MODELADO DE PROCESOS BIOTECNOLÓGICOS

PRÁCTICA

PRODUCCIÓN DE ENZIMAS DE RELEVANCIA INDUSTRIAL: DESDE LA NATURALEZA

HASTA LA INDUSTRIA

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 Strategy to obtain microorganisms with ability to
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produce lipases

13 streak isolation

Consortia inoculation in
plate and flask cultures

Sampling sites:
Thermal springs in province of
Ourense: Cultivation of isolates
As Burgas
A Chavasqueira
O Tinteiro
Lobios
 Part 1: Media Preparation
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-Prepare 200 mL of medium for SOLID CULTURES containing:

- 20 g/L agar
- 10 g/L casein peptone
- 5 g/L yeast extract
- 10 g/L NaCl
- 10 g/L tributyrin

Dissolve the compounds in distilled water, and put in a 500mL-schott bottle, emulsify with an Ultraturrax
and autoclave at 121ºC for 20 min, with blue tips for micropipettes.

-Prepare 1000 mL of medium for LIQUID CULTURES containing:

- 10 g/L casein peptone
- 5 g/L yeast extract
- 10 g/L NaCl

Adjust pH to 7.5, put in three 500 mL-schott bottle and autoclave at 121ºC for 20 min, together
with three 100 mL-cylinder and three Erlenmeyer flasks with the corresponding cellulose stoppers
 Part 1: Plate Cultivation and Flask Cultivation of Consortia
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-After autoclaving, and when medium for SOLID CULTURES is around 60 ºC, pour it on Petri dishes

and leave until total solidification in the laminar flow cabinet. Then, inoculate them with 0.1 mL of

samples from thermal springs. Seal the plates and introduce in the incubator at 50ºC.

- After autoclaving, and when the medium is at room temperature, introduce 50 mL of medium for

LIQUID CULTURES in each of the sterilized Erlenmeyer flasks, through a cylinder. This work must be

done in the laminar flow cabinet. Inoculate each with 0.3 mL of sample from thermal spring, and incubate

it in a shaker at 50ºC and 100 rpm for 24 hours.

-NOTE: Write codes on all the material to identify every sample

 Part 2. Quantification of lipolytic activity (Sigurgisladotir et al, 2003)
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Spectrophotometric assay of p-nitrophenyl esters. Hydrolysis reaction

O OH
O
C CH3 H2O
O H2 + C CH3
10 HO H2
Lipase 10

NO2
NO2

p-nitrophenyl laurate p-nitrophenol Lauric acid

Maximum absorbance at  = 400 nm

(Intense yellow color!)
 Part 2. Quantification of lipolytic activity (Sigurgisladotir et al, 2003)
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Spectrophotometric assay of p-nitrophenyl esters. Assay

Materials:
Sol. A: p-nitrofenol laurato 25 mM in ethanol. Substrate.
Sol. B: Tampón Tris/HCl 50 mM pH 7.5 at 50ºC and CaCl2 40 mM. Buffer solvent.
Sol C: Na2CO3 1 M . Reaction stopper.
Procedure:
Introduce 100 l of Sol A and 800 l of Sol B (in this order) in one eppendorf tube and place it at 50ºC for 5
min.
Add 100 l of sample (always in triplicate) to start reaction (if necessary dilute the sample with solution B).
100 l of Solution B must be added in one eppendorf assay to take it as a blank. The reaction time is 20
min.
250 l of Sol C are added after this time to stop the chemical reaction, and the eppendorfs must be placed in
ice for 10 min. Centrifuge for 10 min at 4ºC and 10000 rpm. Measure absorbance of supernatant at 400 nm.
 Part 2. Quantification of lipolytic activity (Sigurgisladotir et al, 2003)
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Spectrophotometric assay of p-nitrophenyl esters. Assay

 (ABS - ABSblank) 1 mol·cm 1 106 mol  1250 l volume

Activity        100 l sample  dilution
 20 min 17215 l cm 1 mol 

Lipolytic activity: One activity unit is defined as the amount of enzyme that produced

1 µmol of p-nitrophenol per minute under standard assay conditions. The activities were

expressed in (U/L)
 Part 3: Inoculation of isolates and activity measurement
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-Plates containing consortia are screened for microorganisms with transparent halos, and the

colonies are collected for inoculation in other sterile solid cultures following the procedure used in

Day 1 (at 50ºC).

-Flask inoculated must be sampled (1.5 mL in an eppendorf tube).

 Part 3. Protocol for obtaining crude enzyme
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Cultivation in shaker at
50ºC and 100 rpm

Activity of supernatant

Centrifuge at 5000
rpm for 10 min

1 day culture Biomass determination:

Absorbance at 600 nm
 Part 3. Measurement biomass and activity of consortia
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- Measure lipolytic activity of flasks through the spectrophotometric assay of p-

nitrophenyl esters.
 Part 4. Operation at bioreactor scale
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- The first day different parts must be autoclaved: Bioreactor, 100 mL cylinder, air
filter, funnel, 1.7 L of culture medium (liquid).
- The second day, the bioreactor must be connected to the control unit, the agitation
fixed at 300 rpm, the aeration rate at 0.5 vvm (0.7 L/min in bioreactor containing 1,4
L of medium) and the temperature at 50ºC.
- When the bioreactor is ready, the thermophilic microorganism is inoculated (3% v/v,
which means that 51 mL should be added to 1.641 L of medium).
- Samples must be taken at time 0, 4, 8, 12 and 24 h, and biomass (absorbance at
600 nm) and lipolytic activity in the supernatant should be determined.
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Additional Tasks:
- Modelling experimental biomass and enzymatic activity data through Gomperz and
logistic equations
- Discuss the differences observed between maximum biomass and specific growth
rate at flask and bioreactor scale.
- Discuss the existing differences between maximum lipolytic activity and specific
production rate between scales.
- Apply the Luedeking & Piret model and detail the characteristics of the synthesized
lipolytic enzymes.
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Chronogram:
1st session: 14:30 - 16:00: Solid and liquid media preparation and sterilization with material
14:30 - 16:30: Enzyme quantification
16:00 - 17:30: Bioreactor, liquid medium and material sterilization
16:00-17:30: Plates and flasks inoculation
17:30-19:30: Bioreactor assembling
17:30-20:30: Biomass and activity determination of sample 0

2nd session: 8:00 - 8:30: Bioreactor inoculation and sampling of bioreactor and flasks
8:30 - 10:30: Measurement of biomass and enzyme activity of samples
8:30 - 10:30: Isolation of lipase-producing microorganisms in plates
10:30-13:00: Sampling bioreactor and flasks.
11:30 – 13:30: Biomass and activity determination
13:00-14:00: Washing of material and bioreactor.