Construction of Expression Vector for Pichia pastoris Containing Chitosanase Gene from Bacillus amyloliquefaciens ABBD
Chitosan is a polymer of ?-1,4-D-glucosamine which has several bioactivities, such as fungistatic, antitumor, and anticholesterol. This polymer is derived from the deacetylation of chitin (poly-?-1,4-N-acetyl-D-glucosamine) isolated from crustacean shell. Unfortunately, the utilization of chitosan...
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| Format: | Final Project |
| Language: | Indonesia |
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| Online Access: | https://digilib.itb.ac.id/gdl/view/37953 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Chitosan is a polymer of ?-1,4-D-glucosamine which has several bioactivities, such as fungistatic, antitumor, and anticholesterol. This polymer is derived from the deacetylation of chitin (poly-?-1,4-N-acetyl-D-glucosamine) isolated from crustacean shell. Unfortunately, the utilization of chitosan is limited due to its low solubility and high viscosity. Nevertheless, chitosanase can hydrolyze chitosan into chitooligosaccharide which has higher solubility and hence could have broader applications. Chitosanase gene (csn1) of the marine bacterium Bacillus amyloliquefaciens ABBD has been isolated previously. The purpose of this study was to construct a pPICZ?-A recombinant plasmid containing csn1 gene in which the gene is fused with oligonucleotide for MF? signal sequence and regulated by AOX1 promoter from Pichia pastoris. The amplification of csn1 gene was conducted by Polymerase Chain Reaction (PCR) using Bacillus amyloliquefaciens ABBD chromosome as template and a set of primers which was designed based on the published nucleotide sequence of csn1 (GenBank AGT20518.1). The PCR DNA fragment with the size of 737 base pairs (bp) was ligated into the pGEM-T. The resulted pGEMT-csn1 recombinant plasmid was then verified by nucleotide sequence analysis and it was found out that the csn1 has two point mutations with respect to the published csn1 sequence. The pGEMT-csn1 recombinant plasmid was then digested with restriction enzymes EcoRI and XbaI, and subsequently the csn1/ EcoRI-XbaI DNA fragment was ligated into pPICZ?-A which has been cut by similar enzymes. The resulted pPICZ?A-csn1 has been confirmed by restriction enzyme and by nucleotide sequence analysis, thereby it can be used for production of recombinant chitosanase protein in P. pastoris in the future. |
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