LIPASE FROM <i>RHIZOPUS ORYZAE</i> IMMOBILIZED ON CORN COB POWDER: A NEW APPROACH FOR DIETETIC TRIGLYCERIDE SYNTHESIS IN A FIXED BED REACTOR

Pacheco, Bruna Jeanne Soares; Andrade, Grazielle Santos Silva; Paula, Ariela Veloso de

doi:10.21577/0100-4042.20240025

Bruna Jeanne Soares Pacheco

Instituto de Ciência e Tecnologia, Universidade Federal de Alfenas (UNIFAL), 37715-400 Poços de Caldas - MG, Brasil

Grazielle Santos Silva Andrade ^**e-mail: grazielle.andrade@unifal-mg.edu.br

Instituto de Ciência e Tecnologia, Universidade Federal de Alfenas (UNIFAL), 37715-400 Poços de Caldas - MG, Brasil

https://orcid.org/0000-0001-6867-5969

Ariela Veloso de Paula

Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista (UNESP), 14800-903 Araraquara - SP, Brasil

*e-mail: grazielle.andrade@unifal-mg.edu.br

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Figure 1
Scheme of the preparation of enzymes process; carrier treatment and immobilization by physical adsorption

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Figure 2
Response surface plot of immobilized enzyme hydrolytic activity as a function of temperature and pH

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Figure 3
Thermal deactivation profile of free (□○△) and immobilized (■●▲) enzymes at 35, 41 and 45 °C. Nonlinear adjustments of free (dash-dotted) and immobilized (solid dot) enzyme

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Figure 4
Response surface plot of degree of incorporation (ID) of capric acid as a function of grape seed oil/capric acid molar ratio and amount of immobilized biocatalyst in a stirred tank reactor

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Figure 5
Distribution of residence time of the fixed bed reactor packed with corn cob powder

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Figure 6
Average degree of incorporation of capric acid in grape seed oil at a molar ratio of 1:3 and the immobilized biocatalyst of reactions 1 (′) and 2 (≤) containing triple U in a fixed bed reactor in total recycling

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Table 1
Real and coded values of independent variables analyzed using a central composite rotatable design for the immobilized enzyme characterization

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Table 2
Real and coded values of independent variables analyzed using a central composite rotatable design of batch reactions

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Table 3
Fixed asset yield, recovered activity and fixed asset activity of R. oryzae lipase immobilization for different enzyme concentrations

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Table 4
Experimental design matrix for the characterization of immobilized lipase from R. oryzae regarding the influence of temperature and pH on the hydrolytic activity of the immobilized enzyme

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Table 5
Regression coefficients, standard errors, calculated t and p-values for the evaluation of temperature and pH effects on hydrolytic activity of the immobilized enzyme (significance level of 5%, p-value < 0.05)

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Table 6
Results of variance analysis (ANOVA) for the assessment of temperature and pH effects

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Table 7
Kinetic and thermodynamic parameters of thermal inactivation of free and immobilized enzyme of the influence of temperature on its stability

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Table 8
Experimental design matrix for evaluating the influence of acid molar ratio, temperature and amount of biocatalyst on the normalized degree of incorporation of capric acid in triacylglycerols in a stirred tank batch

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Table 9
Regression coefficients, standard errors, calculated t and p-value for the evaluation of acid molar ratio, temperature, and amount of biocatalyst effects on incorporation degree (significance level of 5%, p-value < 0.05)

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Table 10
Results of variance analysis (ANOVA) for the assessment of acid molar ratio, temperature, and amount of biocatalyst effects

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Variables	Level
Variables	-1.41	-1	0	+1	+1.41
Temperature (°C)	24	30	45	60	66
pH	5.0	5.5	7.75	10.0	10.5

Variables	Level
Variables	-1.68	-1	0	+1	+1.68
Molar ratio (v v^-1)	2.64	4	6	8	9.36
Temperature (°C)	28.18	35	45	55	61.82
Biocatalyst (% m m^-1)	6.59	10	15	20	23.41

Parameters	ES (mg mL^-1)
Parameters	10	15	20	30	35
AY (%)	90.4 ± 0.9	90.6 ± 0.9	82.3 ± 0.2	90.8 ± 0.5	90.6 ± 0.2
RA (%)	2.8 ± 0.2	2.4 ± 0.2	5.5 ± 0.4	6.1 ± 0.3	6.5 ± 0.1
AA (U g^-1)	91.1 ± 3.6	99.2 ± 5.1	175.5 ± 11.6	251.6 ± 10.3	273.8 ± 11.2
Fixed asset yield (AY). Recovered activity (RA). Fixed asset activity (AA). Enzyme solution (ES).

Run	Coded variables		Real variables		Response variable
Run	T (°C)	pH	T (°C)	pH	Activity (U g^-1)
1	-1	-1	30	5.5	93.45
2	1	-1	60	5.5	83.10
3	-1	1	30	10	121.52
4	1	1	60	10	83.19
5	-1.41	0	23.79	7.75	112.14
6	1.41	0	66.21	7.75	95.01
7	0	-1.41	45	4.57	104.79
8	0	1.41	45	10.93	83.01
9	0	0	45	7.75	154.44
10	0	0	45	7.75	158.47
11	0	0	45	7.75	158.41
Temperature (T). Hydrolytic activity of the immobilized enzyme (activity).

