IDENTIFICATION OF CGTASE GENE FROM INDONESIAN BACILLUS SP. LT1B AND PT2B ISOLATE BY LINEAR AMPLIFICATION AND SINGLE PRIMER POLYMERASE CHAIN REACTION (LASP-PCR) METHOD

• Main Author: Musliha, Affina
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/78770
• Institution: Institut Teknologi Bandung

Cyclodextrin glucanotransferases (CGTase, EC 2.4. l. 19) is an enzyme that convert starch into cyclodextrin. Cyclodextrin is used in pharmaceutical dosage forms formulation to improve the solubility, to increase stability and to mask unpleasant taste and smell. Bacillus sp. LTIB and PT2B isolate from Indonesia, which collected by Laboratory of Pharmaceutical Biotechnology ITB, were known for their ability to produce CGTase-ß enzyme, stable until 60 o c and pH range 7-10. Nucleotide sequence determination is substantial for large-scale recombinant CGTase production. About 800 bp cgtase internal genes sequences of those isolate have been determined in previous research. Average size of cgtase genes from several Bacillus are in the range of 2000-2500 bp. The aim of this experiment is to identify unknown cgtase gene by using Linear Amplification and Single Primer Polymerase Chain Reaction (LASP-PCR). LASP R1200 and LASP R900 primer were designed to amplify upstream region of cgtase internal gene while For LASP, LASP FA, LASP FC, LASP For Mid and LASP For End were designed to amplify downstream region of cgtase internal gene. LASP-PCR were succesfully amplifying cgtase gene fragment using LASP R 1200 primer at annealing temperature 55 oc, For LASP primer at 60 oc and LASP FA at 55 oc. The PCR products were then cloned into cloning vector. Sequence with 2062 bp of cgtase gene from Bacillus sp. PT2B was identified by assembling sequencing results of recombinant vector using BioEdit software. Promoters sequences, Ribosome Binding Site (RBS), start codon and 1836 bp of Open Reading Frame (ORF) in cgtase gene from Bacillus sp. PT2B were unveiled. Gene sequences were also analyzed by BLASTn and BLASTx resulting 96% of homology with cgtase gene from B. lehensis Gl (Accession Number CP003923.1) and Bacillus sp. Gl-2004 (Accession Number AY770576.1). Amino acid deduction results gave 96 % of homology with CGTase from B. firmus strain 37 (Accession Number AGR66230.1) and Bacillus sp. (Accession Number 2009322A). LASP-PCR condition need further optimization for cgtase gene from Bacillus sp. LTIB.