Recombinant Pepsin Composition for High Activity and Stability

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Solution Overview

Problem

Existing methods for producing pepsin compositions face challenges in achieving high specific activity and stability, particularly in environments outside the acidic conditions of the stomach, limiting their applications in food processing, pharmaceuticals, and biotechnology.

Innovation Solution

A method involving the expression of recombinant pepsinogen in microorganisms, followed by culturing, pH adjustment to activate pepsin, and stabilization at a pH between 5.4 and 7.0, results in a high-activity stable pepsin composition with specific activities exceeding 20,000 FCC units/mg total protein.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If pepsin is extracted from pig stomach under acidic conditions, then the extracted form is primarily active pepsin, but the enzyme lacks stability in non-acidic environments

Engineering Contradiction:

Improveenzyme activityVSAvoidpepsin stability

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent uses pepsinogen as an intermediary substance that can be produced recombinantly with high stability, then converted to active pepsin only when needed through pH reduction. This mediator approach allows the enzyme to maintain stability during production and storage while achieving high activity during application.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent applies parameter changes by controlling pH levels to switch between pepsinogen (stable, inactive form at neutral pH) and pepsin (active form at low pH). This parameter control resolves the contradiction by allowing the enzyme to exhibit stability under one condition and high activity under another.

Inventive Principle:

Principle #35Parameter changes

2Ease of manufacture

If pepsin is produced through traditional extraction methods, then the process is straightforward, but the specific activity remains limited

Engineering Contradiction:

Improveproduction simplicityVSAvoidspecific activity

Core Design Contradiction:

Ease of manufactureVSManufacturing precision

Solution Approach 1:

The patent employs self-service by using the microorganism's own cellular machinery to produce and secrete recombinant pepsinogen into the growth medium. This eliminates the need for complex extraction and purification processes from animal stomachs, simultaneously simplifying manufacture and enhancing specific activity through recombinant expression optimization.

Inventive Principle:

Principle #25Self-service

3Reliability

If pepsinogen is activated at low pH to form pepsin, then enzyme activity increases, but the composition becomes unstable outside acidic conditions

Engineering Contradiction:

Improveenzyme activityVSAvoidcomposition stability

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent applies preliminary action by producing and purifying pepsinogen in its stable, inactive form under neutral pH conditions during the entire manufacturing process. The activation to active pepsin is performed only as a final step immediately before application, ensuring the composition remains stable during production, storage, and transport while achieving high activity when needed.

Inventive Principle:

Principle #10Preliminary action

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 method produces a stable pepsin composition with enhanced specific activity, maintaining effectiveness in a broader pH range, suitable for various industrial and pharmaceutical applications.

Implementation Method 1

providing a microorganism that expresses a recombinant pepsinogen; culturing the microorganism under conditions in which the recombinant pepsinogen polypeptide is expressed and secreted by the microorganism into a growth media

Methodology Applied

Scientific EffectRecombinant protein expression and secretion: Enzyme

Implementation Method 2

Under non-acidic pH condition, pepsinogen is activimmature, due to the presence of the propeptide. Under acidic pH conditions, pepsinogen can unfold and cleave itself to create the mature form of the enzyme which is pepsin

Methodology Applied

Scientific EffectpH-induced conformational change and autocleavage: Enzyme