Ariaeenejad Shohreh, Motamedi Elaheh, Kavousi Kaveh, Ghasemitabesh Rezvaneh, Goudarzi Razieh, Salekdeh Ghasem Hosseini, Zolfaghari Behrouz, Roy Swapnoneel
Department of Systems and Synthetic Biology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran.
Department of Nanotechnology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran.
Front Microbiol. 2023 Jan 4;13:1056364. doi: 10.3389/fmicb.2022.1056364. eCollection 2022.
Some enzymes can catalyze more than one chemical conversion for which they are physiologically specialized. This secondary function, which is called underground, promiscuous, metabolism, or cross activity, is recognized as a valuable feature and has received much attention for developing new catalytic functions in industrial applications. In this study, a novel bifunctional xylanase/β-glucosidase metagenomic-derived enzyme, PersiBGLXyn1, with underground β-glucosidase activity was mined by in-silico screening. Then, the corresponding gene was cloned, expressed and purified. The PersiBGLXyn1 improved the degradation efficiency of organic solvent pretreated coffee residue waste (CRW), and subsequently the production of bioethanol during a separate enzymatic hydrolysis and fermentation (SHF) process. After characterization, the enzyme was immobilized on a nanocellulose (NC) carrier generated from sugar beet pulp (SBP), which remarkably improved the underground activity of the enzyme up to four-fold at 80°C and up to two-fold at pH 4.0 compared to the free one. The immobilized PersiBGLXyn1 demonstrated 12 to 13-fold rise in half-life at 70 and 80°C for its underground activity. The amount of reducing sugar produced from enzymatic saccharification of the CRW was also enhanced from 12.97 g/l to 19.69 g/l by immobilization of the enzyme. Bioethanol production was 29.31 g/l for free enzyme after 72 h fermentation, while the immobilized PersiBGLXyn1 showed 51.47 g/l production titre. Overall, this study presented a cost-effective in-silico metagenomic approach to identify novel bifunctional xylanase/β-glucosidase enzyme with underground β-glucosidase activity. It also demonstrated the improved efficacy of the underground activities of the bifunctional enzyme as a promising alternative for fermentable sugars production and subsequent value-added products.
一些酶能够催化不止一种它们在生理上专门负责的化学转化反应。这种被称为隐秘、混杂、代谢或交叉活性的次要功能，被认为是一项有价值的特性，并在工业应用中开发新的催化功能方面受到了广泛关注。在本研究中，通过计算机筛选挖掘出一种具有隐秘β-葡萄糖苷酶活性的新型双功能木聚糖酶/β-葡萄糖苷酶宏基因组来源的酶PersiBGLXyn1。然后，克隆、表达并纯化了相应的基因。PersiBGLXyn1提高了有机溶剂预处理咖啡渣废弃物（CRW）的降解效率，进而提高了在单独酶水解和发酵（SHF）过程中生物乙醇的产量。经过表征后，将该酶固定在由甜菜粕（SBP）生成的纳米纤维素（NC）载体上，与游离酶相比，这显著提高了该酶在80°C时的隐秘活性达四倍，在pH 4.0时达两倍。固定化的PersiBGLXyn1在70°C和80°C时其隐秘活性的半衰期提高了12至13倍。通过固定化该酶，CRW酶促糖化产生的还原糖量也从12.97 g/l提高到了19.69 g/l。游离酶发酵72小时后生物乙醇产量为29.31 g/l，而固定化的PersiBGLXyn1显示产量为51.47 g/l。总体而言，本研究提出了一种经济高效的计算机宏基因组方法来鉴定具有隐秘β-葡萄糖苷酶活性的新型双功能木聚糖酶/β-葡萄糖苷酶。它还证明了双功能酶隐秘活性的提高效果，是生产可发酵糖及后续增值产品的一种有前景的替代方法。
