H83BCE-E1

The University of Nottingham

DEPARTMENT OF CHEMICAL AND ENVIRONMENTAL ENGINEERING

A LEVEL 3 MODULE, SEMESTER 1, 2016-2017

BIOCHEMICAL ENGINEERING

Time allowed TWO Hours

Candidates may complete the front cover of their answer book and sign their desk card
but must NOT write anything else until the start of the examination period is announced

Answer ONE Question (of TWO) from Section A

and ONE Question (of TWO) from Section B

ANSWER EACH SECTION IN A SEPARATE BOOKLET

Only silent, self-contained calculators with a Single-Line Display or Dual-Line Display

are permitted in the examination.

Dictionaries are not allowed with one exception. Those whose first language is not
English may use a standard translation dictionary to translate between that language
and English provided that neither language is the subject of this examination. Subject
specific translation dictionaries are not permitted.

No electronic devices capable of storing and retrieving text, including electronic

dictionaries, may be used.

DO NOT turn examination paper over until instructed to do so

In this examination candidates are required to answer ONE out of TWO

questions Section A and ONE out of TWO questions in Section B. If a
candidate answers more than the required number of questions, all
questions will be marked and the highest marks will be used in the final
examination mark.

ADDITIONAL MATERIAL: Graph paper and semi-logarithmic graph paper

INFORMATION FOR INVIGILATORS:

All questions to be answered in a separate booklet.

Question papers should be collected in at the end of the exam – do not allow
candidates to take copies from the exam room.
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SECTION A

You must answer ONE question from the TWO in this Section
Use a separate answer book

1. Answer all parts of this question.

Fine chemicals and pharmaceutical precursors have been the major

uptake areas for biocatalysis because of the high chemical- and stereo-
selectivities that can be obtained from enzymatic systems.

Degussa (now Evonik) makes the anti-HIV drug precursor L-tert-leucine

via the following coupled process:

(1)

(2)

(a) The enzymes Leucine dehydrogenase (1) (EC 1.4.1.9) and Formate
dehydrogenase (2) (EC 1.2.1.2) are both oxidases, denoted by the
‘1’ in the first part of the EC number. If you were to use the website
BRENDA to look up the EC numbers, describe briefly at least four
useful sorts of information you might find out about these enzymes.
[4 marks]

(b) NADH is known as a cofactor. Describe what a cofactor is and why it

is necessary for reactions such as those carried out by oxidases.
What is cofactor-recycling and why is it important?
[6 marks]

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(c) Your technical team have provided experimental data for the two
enzymes leucine dehydrogenase (1) and formate dehydrogenase
(2), using the same concentration of enzyme for each assay.

Ketoacid concentration Reaction

(mM) velocity
(mmol/s) (1)
0.00 0.0
2.31 3.11
4.31 5.32
8.68 9.08
16.86 13.72
34.13 18.91

Formic acid Reaction

concentration (mM) velocity
(mmol/s) (2)
0.00 0.0
0.31 9.12
0.63 9.54
1.23 9.76
2.49 9.88
4.95 9.94

The Michaelis-Menten equation has the form:

V = (vmax[S])/(Km + [S])

i. Calculate Km and vmax for both enzymes, showing your working,

and using an appropriate graph to derive these values.
[20 marks]

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ii. From the combination of Km and vmax values you have derived
from part (c)(i) for enzymes (1) and (2):

 state which enzyme is rate limiting under the assay

conditions and why.
 comment on one way by which you might improve enzyme
activity, whilst maintaining the current conditions.
 suggest an alternative change you could make to the
reaction conditions to improve the apparent rate and why
this would work.
[6 marks]

(d) For the process to be economically viable at 50,000 L scale with

respect to separation, a concentration of 40 g/L of the product of
enzyme (1), L-tert-leucine, needs to be reached. The molecular
weight of this derivative is 131.2 g/mol. Calculate, showing your
working, how many days the reaction would take to reach the target
product concentration. Assume that saturation conditions can be
reached for the starting material, and the same concentration of
enzyme is used as for the assay conditions above.
[10 marks]

(e) NADH costs around £200/kg, and formic acid around £400/metric
tonne. This price differential makes the coupled recycling process
look much more attractive than a single enzyme process, but is not
the whole story. Based on the information you have, comment on
the possible additional costs and considerations, including safety and
environmental implications, that the coupled process might present.
[4 marks]

[Total: 50 marks]

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2. Answer all parts of this question.

(a) You find in the patent literature a competing process to the Degussa
production of L-tert-leucine which has been developed, also using a
coupled process but avoiding the necessity for NADH recycling.

(3)

The coupled process is run in a whole cell by using a plasmid with

both the branched-chain aminotransferase (3) (BCAT) and aspartate
aminotransferase (4) (AAT) on it.

i. Describe, illustrating your answer with diagrams, what a

plasmid is, and how it differs from chromosomal DNA. Include
in your answer the advantages and disadvantages of using a
plasmid for protein expression.
[8 marks]

ii. Describe briefly with diagrams the ‘central dogma’ and

comment on how this process relates DNA to protein
production.
[8 marks]

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iii. The process uses the antibiotic kanamycin in the growth

medium for the cells. Explain the term “selection pressure” in
this context.

