23-Chem-B4 Biochemical Engineering · December 2019
Nivaar worked solution (AI-drafted; not reviewed by a licensed engineer)
National Exam 16-Chem-B4, Biochemical Engineering — December 2019. 3 hours, Closed-Book Exam (any non-communicating Casio or Sharp calculator permitted). Per the exam notes, FIVE (5) questions constitute a complete paper and all five must be answered; most require a short-essay-format answer, and clarity/organization of the answer are explicitly marked.
Reference texts: Shuler & Kargi, Bioprocess Engineering: Basic Concepts, 2nd ed.; Bailey & Ollis, Biochemical Engineering Fundamentals, 2nd ed.; Madigan et al., Brock Biology of Microorganisms, 13th ed.
Question text not reproduced: the examination questions are © Engineers and Geoscientists BC. Open the official past paper (linked at the top of this page) to read the question, then follow the worked solution below.
Definition. Immobilization is the physical or chemical confinement of an enzyme (or a whole cell) to a defined region or support — a solid particle, a gel matrix, or a membrane-bound compartment — such that it retains catalytic activity while being spatially separated from the bulk liquid phase, so it can be reused, retained inside a reactor, and easily separated from the product stream.
(a) Adsorption. The enzyme attaches to a solid support (activated carbon, alumina, ion-exchange resin) via weak physical or ionic forces. Simple and inexpensive, and minimally disruptive to the enzyme's native structure, but reversible — the enzyme can desorb (leach) under shear, pH, or ionic-strength changes during operation.
(b) Covalent binding. The enzyme is chemically bonded to a functionalized support surface (e.g. via a carbodiimide or glutaraldehyde linkage). The bond is strong and essentially irreversible, giving excellent operational stability and no leaching, but the coupling chemistry can attack residues in or near the active site, reducing activity, and the support cannot be regenerated once the enzyme's usable life ends.
(c) Entrapment. The enzyme (or whole cell) is physically trapped inside the three-dimensional network of a gel or polymer matrix (calcium alginate, polyacrylamide, κ-carrageenan) formed around it. No chemical modification of the enzyme is needed (preserves native activity well) and it suits whole-cell immobilization particularly well, but substrate and product must diffuse through the matrix to reach the enzyme, adding an internal mass-transfer resistance.
(d) Encapsulation (membrane confinement). The enzyme is held in free solution behind a semi-permeable membrane (a microcapsule or hollow-fibre device) that lets small substrate/product molecules pass but retains the larger enzyme. This avoids any direct chemical or physical contact between enzyme and support, preserving activity well, but the membrane itself is an added mass-transfer barrier and can foul or rupture in service.
| Advantages | Limitations |
|---|---|
| Enzyme is reused across many batches/continuous operation, greatly lowering enzyme cost per unit product | Immobilization itself can reduce specific activity (steric/diffusional/chemical effects, depending on method) |
| Easy separation of enzyme from product stream (no downstream enzyme-removal step) | Internal/external mass-transfer resistance can markedly slow the immobilized system vs. free enzyme at the same loading |
| Often improved operational/thermal stability vs. free enzyme in solution | Added capital/process cost of the support, coupling chemistry, or membrane |
| Enables continuous packed-bed or fluidized-bed reactor operation | Support fouling, bead attrition, or membrane rupture over extended operation |
A classical industrial application is immobilized penicillin G acylase (also called penicillin amidase), used at large scale to hydrolyze penicillin G into 6-aminopenicillanic acid (6-APA), the core nucleus from which essentially all semisynthetic penicillins (ampicillin, amoxicillin, and others) are chemically derivatized. The enzyme is immobilized — commonly by covalent binding or entrapment onto a support — and typically run in stirred batch reactors fitted with a sieve that retains the enzyme particles (or in fixed beds); because the immobilized enzyme is recovered and reused over hundreds of reaction cycles rather than consumed in a single pass, enzymatic 6-APA production is economically viable at the scale the pharmaceutical industry requires, while also replacing the older chemical deacylation route (low-temperature silylation/PCl5 chemistry in chlorinated solvents) that immobilized-enzyme processing has largely displaced.
