Being able to recombine more than two genes with four or more crossover points in a sequence independent manner is still a challenge in protein engineering and limits our capabilities in tailoring enzymes for industrial applications. By computational analysis employing multiple sequence alignments and homology modeling, five fragments of six phytase genes (sequence identities 31–64 %) were identified and efficiently recombined through phosphorothioate-based cloning using the PTRec method. By combinatorial recombination, functional phytase chimeras containing fragments of up to four phytases were obtained. Two variants (PTRec 74 and PTRec 77) with up to 32 % improved residual activity (90 °C, 60 min) and retained specific activities of > 1100 U/mg were identified. Both variants are composed of fragments from the phytases of Citrobacter braakii, Hafnia alvei and Yersinia mollaretii. They exhibit sequence identities of ≤ 80 % to their parental enzymes, highlighting the great potential of DNA recombination strategies to generate new enzymes with low sequences identities that offer opportunities for property right claims.

能够以序列无关的方式重组两个以上具有四个或更多交叉点的基因仍然是蛋白质工程中的一个挑战，并限制了我们为工业应用定制酶的能力。通过采用多序列比对和同源性建模的计算分析，使用 PTRec 方法通过基于硫代磷酸酯的克隆鉴定并有效重组了六个植酸酶基因的五个片段（序列同一性 31-64%）。通过组合重组，获得了含有多达四种植酸酶片段的功能性植酸酶嵌合体。鉴定出两种变体（PTRec 74 和 PTRec 77），其残留活性提高了 32%（90 °C，60 分钟），保留的比活性 > 1100 U/mg。两种变体均由来自植酸酶的片段组成Citrobacter braakii、Hafnia alvei和Yersinia mollaretii。它们与其亲本酶的序列同一性≤80%，突出了 DNA 重组策略在生成具有低序列同一性的新酶方面的巨大潜力，从而为产权主张提供了机会。