Directed evolution for increased chitinase activity
Directed evolution for increased chitinase activity
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DOI：
10.1007/s00253-007-0996-7
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发表时间：
2007-08-01
影响因子：
5
通讯作者：
Pei, Yan
中科院分区：
文献类型：
--
作者：
Fan, Yanhua;Fang, Weiguo;Pei, Yan
通过DNA改组和筛选的定向进化，提高了一种真菌球孢白僵菌几丁质酶Bbchitl的催化能力。Bbchit基因首先与各种原核信号序列连接并在大肠杆菌中表达。来自Erwinia carolovora的信号肽PelB导致最大的几丁质酶分泌到肉汤中。然后将表达PelB信号肽的核苷酸序列掺入E.大肠杆菌载体，以表达通过三轮DNA改组产生的Bbchitl变体。构建了具有150，000个变体的Bbchitl文库，其中核苷酸点突变频率为0.6%，并筛选了几丁质分解活性。选择两种Bbchitl变体（SHU-1和SHU-2），其与野生型相比显示出增加的几丁质分解活性。这些变体的序列分析揭示了氨基酸残基中的突变，这些突变通常不会被考虑用于改进几丁质酶活性的合理设计。氨基酸取代发生在两个推定的底物结合位点和催化区域之外。
Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchitl. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carolovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchitl variants generated by three rounds of DNA shuffling. A Bbchitl library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchitl variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.