Open Access Journal

ISSN : 2394-6849 (Online)

International Journal of Engineering Research in Electronics and Communication Engineering(IJERECE)

Monthly Journal for Electronics and Communication Engineering

Open Access Journal

International Journal of Science Engineering and Management (IJSEM)

Monthly Journal for Science Engineering and Management

ISSN : 2456-1304 (Online)

Isolation, Screening and Molecular Characterization of Polygalacturonase Enzyme from Bacillus Species using RAPD Markers

Author : Dhananjaiah T M 1 Dr.Prasad.M.P 2

Date of Publication :15th July 2020

Abstract: Pectic substances form the major components of the plant cell wall. Polygalacturonase is a hydrolytic enzyme, which acts on polygalacturonic acid (PGA), hydrolyzing α-1,4 glycosidic bonds of pectic acid cleaving the α (1-4) bonds between adjacent galacturonic acid within the homogalacturonic acid backbone of pectin. Pectin modifying enzymes are formed in huge amount by plant related Microbes. Polygalacturonase enzyme has vital role in extraction and softening of fruits, and also plays a major role in agriculture and environmental sectors. The potential of using microorganisms as a biotechnological source in the production of industrially relevant food and textile processing enzymes, has stimulated renewed interest in the exploration of extracellular enzymatic activity. Production and scale up of Bacterial enzymes are easy, low cost and can act on a wider range of pH and environment. The current research was aimed at isolating pectinolytic Bacillus sp. from municipal dump sites, screen for polygalacturonase enzyme production and to determine the genetic variability. Six different soil samples were collected and plated on nutrient agar and the isolated colonies were screened for polygalacturonase production on modified MS medium. Of the 20 isolates screened, 8 isolated exhibited clear zones of hydrolysis indicating positive activity for polygalacturonase enzyme. The organisms were identified as Bacillus sp. based on colony, cell and biochemical characterization. The genetic variability of these polygalacturonase enzyme producing Bacillus sp. were analyzed using PCR based RAPD technique using four primers RBA1, RBA2, RBA3, RBA4. Three different groups of RAPD profiles were determined within the eight species based on the distance calculated by Euclidean Similarity matrix. The First clade has 4 organisms with ORG13 and ORG18 closely related followed by ORG1 and ORG15 and in the second clade ORG4 and ORG12 are closely related and according to the third clade ORG16 and ORG17 were found to have close evolutionary relationship. The genetic diversity is evident based on the percentage similarity between the isolates.

Reference :

    1. Aarti. C, and K. A. 2015. Discovery of polygalacturonase producing Bacillus tequilensis strain ARMATI using 16s rRNA gene sequencing. Asian Journal of Pharmaceutical and Clinical Research, 8(5): 58-62,
    2. Bibi, Nowshad, Ali, Sikander, & Tabassum, Romana. 2018. Isolation and Identification of Novel IndigenousBacterial Strain as a Low Cost Pectinase Source. Brazilian Archives of Biology and Technology, 61
    3. Gaurav Pant, Anil Prakash, J.V.P. Pavani, Sayantan Bera, G.V.N.S. Deviram, Ajay Kumar, Mitali Panchpuri & Ravi Gyana Prasuna. 2015. Production, optimization and partial purification of protease from Bacillus subtilis, Journal of Taibah University for Science, 9(1): 50-55.
    4. Greg Tucker, Xueren Yin, Aidi Zhang, MiaoMiao Wang, Qinggang Zhu, Xiaofen Liu, Xiulan Xie, Kunsong Chen, Don Grierson, 2017. Ethylene and fruit softening, Food Quality and Safety, 1(4): 253– 267.
    5. Jayani, Ranveer & Gupta, Reena. (2010). Screening of Bacterial Strains for Polygalacturonase Activity: Its Production by Bacillus sphaericus (MTCC 7542). Enzyme research. 306785. 10.4061/2010/306785.
    6.  Kwon GH, Lee HA, Park JY, et al., 2009. Development of a RAPD-PCR method for identification of Bacillus species isolated from Cheonggukjang. Int J Food Microbiol. 129(3):282- 287.
    7. Lü F, Wang J, Shao L, He P. Enzyme disintegration with spatial resolution reveals different distributions of sludge extracellular polymer substances. Biotechnol Biofuels. 9:29
    8. Mahdi Eskandarian Boroujeni, Arijit Das, K. Prashanthi, Sandeep Suryan and Sourav Bhattacharya, 2012. Enzymatic Screening and Random Amplified Polymorphic DNA Fingerprinting of Soil Streptomycetes Isolated from Wayanad District in Kerala, India. Journal of Biological Sciences, 12: 43-50.
    9. Mkada-Driss I, Lahmadi R, Chakroun AS, et al., 2014. Screening and characterization of RAPD markers in viscerotropic Leishmania parasites. PLoS One. 9(10):e109773.
    10. Patidar MK, Nighojkar S, Kumar A, Nighojkar A. 2018. Pectinolytic enzymes-solid state fermentation, assay methods and applications in fruit juice industries: a review. 3 Biotech. 8(4):199.
    11. Perez KJ, Viana JD, Lopes FC, et al., 2017. Bacillus spp. Isolated from Puba as a Source of Biosurfactants and Antimicrobial Lipopeptides. Front Microbiol. 8:61.
    12. Preeti G. Dharmik, Dr. Ashok V. 2013. Gomashe Bacterial Polygalacturonase (PG) Production from Agro Industrial Waste by Solid State Fermentation. Indian Journal of Applied Research, Vol.III, Issue.VI.
    13. Rahman MS, Choi YS, Kim YK, Park C, Yoo JC. 2019. Production of Novel Polygalacturonase from Bacillus paralicheniformis CBS32 and Application to Depolymerization of Ramie Fiber. Polymers (Basel). 11(9):1525
    14. Swain MR, Kar S, Ray RC. 2009. Exopolygalacturonase production by Bacillus subtilis CM5 in solid state fermentation using cassava bagasse. Braz J Microbiol. 40(3):636-648.
    15. Verma KS, Ul Haq S, Kachhwaha S, Kothari SL. 2017. RAPD and ISSR marker assessment of genetic diversity in Citrullus colocynthis (L.) Schrad: a unique source of germplasm highly adapted to drought and high-temperature stress. 3 Biotech. 7(5): 288.
    16. Voragen, A.G.J., Coenen, G., Verhoef, R.P. et al., 2009. Pectin, a versatile polysaccharide present in plant cell walls. Struct Chem. 20, 263.
    17. Williams & Wilkins. Bergey's manual of systematic bacteriology, 1986, 2.
    18. Xavier AREO, Lima ER, Oliveira AME, et al., 2017. Genetic diversity of Bacillus sp producers of amylase isolated from the soil. Genet Mol Res. 16(3):10.4238/gmr16039771
    19.  Zeni J, Cence K, Grando CE, et al., 2011. Screening of pectinase-producing microorganisms with polygalacturonase activity. Appl Biochem Biotechnol. 163(3):383-392.

Recent Article