Abstract
Development of green hydrogen production is one of sustainable approaches to replace fossil fuels. An in vitro cell-free synthetic enzymatic pathway (SyPaB) for hydrogen production is an alternative to less effective microbial fermentation. To increase the production of xylulose 5-phosphate (Xu5-P); the rate-limiting step's product in the ATP-free SyPaB for biohydrogen production powered by xylooligomers, immobilization of cellodextrin phosphorylase, phosphopentomutase, and ribose-5-phosphate isomerase, on silica nano-particles (SiNPs) was evaluated. Immobilization of 1 Unit/mL of each enzyme on 15 to 20 mg of amorphous SiNPs for 120 min, at 4 °C gave the highest immobilization yield. Immobilized enzymes along with other 10 free enzymes gave maximum hydrogen generation rate of 3.618 mmole/L.hr after 0.8 h which 1.3 fold higher than using 13 free enzymes at the same conditions. The study reveals that immobilized enzymes efficiently produce Xylulose 5-phosphate, and its application in effective biohydrogen production, indicating potential for future enzyme immobilization.
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