed into tubes and sterilized by autoclaving. The pure culture of the isolates was incubated at 27⁰C till 72 hr. in the tubes and the result was indicated by change of color from red to yellow (Shrestha and Sharma, 1995). Control tubes were used in each set to monitor contamination.

Yeast Propagation: Sterile basal media containing ammonium sulphate, yeast extract and potassium dihydrogen phosphate was prepared with pH 4.5. Filter sterilized lactose (0.05M) was added to the 100 ml broth and yeast was inoculated and was incubated in waterbath shaker at 30°C for 48 hour.

Enzyme Extraction: The broth was centrifuged at 7000 rpm for 10 min. and the pellet was resuspended in 80ml phosphate buffer of pH 6.8. Chloroform (2%, v/v) was added and incubated overnight at 28°C. Incubated cells were centrifuged at 10000 rpm for 20 min. Supernatant was collected and one volume of acetone was added. The mixture was shaken in vertex shaker and centrifuged at 10000 rpm for 20 min. Pellets were collected and dissolved in 16ml of phosphate buffer with pH 6.8.

Enzyme Activity: β-galactosidase assay was done with O-nitrophenol β-D-galactopiranoside (ONPG) as substrate. Two test tubes were filled with 3ml of 0.01M of ONPG prepared in supplemented phosphate buffer of pH 6.8. Enzyme extract (1ml) was added to the test solution and reaction progress was determined spectrophotometrically against the blank tube at 420nm for every 30 min. for 3 hours. The reaction was quenched by adding 1ml of 0.5M sodium carbonate.

Determination of optimum time duration for reaction: Seven test tubes were filled with 3ml of 0.01M ONPG prepared in supplemented phosphate buffer of pH 6.8. Enzyme extract (1ml) was added in each tube and incubated at 37°C. By adding 1ml of 0.5M sodium carbonate, the reaction was quenched at every 30 min interval for respective tube and reaction progress was measured at 420nm against the blank.

Determination of optimum pH: Seven different test tubes were taken and 3ml of 0.01M ONPG prepared in buffer of pH 5.5, 6.0, 6.2, 6.4, 6.6, 6.8 and 7.0 was added to each respective test tube. Enzyme extract (1ml) was added to each test tube and incubated at 37°C for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Determination of optimum temperature: Seven test tubes were taken and 3ml of 0.01M ONPG prepared in phosphate buffer of pH 6.6 was added to each test tube. Enzyme extract 1ml was added to each test tube and incubated separately at 0°C, 15°C, 28°C, 37°C, 40°C, 45°C and 50°C respectively for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Determination of K_M value: ONPG solution (0.01M) was prepared in phosphate buffer of pH 6.6, and 0.6ml, 1.2ml, 1.8ml, 2.4ml, 3.0ml of the substrate was added in a series of five test tubes respectively. The final volume was set to 3ml by adding phosphate buffer of pH 6.6. Enzyme extract 1ml was added to each test tube and incubated at 37°C for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Results:

L4 strain those posses highest lactose positive activity was selected for production, partially purification and characterization of β-galactosidase. The enzyme was purified with Acetone precipitation method to 7.7 fold activity with 77.6% yield; specific activity was found to be 0.054 nmol/min/mg of protein (Table 1). Optimum pH and temperature were determined to be 6.6 (Fig. 1) and 37°C (Fig. 2) respectively with the reaction time period 180 min (Fig. 3). Michaelis-Menten constant (Km) value of the enzyme was found to be 8.798 mM of O-nitrophenyl-β-D-galactopyranoside (Fig. 4).

Discussion:

Yeasts are one of the important organisms having a wide industrial application. Most of the genera are unable to assimilate lactose as carbon source (Barnett et al., 1990). A higher abundance of the yeasts capable to produce β-galactosidase and thus assimilate lactose explored the importance of Nepalese yeast cakes (Murcha). Nahvi and Moeini (2004) reported three (3/30, 10.0%) β-galactosidase positive strains in Iran. In this study out of 148 isolates, only 11 were lactose positive.

The β-galactosidase from the yeast isolated from marcha on this study was characterized by their pH optimum at neutral or on the weekly acidic side. In general, β-galactosidase from yeast and bacteria have pH optimum near the neutral region, whereas the enzyme from molds act well at more acidic level as reported by Wierzbicki and Kosikowski (1973). The β-galactosidase of yeast was relatively heat labile, but they hydrolyze substrate well at lower temperature. The optimal hydrolysis was attended at 37°C and nearly 60% of hydrolyzing ability was maintained at 14°C. An enzyme workable at low temperature as possible is preferred for treatment of foods like milk. The Km value with ONPG as a substrate is slightly higher than reported in K. lactis by Kim and Lim (1981).

