Part:BBa_K5487001

Design Notes Enhanced Soluble Protein Yield:

Rationale: Industrial applications require high levels of soluble enzyme for cost-effective production. Approach: Engineered Z1-PETase by introducing mutations that improve the protein's folding and reduce aggregation, thereby increasing the soluble protein yield. Low-Temperature Activity:

Rationale: Enzymes that maintain activity at lower temperatures are desirable for energy efficiency in industrial processes. Approach: Selected mutations that enhance the enzyme's flexibility and activity at temperatures below the optimal range while not compromising its stability. Thermal Stability (TmD Value):

Rationale: Higher thermal stability ensures enzyme longevity and activity under various conditions, including higher temperatures. Approach: Incorporated additional disulfide bonds and stabilizing mutations to enhance the enzyme's structural integrity without hindering its catalytic activity. Specific Activity:

Rationale: High catalytic efficiency is crucial for the enzyme's effectiveness in PET degradation. Approach: Carefully selected mutations to maintain or improve the enzyme's active site functionality, ensuring high substrate turnover rates. Structural Interpretation:

Rationale: Understanding the enzyme's structure is essential for guiding mutations that will enhance stability and activity. Approach: Utilized structural bioinformatics and X-ray crystallography to model and analyze the effects of mutations on the enzyme's structure. Mutagenesis Strategy:

Rationale: Systematic and targeted mutagenesis is necessary to achieve desired enhancements without introducing detrimental changes. Approach: Employed site-directed mutagenesis and combinatorial engineering strategies to introduce multiple beneficial mutations. Protein Expression and Purification:

Rationale: The enzyme must be easily produced and purified for practical application. Approach: Designed the enzyme for expression in commonly used host systems like E. coli and included purification tags for straightforward protein isolation. Enzyme Kinetics and Mechanism:

Rationale: A thorough understanding of the enzyme's kinetics and mechanism of action is necessary for optimizing its performance. Approach: Conducted detailed enzyme kinetics studies and structural analyses to inform the design and ensure the mutations enhance the enzyme's function. Scalability and Industrial Viability:

Rationale: The enzyme must be suitable for scale-up and industrial implementation. Approach: Considered the enzyme's performance in a bioreactor setting and its ability to function in continuous processing conditions. Environmental Impact:

Rationale: The engineered enzyme should contribute positively to environmental sustainability. Approach: Focused on enhancing the enzyme's ability to depolymerize PET, a major contributor to plastic pollution, into monomers that can be recycled.

Source Original Organism: Ideonella sakaiensis

Genomic Sequence: The gene encoding the Z1-PETase enzyme is derived from the Ideonella sakaiensis genome, where the native PETase enzyme (IsPETase) was identified. This native enzyme exhibits high PET hydrolysis activity but has limitations in stability, which has been addressed through engineering efforts.

Engineering Process: Z1-PETase is a result of a targeted protein engineering approach that involved site-directed mutagenesis and multiple rounds of selection to enhance the enzyme's soluble protein yield, low-temperature activity, and overall durability. The specific mutations introduced in the engineering process are detailed in the research article "Three-directional engineering of IsPETase with enhanced protein yield, activity, and durability," published in the Journal of Hazardous Materials.

Expression System: The engineered Z1-PETase gene is typically expressed in a heterologous host, such as Escherichia coli, which is a common expression system for recombinant proteins. The gene is cloned into an expression vector, which is then introduced into the host cells for protein production.

References Research Article Reference: For detailed information on the source, mutagenesis, and characterization of Z1-PETase, refer to the research article mentioned above, which provides insights into the enzyme's structural and functional properties.