Response-Surface Statistical Optimization of Submerged Fermentation for Pectinase and Cellulase Production by Mucor circinelloides and M. hiemalis

Abstract

Cellulase and pectinase are degrading cellulosic and pectic substances that form plant cell walls and, thereby, they have a wide range of applications in the agro-industrial by-products recycling and food industries. In the current research, Mucor circinelloides and M. hiemalis strains were tested for their ability to produce cellulase and pectinase from tangerine peel by submerged fermentation. Experiments on five variables: temperature, pH, incubation period, inoculum size, and substrate concentration, were designed with a Box–Behnken design, as well as response surface methodology (RSM), and analysis of variance was performed. In addition, cellulase and pectinase were partially purified and characterized. At their optimum parameters, M. circinelloides and M. hiemalis afforded high cellulase production (37.20 U/mL and 33.82 U/mL, respectively) and pectinase (38.02 U/mL and 39.76 U/mL, respectively). The partial purification of M. circinelloides and M. hiemalis cellulase produced 1.73- and 2.03-fold purification with 31.12 and 32.02% recovery, respectively; meanwhile, 1.74- and 1.99-fold purification with 31.26 and 31.51% recovery, respectively, were obtained for pectinase. Partially purified cellulase and pectinase from M. circinelloides and M. hiemalis demonstrated the highest activity at neutral pH, and 70 and 50 °C, for cellulase and 50 and 60 °C, for pectinase, respectively. Moreover, 10 mM of K+ increased M. circinelloides enzymatic activity. The production of cellulase and pectinase from M. circinelloides and M. hiemalis utilizing RSM is deemed profitable for the decomposition of agro-industrial wastes.

1. Introduction

Filamentous fungi biotechnological processes have enabled the industrial exploitation of their capability to produce profitable enzymes, due to their easy propagation and high production of extracellular enzymes with particular properties such as stability in wide ranges of temperature and pH [1,2,3]. The genus Mucor belonging, to the class Zygomycetes, shows a variety of bioactivities such as multi-metal remediation by M. hiemalis [4]; production of biosurfactants [5], fungal chitosan [6] and bio-oil by M. circinelloides [7,8]; the yielding of ethanol by M. indicus [9] along with the potential to produce extracellular hydrolytic enzymes on various agro-industrial wastes that can be harnessed in diverse applications in industry [10]. These enzymes include milk-clotting proteases [11], malic enzyme [12] and polygalacturonase by M. circinelloides [13], ferulic acid esterase from M. hiemalis [14], lipase by M. geophillus [15], amylase [16], xylanase by M. indicus and M. hiemalis [17], and endoglucanase by M. racemosus [18].

The increasing expansion of agricultural-waste activity has led to the accumulation of a large quantity of lignocellulosic residues across the world [19]. Lignocellulosic plant biomass is mainly constituted of hemicellulose, cellulose, and lignin [20]. Cellulases are a group of synergetic enzymes that catalyze the degradation of cellulose into fermentable sugars and can be divided into three major components, namely, endoglucanase, exoglucanase and β-glucosidase. Furthermore, endoglucanase is considered the most economical type to create free end groups on the cellulose, thereby producing starting points for the other cellulase synergetic components [21,22]. Fungi have the capability to produce higher quantities of cellulases as compared to other organisms. Cellulase has been included in a wide range of industrial applications for alcohol fermentation, biofuel and starch production, juices extraction, animal-feed processing, and textile and paper manufacturing [23].

Pectinase is involved in the hydrolysis of pectin present in the middle lamella and primary cell wall of vegetables and fruits into D-galacturonic acid by breaking down α-1-4 chains [24]. Pectic compounds are copious in the plant biomass composition; their levels are between 4 and 30% in the pulp of beet and the peel of citrus fruits [25]. Pectinase has a broad domain of implementations and plays critical roles in the food industry, such as the clarification of fruit juice, oil squeezing, the beverage industry, waste management, tea fermentation, the paper and pulp industry, and softening plant-based fibers [26]. Genera of Zygomycetes and Ascomycetes could be a preferred source of pectinase, as 50% of total pectinases are obtained from fungi, because of their easy growth, high productivity rate, being cost effective and having a short life span [18].

