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Graphical abstract Highlights • Novel R. ruber chaperones were identified by transcriptome analysis. • R. ruber chaperone GroEL2 was distinguished from conventional GroEL. • GroEL2 showed high self-thermal stability and protection effect on other proteins. • NHase-GroEL2 fusion chimera enhanced the activity of NHase by 63.6%. • It also enhanced thermal stability by 2.9× and acrylamide resistance by 1.1×. Abstract Rhodococcus ruber harboring intracellular nitrile hydratase (NHase) is widely used in large-scale acrylamide production. Transcriptome analyses of R. ruber under urea induction and heat shock revealed the novel chaperones GroEL2 and GroES. M. jannaschii chaperone rTHS, functional in organic solvent as reported in literature, was selected as control. In vitro experiments (70 °C/90 °C incubation) showed that GroEL2 from R. ruber was highly thermostable and can stabilize other proteins as well. GroEL2 was co-expressed with NHase in E. coli in three ways: (a) monocistronic expression with one T7 promoter, (b) bicistronic expression with double T7 promoters, and (c) fusion expression with one T7 promoter driving the NHase-GroEL2 chimera. Experimental results showed that the NHase-GroEL2 chimera was the most successful expression strategy. Maximal NHase activity was enhanced by 63.6% compared with the single NHase control. A stability assessment showed that the residual activity levels after heat shock and acrylamide (AM) immersion increased by 2.9× and 1.1×, respectively. For the fusion chimeras NHase-GroES and NHase-rTHS, their thermal stability also significantly enhanced by 1.6× and 64.0%, respectively; but their AM resistance both reduced (36.2% and 29.0%, respectively). In vivo heat inactivation curves further confirmed thermal stability enhancement of NHase by chaperone fusion, in which NHase-GroEL2 was the most stable one. Its inactivation constant k d was only ∼1/4 that of the single NHase. The K cat of the intracellular NHase-GroEL2 was also remarkably higher than that of the NHase control. [ABSTRACT FROM AUTHOR]

• An amyloid-like protein nanofilm coated around CPO@DSP can enhance its reusability. • Distribute CPO inside/outside of DSP simultaneously can increase its activity and reuse. • SP should keep accessible internal areas for the substrates to be efficiently transformed. • Over 90% of antibiotics in wastewater can be degraded in 30 min by CPO@PTL-CPO@DSP. Dendritic silica particles (DSP) with center-radial vertical pore were prepared for entrapping chloroperoxidase (CPO) to construct CPO@DSP. This enzymatic composite is very stable in the presence of heat and organic solvents, remaining 90.74% activity at 80 °C after 3 h while the free CPO can keep only 14.28% activity at the same condition. Moreover, it was found the pore size of DSP can influence the catalytic activity of the composite. The pore should keep the accessible internal areas for the substrates besides accommodating the enzyme itself. A novel amyloid-like protein nanofilm formed by phase transition lysozyme (PTL) was coated around the surface of CPO@DSP (expressed as PTL-CPO@DSP), and then the PTL was cross-linked with another layer of CPO through glutaraldehyde to construct CPO@PTL-CPO@DSP. In this way, the CPO was widely distributed both in mesoporous channel and on the surface of DSP simultaneously. Both the enzyme loading and the reuse of CPO@PTL-CPO@DSP was greatly improved compared with CPO@DSP. After 20 cycle of use, CPO@PTL-CPO@DSP can maintain over 80% of its original activity. CPO@PTL-CPO@DSP were very efficient when applied in the degradation of the antibiotic levofloxacin and rifaximin in wastewater. Over 88% of both antibiotics can be degraded within 30 min at concentration of 20 μg·mL−1 in wasterwater. [ABSTRACT FROM AUTHOR]