β-galactosidase commercially can be extracted from yeast. Its primary commercial use is to break down lactose in milk to make it suitable for people with lactose intolerance. β-galactosidase is also used in the manufacture of ice cream. Because glucose and galactose are sweeter than lactose, β-galactosidase produces a more pleasant taste. Lactose positive yeast strains can be used for removal of whey pollutants, Single cell protein (SCP) and ethanol production and treatment of lactose in dairy industry (Nahvi and Moeini, 2004).

Whey is the aqueous fraction of milk generated a by-product of cheese manufacturing which is produced in large amounts. The main solute in cheese whey is lactose present at a concentration of about 4.5-5% (Rohm et al., 1992; Zadow, 1992). Because of its high organic content, dumping directly to the environment causes serious contamination problems. As a solution, bioconversion of whey into SCP or ethanol has been performed in several countries (Gonzales, 1996; Irvine and Hill, 1985; Mawson, 1994). SCP could be produced from whey with employing of yeasts from different species including Kluyveromyces spp., Candida spp. and Trichosporon spp. as they are naturally able to metabolize lactose (Castillo, 1990). Although species of yeasts may differ considerably in their physiology, those of industrial importance have enough physiological characteristics in common to permit generalizations, provided that it is kept in mind that there will be exceptions to every statement made (Frazier and Westhoff, 1995). The findings explored that these yeasts may be new strains and lead to a search of yeast strains having high β-galactosidase activity (Nahvi and Moeini, 2004) as more efficient bioactive agents.

References

Barnett, J.A., D. Yarrow and R.W. Payne. 1990. The yeasts: Classification and identification. 2^nd Ed., Cambridge University Press. pp. 50-77.

Castillo, F. 1990. Lactose metabolism by yeast. In Verachtert, H. and R. DeMot (eds.) Yeast: Biotechnology and Biocatalysis. Marcel Dekker, New York; pp.: 297-320.

Frazier, W.C. and D.C. Westhoff. 1995. Food Microbiology. 4^th Ed., Tata McGraw-Hill Publishing Company Ltd. p10.

Gonzales, S.M.I. 1996. The biotechnological utilization of cheese whey. A review. Biores. Technol. 57: 1-11.

Irvine, D.M. and R.M. Hill. 1985. Cheese technology. In: Moo-young M (Ed.) Comprehensive Biotechnology. Pergman, Oxford; pp: 523-526.

Kim S.H. and Lim K.P. 1981. Differences in the hydrolysis of lactose and other substrates by β-D-Galactosidase from Kluyveromyces lactis.

Lodder, J. 1984.General classification of yeasts. In J Lodder (ed.), The yeasts. 3^rd ed., North-Holland Publishing Co., Amsterdam.

Mawson, A.J. 1994. Bioconversions for whey utilization and waste abatement. Biores. Technol. 47: 195-203.

Nahvi, I. and H. Moeini. 2004. Isolation and identification of yeast strains with high beta-galactosidase activity from diary products. Biotechnol. 3: 35-40.

Rohm, H., F. Eliskases-Lechner and M. Brauer. 1992. Diversity of yeasts in selected dairy products. J. Applied Bacteriol. 72: 370-376.

Sarova, I. and M. Nikolova. 2002. Isolation and taxonomic study of yeast strain from Bulgarian diary products. J. Culture Collection. 3: 59-65.

Shrestha, B. and A.P. Sharma. 1995. Manual on practical pharmaceutical microbiology. 1^st Ed., pp. 83-84.

Tamang, J.P. and P.K. Sarkar. 1995. Microflora of Murcha: an amylolytic fermentation starter. Microbiol. 81: 115-122.

Tiwari KB, Shrestha ML and Agrawal VPA. 2009. Abundance of lactose utilizing yeasts from Nepalese Murcha (Yeast Cakes). Nepalese Journal of Microbiology. In press

Tsuyoshi, N., R. Fudou, S. Yamanaka, M. Kozaki, N. Tamanag, S. Thapa and J. P. Tamang. 2005. Identification of yeast strains isolated from murcha in Sikkim, a microbial starter for amylolytic fermentation. Int. J. food Microbiol. 99: 135-146.

