Study on the Cloning Methods of Debitterness Enzyme -alfa-Rhamnosidase Used in Grapefruit Juice Industry

• Main Authors: Chien, Po-jung, 簡伯容
• Other Authors: Ynan-Tay Shyu
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/78363401855956792798

碩士 === 國立臺灣大學 === 園藝學系 === 85 === alfa-Rhamnosidase, the key enzyme of grapefruit debittering process industry, can hydrolyze naringin which is the major bitter contributor in grapefruit juice. Object of this study is to evaluate the methods for alfa- rhamnosidase gene cloning from Penicillium decumbens. Polyclonal antibody with high specificity and high titer was yielded from immunized rabbit. The antibody can clearly reacted with 20 mg/mL alfa-rhamnosidase after 12,000X diluted. Penicillium decumbens really containing alfa-rhamnosidase was also proved by antibody. The result of N-terminal sequencing showed the N-terminal sequences of alfa-rhamnosidase is Ala-Ser-Val-Pro-Tyr-Gly-Glu-Tyr-Ile-Leu- Ala-Pro-Pro-Ser-Ile. The method of RNA preparation by guanidinium-acid-phenol exraction from Penicillium decumbens was compared with RNA isolation by RNeasy kit, and no clearly difference was showed between those two methods. The nucleotide codon usage table of Penicillium Spp. was caculated from GenBank database. Two groups of primers, one with all possibile primers mixed and the other one containing five maximum probability primers, were designed by referening N-terminus sequences and the codon usage table descripted above. No DNA fragment was got from mixed primered after PCR, and four DNA fragments (named rha1, rha2, rha3, rha4) were obtained from the other primer group. Results indicated that RACE technique is more successful than RT-PCR by only one specific primer applied. The four genes were introduced into E.coli XL1-Blue and the sequences of each gene were also sequenced, too. The translated amino acid sequences of all four gene cloned were not fully compatible with alfa-rhamnosidase. Compared by EMBL/GenBank/DDBJ database, the translated proteins of rha1, rha2, rha3 were similar to genome polyprotein, and the translated protein of rha4 was most similar to phosphomannomutase.