Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India.
Microbiology & Fermentation Technology Department, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, India.
J Appl Microbiol. 2020 Dec;129(6):1644-1656. doi: 10.1111/jam.14757. Epub 2020 Jul 16.
Aspergillus niger is well established for secreting α-glucosidase having transglycosylation activity, which is used as processing aid for synthesis of isomaltooligosaccharides. The present study focuses on identification and characterization of a non-niger Aspergillus isolate and its gene conferring strong transglycosylation activity.
The soil isolate was identified as Aspergillus neoniger belonging to Aspergillus section Nigri using ITS (internal transcribed spacer) and β-tubulin analysis. The sequence analysis of gene responsible for α-glucosidase synthesis revealed significant nucleotide variations when compared to other Aspergillus species. Molecular docking studies using the homology model revealed the presence of threonine at 694 subsite position instead of asparagine as in case of A. niger's α-glucosidase. The enzyme was purified to several fold using DEAE Sepharose-CL6B column and on SDS-PAGE analysis, it was found to be 145 kDa. MS/MS analysis of the purified enzyme validated the presence of threonine at 694 position. Commercial α-glucosidase (Transglucosidase L 'Amano') derived from A. niger and the α-glucosidase from isolate were compared for transglycosylation activity using constant test conditions. α-glucosidase from the isolate produced 27·4% higher panose when compared to that of commercial enzyme. Moreover, the rate of secondary hydrolysis of panose is much lower in case of the isolate's enzyme.
Fungal isolate A. neoniger was characterized, and its gene conferring α-glucosidase activity was established for strong transglycosylation activity having higher panose yields.
To the best of our knowledge, this is the first report to establish a variant of α-glucosidase having strong transglycosylation activity from A. neoniger strain. We have demonstrated that this enzyme when used as processing aid could improve panose significantly, which is a potential prebiotic. Also, the sequence analysis established in our studies could provide pointers for directed evolution of this enzyme to further improve transglycosylation activity.
黑曲霉已被广泛用于分泌具有转糖苷活性的α-葡萄糖苷酶，该酶被用作异麦芽低聚糖合成的加工助剂。本研究集中于鉴定和表征一种非黑曲霉的分离株及其赋予强转糖苷活性的基因。
通过 ITS（内部转录间隔区）和β-微管蛋白分析，将土壤分离株鉴定为属于黑曲霉节的新黑曲霉。与其他曲霉属物种相比，负责α-葡萄糖苷酶合成的基因序列分析显示出显著的核苷酸变异。使用同源建模进行的分子对接研究表明，在 694 亚位点处存在苏氨酸而不是天冬酰胺，如黑曲霉α-葡萄糖苷酶的情况。该酶使用 DEAE Sepharose-CL6B 柱进行了几倍的纯化，并且在 SDS-PAGE 分析中，发现其分子量为 145 kDa。纯化酶的 MS/MS 分析验证了 694 位存在苏氨酸。从黑曲霉衍生的商业α-葡萄糖苷酶（Transglucosidase L 'Amano'）和分离株的α-葡萄糖苷酶在恒测试条件下比较转糖苷活性。与商业酶相比，分离株的α-葡萄糖苷酶产生的潘糖高出 27.4%。此外，分离株酶的潘糖的二次水解速率要低得多。
对真菌分离株 A. neoniger 进行了表征，并建立了赋予其α-葡萄糖苷酶活性的基因，该基因具有较强的转糖苷活性，具有更高的潘糖产率。
据我们所知，这是首次从 A. neoniger 菌株中建立具有强转糖苷活性的α-葡萄糖苷酶的变体的报道。我们已经证明，当用作加工助剂时，该酶可以显著提高潘糖产量，这是一种潜在的益生元。此外，我们的研究中的序列分析可以为该酶的定向进化提供指导，以进一步提高转糖苷活性。