Wierzbicki, L. E., and F. V. Kosikowski. 1973. Food syrups from acid whey treated with/3-galactosidase of Aspergillus niger. J. Dairy Sci. 56:1182.

Zadow, J.G. 1992. Whey and lactose processing. Elservier Applied Science, London and New York.

Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2)

Streptomyces. These bacteria are widely distributed in nature, especially in the soil. The characteristic earthy smell of freshly plowed soil is actually attributed to the aromatic terpenoid geosmin produced by species of Streptomyces. There are currently 364 known species of this genus, many of which are the most important industrial producers of antibiotics and other secondary metabolites of antibacterial, antifungal, antiviral, and antitumor nature, as well as immunosuppressants, antihypercholesterolemics, etc. Streptomycetes are crucial in the soil environment because their diverse metabolism allows them to degrade the insoluble remains of other organisms, including recalcitrant compounds such as lignocelluloses and chitin. Streptomycetes produce both substrate and aerial mycelium. The latter shows characteristic modes of branching, and in the course of the streptomycete complex life cycle, these hyphae are partly transformed into chains of spores, which are often called conidia or arthrospores. An important feature in Streptomyces is the presence of type-I peptidoglycan in the cell walls that contains characteristic interpeptide glycine bridges. Another remarkable trait of streptomycetes is that they contain very large (~8 million base pairs which is about twice the size of most bacterial genomes) linear chromosomes with distinct telomeres. The linear chromosomal DNA is highly unstable and frequently undergoes large rearrangements at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, and lead to metabolic diversity within the Streptomyces group. Sequencing of several strains of Streptomyces is aimed partly on understanding the mechanisms involved in these diversification processes.

Streptomyces coelicolor. This bacterium is a soil-dwelling filamentous organism responsible for producing more than half of the known natural antibiotics. It is a well-studied species of Streptomyces and genetically is the best known representative.

Streptomyces coelicolor strain A3(2) M145. This strain is a derivative of the laboratory strain A3(2) lacking its two plasmids SCP1 and SCP2 which were sequenced separately after being isolated from the original strain A3(2).

SOURCE: http://www.blogger.com/post-create.g?blogID=8431478503600028158

Links:

1. http://www.blogger.com/post-create.g?blogID=8431478503600028158

2. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=242

3. http://www.ncbi.nlm.nih.gov/nuccore/30407153?report=genbank

4. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=238

5. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=13298

6. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=238

7. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=241

8. http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=16388

GENETIC NOMENCLATURE

GENETIC NOMENCLATURE

Source: http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/mutations/nomenclature-v3.pdf

Strain collections. The ease of rapidly accumulating a large number of mutants requires careful bookkeeping to avoid confusing one mutant with another. Each mutant should be assigned a strain number. Strain numbers usually consist of 2-3 capital letters designating their source and a serial numbering of the strains in a central laboratory collection. It is a good idea to check existing genetic resources to avoid the potential confusion that can result from assigning different genes the same name.

In addition to genome databases, good resources for gene names include the Salmonella Genetic Stock Centre (http://www.ucalgary.ca/~kesander/), the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu/), and the Bacillus subtilis Genetics web site (http://www1.rhbnc.ac.uk/biological-sciences/cutting/index.html).

Nomenclature. Through the 1960’s, genetic nomenclature was a “tower of babel”. Due to the absence of clear rules for naming genes, each investigator assigned new names haphazardly, often resulting in the same name being applied to different genes or different names being applied to the same gene. To eliminate the resulting confusion, Demerec et al. (1966, 1986) developed a standard nomenclature for bacterial genes. With the development of new genetic tools, some modifications have been required. A detailed description of these rules can be found in the instructions to authors for the J. Bacteriol. (http://jb.asm.org/misc/ifora.shtml). The basic rules are described below.

GENOTYPE:

1. Genes. Each gene is assigned a three-letter designation, usually an abbreviation for the pathway or the phenotype of mutants. When the genotype is indicated, the three-letter designation is written in lower case. Different genes that affect the same pathway are distinguished by a capital letter following the three-letter designation.

For example, mutations affecting pyrimidine biosynthesis are designated pyr; the pyrC gene encodes the enzyme dihydroorotase and the pyrD gene encodes the enzyme dihydroorotate dehydrogenase.

