Expression and production of thermophilic alginate lyases in Bacillus and direct application of culture supernatant for seaweed saccharification

Nanna Rhein-Knudsen,Chengran Guan,Geir Mathiesen,Svein Jarle Horn

doi:10.1016/j.algal.2021.102512

Nanna Rhein-Knudsen, Chengran Guan + Show 2 more

Open Access

https://doi.org/10.1016/j.algal.2021.102512

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Abstract

Brown seaweeds are rich in carbohydrates and may be used as a source of fermentable sugars. Saccharification of the seaweed biomass can be carried out enzymatically by a combination of cellulases and alginate lyases. In this study, thermotolerant exo- and endo alginate lyases were cloned and expressed in Bacillus subtilis. The lyases were secreted to the culture supernatant and used directly together with a commercial cellulase preparation to saccharify Saccharina latissima biomass. The results showed that the strategy of using the culture supernatants directly as a source of alginate lyases worked very well, releasing glucose, mannitol, and uronic acids. The ratio between the exo- and endo-acting alginate lyases proved to be very important for saccharification yield, and under optimal reaction conditions the use of culture supernatants containing alginate lyases improved final glucose concentration by 73%, when compared to only applying cellulases. This direct use of culture supernatants as a source of alginate lyases shows that enzyme purification steps are not needed, saving seaweed processing costs and points to the possibility of a relatively simple on-site enzyme production for seaweed biorefining.

Highlights

Seaweeds are marine plant-like multicellular organ isms that predominantly grow in the ocean
The optimal ratio of the endo- and exo-acting alginate lyases (PL7/ PL17 ratio) during seaweed saccharification was determined by saccharification studies using 10% w/w dry matter (DM) seaweed with a constant concentration of 10 mg Cellic® CTec2 per g of dry seaweed and varying ratios of the two alginate lyses (0.05 mg alginate lyases in total per g of dry seaweed)
B. subtilis harboring AMOR-PL7, AMOR-PL17 or the empty plasmid pBE were cultivated on Terrific Broth (TB) medium in shake flaks

Summary

Introduction

Seaweeds (macroalgae) are marine plant-like multicellular organ isms that predominantly grow in the ocean. The secreted alginate lyases were used directly as culture su pernatants in combination with the commercial cellulase mixture Cel lic® CTec for enzymatic saccharification of the brown seaweed S. latissima at 55 ◦C This proof-of-concept study demonstrates that alginate lyases can be successfully produced in a Bacillus expression system and used directly as culture supernatants for saccharification of brown seaweed biomass without the need of protein purification. This points to the possibility of relatively simple on-site production of algi nate lyases for use in seaweed biorefining processes

Bacterial strains and growth conditions

Cloning strategy and plasmid construction

Expression and activity

Monosaccharide analysis

Statistics

Growth and expression in Bacillus subtilis