Immobilization of Pullulanase from Local Isolate Bacillus subtilis MF467279 Strain onto Magnetic@TEMPO Cellulose Nanospheres for Hydrolysis of Pullulan

Document Type : Original Article

Authors

1 Biochemisrty Department- Biotechnology Research Institute-National Research Centre

2 Microbial Chemistry Department Biotechnology research institute

3 Cellulose and Paper Department Chemical Industries Research Institute

Abstract

Bacillus subtilis MF467279 was isolated from the desert plant species Salsola imbricate, that collected from Tur Sinai, Egypt, for production of pullulanase using growth on optimized liquid medium. Pullulanase was partially purified using fractionation by using 20-80% ammonium sulfate with 85% yield and 4.47 purification fold. The partially purified pullulanase was immobilized onto a biopolymer carrier (magnetic@TEMPO cellulose nanospheres) with 90% immobilization efficiency and 85 % immobilization yield. Kienetic parameters (Km and Vmax) for pullulan, soluble starch and amylopectin was achieved. The optimal temperature for free and immobilized enzymes was 40 and 60°C respectively with broad range (4-10) of pH activity for immobilized pullulanase. Characterizations revealed that immobilization highly improved thermostability, reusability and shelf stability. The free and immobilized pullulanse were able to hydrolyze soluble starch, amylopectin glycogen, dextrin and dextran. Studying the end products resulted from hydrolysis of pullulan and starch by free and immobilized enzyme via thin layer chromatography (TLC) indicated that Bacillus subtilis enzyme is pullulanase hydrolase type III which consider the most important hydrolase in starch saccharification process.



Key words. Pullulanase - Bacillus subtilis MF46727- Immobilization- magnetic@TEMPO cellulose nanospheres- Thin Layer chromatography (TLC).

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History

  • Receive Date: 04 September 2024
  • Revise Date: 28 September 2024
  • Accept Date: 08 October 2024

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