Cloning of cellulose microfibril swelling (cms) gene from Bacillus sp. AY8 to reduce the recalcitrance of cellulose
cellulose recalcitrance H-bonds Cellulose swelling Bacillus sp Cellulose microfibril swellase gene solid state NMR saccharification;
- 원문 URL
The hydrogen (H) bond in cellulose microfibrils are recalcitrance for cellulase. The discovery of cellulose microfibril swelling enzyme from bacteria can provide advantages over plant and fungal proteins in terms of their production and relative ease of protein engineering. In this study, a Gram positive and rod shaped bacterial strain was isolated from pulp sludge of paper mill (Moorim Paper, Jinju). The bacterium showed the highest phylogenetic relationships with Bacillus sp. that was nomenclatured to Bacillus sp. AY8. Extracellular enzyme of the strain AY8 could swell filter paper without showing detectable cellulase activities. A colorimetric method was developed to characterize the cellulose swelling degree since there is no suitable method to characterize the cellulose swelling enzyme yet. Here, cotton cellulose was swollen after treating with 2 to 10% NaOH solution at -20 °C for 60 min. The amount (μM) of Congo red (CR) adsorption was increased gradually in 2 to 8% NaOH treated cotton and decreased over 8% NaOH treated cotton when dyeing with 100 µM CR solution. Therefore, swelling degree can be determined by the amount of CR adsorption. The cellulose swelling enzyme of the Bacillus sp. AY8 was purified using DEAE-Sephadex A50 column chromatography using 1M NaCl gradient. The enzyme treated cotton macrofirbils were amorphic and wide range reduced from 320~425 nm to 50~70 nm which was observed using field emission scanning electron microscopy (FE-SEM) and atomic force microcopy (AFM). In Fourier transform infrared spectroscopy (FT-IR) spectral analysis, the peak intensity around 3000–4000 cm-1 was reduced in the enzyme treated cotton indicating disruption of the intra-chain and inter-chain hydrogen (H) bonds. The enzyme treated cotton crystalline index (CrI) was reduced by 2.07% compared with that of untreated one that observed using X-ray diffraction (XRD) analyses indicating the breaking of inter-sheet H-bonds. In solid state NMR analyses, new resonances were appeared in the C1 to C6 ranges and were shifted to the upfield for the signal assigned to C1, C2, C3, C4, and C6 and down field for C5 in the enzyme treated cotton indicating the breaking of inter-sheet H-bonds. Therefore, the enzyme was nomenclatured to cellulose microfibril swellase (Cms). The Cms treated cotton cellulose adsorbs more CR red compared to untreated one. Thus, quantitative characterization of Cms activity was developed based on CR colorimetric method. The Cms enzyme showed maximum activity at 37 °C temperature and pH 7. The activity of Cms enzyme was increased almost two fold in presence of Mn2+, Cu2+, and Zn2+ ions but slightly increased in presence of Ca2+ and Mg2+ ions. The Cms treated avicel yielded 212 mg/g more reducing sugar than control avicel after enzymatic saccharification. A cellulose microfibril swelling gene was cloned from the isolated Bacillus sp. AY8. The gene was targeted based on the molecular mass of the purified Cms enzyme and protein database analysis of the nearest phylogeny of Bacillus sp. AY8. The targeted gene sequence was deduced by using primer sets of conserved region of the gene. Later, the complete open reading frame (ORF) of the gene was deduced by chromosomal walking. The cloned cms gene has ATG start codon, TGA stop codon, 679 bp which encodes 225 amino acids. The product of cms gene has NlpC/P60 conserved region and has 98% homology with the NlpC/P60 family protein of Burkoholderia lata and Burkholderia vietnamiensis strains. The Cms enzyme conserved region did not match with the expansins and swollenin proteins.To date, this study first reports a cellulose microfibril swellase enzyme which supposes to be applicable as a potential additive in the production of fermentable sugars from cellulose.