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.

Generation of actinomycete mutants by exposing corresponding tolerant wild type strains from Khumbu, Everest Base Camp

Here goes below an abstract of work of Junu Hamal and Shruti Regmi 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 generate mutants of tolerant wild type actinomycete 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. With the International Foundation for Science (IFS)-Grant 2008, this work was done to expand the limited number of mutants of tolerant actinomycetes (50-100ppm) of sodium azide. Actinomycetes were isolated in Starch Casein Agar (SCA) from the soil samples and subsequently purified. Of the total 36, 12 were found to be sensitive upto 100 ppm of sodium azide and were selected to generate mutants. Based on differences in colonial characteristics compared from the 12 wild strains, 36 mutants (each from 50ppm, 75ppm and 100ppm of sodium azide) were selected and purified in an agar medium without the mutagen. Individual mutants were described for their colonial morphologies.

Actinomycete Nomenclature system in RLABB

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. With the International Foundation for Science (IFS)-Grant 2008, this work was done to expand the limited number of actinomycete mutants generated by sodium azide exposure. Feeling the need to develop the nomenclature system in order to designate and discriminate the vast array of the organisms’ database, we developed a “Guide for nomenclature of wild types and corresponding mutants of actinomycetes generated by exposure of sodium azide in RLABB” as follows:
1. Section 1: The wild strains are started with the capital initial of the region of isolation. E.g., S from Sagarmatha National Park, J from Jorsela, K from Kalapatthar and N from Namche.
2. Section 2: The capital letter for the wild type strain is followed by serial number which explains the different sites from the same region. E.g., S16 and S18 were isolated from two different sites of Sagarmatha National Park.
3. Section 3: The serial number may be tagged with decimal serial numbers to distinguish the soil samples from the same site from the same region. E.g., K70.1, K70.3 and K70.5 were the three isolates from corresponding three soil samples from the site (70th) of Kalapatthar region.
4. Section 4: The isolates from a soil samples are distinguished with the small letter tag after the serial numeral(s) described in sections 2 and 3 above. E.g., J1b and J1d are two isolates from the site (1st) from Jorsela region.
5. Section 5: Initials of the corresponding mutants are carried from the complete name of the respective wild types and followed by decimal and the concentration of sodium azide from which they were picked up. E.g., S18a.10 and S18a.20 were two mutants generated from S18a isolates exposed to 10ppm and 20ppm of sodium azide.
6. Section 6: Different mutants from the same concentration of sodium azide for the same wild type strain follows a decimal and i, ii, iii etc. E.g., S18a.10.i and S18a.10.ii were two mutant isolates from the S18a exposed to 10ppm of sodium azide.