Name	Coefficient	Standard error	t-calculated	p-value
Mean	157.11	5.94	26.47	0.0000
x₁	-9.11	3.64	-2.51	0.0406
x₁²2 López-Fernández, J.; Benaiges, M. D.; Valero, F.; Catalysts 2020, 10, 1277. [Crossref] Crossref...	-27.62	4.33	-6.38	0.0004
x₂²2 López-Fernández, J.; Benaiges, M. D.; Valero, F.; Catalysts 2020, 10, 1277. [Crossref] Crossref...	-32.46	4.33	-7.50	0.0001
Linear effect temperature (x₁). Quadratic effect temperature (x₁²2 López-Fernández, J.; Benaiges, M. D.; Valero, F.; Catalysts 2020, 10, 1277. [Crossref] Crossref... ). Quadratic effect pH (x₂²2 López-Fernández, J.; Benaiges, M. D.; Valero, F.; Catalysts 2020, 10, 1277. [Crossref] Crossref... ).

Source of variation	Sum of squares	Degrees of freedom	Medium square	F_calc	p-value
Regression	8633.6	3	2877.9	27.2	0.00031
Residue	740.0	7	105.7	-	-
Lack of fit	729.3	5	145.9	27.3	0.03565
Pure error	10.7	2	5.3	-	-
Total	9373.6	10	-	-	-

Parameter	Enzyme	Temperature (°C)
Parameter	Enzyme	35	41	45
k_d (min^-1)	free	0.0061 ± 0.0024	0.0096 ± 0.0000	0.0307 ± 0.0041
k_d (min^-1)	immobilized	0.0038 ± 0.0000	0.0066 ± 0.0010	0.0275 ± 0.0021
R²	free	0.7075	0.9878	0.9686
R²	immobilized	0.9516	0.9317	0.9873
t_1/2 (min)	free	113.63	72.20	22.58
t_1/2 (min)	immobilized	177.73	106.64	25.21
SF		1.56	1.48	1.12
E_d ( kJ mol^-1)	free	125.51 (R² = 0.8680)
E_d ( kJ mol^-1)	immobilized	151.27 (R² = 0.8538)
∆H (kJ mol^-1)	free	125.51	125.51	125.51
∆H (kJ mol^-1)	immobilized	151.27	151.27	151.27
∆G (kJ mol^-1)	free	99.11	99.90	98.13
∆G (kJ mol^-1)	immobilized	100.32	100.88	98.42
∆S (kJ mol^-1 K^-1)	free	0.0857	0.0815	0.0861
∆S (kJ mol^-1 K^-1)	immobilized	0.1654	0.1584	0.1661
Thermal deactivation constant (k_d). Half-life time (t_1/2). Stability factor (SF). Deactivation energy (E_d). Enthalpy (∆H). Gibbs free energy (∆G). Entropy (∆S).

Run	Coded variables			Real variables			Response
Run	MR	T (°C)	Biocat (%)	MR	T (°C)	Biocat (%)	ID (%)
1	-1	-1	-1	4	35	10	55.38 ± 1.82
2	1	-1	-1	8	35	10	72.16 ± 3.47
3	-1	1	-1	4	55	10	49.85 ± 1.49
4	1	1	-1	8	55	10	73.15 ± 0.80
5	-1	-1	1	4	35	20	54.33 ± 4.26
6	1	-1	1	8	35	20	81.27 ± 2.06
7	-1	1	1	4	55	20	58.16 ± 1.73
8	1	1	1	8	55	20	84.68 ± 2.14
9	-1.68	0	0	2.63	45	15	42.62 ± 0.17
10	1.68	0	0	9.36	45	15	86.93 ± 2.25
11	0	-1.68	0	6	28.18	15	61.83 ± 2.15
12	0	1.68	0	6	61.81	15	66.07 ± 2.63
13	0	0	-1.68	6	45	6.59	59.94 ± 0.94
14	0	0	1.68	6	45	23.40	72.10 ± 2.06
15	0	0	0	6	45	15	66.34 ± 0.91
16	0	0	0	6	45	15	64.41 ± 1.73
17	0	0	0	6	45	15	66.85 ± 2.63
Molar ratio (MR). Temperature (T). Amount of biocatalyst (Biocat). Normalized incorporation degree (ID).

Name	Coefficient	Standard error	t-calculated	p-value
Mean	65.65	0.45	146.96	0.0000
x₁	12.30	0.50	24.69	0.0000
x₃	3.54	0.50	7.10	0.0000
x₁.x₃	1.67	0.65	2.57	0.0246
x₂.x₃	1.47	0.65	2.26	0.0433
Linear effect molar ratio (x₁). Linear effect amount of biocatalyst (x₃). Interaction between molar ratio and amount of biocatalyst (x₁.x₃). Interaction between molar ratio and temperature (x₁.x₂).

Source of variation	Sum of squares	Degrees of freedom	Medium square	F_calc	p-value
Regression	2278.7	4	569.7	167.9	0.00000
Residue	40.7	12	3.4	-	-
Lack of fit	37.4	10	3.7	2.2	0.34680
Pure error	3.3	2	1.7	-	-
Total	2319.4	16	-	-	-

[1] *e-mail: grazielle.andrade@unifal-mg.edu.br

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LIPASE FROM RHIZOPUS ORYZAE IMMOBILIZED ON CORN COB POWDER: A NEW APPROACH FOR DIETETIC TRIGLYCERIDE SYNTHESIS IN A FIXED BED REACTOR