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Investigations of heat treatment to improve the isolation of intracellular enzymes

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Abstract

Heating, an old fashioned method for enzyme purification, was re-investigated as a continuous process in a jacketed tube heat exchanger. Biological, physical and process-engineering parameters were studied with regard to heating during the isolation of alcohol-dehydrogenase from cell homogenates of baker's yeast. The heat treatment served to coagulate cell debris and protein, thereby increasing not only the specific activity of the target enzyme in solution, but at the same time the particle size of the solids. The most important parameters of the process are the selected feed temperature, pH-value, volumetric flow rate and cell concentration. At optimal conditions a clear supernatant after centrifugation, (RCF 7000), is achieved containing only 50–40% of the soluble protein and 85–75% enzyme activity, resulting in a two-fold enrichment compared to the unheated crude extract. The experiments demonstrate that a continuous heating process can be applied for pre-purification and conditioning. Enzymes with better heat stability will give even higher yields.

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Abbreviations

A 0 U/cm3 or %:

initial activity (before heating)

A HE m2 :

heating area of heat exchanger

BM %:

biomass concentration

c/c 0 :

dimensionless marker concentration

c H kJ/kg K:

specific heat capacity of heating fluid

c S kJ/kg K:

specific heat capacity of feed

D 1/s:

shear rate

EF :

enrichment factor

d m:

inner tube diameter

k (T) %/s:

inactivation rate

k HE kJ/m2h K:

heat transfer coefficient

l m:

tube length

Q kJ/h:

heat rate

R kJ/mol K:

universal gas constant=8.31·10−3

r m:

inner tube radius

RA %:

remaining activity (after heating)

RCF g:

relative centrifugal force

RP %:

remaining protein (after heating)

Re:

Reynold's number

t G s :

total residence time

t (H) s :

heating time

t C s :

cooling time

T °C or K:

temperature

T m °C or K:

mean logarithmic temperature

V H dm3/h:

volumetric flow rate of heating fluid

V HE m3 :

volume of heat exchanger

V (S) dm3/h:

volumetric flow rate of feed

w m/s:

velocity of feed

η mPas:

apparent viscosity

ϱ H kg/dm3 :

density of heating fluid

ϱ S kg/dm3 :

density of feed

Θ :

dimensionless time (Θ=V·t/V HE )

ΔT H °C or K:

temperature difference of heating fluid

ΔT S °C or K:

temperature difference of feed

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Authors and Affiliations

  1. Institut für Enzymtechnologie der Universität Düsseldorf in der KFA Jülich, P.O. Box 2050, D-5170, Jülich, FRG

    J. Schnell &  M. R. Kula

Authors
  1. J. Schnell
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  2. M. R. Kula
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Schnell, J., Kula, M.R. Investigations of heat treatment to improve the isolation of intracellular enzymes. Bioprocess Engineering 4, 129–138 (1989). https://doi.org/10.1007/BF00369762

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