An Isolate of Bacillus clausii Appears to Possess Four Distinct Pectinolytic Activities

Year 2021, , 635 - 644, 01.03.2021

Sevinç Berber , Serap Çetinkaya

https://doi.org/10.21597/jist.768556

Abstract

For the bacterial source, Koyulhisar (Sivas, Turkey) apple orchard soil samples were used. Obtained isolates were identified at the species level by partial protein- and 16S rRNA gene sequence homology All of them were determined to be Bacillus clausii. Cultures were prepared with these isolates and after removing the cells, the proteins in the upper fluid were precipitated with the help of an organic solvent. While performing pectinase screening, it was noticed that an existing protease digested other proteins. This proteolytic activity was eliminated by increasing the incubation temperature to 55oC. Four different pectinase activities, polygalacturonase, pectin lyase, pectate lyase, and pectin methylesterase, were then demonstrated. Four protein bands corresponding to these activities visualised by SDS-PAGE were 80 kDa, 58 kDa, 46 kDa and ~ 28 kDa, respectively. Spectroscopic methods were employed for the identification of the enzymes. The smallest protein band (28 kDa) was found to be pectin methylesterase. Km and Vmax values of this enzyme were 0.4 and 217U, respectively.

Keywords

Bacillus clausii, pectate lyase, pectin lyase, pectin methylesterase, polygalacturonase

Supporting Institution

Sivas cumhuriyet Üniversitesi (CÜBAP)

Project Number

F-558 and F-597

Thanks

This work (F-558) and (F-597) were supported by CUBAP, Sivas Cumhuriyet University.

Bir Bacillus clausii İzolatı Dört Farklı Pektinolitik Aktiviteye Sahip Görünmektedir

Abstract

Bakteri kaynağı olarak Koyulhisar (Sivas, Türkiye) elma bahçesi toprak örnekleri kullanıldı. Elde edilen izolatlar tür düzeyinde kısmi protein- ve 16S rRNA gen dizisi homolojisi ile belirlendi. Hepsinin Bacillus clausii olduğu belirlendi. Bu izolatlar ile kültürler hazırlandı ve hücreler uzaklaştırıldıktan sonra üst sıvıdaki proteinler organik bir çözücü yardımı ile çöktürüldü. Pektinaz taraması yapılırken, mevcut bir proteazın diğer proteinleri sindirdiği fark edildi. Bu proteolitik aktivite, inkübasyon sıcaklığının 55oC'ye çıkarılmasıyla engellendi. Dört farklı pektinaz aktivitesi, poligalakturonaz, pektin liyaz, pektat liyaz ve pektin metilesteraz bundan sonra saptanabildi. SDS-PAGE'de görüntülenen ve bu aktivitelere karşılık gelen dört protein bandı, sırasıyla 80 kDa, 58 kDa, 46 kDa ve ~ 28 kDa idi. Enzimlerin tanımlanması için spektroskopik yöntemler kullanıldı. En küçük protein bandının (28 kDa) pektin metilesteraz olduğu bulundu. Bu enzimin Km ve Vmax değerleri sırasıyla 0.4 ve 217U idi.

Keywords

Bacillus clausii, pektat liyaz, pektin liyaz, pektin metilesteraz, poligalakturonaz

