Molecular Cloning, In Silico Analysis, and Characterization of a Novel Cellulose Microfibril Swelling Gene Isolated from Bacillus sp. Strain AY8

Md Azizul Haque; Dhirendra Nath Barman; Aminur Rahman; Md Shohorab Hossain; Sibdas Ghosh; Most Aynun Nahar; Mst Nur-E-Nazmun Nahar; Joyanta K Saha; Kye Man Cho; Han Dae Yun

doi:10.3390/microorganisms11122857

Molecular Cloning, In Silico Analysis, and Characterization of a Novel Cellulose Microfibril Swelling Gene Isolated from Bacillus sp. Strain AY8

Microorganisms. 2023 Nov 24;11(12):2857. doi: 10.3390/microorganisms11122857.

Authors

Affiliations

¹ Department of Biochemistry and Molecular Biology, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200, Bangladesh.
² Department of Biotechnology and Genetic Engineering, Noakhali Science and Technology University, Noakhali 3814, Bangladesh.
³ Department of Biomedical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa 31982, Saudi Arabia.
⁴ Department of Biochemistry and Molecular Biology, Trust University, Barisal 8200, Bangladesh.
⁵ Department of Biological Sciences, College of Arts and Sciences, Carlow University, 3333 Fifth Avenue, Pittsburgh, PA 15213, USA.
⁶ Department of Chemistry, Jagannath University, Dhaka 1100, Bangladesh.
⁷ Department of GreenBio Science and Agri-Food Bio Convergence Institute, Gyeongsang National University, Jinju 52725, Republic of Korea.
⁸ Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju 52725, Republic of Korea.

Abstract

A novel cellulose microfibril swelling (Cms) gene of Bacillus sp. AY8 was successfully cloned and sequenced using a set of primers designed based on the conserved region of the gene from the genomic database. The molecular cloning of the Cms gene revealed that the gene consisted of 679 bp sequences encoding 225 amino acids. Further in silico analysis unveiled that the Cms gene contained the NlpC/P60 conserved region that exhibited a homology of 98% with the NlpC/P60 family proteins found in both the strains, Burkholderialata sp. and Burkholderia vietnamiensis. The recombinant Cms enzyme had a significant impact on the reduction of crystallinity indices (CrI) of various substrates including a 3%, a 3.97%, a 4.66%, and a substantial 14.07% for filter paper, defatted cotton fiber, avicel, and alpha cellulose, respectively. Additionally, notable changes in the spectral features were observed among the substrates treated with recombinant Cms enzymes compared to the untreated control. Specifically, there was a decrease in band intensities within the spectral regions of 3000-3450 cm^-1, 2900 cm^-1, 1429 cm^-1, and 1371 cm^-1 for the treated filter paper, cotton fiber, avicel, and alpha cellulose, respectively. Furthermore, the recombinant Cms enzyme exhibited a maximum cellulose swelling activity at a pH of 7.0 along with a temperature of 40 °C. The molecular docking data revealed that ligand molecules, such as cellobiose, dextrin, maltose 1-phosphate, and feruloyated xyloglucan, effectively bonded to the active site of the Cms enzyme. The molecular dynamics simulations of the Cms enzyme displayed stable interactions with cellobiose and dextrin molecules up to 100 ns. It is noteworthy to mention that the conserved region of the Cms enzyme did not match with those of the bioadditives like expansins and swollenin proteins. This study is the initial report of a bacterial cellulose microfibril swellase enzyme, which could potentially serve as an additive to enhance biofuel production by releasing fermentable sugars from cellulose.

Keywords: H-bonds; cellulose microfibril swelling gene; cloning and expression; in silico analysis; molecular cloning; recalcitrance; structural analysis.

Grants and funding

GRANT 4264/Ministry of Education in Saudi Arabia