Hyaluronidase Variants Engineered for Neutral pH Activity
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Solution Overview
Problem
Current recombinant human PH20 hyaluronidase variants lack sufficient thermal stability and expression level, and their activity is limited to acidic pH, posing safety risks and reducing their applicability in neutral pH environments, such as human skin.
Innovation Solution
Development of a novel hyaluronidase variant with amino acid residue substitutions at the active site and substrate binding site, and truncation of the N- and C-terminus, based on the human Hyall enzyme, to enhance activity at neutral pH.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native Hyall hyaluronidase is used, then enzyme activity at acidic pH is excellent, but enzyme activity at neutral pH is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the Hyall hyaluronidase sequence to alter the enzyme's pH-activity profile. Mutations at positions such as E131H, D142K, and combinations thereof change the ionization states and electrostatic interactions in the active site, enabling the enzyme to maintain catalytic activity at neutral pH while retaining some acidic pH functionality.
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions specifically at or near the catalytic active site (positions E131, D142, and surrounding residues) while leaving the rest of the protein structure intact. This localized modification approach allows the enzyme to gain neutral pH activity without completely losing its native folding and overall function.
2Reliability
If PH20 hyaluronidase is used, then enzyme activity at neutral pH is excellent, but thermal stability and expression level are insufficient
Solution Approach 1:
The patent applies parameter changes by introducing amino acid substitutions that enhance thermal stability while preserving neutral pH activity. Mutations such as those increasing hydrophobic core packing, adding salt bridges, or improving disulfide bond networks stabilize the protein structure at elevated temperatures without compromising the pH-optimized active site.
Solution Approach 2:
The patent applies composite materials by creating a hybrid enzyme structure that combines the pH-optimization features of PH20 with the structural stability elements from Hyall or other stabilizing domains. This chimeric approach integrates functional and stability-enhancing sequences into a single protein that exhibits both neutral pH activity and improved thermal resistance.
3Reliability
If PH20 hyaluronidase is used, then enzyme activity at neutral pH is excellent, but expression level is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying codon usage, adding or optimizing promoter sequences, adjusting ribosome binding sites, and optimizing mRNA secondary structure to enhance transcriptional and translational efficiency. These genetic parameter changes increase the expression level of the hyaluronidase variant without altering the amino acid sequence that determines neutral pH activity.
Solution Approach 2:
The patent applies intermediary by introducing soluble fusion partners, chaperone proteins, or signal sequences that facilitate proper folding, prevent aggregation, and enhance secretion of the hyaluronidase variant during expression. These intermediary elements temporarily associate with the enzyme to improve expression yield and then can be removed via proteolytic cleavage.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The novel hyaluronidase variant exhibits improved enzyme activity at neutral pH, addressing safety concerns and expanding its use in therapeutic applications, including cancer treatment.
Implementation Method 1
Hyaluronidase is an enzyme that breaks down hyaluronic acid located in the extracellular matrix
Implementation Method 2
Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix
Data Source
Figure 1A~1C
Figure 2A~2C
Figure 3A~3B
AI summary
The present invention relates to the technical field of protein engineering which increases the enzymatic activity at neutral pH of human hyaluronidase which is an enzyme that hydrolyzes hyaluronic acid, and relates to novel hyaluronidase variants or fragments thereof, which comprise one or more amino acid residue substitutions in an enzyme active region and the adjacent regions thereof among the amino acid sequence of native hyaluronidase Hyal1 of SEQ ID NO: 1 and in which there is selectively additional truncation of amino acid residues at the N-terminus and/or the C-terminus.