Multifunctional alkalophilic \u03b1-amylase with diverse raw seaweed degrading activities

Xiaoqian Gu,Liping Fu,Jiang Li,Yuanyuan Gui,Aihong Pan,Qian Zhang

doi:10.1186/s13568-021-01300-x

Abstract

Uncultured microbes are an important resource for the discovery of novel enzymes. In this study, an amylase gene (amy2587) that codes a protein with 587 amino acids (Amy2587) was obtained from the metagenomic library of macroalgae-associated bacteria. Recombinant Amy2587 was expressed in Escherichia coli BL21 (DE3) and was found to simultaneously possess α-amylase, agarase, carrageenase, cellulase, and alginate lyase activities. Moreover, recombinant Amy2587 showed high thermostability and alkali resistance which are important characteristics for industrial application. To investigate the multifunctional mechanism of Amy2587, three motifs (functional domains) in the Amy2587 sequence were deleted to generate three truncated Amy2587 variants. The results showed that, even though these functional domains affected the multiple substrates degrading activity of Amy2587, they did not wholly explain its multifunctional characteristics. To apply the multifunctional activity of Amy2587, three seaweed substrates (Grateloupia filicina, Chondrus ocellatus, and Scagassum) were digested using Amy2587. After 2 h, 6 h, and 24 h of digestion, 121.2 ± 4 µg/ml, 134.8 ± 6 µg/ml, and 70.3 ± 3.5 µg/ml of reducing sugars were released, respectively. These results show that Amy2587 directly and effectively degraded three kinds of raw seaweeds. This finding provides a theoretical basis for one-step enzymatic digestion of raw seaweeds to obtain seaweed oligosaccharides.

Highlights

Introduction αAmylases hydrolyze their substrate by breaking the α-1,4-glycosidic bonds in starch molecules releasing glucose, maltose, and oligosaccharide chains as the products (Gupta et al 2003; Kandra 2003; Rajagopalan and Krishnan 2008)
Study showed a bifunctional enzyme from the hyperthermophilic bacterium Caldicellulosiruptor bescii, has two catalytic domain, which are responsible for different function, respectively (Ye et al 2012), but most of reported multifunctional enzymes only have one catalytic domain, which can play multifunction simultaneously (Huy et al 2013; Xue et al 2015; Yang et al 2015)
Substrate specificity of Amy2587 To determine the multifunctionality of Amy2587, we studied its substrate specificity. 100 μL purified Amy2587 (0.2 mg/mL) and 900 μL substrate (0.1% soluble starch, 0.1% agarose, 0.1% carrageen, 0.1% sodium cellulose, and 0.1% alginate) were incubated for 40 min at 50 °C, and the Amy2587 activity was measured by the 3,5-dinitrosalicylic acid (DNS) method (Chi et al 2014)

Summary

Introduction

Amylases hydrolyze their substrate by breaking the α-1,4-glycosidic bonds in starch molecules releasing glucose, maltose, and oligosaccharide chains as the products (Gupta et al 2003; Kandra 2003; Rajagopalan and Krishnan 2008). This enzymatic process is one of the earliest to be applied industrially for the production of sugars. To identify the possible functional domain responsible for the multifunctional property of Amy, three successive conserved domains were deleted one by one, even though the result showed that GH70 homologs might play an important role in the multifunctionality of Amy, but the exact relationship between catalytic domain and the multifunctionality of Amy need to be elucidated further (Liu et al 2016)

Methods

Results

Conclusion