Standardization of the Cultivation of Rhizopus arrhizus Using Agroindustrial Residues: High Production of Amylases in Pineapple Peel

Lopes, Paulo Henrique Silva; Pasin, Thiago Machado; Benassi, Vivian Machado; Nelson, David Lee; Oliveira, Tássio Brito de; Polizeli, Maria de Lourdes Teixeira de Moraes

doi:10.1590/1678-4324-2024240293

Acessibilidade / Reportar erro

Brasil

Brazilian Archives of Biology and Technology

Español English

Brasil

Español English

sumário « anterior atual seguinte »

Sumário

Article - Agriculture, Agribusiness and Biotechnology • Braz. arch. biol. technol. 67 • 2024 • https://doi.org/10.1590/1678-4324-2024240293 copy

Standardization of the Cultivation of Rhizopus arrhizus Using Agroindustrial Residues: High Production of Amylases in Pineapple Peel

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Amylases, crucial in various industries such as food, textile, and biofuels, require optimized production and biochemical characterization. Thus, this study aimed to produce amylases from Rhizopus sp. I 1.2.1, characterize the cultivation conditions and the synthesized enzyme. The fungus was molecularly identified with 99.06% homology in the LSU gene, 98.08% identity in the ITS region, and placed phylogenetically closer to Rhizopus arrhizus CBS 112,07. The LSU gene and ITS region sequences were deposited in GenBank. The CP medium was optimal for amylase production by Rhizopus arrhizus, with peak activity on the 6th day. Supplementation with urea significantly increased amylolytic activity by 110-fold. CP salts outperformed other salts in enzyme production, achieving a maximum amylase activity of 15.5 U/ml. The pH 6.0 was optimal for amylase production, with higher specific activity at pH 5.0. Pumpkin peel and pineapple peel were the best carbon sources for amylase production with an amylolytic activity of 18 and 16 U/mL, respectively. The amylase production showed a significant increase when pineapple peel was used in the presence of glucose 0.5% showing an activity of 22.4 U/mL, representing a 1.5- and 1.6-fold increase over the control. The optimal reaction of the amylase was observed at pH 6.0 and 60 °C. The enzyme remained stable for 240 minutes at 50-55 °C and at pH 4.0-7.0. Amylase was inhibited by glucose concentration and certain salts, but EDTA and K₂SO₄ increased activity by 25%. These results suggest industrial potential based on residual carbon source use, cultivation conditions, and crude enzyme stability.

Keywords:
Agroindustrial residues; Amylases; filamentous fungus; Rhizopus arrhizus; stability

HIGHLIGHTS

Maximum production of amylases with residual carbon sources.

Amylase production with less expensive nitrogen sources.

Amylolytic activity is independent of the inoculum diameter.

Crude amylases are stable for over two hours.

GRAPHICAL ABSTRACT

¹ Variables	Levels
¹ Variables	-1,41	-1	0	+1	+1,41
Temperature	32	40	60	80	88
pH	3.2	4.0	6.0	8.0	8.8

Nitrogen Source¹	Activity (U.mL^-1)	Protein (mg.mL^-1)	Specific Activity (U.mg^-1)
E (control)	0.185 ± 0.019 ^c	0.289 ± 0.027 ^c	0.645 ± 0.096 ^c
P	4.319 ± 0.564 ^b	0.334 ± 0.013 ^c	12.922 ± 1.347 ^b
U	17.738 ± 3.268 ^a	0.178 ± 0.017 ^c	99.251 ± 12.437 ^a
P+U	0.743 ± 0.146 ^c	2.974 ± 0.720 ^b	0.258 ± 0.067 ^c
Y+U	1.181 ± 0.164 ^c	3.595 ± 0.230 ^a	0.329 ± 0.049 ^c
Y+P	4.216 ± 0.474 ^b	0.319 ± 0.042 ^c	13.341 ± 2.091 ^b
Y+P+U	0.970 ± 0.103 ^c	3.489 ± 0.286 ^a	0.277 ± 0.008 ^c

Salts	Activity (U.mL^-1)	Protein (mg.mL^-1)	Specific Activity (U.mg^-1)
CP (control)	15.527 ± 0.945 ^a	0.467 ± 0.008 ^d	33.291 ± 2.270 ^a
SR	1.053 ± 0.071 ^b	8.185 ± 0.943 ^a	0.130 ± 0.020 ^b
Khanna	1.395 ± 0.095 ^b	8.883 ± 0.657 ^a	0.157 ± 0.001 ^b
Wesson	0.366 ± 0.019 ^b	1.438 ± 0.224 ^c	0.257 ± 0.025 ^b
Modified Vogel	2.076 ± 0.648 ^b	3.654 ± 0.192 ^b	0.573 ± 0.196 ^b

Initial pH	Activity (U.mL^-1)	Protein (mg.mL^-1)	Specific Activity (U.mg^-1)
6.0 (control)	14.374 ± 0.154 ^a	0.623 ± 0.047 ^b	23.009 ± 1.550 ^c
4.5	13.003 ± 0.825 ^a	1.074 ± 0.063 ^a	12.141 ± 1.096 ^c
5.0	13.695 ± 2.352 ^a	0.201 ± 0.019 ^c	68.394 ± 11.379 ^a
5.5	11.799 ± 0.200 ^b	0.843 ± 0.283 ^b	14.961 ± 4.379 ^c
6.5	10.172 ± 1.111 ^b	0.211 ± 0.044 ^c	49.075 ± 6.989 ^b

