FEMS 0821 Poster

Maastricht, June 7-11/2015, The Netherlands
BN:230
FEMS 6th Congress of European Microbiologist
Sequential statistical designs for improvement a recombinant

laccase production in Pichia pastoris immobilized cells
1
Rivera-Hoyos ,
2,3
Morales-lvarez ,
1
Rojas-Fajardo ,
Claudia M.
Edwin D.
Mara F.
Luis M. Chves1
1
1
4
Tequia , Angela M. Cardozo-Bernal , Ral A. Poutou-Piales , Eliana M. Gonzlez-Neira , Aura M.
2
Pedroza-Rodrguez
1Laboratorio
de Biotecnologa Molecular, Grupo de Biotecnologa Ambiental e Industrial. Departamento de Microbiologa. Facultad de

Ciencias. Pontificia Universidad Javeriana. Bogot, D.C., Colombia.
2 Laboratorio de Microbiologa Ambiental y de Suelos, Grupo de Biotecnologa Ambiental e Industrial. Departamento de Microbiologa.
Facultad de Ciencias. Pontificia Universidad Javeriana. Bogot, D.C., Colombia.
3Departamento de Qumica y Grupo de Investigacin en Gentica, Biodiversidad y Manejo de Ecosistemas (GEBIOME), Facultad de Ciencias
Exactas y Naturales, Universidad de Caldas, Manizales-Caldas, Colombia.
4 Departamento de Ingeniera Industrial. Facultad de Ingeniera. Pontificia Universidad Javeriana. Bogot, D.C., Colombia.
INTRODUCTION
Laccases are multicopper oxidases widely distributed in nature and catalyze the transformation of aromatic and non-aromatic compounds with
reduction of molecular oxygen to water 1. We previously cloned the syntetic optimized gene, POXA 1B from Pleurotus ostreatus in Pichia pastoris. In
expression experiments with free cells, we obtained an enzymactic activity of 451.08 6.46 UL-1 2 at 168h of culturing. In other previous work we
obtained by using immobilized cells of the same clone, an enzyme activity of 14.4 2.60 UL-1 3 at 156h of batch culture. We are planing to employ
recombinant immobilized cells to the continuous process removal of dyes in textil effluents. The aim of this work was to improve the enzyme activity
by optimizing the batch culture media components.
MATERIALS AND METHODS
Strain
We used Pichia pastoris X33 containing the integrative expression vector pGAPZA-LaccPost-Stop 2.
Statistical design
We used a Plackett-Burman experimental design to evaluate media volume, copper, glucose, NH4SO4, peptone and yeast extract concentration, each
one of them with two levels, to detect the positive or negative influence and contribution percentage of each one. After that, a Box-Behnken design
allowed us to optimize the more influent factor by analizing the three levels of factor interactions throught a response surface methodology (RSM).
Finaly the best treatment in Box-Behnken design was repeated to analyse treatment consistence. In all assays as response variables enzyme activity
(UL-1), specific activity (UL-1mg-1) and productivity (UL-1h-1) were used. Assays were followed too by glucose (gL-1) and extracelular protein
concentration (mgmL-1). Shake flask (500 mL Erlenmeyer) culture conditions were, 30C, 180rpm, during 168h.
RESULTS
Treatment 11
20
10
10
10
0.5
0.0
1.0
T1
0
0
20
40
60
80
100
120
140
160
180
T11
5
0.05
20
10
0
0
20
40
60
1.5
20
1.0
10
10
0.5
0.0
80 100 120 140 160 180
0.00
Time (h)
20
40
60
80
100
120
140
160
180
Time (h)
-1
Enz. Act (UL )

