Difference between revisions of "Part:BBa K3979004"

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Bacterial Chitinase Combo 1

Bacterial Chitinase Combo 1(BC1) is recombinant 1, from ChiB (Serratia marcescens QMB1466) and ChiA(Pseudoalteromonas sp. DL-6). The molecular size and weight of the protein are 1818 bp and 55.440 kDa, respectively.

Sequence and Features

Overview

The chitinase enzyme hydrolyzes insoluble chitin to its oligo and monomeric components. Chitinase proteins are abundant in microorganisms such as bacteria, which use these enzymes to degrade chitin for nutrition. Chitinases are classified as endochitinases or exochitinases. Endochitinases cleave chitin at internal sites to produce GlcNAc multimers. Exochitinases catalyze the progressive hydrolysis of chitin to produce GlcNAc, chitobiose, or chitotriose. Chitinases are classified into different glycoside hydrolase (GH) families based on their amino acid sequences, such as GH18, GH19, and GH20. The GH18 family contains the majority of bacterial chitinases. Based on amino acid sequence homology of the individual catalytic domains, bacterial GH18 chitinases are classified into three major subfamilies, A, B, and C. Chitinolytic bacteria are found in a variety of habitats and decompose chitin in both aerobic and anaerobic conditions. Chitinases from Serratia marcescens are well characterized and found to kill fungi from different genera like Aspergillus, Rhizopus. X.Wang et al. discovered that ChiA from the marine bacteria Pseudoalteromonas sp. DL-6 has better activity in the body and room temperature ranges, as well as an optimum pH around neutral, with efficient activity against a variety of fungal species. Here, we are using chitinase domains from both these bacterial species to produce a combinatorial chitinase gene for enhanced activity.

Primer Sequences Used

Fig. 1. BC1 Forward Primer

Fig. 2. BC1 Reverse Primer

Protein Structure from RaptorX

The annotated sequence was input into the RaptorX server to give us the predicted 3D structure in the form of a PDB file. File:BC1.mp4

Autodock Results

The receptor, here, is our engineered chimeric chitinase and the ligand is the Chitin octamer(CID 24978517). The threshold binding energy is -6kcal/mol which is generally accepted as the cut-off in ligand-binding /docking studies, any value more negative than this is considered significant. So, this protein will show binding with the chitin polymer. The protein structures were prepared before docking by removing water molecules, adding polar hydrogen atoms, and adding Kollman charges. A grid box was created so as to eliminate any surface binding and provide us with better and more reliable results. These modifications are necessary for the efficient binding of the ligand to the protein through non-covalent interactions.

References

• Pseudoalteromonas sp DL-6: Wang X, Zhao Y, Tan H, Chi N, Zhang Q, Du Y, Yin H. Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium. Int J Biol Macromol. 2014 Sep;70:455-62. doi: 10.1016/j.ijbiomac.2014.07.033. Epub 2014 Jul 23. PMID: 25064555.
• Serratia marcescens: Li J, Zheng J, Liang Y, Yan R, Xu X, Lin J. Expression and characterization of a chitinase from Serratia marcescens. Protein Expr Purif. 2020 Jul;171:105613. doi: 10.1016/j.pep.2020.105613. Epub 2020 Feb 23. PMID: 32097727.
• Saima, & Kuddus, Mohammed & Roohi, & Ahmad, Iffat. (2013). Isolation of novel chitinolytic bacteria and production optimization of extracellular chitinase. Journal of Genetic Engineering and Biotechnology. 11. 39–46. 10.1016/j.jgeb.2013.03.001.