2. Allele numbers. Each mutation in the pathway is consecutively assigned a unique allele number. A separate series of allele numbers is used for each three-letter locus designation. If there is no capital letter designating a specific gene, insert a dash before the allele number.

For example, pyrC19 refers to a particular pyr mutation that affects the pyrC gene. In order to distinguish each mutation, no other pyr mutation, regardless of the gene affected, will be assigned the allele number 19. A separate series of allele numbers is used for each three-letter locus designation. Allele numbers should be used sequentially and carefully monitored to insure that two different mutations are not named with the same allele numbers. The entire genotype is italicized or underlined (e.g. pyrC19).

3. Insertions. Transposable elements or suicide plasmids can insert in known genes or in a site on the chromosome where no gene is yet known. When an insertion is in a known gene, the mutation is given a three-letter designation, gene designation, and allele number as described above, followed by a double colon then the type of insertion element. DO NOT leave blank spaces between the letters or numbers and the colon. For example, a particular Tn10 insertion within the pyrC gene (mutant allele number 103) may be designated pyrC103::Tn10.

When a transposon insertion is not in a known gene, it is named according to the map position of the insertion on the chromosome. Such insertions are named with a three-letter symbol starting with z. The second and third letters indicate the approximate map position in minutes: the second letter corresponds to 10-minute intervals of the genetic map numbered clockwise from minute 0 (a = 0-9; b = 10-19; c = 20-29, etc.); the third letter corresponds to minutes within any 10-minute segment (a= 0; b = 1; c = 2; etc). For example, a Tn10 insertion located near pyrC at 23 minutes is designated zcd::Tn10.

Allele numbers are assigned sequentially to such insertions regardless of the letters appearing in the second and third positions, so if more refined mapping data suggests a new threeletter symbol, the allele number of the insertion mutation is retained. This nomenclature uses zaa (0 min) to zjj (99 min). Insertion mutations on extrachromosomal elements are designated with zz, followed by a letter denoting the element used. For example, zzf is used for insertion mutations on an F' plasmid. Insertions with an unknown location are designated zxx.

zaa = insertion at 0-1 min
zab = insertion at 1-2 min
zac = insertion at 2-3 min
zad = insertion at 3-4 min
zae = insertion at 4-5 min
zaf = insertion at 5-6 min
zag = insertion at 6-7 min
zah = insertion at 7-8 min
zai = insertion at 8-9 min
zaj = insertion at 9-10 min
zaa = insertion at 0 min
zba = insertion at 10 min
zca = insertion at 20 min
zda = insertion at 30 min
zea = insertion at 40 min
zfa = insertion at 50 min
zga = insertion at 60 min
zha = insertion at 70 min
zia = insertion at 80 min
zja = insertion at 90 min
zxx = insertion with unknown location
zzf = insertion on F-plasmid

Some commonly used mini-transposon derivatives are designated as follows:

Tn10dTet = Tet resistance, deleted for Tn10 transposase
Tn10dCam = Derived from Tn10dTet, Cam resistance substituted for Tet resistance
Tn10dKan = Derived from Tn10dTet, Kan resistance substituted for Tet resistance
Tn10dGen = Derived from Tn10dTet, Gen resistance substituted for Tet resistance
MudJ = Kan resistance, forms lac operon fusions, deleted for Mu transposase
MudJ-Cam = Derived from MudJ, Cam resistance marker disrupts Kan resistance
MudCam = Cam resistance substitution between ends of Mu

4. Plasmids. Plasmids should be indicated by a / slash after the genotype. Indicate the name of the plasmid, the plasmid origin, and the relevant genotype or phenotype carried by the plasmid. Insertions of suicide plasmids into the chromosome can be indicated as described for transposons. If a duplication is generated it can be described as indicated under chromosomal rearrangements.

5. Phage. Prophages or plasmids integrated into an attachment site can be indicated by the name of the attachment site followed by a double colon and the phage genotype indicated in brackets. For example, att::[P22 mnt::Kan].

6. Chromosome rearrangements. Chromosome rearrangements including deletions, duplications, and inversions should be indicated by a three letter symbol indicating the type of rearrangement, followed by the genes involved indicated in parenthesis, followed by the allele number.

