| A systematic research of directed evolution in vitro of α -amylase has been made in the article, including cloning of α-amylase genes, random mutagenesis in vitro, DNA shuffling, relationships of amylase structure and function, characteristics of mutant enzyme as well. The most plentiful results so far on a -amylase in basic field in domestic have also been made as follows:The properties of the a -amylase from strain Xanthomonas compestris 8004 was studied and its gene was cloned as well as the a -amylase gene from strain Bacillus subtilisAS 1.108-It was demonstrated through enzymatic location analysis that strainXanthomonas compestris 8004 only produced one type of a -amylase which was secreted outside the cell. The optimal temperature and pH of the a -amylase were 50℃ and pH6.2 respectively. The enzyme was not thermostable as it nearly lost all activity after being incubated at 60℃ for 30min, yet it was quite stable in wide pH range of 4.86~10.47. Its molecular weight was estimated as 50kDa by SDS-PAGE. The gene of the enzyme was cloned smoothly by shotgun method with an accession number AF482991 in NCBI. The whole coding region was 1425bp containing the typical four conserved regions common in a -amylases from different sources. The a -amylase precursor speculated by nucleotide sequence consisted of 475 aa with the first 35 aa in N-terminal being signal peptide, and so the mature a -amylase consisted 440 aa whose calculated molecular weight was equivalent to that of determined by experiment. The a -amylase gene of Bacillus subtilis was similarly cloned by shotgun method with the accession number AY376455 in NCBI. The ORF of the gene was 1947bp. The a -amylase precursor speculated by nucleotide sequence consisted of 649 aa with the first 33 aa in N-terminal being signal peptide, and so the mature a -amylase consisted 616 aa whose calculated molecular weight was 67 kDa .A new method of gene mutagenesis in vitro was set up with the use of a PCRtechnique in which the deoxythymidine triphosphate (dTTP) was partially replaced by 5-bromo-2 ' -deoxyuridine-5 ' -triphosphate (5-BrdUTP), and a high activity mutant ofXanthomonas compestris a -amylase gene was obtained-5-BrdUTP is the analogue ofdTTP, so it can substitute dTTP and participate in the DNA, and consequently caused mutation. Experiments showed that 0.1% 5-BrdUTP substitution ratio was optimal in mutagenesis in vitro suitable for acquirement of high activity mutant a -amylase gene. Too high ratio would lead to too many inactive genes and too low ratio would not be enough to form a large mutant library. It was also demonstrated that the method in which dTTP was partially replaced by 5-BrdUTP in PCR system was convenient and effective for gene mutagenesis in vitro as it could induce not only base substitution but also base deletion or addition, furthermore the mutations it induced randomly happened among the whole gene fragment. Using this method after 2 rounds of mutagenesis and screening, we obtained a high activity mutant pHNH003 which showed 10 times α-amylase activity of the wild gene. Nucleotide sequence analysis revealed that due to frameshift translation resulting from a base C deletion the mutant had a totally different amino acid sequence with wild type from the 390th amino acid. Based on these experiments we raised a new hypothesis of gene molecular evolution that genes differentiated from the DNA fragment unstable and potential to mutation (called archaeonucleic acid ).α -Amylase gene of Xanthomonas compestris was subjected to DNA shuffling both asa single gene and coupled with a -amylase gene of Bacillus subtilis-In monogeneDNA shuffling, only after 2 rounds of shuffling and screening a mutant p103 with 2 times higher activity than wild one was obtained which had 2 amino acid substitutions, Ser310Phe and Ala324Ser, demonstrating DNA shuffling an evolution tool in vitro with the advantage of effective combination of DNA recombination and mutation. With the hope of getting an chimeric gene, two α-amylase genes of Xanthomonas compestris...
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