Glucose (gL-1)
Prot. Conc.(mg mL-1)
Spec. Act.
Productivity (UL-1h-1)
-1
30
30
Glucose (gL )
40
1000
800
20
600
400
20
10
10
Spec. Act.
-1
-1
1200
40
81.640
81.640
128.996
0.0015
46.454
6.585
16.728
38.265
148.143
1
1
1
1
1
46.454
6.585
16.728
38.265
148.143
73.401
10.404
26.432
60.461
234.075
0.0033
0.0484
0.0143
0.0044
0.0006
71.203
1.899
765.030
1
3
15
71.203
112.505
0
0
20
40
60
80
100
120
140
160
F Value
0.633
Treatment 3
2,09E+09
6,97E+08
51.45
<0.0001
5010.91
5010.91
0.37
0.5554
64839.07
2,02E+09
1,49E+08
1
1
11
64839.07
2,02E+09
13547.54
9
2
14
1600
T3
0
0
20
40
60
80
100 120 140 160 180
Time (h)
6
4
2
0
Laccase Act. (UL )
-1
h )
T6
Productivity (UL
-1
200
40
30
20
10
0
Table 3. BBED matrix for comparing observed and predicted results.
1400
50
40
1200
30
30
1000
Box-Behenken
800
20
600
400
10
20
10
200
0
0
0
20
40
60
80
100
Time (h)
Figure 2. Box-Behnken experimental design

(BBED). a. Enzyme activity results (12 treatments).
b. Treatmen 12 results [BBED-T12: 500mL shake flask
(350mL, 0.05mM CuSO4, 30gL-1 glucose, 2.5mM
NH4SO4, 10gL-1 peptone, 30gL-1 yeast extract), Enz.
Act. 1300.34 125.3 UL-1, 168h]. c. Treatmen 3
results [BBED-T3: 500mL shake flask (300mL,
0.05mM CuSO4, 30gL-1 glucose, 2.5mM NH4SO4, 5gL1 peptone, 30gL-1 yeast extract), Enz. Act. 1079.64
125.3 UL-1, 168h].
120
140
160
180
Enz. Act. (UL-1)

Glucose (gL-1)
Prot. Conc. (mg mL-1)
Spec. Act.
Productitivy (UL-1 h-1)
Spec. Act
400
40
Treatment
Design
point
T1
Factorial
T2
Factorial
T3
Factorial
T4
Factorial
T5
Factorial
T6
Factorial
T7
Factorial
T8
Factorial
T9
Factorial
T10
Factorial
T11
Factorial
T12
Factorial
Central Point
Central Point
Central Point
Volume
of culture Yeast extact
media
(gL-1)
(mL)
300
10
400
10
300
30
400
30
300
20
400
20
300
20
400
20
350
10
350
30
350
10
350
30
350
20
350
20
350
20
0.0512
<0.0001
Observed Enz Predicted Enz

Peptone
Act. (UL-1)
Act. (UL-1)
-1
(gL )
168h
168h
5
5
5
5
0
0
10
10
0
0
10
10
5
5
5
5.902
97.497
1.079.640
850.400
448.810
407.280
639.360
618.316
43.856
944.255
6.399
1.300.340
542.662
344.710
368.032
35.570
-14.486
1.040.810
990.759
448.164
398.109
628.363
578.164
-79.485
925.759
100.569
1.105.810
513.164
513.164
513.164
1.19
62
60
Glucose (gL )
60
4.79
149.18
13957.19
11704.09
Time (h)
-1
10
Table 2. Main effect on Enz. Act.