Deletions = DEL(genes)allele number
Inversions = INV(join point gene #1 – join point gene #2)allele number
Duplications = DUP(gene #1*join point*gene #2)allele number

PHENOTYPE:

1. Growth phenotypes. It is often necessary to distinguish the phenotype of a strain from its genotype. The phenotype is usually indicated with the same three-letter designation as the genotype but phenotypes start with capital letters and are not underlined. (For example, strain TR251 [hisC527cysA1349 supD] has a Cys+ His+ phenotype because the supD mutation suppresses the amber mutations in both the cysA and the hisC genes.)

2. Antibiotic resistance. Both two and three letter designations are commonly used for antibiotic resistance markers. Both are acceptable, but it is essential to be consistent. Resistance and sensitivity is indicated with a superscript but on the computer it is often simpler to indicate resistance with (R) and sensitivity with (S).

Amp = Ampicillin
Cam = Chloramphenicol
Gen = Gentamicin
Kan = Kanamycin
Neo = Neomycin
Spc = Spectinomycin
Str = Streptomycin
Tet = Tetracycline
Zeo = Zeomycin
XG = X-gal
XP = X-phosphate

3. Conditional alleles. Conditional alleles indicated by the genotype including allele number followed by the two letter designation for the conditional phenotypes shown in parenthesis. For example, leuA414(Am). Note that because this is a phenotype it begins with a capital letter.

(Ts) = Temperature sensitive mutation
(Cs) = Cold sensitive mutation
(Am) = Amber mutation
(Op) = Opal mutation
(Oc) = Ochre mutation

REFERENCES:

Demerec, M., E. Adelberg, A. Clark , and P. Hartman. 1966. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54(1):61-76.
Demerec, M., E. Adelberg, A. Clark , and P. Hartman. 1968. A proposal for a uniform nomenclature in bacterial genetics. J Gen Microbiol. 50(1):1-14.
Maloy, S., J. Cronan, and D. Friefelder. 1994. Microbial Genetics, Second edition. Jones and Bartlett, MA.
Maloy, S., V. Stewart, and R. Taylor. 1996. Genetic Analysis of Pathogenic Bacteria. Cold Spring Harbor Laboratory Press, NY.
Journal of Bacteriology Instructions to Authors. 2001. http://jb.asm.org/misc/ifora.shtml

Sodium Azide induced enhancement of antimicrobial property of previously selected actinomycetes (SIMA)

A proposal on
Sodium Azide induced enhancement of antimicrobial property of previously selected actinomycetes (SIMA)

Principle Investigator: Pragya Sharma and Mandira Manandhar
Students from eighth semester
Universal Science College
Pokhara University
Maitidevi, Kathmandu 2009

Supervisor: Kiran Babu Tiwari
Universal Science College
Pokhara University
RLABBMaitidevi, Kathmandu

Research Lab
Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu

Introduction:
Actinomycetes, mostly known for their antibiotic activity, resemble both bacteria (prokaryotes) and fungi (eukaryotes). They have cell wall similar to gram positive bacteria with high G+C (>55%) content and also have 70S ribosome like that of bacteria. But their filamentous form, sporulating form and histone like protein (that are not found in prokaryotes) shows fungal characters. According to Bergey's Manual of Systematic Bacteriology, actinomycetes are divided into eight diverse families: Actinomycetaceae, Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae (Holt, 1989) and they comprise 63 genera (Nisbet and Fox, 1991). Characteristic biological aspects of actinomycetes is its ability to produce a wide variety of secondary metabolites including most of the antibiotics. Some examples of antibiotics produced actinomycetes are streptomycin, aureomycin, terramycin and chloromycetin.

Background:
In RLABB, Bhattarai and Tiwari (2006) developed a prototype methodology to explore mutagenic effects of sodium azide in Streptomyces spp., viz. both for loss of function (LOF) and gain of function (GOF) effects. The GOF is of particular importance and this type of work will be a good example of the foundation of applied research. The study should be extended in order to cover more parameters of actinomycetes, which helps to understand their physiology with respect to enhanced antibacterial properties among the mutants compared to respective wild types.

General objectives:
To study sodium azide induced enhancement of antimycrobial property of actionmycetes strain

Specific Objectives:
To revive and purify actinomycetes from preserved sample
To screen antibacterial activities of the strains by primary screening method
To verify the antibacterial activities of the strains by secondary screening method

Methodology Revival of the preserved actinomycetes: To revive them, they will be incubated at 28°C for one hour and then streaked on starch casein agar plates and further incubated at 28°C for 7 days.

