| Glucose amylase is simply called glucoamylase, which can hydrolyze starch, dextrin, hepatin and so on. The hydrolyzate is glucose. Catalytic action depending on glucoamylase is essential in industrial fermentation process which can produce ethanol, sugar, organic acid, amino acid and so on. All the manufactories try to increase glucoamylase yield. Resistence of glucoamylase secreted by filamentous fungus Aspergillus niger against heat and acid is excellent. A. niger had become primary strain in industrial production. Recently, in order to illustrate the secretive mechanism of glucoamylase, research focus on using new techniques such as molecular biology, biochemistry and gene engineering to study biosynthetic pathway, gene and genetic regulation of glucoamylase.As a sort of food additives, D-isoascorbic acid has a broad market. Pseudomonas fluorescens can produce 2-keto-D-gluconic acid in industrial fermentation process. In this process, starch is catalysed by glucoamylase, and change into glucose. Then glucose is changed into 2-keto-D-gluconic acid by P. fluorescens fermentation . Finally 2-keto-D-gluconic acid is used to produce D-isoascorbic acid . At present , the 2-keto-D-gluconic acid production in China has occupied over 50 percent of the total production in the world(Sun et al, 2003).Transforming glucoamylase gene into P. fluorescens is the aim of this research, hoping this P. fluorescens can secret glucoamylase. Thus it can use starch, insteading of glucose, to produce 2-keto-D-gluconic acid . We expect to omit the catalysing process by glucoamylase, to decrease fermentation time, to save costs and to increase economic income. In this study, glucoamylase gene cDNA was subcloned into prokaryotic broad host range plasmids pBBRlMCS-2, pUCP19-K with correct reading frame to obtain recombinant plasmids pBBRlMCS-2GA, pUCP19-KGA, then were transformed into P. fluorescens AR4 respectively which was a high yielding strain of 2-keto-D-gluconic acid. Finally we obtained the recombinant P .fluorescens ARW4, ARH4. This research expected to obtain glucoamylase secreting from ARW4, ARH4. SDS-PAGE gel electrophoresis showed that one more strip of protein was found in total proteins of P. fluorescens ARW4 comparing with P. fluorescens AR4. The molecular weight of this protein was 70.5kD which was the same as the molecularweight of A. nigex glucoamylase protein. We estimated that it should be the glucoamylase expressed in the P. fluorescens ARW4. In order to determined the BAPT expression in transgenic cell , we immunized rabbit to produce polyclonal antibodies against glucoamylase. The result of the Western blot shows that one more strip of protein as 70.5kD was printed with polyclonal antiserum comparing with normal serum. We confirmed that the recombinant glucoamylase protein has expressed effectively in ARW4. But it existed in the form of inclusion which was inactive. Although we tried to change and optimize the culture conditions of the engineering P. fluorescens, the soluble protein was not obtained in the end. We did not obtain the active glucoamylase secreted by P. fluorescens which still needed to be further investigated.In this research A . nigex glucoamylase gene cDNA was first transformed into P. fluorescens, and was successfully expressed in the form of inclusion. We did the basic research for the active glucoamylase secreted by P. fluorescens in the future study.Although this research did not achieve our anticipative purpose, we made a exploration on heterogenous gene expression system of P.fluorescens. Moreover, we used PCR technique to turn plasmid pUCP19(Ampr)into pUCP19-K (Kanr). Successfully gainning plasmid with kanamycin resistance gave us an confidence to explore reconstruction of plasmid with the aim of creating diverse vectors.
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