Department of Biology, Federico II University of Naples, Via Cinthia 4, 80126, Naples, MSA, Italy.
Institute of Biosciences and BioResources, CNR, Naples, Italy.
Microb Cell Fact. 2017 Nov 28;16(1):218. doi: 10.1186/s12934-017-0833-3.
The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-D-xylans to remove successive D-xylose residues from the non-reducing termini.
We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation.
Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.
枯草芽孢杆菌孢子长期以来一直被用于展示抗原和酶。孢子展示可以通过重组和非重组方法来完成，后者被证明比前者更有效。我们使用非重组方法独立吸附两种嗜热酶，来自极端嗜热古菌的内切 1,4-β-木聚糖酶（EC 3.2.1.8）GH10-XA 和来自嗜热栖热菌的β-木糖苷酶（EC 3.2.1.37）GH3-XT。这些酶分别催化木聚糖中（1-4）-β-D-木糖苷键的内切水解和（1-4）-β-D-木聚糖的水解，从非还原端逐个去除 D-木糖残基。
我们报告说，两种纯化的酶都可以独立地吸附在枯草芽孢杆菌的纯化孢子上。吸附非常紧密，两种酶都保留了部分特异性活性。当显示 GH10-XA 或 GH3-XT 的孢子混合在一起时，木聚糖的水解效率比两种游离的、未吸附在孢子上的酶的混合物更高。吸附在孢子上的酶的总活性很高，很可能是由于酶的稳定性增加所致，吸附在孢子上的酶在反应条件下保持活性的时间比游离酶更长。收集两步反应后的吸附酶，并用新鲜底物孵育，仍然具有活性，并能够继续进行木聚糖降解。混合吸附酶的循环允许木聚糖降解的强烈增加。
我们的结果表明，两步法降解木聚糖可以通过混合显示两种所需酶之一的孢子来完成。两步法比两种未吸附的游离酶更有效，吸附的孢子至少可以再用于另一个反应循环。该过程的效率、吸附酶的可重复使用性以及枯草芽孢杆菌孢子良好的记录稳定性表明，孢子是一种适合展示用于单步和多步反应的酶的合适平台。