Purification of actinomycetes : Streak plate method will be used to purify cultures of actinomycetes contaminated by bacteria and fungi (Williams and Cross, 1971, Singh and Agrawal 2002; Agrawal 2003). After isolation of the pure colonies based on their colonial morphology, colour of hyphae, color of aerial mycelium, they will be individually plated on another but the same agar medium.

Primary screening : Antibacterial activity of pure wild type and corresponding mutants will be determined by perpendicular streak method on Nutrient agar (NA). Test actinomycete strain will be streaked on the middle part of the NA plate and incubated for 5 - 7 days in room temparature. The test organisms to be used will be: Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Klebsiella species, Proteus species, Pseudomonas species, Salmonella typhi and Shigella species. Log phase culture (4hrs of NB at 37C) of the bacteria will be straeked perpendicular to the actinomycete streak line and incubated for overnight.

Secondary screening : Fresh and pure culture of each isolate selected from the primary screening will be inoculated in starch casein broth and incubated at 28°C for 7 days in water bath shaker . Growth of the organism in the flask will be confirmed by the visible pellets, clumps or aggregates and turbidity in the broth. Contents of flasks will be filtered through Whatman no.1 filter paper aseptically. The filtrate will be used for the determination of antibacterial activity against the standard test organisms by agar well-cut method.

Expected outcome: It has been found that azide induces gain of function and loss of function (Bhattarai and Tiwari, 2006). Enhanced antibacterial activity that they reported on primary screening (Bhattarai et al. 2007) is of particular importance; and hence, further elaboration of these applied aspects can be verified with secondary screening method. This study may explore some mutants with enhanced antibacterial properties compared to their respective wild types as a way of strain improvement among actinomycetes.

References:
Agrawal, V. P (2003) Biodiversity of Khumbu Region : Population Study of Actinomycetes, a Project Report Submitted to the Royal Nepal Academy of Science and Technology, Khumaltar, Lalitpur, Nepal "R"

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Enhanced antibacterial activity of sodium azide treated mutant Streptomyces strain. Journal of Nepal Association for Medical Laboratory Sciences, 8(1): 67-8.

Bhattarai, K., Tiwari, K.B. and Agrawal, V.P. (2007). Loss-of-function (LOF) and gain-of-function (GOF) mutation of sodium azide in Streptomyces spp. Journal of Nepal Biotechnology Association. (accepted).

Holt, J.G., (1989) Bergey’s manual of systematic bacteriology, vol. 4, ed. S.T.Williams and M.E.Sharpe, Baltimore, Md : Williams and Williams.

Biochemical characterization of mutants generated by sodium azide treatment of corresponding sensitive wild Actinomycete strains isolated from Khumbu

Biochemical characterization of mutants generated by sodium azide treatment of corresponding sensitive wild Actinomycete strains isolated from Khumbu, Everest Base Camp

Here goes below an abstract of work of Mamata Khatri and Yurika Rajbhandari who pursued their thesis conducted under my supervision and Prof. Agrawal as a partial fulfillment of their B. Sc. Biochemistry degree from Universal Science College, Pokhara University. They jointly worked to characterize biochemically the actinomycete mutants generated by sodium azide treatment of corresponding sensitive wild strains isolated from Khumbu, Everest Base Camp. The work is granted by International Foundation for Science (IFS) – 2008. This endeavor followed the findings of previous student, Keshav Bhattarai for his thesis work. With his work, we discovered various Gain-of-Function and Loss-of-Function mutations in a Streptomyces strain isolated from a soil sampele from Everest Base Camp.

Abstract: Sodium azide is being used to generate mutants of Actinomycetes isolated in Research Laboratory for Biotechnology and Biochemistry (RLABB) from soil samples from Khumbu, Mount Everest Base camp. This work was done to expand the limited number of actinomycete mutants sensitive 10-50ppm of sodium azide and characterize them biochemically alongwith the corresponding wild strains. Actinomycetes were isolated in Starch Casein Agar (SCA) from the soil samples and subsequently purified. Of the total 36 strains from RLABB, 24 were found to be sensitive to 50 – 100 ppm of sodium azide and were selected to generate mutants. Based on differences in colonial characteristics compared to the wild strains, 32 of mutants were selected and purified in an agar medium without the mutagen. The mutants and corresponding wild types were characterized biochemically and observed various Gain-of-Function (GOF) and Loss-of-function (LOF) mutation.