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035 _a.b11861435
099 9 _aAIT Thesis no. BP-00-10
100 1 _aShrestha, Binesh
245 1 0 _aCloning of fungal chitin deacetylase gene into yeast Pichia pastoris
260 _aBangkok :
_bAsian Institute of Technology,
_c2000
300 _a82, [2] leaves
490 1 _aThesis ;
_vno. BP-00-10
500 _aA thesis submitted in partical fulfillment of the requirements for the degree of Master of Science, School of Environment, Resources and Development
502 _aThesis (M.Sc.) - Asian Institute of Technology, 2000
520 _aChitin deacetylase (CDA) gene has been amplified from Colletotrichum lindemuthianum UPS9 DNA and cloned into one of the E.coli-Pichia pastoris shuttle vector (pPlC9K). The sequence analysis of cloned gene showed 98% homology with that of C. /indemuthianum ATCC56676. The gene was cloned into pPIC9K in frame with the asecretion signal factor present in the vector. Furthermore, the gene was transformed into Pichia pastoris expression host in order to obtain overexpressing strains. Two strains of Pichia namely GS115 (fast methanol utilizing, Mut+) and KM71 (slow methanol utilizing, Mut5 ) were selected as a host for the exrpession of CDA gene. Pichia transformants were selected on the basis of Histidine synthesizing phenotype and were screened for transformants carrying multi copy inserts of CDA gene on the basis of degree of resistance to G418 (Geneticine ). Transformants carrying resistance upto 3 mg/ml of G418 were screened out which might carry several copies of cloned CDA gene. The expression of cloned CDA gene in Pichia transformants were studied by analysing culture supernatant on SOS-PAGE for excreted protein expression.
650 1 0 _aMolecular cloning
650 1 0 _aChitin
700 1 _aStevens, Willem Frans,
_eChairperson
700 1 _aHegarat, Francoise Le,
_eCo-Chairperson
700 0 _aSuwalee Chandrkrachang,
_eExamination committee
700 0 _aPakorn Nuchnoi,
_eExamiantion Committee
700 0 _aSunee Nitisinprasert,
_eExamination committee
710 2 _aH.M. the King of Thailand,
_eScholarship donor
810 2 _aAsian Institute of Technology.
_tThesis ;
_vno. BP-00-10
856 _3Full-Text
_uhttp://203.159.5.9/ait-thesis/detail.php?q=B06980
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