The amount of fungal-enzyme manufacturing relies on the conditions of the fermentation process, along with the necessity to optimize these conditions in order to reduce enzyme-production cost [27]. Fungal-enzyme manufacturing is prevalently implemented by submerged or solid-state fermentations [28]. Submerged fermentation, reported in 90% of the industrial enzymes production, occurs in the presence of excess water, thus offering easy handling and better monitoring [29,30]. Concerning the accumulation of vast bulks of agro-industrial by-products around the world, this study aimed to evaluate the production of high-value, partially purified pectinase and cellulase by Mucor circinelloides and M. hiemalis strains, using agro-industrial by-products as a cheap substrate in submerged-fermentation conditions. The optimization of cellulase and pectinase production and characterization of partially purified enzymes were investigated.

2. Materials and Methods

2.1. Tested Fungi

Mucor circinelloides AUMC 6696.A (Accession no. MT509983) and M. hiemalis AUMC 6031 (Accession no. MT365791) [31] were utilized in the in the current search for pectinase and cellulase production. Pure cultures were kept in potato-dextrose-agar (PDA) tubes and preserved at 4 °C for further use.

2.2. Enzymes Preliminary Screening

2.2.1. Screening on Agar Plates

Czapek’s agar medium (g/L: KH₂PO₄, 1; NaNO₃, 2; MgSO₄.7H₂O, 0.5 and CaCl₂.2H₂O, 0.5) was supplemented with 10 g/L of pectin and carboxymethylcellulose as carbon source for pectinase and cellulase production. The pH was set to 7 and the inoculated plates were incubated at 28 ± 2 °C for 5 days, and then screened for enzyme production [32]. After flooding the cultured agar plates in iodine solution for 15 min, they were checked for the appearance of a clear zone.

2.2.2. Screening by Submerged Fermentation (SmF)

Wheat straw, pomegranate, and tangerine peels were desiccated at 65 °C for 24 h, then grounded to fine powders and passed through a sieve of mesh size 600 µm and used, subsequently, as a carbon source (10 g/L) in Czapek’s mineral-salts broth. Before sterilization, the broth-containing flasks were adjusted to pH 7.0 using potassium phosphate buffer (pH 7.0, 0.05 M) [33], and then autoclaved at 121 °C, 1.5 bar for 20 min. The strains’ spores were removed from the colony surface into suspensions of 10 mL sterile distilled water containing 0.1% Tween-80. The spores’ concentration was prepared by measuring and calculating with a haemocytometer and a binocular microscope, then dilution was performed to give a final concentration of approximately 1 × 10⁷ spores/mL which was then utilized to inoculate 250 mL flasks holding 100 mL liquid culture medium. The inoculated flasks were incubated at 28 ± 2 °C for seven days on both shaking at 120 rpm and static conditions. Subsequently, broth media were centrifuged at 10,000 rpm under cooling, and supernatants were maintained at 4 °C for further enzymatic analysis.

2.2.3. Quantitative Screening of Cellulase and Pectinase

Cellulase and pectinase activities were measured according to Miller [34]. In addition, 0.5 mL culture supernatant was added to 0.5 mL pectin or carboxymethylcellulose (1% w/v) in acetate buffer (pH 4.8, 0.05 M) [33], and the mixture was incubated at 50 °C for 30 min. Afterward, the interaction was intercepted by appending 1 mL of 3,5-dinitrosalicylic acid reagent and incubated at 100 °C for 10 min. After cold dishing, the absorbance was measured at 570 nm using a spectrophotometer (Jenway 7315, UK). The amount of reducing sugars was determined using glucose as a standard for plotting the calibration curve (Figure 1). All the tests were carried out three times, and the production was expressed as an average value. A unit of the enzyme was acquainted as the quantity of the enzyme per one mL required to release one μmol of reducing sugar from a substrate per 60 s under the optimum trial conditions, including pH, temperature, and incubation time [35].