Comment on the potential safety and environmental issues of

using antibiotics to induce selection pressure.
[6 marks]

(b) BCAT (3) and AAT (4) essentially carry out the same reaction but
on different substrates. Explain how such selectivity can be
achieved in enzymatic catalysts, illustrating your answer with
diagrams and highlighting specific interactions you might expect to
be different.
[5 marks]

(c) BCAT (3) has a pH optimum recorded in the BRENDA database of

8.5. In contrast, AAT (4) has a pH optimum reported as 7.5. Explain
the importance of pH in enzyme catalysis, illustrating your answer
with graphs as appropriate. To improve the reaction process
containing both BCAT (3) and AAT (4), suggest two approaches you
could use to avoid pH conflicts.
[6 marks]

(d) There is interest in running this reaction as a flow process, using

immobilised enzymes in a column.

i. Describe at least one physical and one chemical approach to

immobilisation, illustrating your answer with diagrams. List two
key advantages of immobilisation and two disadvantages.
[8 marks]

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ii. Given a vmax value for BCAT for the ketoacid substrate of
50 mmols-1, calculate the number of hours it would take to
generate 100 kg of L-tert-leucine (MW 131.2 g/mol) under
saturating conditions of starting material, showing your
working.
[6 marks]

iii. What advantage might a flow-based system have over a

standard batch reactor? Illustrate your answer with diagrams of
each system.
[3 marks]
[Total: 50 marks]

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SECTION B

You must answer ONE question from the TWO in this Section
Use a new answer book

3. Answer all parts of this question.

Cupriviadus necator is a Gram negative soil bacterium capable of growing

on CO2. You have expressed the efe (ethylene forming enzyme) gene from
Pseudomonas in C. necator. As proof of principle and to establish if you
can make ethylene in C. necator you are initially growing it in FGN
minimal media with fructose and glycerol, where the following data were
recorded:

Time (hours) OD after growth on:

Fructose Glycerol
0 0.08 0.07
2 0.08 0.072
4 0.08 0.078
6 0.145 0.08
8 0.146 0.09
10 0.15 0.12
12 0.40 0.15
14 0.60 0.2
16 1.2 0.34
18 1.7 0.6
20 1.8 0.94
22 1.83 0.96
24 1.83 0.96

OD means optical density; this is a quick way of measuring the cell

concentration in the culture. It involves measuring light scattering using a
spectrophotometer. One OD unit is equal to a cell dry weight of 0.5 g/L.

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In the culture grown with fructose, the fructose concentration at time zero
was 4.0 g/L, and the final fructose concentration was 0.15 g/L. In the
culture grown with glycerol the glycerol concentration at time zero was
also 4.0 g/L and the final concentration was 0.30 g/L. The molecular
formula of fructose is C6H12O6 and its molecular weight is 180 g/mol.
Glycerol is C3H8O3 its molecular weight is 92.09 g/mol. The molecular
weight of ethylene (C2H4) is 28.05 g/mol and it has a density of
1.18 kg/m3.

Answer all of the following questions:

(a) For each culture, calculate the specific growth rate (). Show your
working and explain the derivation of each calculation. Highlight
where  should be calculated from on your graph.
[20 marks]

(b) For each culture, illustrate the five phases of growth that occurred
by labelling a plot of cell number versus time at key points. Include
a description of what happens to the cells at each phase.
[10 marks]

(c) Calculate the growth-yield on:

i. Fructose.

ii. Glycerol.
[10 marks]

(d) Having generated an initial yield of 140 nmols/OD600/mL of

ethylene in C. necator you would now like to increase productivity.
Suggest two approaches that you could use to improve your yield.
Include in your answer details of each approach.
[10 marks]

[Total: 50 marks]

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4. Answer all parts of this question.

(a) Cells are essential components of living systems and can be

considered the basic units of life. There are several types of cell.
Explain the key differences/similarities between prokaryotic and
eukaryotic cells illustrating your answer with clear sketches.
[12 marks]

(b) Describe the different membranes found in different types of

prokaryotic and eukaryotic cells and highlight the key components
of cellular membranes.
[7 marks]

(c) Phospholipids are one of the key components of membranes. Draw a

sketch of a phospholipid, highlighting the key structural features.
[4 marks]

(d) Describe the role that membranes play in the cell, highlighting the
mechanisms of transport for different types of molecules.
[7 marks]

(e) A new start-up company has approached you with a significant

problem during the microbial production of n-hexane. Accumulation
of the n-hexane is negatively impacting on cultivation of their host
strain, with a 104-fold decrease in colony forming units. This is now
limiting production levels. Suggest how tolerance engineering could
improve productivity, giving clear examples of different types of
changes that could be made to increase the tolerance of the strain
and how they work.
[20 marks]

[Total: 50 marks]

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