Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc

Abstract

Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N′-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.

High-Level Extracellular Expression of a New β-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc

“…This is slightly less than Fu et al's (2014) 92.6% and Wang et al's (2018) 95% conversion ratio. But the reaction time greatly reduced from more than 24 to 3.5 h by using enzymes with higher activities, which is also far lower than our previously reported 12 h (Song et al, 2020).…”

“…In our research, BlNagZ has the ability to hydrolyze chitin and produce GlcNAc with very low efficiency ( Figure 5D). Combining with chitinase ChiA from B. licheniformis (Song et al, 2020), the yield rate of GlcNAc increased to 89.2%. This is slightly less than Fu et al's (2014) 92.6% and Wang et al's (2018) 95% conversion ratio.…”

“…To increase the output of GlcNAc, 30% colloidal chitin was firstly hydrolyzed with chitinase ChiA (Song et al, 2020) for 3 h, and then hydrolyzed with different amount of BlNagZ for 30 min. The results showed that BlNagZ can effectively hydrolyze N, N -Diacetylchitobiose produced by ChiA into GlcNAc, and 2.6 U/ml of BlNagZ could convert the hydrolysis product of ChiA into GlcNAc completely.…”

“…The complete degradation of chitin to GlcNAc requires the synergistic action of chitinases and β-N-Acetylglucosaminidases (NAGases), that is, the colloidal chitin was hydrolyzed into chitin-oligosaccharides by chitinases, and further hydrolyzed into GlcNAc by NAGases (Du et al, 2019). Chitinase, such as ChiA, had achieved high-efficiency expression in our lab (Song et al, 2020). Therefore, it is essential to develop new NAGases with low cost for industrial use.…”

“…The recombinant BlNagZ showed the maximum activity of 13.05 U/mg at pH 5.5 and 60 • C. The expression level was improved by 4.25 times after optimizing the fermentation conditions. Combined with the reported chitinase 1 http://www.cazy.org/ (Song et al, 2020), recombinant BlNagZ can hydrolyze colloidal chitin to obtain GlcNAc with 89.2% conversion ratio within 3.5 h.…”