Cheng Tao,Liu Hui,Zou Huibin,Chen Ningning,Shi Mengxun,Xie Congxia,Zhao Guang,Xian Mo
Background As an important bulk chemical for synthetic rubber, isoprene can be biosynthesized by robust microbes. But rational engineering and optimization are often demanded to make the in vivo process feasible due to the complexities of cellular metabolism. Alternative synthetic biochemistry strategies are in fast development to produce isoprene or isoprenoids in vitro. Results This study set up an in vitro enzyme synthetic chemistry process using 5 enzymes in the lower mevalonate pathway to produce isoprene from mevalonate. We found the level and ratio of individual enzymes would significantly affect the efficiency of the whole system. The optimized process using 10 balanced enzyme unites (5.0 µM of MVK, PMK, MVD; 10.0 µM of IDI, 80.0 µM of ISPS) could produce 6323.5 µmol/L/h (430 mg/L/h) isoprene in a 2 ml in vitro system. In a scale up process (50 ml) only using 1 balanced enzyme unit (0.5 µM of MVK, PMK, MVD; 1.0 µM of IDI, 8.0 µM of ISPS), the system could produce 302 mg/L isoprene in 40 h, which showed higher production rate and longer reaction phase with comparison of the in vivo control. Conclusions By optimizing the enzyme levels of lower MVA pathway, synthetic biochemistry methods could be set up for the enzymatic production of isoprene or isoprenoids from mevalonate.
Millions of tons of shrimp and crab waste, rich in chitin, are produced annually worldwide. To efficiently utilize this resource and address the contamination caused by traditional chitin treatment, a high-chitinase-producing strain, GXMU-J23.1, was isolated from the marine environment and identified as Bacillus paralicheniformis . Genome sequencing revealed several chitinolytic enzymes, such as chitinase, chitin deacetylase, and polysaccharide monooxygenases. Under optimal conditions, the chitinase activity increased 9.1-fold to 356.32 ± 1.21 U/mL. The purified chitinase Chi23 exhibited optimal activity at 50 °C and pH 5.0, degrading various chitin substrates. Metal ions such as Ca 2+ and reagents such as EDTA increased the activity, whereas Fe 2+ and Zn 2+ inhibited the activity. Chi23, an endochitinase, converts chitin into chitotriose and diacetylchitobiose. Based on the structural reconstruction and molecular docking of Chi23, the potential enzyme–substrate mode of action was elucidated, which will support subsequent enzyme modification and in-depth development of enzyme systems assisting in chitin degradation.
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