Project Number

F-558 and F-597

References

• Ahlawat S, Mandhan RP, Dhiman SS, Kumar R, Sharma J, 2008. Potential application of alkaline pectinase from Bacillus subtilis SS in pulp and paper industry. Applied Biochemistry and Biotechnology, 149:287–293.
• Akbalık G, 2003. Screening for Industrially Important Extracellular Enzymes from Alkolophilic Bacillus Genus. İzmir Institute of Technology (Master of Science Thesis), 99p, İzmir.
• Berber S, 2018. Productıon, Isolatıon and Characterızatıon of Extracellular Pectinase from Soil Bacteria. Cumhuriyet University Institute of Science (Master's Thesis), 21s.
• Cao J, Zheng L, Chen S, 1992. Screening of pectinase producer from alkalophilic bacteria and study on its potential application in degumming of ramie. Enzyme and Microbial Technology, 14:1013–1016.
• Giacobbe S, Pepe O, Ventorino V, Birolo L, Vinciguerra R, Faraco V, 2014. Identification and characterisation of a pectinolytic enzyme from Paenibacillus xylanolyticus. BioResources, 9: 4873–4887.
• Gonzalez SL, Rosso ND, 2011. Determination of Pectin Methylesterase Activty in Commercial Pectinases and Study of the Inactivation Kinetics Through Two Potentiometric Procedures. Food Science and Technology, 31(2): 412-417.
• Khairnar Y, Krishna VK, Borast A., Gupta , Trivedi S, Patli P, Gupta G, Gupta M, Jhadav A, Mujapara A, Joshi B, Mishra D, 2009. Study of Pectinase Production in Submerged Fermentation Using Different Strains of Aspergillus niger. International Journal of Microbiology Research, 1: 13-17.
• Khan MM, Choi, YS, Kim, YK, Yoo, JC, 2018. Immobilization of an alkaline endo polygalacturonase purified from Bacillus paralicheniformis exhibits bioscouring of cotton fabrics, Bioprocess and Biosystems Engineering, 41: 1425–1436.
• Khan M, Nakkeeran E, Umesh-Kumar S, 2013. Potential application of pectinase in developing functional foods. Annual Review of Food Science and Technology, 4:21–34.
• Kamijo J, Sakai K, Suzuki H, Suzuki K, Kunitake E, Shimizu M, Kato M, 2019. Identification and characterization of a thermostable pectate lyase from Aspergillus luchuensis var. saitoi, Food Chemistry, 276: 503-510.
• Kashyap DR, Vohra PK., Chopra S, Tewari R, 2001. Applications of Pectinases in the Commercial Sector: A Review. Bioresource Technology, 77: 215-227.
• Kavuthodi B, Sebastian D, 2018. Review on Bacterial Production of Alkaline Pectinase with Special Emphasis on Bacillus Species. Bioscience Biotechnology Research Communications, 11: 18-30.
• Li Z, Bai Z, Zhang B, Li B, Jin B, Zhang , Lin F, Zhang H, 2012. Purification and Characterization of Alkaline Pectin Lyase from a Newly Isolated Bacillus clausii and Its Application in Elicitation of Plant Disease Resistance. Applied Biochemistry and Biotechnology. 167: 2241-2256.
• Pedrolli DB, Monteiro AC, Gomes E, Carmona EC, 2009. Pectin and Pectinases: Production, Characterization and Industrial Application of Microbial Pectinolytic Enzymes. The Open Biotechnology Journal, 3: 9-18.
• Prathyusha K, Suneetha V, 2011. Bacterial pectinases and their potent biotechnological application in fruit processing/ juice production industry: a review. Journal of Phytological Research, 3:16–19
• Priya V, Sashi V, 2014. Pectinase producing microorganisms. International Journal of Scientific and Research Publications, 4: 1–4.
• Raju EVN, Divakar G, 2013. Production of Pectinase by Using Bacillus circulans Isolated From Dump Yards of Vegetable Wastes. International Journal of Pharmaceutical Sciences and Research, 4: 2615-2622.
• Rebello S, Mohandas A, Mathachan Aneesh E, Sindhu R, Binod P, Pandey A, 2017. Recent advancements in the production and application of microbial pectinases: an overview. Reviews in Environmental Science and Bio/Technology, 16: 381-394.
• Rukmini K, Korivi M, Devi PS, 2016. Biochemical Characterization of Pectinase in Bacillus pumilus Isolated From Fruit Waste. International Journal of Pharmacy and Pharmaceutica,. 3: 25-29.
• Saharan R, Sharma KP, 2019. Production, purification and characterization of pectin lyase from Bacillus subtilis isolated from moong beans leaves (Vigna radiata). Biocatalysis and Agricultural Biotechnology, 21: 101306.
• Sharma N, Rathore M, Sharma M, 2012. Microbial Pectinase: Sources, Characterization and Applications. Reviews in Environmental Science and Bio/Technology, 12: 45-60.
• Urdaci M, Bressollier P, Pinchuk I, 2004. Bacillus clausii Probiotic Strains Antimicrobial and Immunodulatory Activities. Journal of Clinical Gastroenterology, 38: 86-90.
• Urena IC, 2016. A Review of natural and engineered enzymes involved in bioethanol production. Graduate Student Theses, Dissertations, & Professional Papers.
• Zhou C, Xue Y, Ma Y, 2017. Cloning, Evaluation, and High-Level Expression of a Thermo-Alkaline Pectate Lyase from Alkaliphilic Bacillus clausii with Potential in Ramie Degumming. Applied Microbiology and Biotechnology, 101: 3663-3676.