Studies on Cloning and Expression of a-Amylase Gene from Pseudomonas vesicularis MA103 to Lactococcus lactis NZ3900 and Characterization of Recombinant a-Amylase

• Main Author: 蘇明璁
• Other Authors: Chorng-Liang Pan
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/47880887712464116994

碩士 === 國立臺灣海洋大學 === 食品科學系 === 99 === The purpose of this thesis is using -amylase gene amy-00047 from Pseudomonas vesicularis MA103 as a reporter gene combine with plasmid pNZ8149 from nisin-controlled expression system (NICE system) and transgenic to Lactococcus lactis NZ3900 host cells, then evaluate the characterization of nisin-induced recombinant crude enzyme, the composition of oligosaccharide from recombinant enzyme-hydrolyzed soluble starch and the bioactivity of hydrolysate. According to amy-00047 amylase gene sequence a pair of primers pNZ8149-Amy-for and pNZ8149-Amy-rev was designed, and the primer pair were used in PCR to acquire 1074 bps recombinant sequence containing -amylase gene amy-00047 with SpeI restriction enzyme site on 5’-ter and XbaI restriction enzyme site on 3’-ter. Using restriction enzyme SpeI and XbaI to insert amy-00047 recombinant sequence into plasmid pNZ8149 and was transformed into Lc. lactis NZ3900 by electroporation. The plasmid accepted colony Lc. lactis NZ3900-Amy were selected by M17L plate and can confirm the length of 1074 bps inserted gene sequence exist by using PCR. The growth of Lc. lactis NZ3900-Amy will be limit by concentration up to 1000 ng/mL of nisin. Using ultrasonic to broken cells successfully obtained single protein band by SDS-PAGE, named as Pv-AM-R and the molecular weight was approximately 39 kDa. Optimal induced recombinant -amylase Pv-AM-R specific activity 2.6 U/mg is obtained by induction at 0.4 to 0.5 OD600nm cell density with 10 ng/mL nisin after 3 hr cultivation at 30oC. The optimal reaction pH and temperature of recombinant amylase Pv-AM-R were pH 7.0 and 40oC. Ca2+ was able to raise the relative activity of Pv-AM-R (114%), on the other side, Sn2+ was with the ability to completely inhibit the enzyme activity of enzymes. On substrate specificity, Pv-AM-R can hydrolyze soluble starch solution, but no obvious hydrolyze showed on Ulva sp. polysaccharide. Analyzing the hydrolysate of Pv-AM-R by HPLC and TLC, compared to the standards, discovered that the main hydrolysate were assumed to be maltose, DP3, DP4 and oligosaccharide greater than DP7. This study provides optimal parameters for the NICE system in Lc. lactis NZ3900 as well as a means to construct genetically modified probiotic bacteria with the ability to produce functional maltooligosaccharides.