The Directed Molecular Evolution Of α-Amylase And The Analysis Of The Mutants

We describe a new method for random mutagenesis of DNA based on the use of a mixture of triphosphates of nucleoside analogues. The method relies on DNA amplification in vitro with Taq polymerase and in the presence of the 5'-triphosphates of 5-bromo-2'-deoxyuridine-5'-triphosphate (5-BrdUTP). It is incorporated in place of TTP and, with a lower efficiency, in place of dCTP. After 30 cycles of DNA amplification, 5-BrdUTP yield 8 of 12 totally impossible mutation types (A:T, A:G, A:C, T:C, G:A, G:C, C:A and C:T). It mainly induced the A:T→G:C transition pathway (72.9%). The insertions and deletions (including fragment deletions) were also detected in 5-BrdUTP induced mutations. The results described differ from previous reported 5-BrdUTP induced mutagenesis result in three respects: (1) it enables hot spot of mutations in Xanthomonas campestris wild typeα-amylases gene, which is firstly reported in this research. (2) It allows control of the mutational load via the concentration of 5-BrdUTP used in PCR reaction mixture and (3) it yields both transition and transversion mutations. The procedure may find application in the generation of libraries of DNA and protein mutants from which species with improved or novel activities may be selected.A truncated mutantα-amylase, Xa-S2, was obtained from wild typeα-amylases (Xa-WT) through random mutagenesis. The amino acid sequence deduced from the nucleotide sequence of the Xa-S2 gene indicated that it only contains 167 amino acid residues (approximately 65% shorter than that of Xa-WT). Secondary structure prediction implies that Xa-S2 is unable to form the whole (β/α) 8-barrel catalytic domain and doesn't have the three conserved catalytic residues of wild typeα-amylase, but it still displays the starch hydrolyzing activity. Xa-S2 has been prepared, characterized and compared to the recombinant wild-type enzymes. It hydrolyzed starch with a Km value of 32 mg/mL and activity was optimal at pH 6.2 and 30°C. In contrast, the Km value for Xa-WT was 8 mg/mL and optimal enzyme activity was at pH 5.9~6.2 and 45~50°C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates ofα-1, 4-glucosidic bonds. A mutatedα-amylase gene with encoded higher activity enzyme product obtained from the mutant library was cloned to the Saccharomyces cerevisiae expression vector pHBM368---a vector system for high-copy-number integration into the ribosomal DNA of the yeast. The recombinant plasmid was resigned pHBM368XA and transformed into an S. cerevisiae INVScI strain. Three transformants were selected, in which the integrated sequences were stable even after 80 generations of growth in nonselective medium cultivation. The transformants expressed and secretedα-amylase with a secretion efficiency of about 50%.The starch-binding domain of Aspergillus niger glucoamylase (GA-I) was introduced into the C-terminal end ofα-amylase (XA) to generate a chimeric enzyme (XAGsbd) with raw-starch-binding and -digesting activity. The fusion gene was constructed to Pichia pastoris expression plasmid pPIC9K, to yield the recombinant plasmid which is resigned as pPICXAGsbd. pPICXAGsbd plasmid was transformed into Pichia pastoris GS115 and XAGsbd, with an apparent molecular mass of approximately 64kDa (585 residues), was expressed in Pichia pastoris GS115, secreted using theα-signal sequence and purified using raw starch affinity method. The chimeric enzyme showed an optimum pH and temperature of 5.9~6.2 and 45~50°C, respectively, and is very similar to wild type enzyme. SDS-PAGE analyses revealed that the purified fusion protein has a molecular mass of approximately 80kDa in virtue of over glycosylated by the host. Starch-binding assays showed that the adsorbed enzyme could be eluted from the adsorbent by 2%β-Cyclodextrin. Remarkably, XAGsbd can hydrolyze raw corn starch granules with the highest rate of digestion at 21%( 30h).