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
Prot. Conc. (mg mL-1)

T13

20

30

Sum of
Squares
691.929

Plackett-Burman
Mean
df
F Value
Square
11
62.903
99.390

Productivity (UL-1h-1)

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

Prot. Conc. (mg mL )

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

Prot. Conc. (mg mL )

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)
Prot. Conc. (mg mL-1)
Spec. Act. (UL-1 mg-1)
Productivity (UL-1h-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