Plackett-Burman
1,26E+08
23408.18
2,24E+09
R2: 0.9335
Adj. R2: 0.9153
Pred. R2: 0.8798
Adeq. Precision: 19.720
Adeq. Precision: 7.663
180
p-value
Prob > F
0.0018
200
Sum of Squares
600
T10
T2
10
800
T9
T7
20
-1
T11
30
-1
T5
Prot. Conc. (mg mL )
T4
Enz. Act. (UL )
T1
40
Treatment 12
1400
Laccase Act. (UL )
-1
1000
T12
T8
Protuctivity (UL
1200
1600
50
-1
-1
h )
1400
60
Prot. Conc . (mg mL )
10
Model
Lineal Model
A- Culture media
Volume
B- CuSO4
C- Glucose
D- NH4SO4
E- Peptone
F- Yeast extract
Residual
Curvature
Lack of Fit
Pure Error
Cor Total
R2: 0.9044
p-value
Prob > F
0.0015
Box-Behnken
Mean
df
Square
5
4
3
2
0.5365
Effect
Sum of
%
squares Contribution
A-Culture media
Volume
5.217
81.640
11.44
B- CuSO4
-3.935
46.454
6.51
C-Glucose
1.482
6.585
0.92
D-NH4SO4
-2.361
16.728
2.34
E-Peptone
-3.571
38.265
5.36
F-Yeast Extract
7.027
148.143
20.75
Factor
1400
1.6
1200
1.4
58
T13
20
30
Sum of
Squares
691.929
Plackett-Burman
Mean
df
F Value
Square
11
62.903
99.390
T12
0.10
30
30
2.0
Spec. Act.
10
40
40
Glucose (gL-1)
T10
40
Enz. Act. (UL-1)
0.15
-1
T9
15
-1
T8
50
0.20
T6
Source
Treatment 2
-1
T5
Time (h)
20
Productivity (UL h )
Laccase (UL
-1
T3
T4
0.00
25
T2
54
52
50
1.0
800
0.8
600
0.6
400
46
44
20
50
15
10
40
30
0.4
48
60
1.2
1000
56
25
Enz. Act. VC (%)
20
Spec. Act. (UL-1mg-1)
20
Spec. Act.
30
Glucose (gL-1)
1.5
-1
-1
0.05
30
Figure 1. Plackett-Burman experimental design (PBED). a. Enzyme activity results (12 tratments). b. Treatmen 11 results [PBED-T11: 500mL
shake flask (300mL media, 0.1mM CuSO4, 30gL-1 glucose, 5mM NH4SO4, 20gL-1 peptone, 10gL-1 yeast extract), Enz. Act. 29.5 0.80 UL-1, 168h]. c.
Treatment 2 results [PBED-T2: 500mL shake flask (150mL media, 0.1mM CuSO4, 10gL-1 glucose, 5mM NH4SO4, 10gL-1 peptone, 5gL-1 yeast
extract), Enz. Act. 18.60 0.80 UL-1, 168h].
Table 1. ANOVA for selected factorial model.
Enz. Act. (UL-1)
30
30
Glucose (gL )
0.10
2.0
b
Enz. Act. (UL-1)
35
40
40
40
0.15
50
-1
-1
Productivity (UL h )
0.20
200
0.2
0.0
180
80
100
120
140
Time (h)
Figure 3. Consistence assay. Average

of the 5 repetitions of treatment
T12, selected in the Box-Behnken. It
is noted that the coefficient of
variation for enzyme activity to 168h
is less than 20%.
160
20
10
Enz. Act. (UL-1)

Glucose (gL-1)
Spec. Act. (UL-1 mg-1)
Enz. Act. VC (%)
CONCLUSIONS
We improved the enzymatic activity from 14.4 UL-1 at 156h to 1300 UL-1 at 168 h of immobilized cells bath culture, after sequential statististical optimization;
meaning a 90.3-fold increase enzyme activity. The activity of the recombinant enzyme POXA 1B produced by using immobilized cells; exceeds the maximum activity
obtained in previous free cells trials 451.08 UL-1; meaning a 2.88-fold increase enzyme activity at 168 hours. After consistence assay we get an Enz. Act. 989.31
187.45 UL-1 which increased 70-Fold times the preliminary assay.
1.
References
Rivera-Hoyos, et al. (2013) Fungal Biology Reviews. 27(3-4): 67-82.
2. Rivera-Hoyos, et al. (2015) Plos One. 10(1): e0116524.
3. Unpublished data

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Maastricht, June 7-11/2015, The Netherlands

FEMS 6th Congress of European Microbiologist

Sequential statistical designs for improvement a recombinant

de Biotecnologa Molecular, Grupo de Biotecnologa Ambiental e Industrial. Departamento de Microbiologa. Facultad de

80 100 120 140 160 180

Enz. Act (UL )

100 120 140 160 180

Laccase Act. (UL )

Table 3. BBED matrix for comparing observed and predicted results.

Figure 2. Box-Behnken experimental design

Enz. Act. (UL-1)

Observed Enz Predicted Enz

Table 2. Main effect on Enz. Act.

Adeq. Precision: 7.663

Prot. Conc. (mg mL )

Enz. Act. (UL )

Prot. Conc . (mg mL )

Enz. Act. (UL-1)

Prot. Conc. (mg mL )

Enz. Act. VC (%)

Spec. Act. (UL-1mg-1)

Enz. Act. (UL-1)

Prot. Conc. (mg mL )

Figure 3. Consistence assay. Average

Enz. Act. (UL-1)

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