Inoculum diameters¹	Activity (U.mL^-1)	Protein (mg.mL^-1)	Specific Activity (U.mg^-1)
1.0'' (control)	14.989 ± 0.915 ^a	0.800 ± 0.037 ^d	18.763 ± 1.440 ^b
0.5''	14.515 ± 0.810 ^a	0.433 ± 0.106 ^d	34.719 ± 7.623 ^a
1.5''	12.286 ± 0.847 ^b	9.707 ± 0.820 ^b	1.268 ± 0.052 ^c
1.5'' halved	14.412 ± 1.132 ^a	5.115 ± 0.577 ^c	2.825 ± 0.119 ^c
2.0''	13.938 ± 0.256 ^a	9.752 ± 0.518 ^b	1.431 ± 0.067 ^c
2.0'' halved	13.925 ± 1.170 ^a	15.780 ± 2.167 ^a	0.893 ± 0.140 ^c

Carbon Source¹	Activity (U.mL^-1)	Protein (mg.mL^-1)	Specific Activity (U.mg^-1)
Soluble starch w/o glucose	14.545 ± 0.027 ^b	0.162 ± 0.013 ^e	89.870 ± 7.361 ^b
Pumpkin peel w/o glucose	16.507 ± 0.772 ^b	2.333 ± 0.050 ^d	7.757 ± 0.155 ^c
Soluble starch	12.817 ± 2.147 ^b	0.090 ± 0.039 ^e	151.466 ± 42.537 ^a
English potato peel	19.985 ± 2.555 ^a	5.732 ± 0.018 ^a	3.486 ± 0.435 ^c
Pineapple peel	22.848 ± 0.788 ^a	4.271 ± 0.966 ^c	5.469 ± 1.053 ^c
Pumpkin peel	13.009 ± 2.147 ^b	2.970 ± 0.098 ^d	4.394 ± 0.868 ^c
Sweet potato peel	21.733 ± 3.940 ^a	4.264 ± 0.126 ^b	5.113 ± 1.076 ^c
Ground corn	10.665 ± 4.158 ^b	0.785 ± 0.032 ^e	13.487 ± 4.573 ^c
Pumpkin peels/seeds	10.319 ± 0.951 ^b	5.681 ± 0.108 ^a	1.815 ± 0.133 ^c

Variation source¹	Sum of squares	Degrees of freedom	Mean square	Fcalc	p-value
Regression	473.9	3	158.0	95.0	<0.01
Residuals	11.6	7	1.7	-	-
Lack of fit	8.1	5	1.6	0.9	0.59044
Pure error	3.5	2	1.7	-	-
Total	485.5	10	-	-	-

Salts (5 mM)	Relative Activity (%)	Salts (5 mM)	Relative Activity (%)
Control	100 (± 2.01) ^c	Ca(CH₃COO)₂.H₂O	96.664 (± 4.61) ^d
K₂SO₄	126.459 (± 5.146) ^a	Na₂B₄O₇.10H₂O	96.74 (± 1.715) ^d
EDTA	125.095 (± 4.074) ^a	Na₂CO₃	93.404 (± 3.431) ^d
BaCl₂	121.683 (± 4.825) ^a	KH₂PO₄	88.931 (± 5.683) ^e
KI	122.517 (± 3.86) ^a	Na₃PO₄	87.87 (± 4.182) ^e
MgSO₄.7H₂O	123.275 (± 2.788) ^a	NH₄Cl	86.96 (± 2.895) ^e
Na₂SO₄	117.892 (± 4.182) ^a	C₄H₁₁NO₃.HCl	84.382 (± 4.825) ^e
Ba(NO₃)₂	112.813 (± 5.146) ^b	K₂HPO₄	85.368 (± 2.788) ^e
Mg(NO₃)₂.6H₂O	112.661 (± 2.788) ^b	KMnO₄	82.866 (± 4.825) ^e
Na₂HPO₄.7H₂O	110.462 (± 3.967) ^b	NaCl	83.7 (± 2.573) ^e
NaNO₃	109.553 (± 4.825) ^b	ZnSO₄.7H₂O	83.169 (± 2.466) ^e
FeSO₄.7H₂O	104.094 (± 7.827) ^c	CaCl₂	84.155 (± 1.072) ^e
NH₄CH₃COOH	110.614 (± 0.965) ^b	CuSO₄	76.725 (± 3.431) ^f
Na₂S₂O₃.5H₂O	106.293 (± 2.359) ^c	NaHCO₃	77.559 (± 2.466) ^f
KCl	105.838 (± 1.715) ^c	Ca(H₂PO₄)₂.H₂O	77.483 (± 2.359) ^f
(NH₄)₂CO₃	104.397 (± 1.823) ^c	CaCO₃	77.255 (± 2.037) ^f
NaH₂PO₄	100.531 (± 4.932) ^c	NH₄H₂PO₄	66.49 (± 0.107) ^g
CH₃COONa.3H₂O	96.816 (± 6.111) ^d	Ca(NO₃)₂.4H₂O	59.818 (± 5.254) ^g
Na₃C₆H₅O₇	96.285 (± 5.361) ^d

Instituto de Tecnologia do Paraná - Tecpar Rua Prof. Algacyr Munhoz Mader, 3775 - CIC, 81350-010 Curitiba PR Brazil, Tel.: +55 41 3316-3052/3054, Fax: +55 41 3346-2872 - Curitiba - PR - Brazil
E-mail: babt@tecpar.br

Acompanhe os números deste periódico no seu leitor de RSS

[1] *Correspondence: thiagopasin@hotmail.com; (T.M.P.).