2.3. Optimization of Enzymatic Productivity under Submerged Fermentation (SmF)

Response-surface methodology (RSM) tactic using Box–Behnken design (BBD) was used to determine the optimum factors for boosted cellulase and pectinase production including A, temperature; B, pH; C, incubation period; D, inoculum size and E, substrate concentration (

Table 1. Box–Behnken design levels of independent factors.

No.	Factor	Variables	Units	Range
				Minimum	Maximum	Mean
1	A	Temperature	°C	20	40	30
2	B	pH	-	5	9	7
3	C	Incubation period	day	5	9	7
4	D	Inoculum size	mL	1	5	3
5	E	Substrate concentration	g	1	5	3

). Different pH values were adjusted using buffer system including: sodium acetate buffer (pH 5.0, 0.05 M); potassium phosphate buffer (pH 7.0, 0.05 M); glycine-NaOH buffer (pH 9.0, 0.05 M) [33]. The strains’ spore suspensions, of approximately 1 × 10⁷ spores/mL of varied sizes, were utilized to inoculate 250 mL flasks holding 100 mL liquid culture medium with different tangerine-powder concentrations and incubated under static conditions at different tested temperatures for diverse incubation periods. Forty-six experiments with the central points were employed to satisfy the polynomial pattern which is established on a Box–Behnken design (BBD, 5 variables) attained by Minitab 18^® software (Version18.1.1.0. LLC., Pennsylvania State University, State College, PA, USA). A three-level and five-factors experimental BBD was examined, and the number of the tests (N) was determined corresponding to the subsidiary equation:

N = 2k × (k − 1) + C0

(1)

where k is the digit of factors and C0 is the digit of central points, which equal to 6.

The impact of variables on the simulation (Y) was construed by employing a second-order polynomial equation that was utilized to foretell the quixotic states of the cellulase and pectinase biosynthesis.

Y = β₀ + β_AA + β_BB + β_CC + β_DD + β_EE + β_AAA² + β_BBB² + β_CCC² + β_DDD² + β_EEE² + β_ABAB + β_ACAC + β_ADAD + β_AEAE + β_BCBC + β_BDBD + β_BEBE + β_CDCD + β_CECE + β_DEDE

(2)

where Y is response variable; β₀ intercept; β_A, β_B, β_C, β_D and β_E are linear coefficients; β_AA, β_BB, β_CC, β_DD, and β_EE are square coefficients; β_AB, β_AC, β_AD, β_AE, β_BC, β_BD, β_BE, β_CD, β_CE, and β_DE are interaction coefficients; and A, B, C, D, E, A², B², C², D², E², AB, AC, AD, AE, BC, BD, BE, CD, CE and DE are levels of independent variables. The corresponding coefficients of variables, interaction variables, and contour graphs were obtained by Minitab 18^® software. By analyzing the regression equation and constructing the response plots, the ideal values of the tested variables were secured. The coefficient of limitation R² was used to express the fineness of profit of the polynomial equation, and the F test was used to determine its statistical significance level.

2.4. Partial Purification of Cellulase and Pectinase from Mucor Strains

After incubation period under optimum conditions obtained from runs no. 20 and 36 for cellulase from M. circinelloides and M. hiemalis, respectively, and runs no. 18 and 40 for pectinase from M. circinelloides and M. hiemalis, respectively, the contents of the broth culture were centrifuged at 10,000 rpm under cooling, and the supernatant was utilized for enzymes assay.

Crude enzymes solutions were partially purified by precipitation using cold acetone. Pre-cooled acetone (−20 °C) was subjoined to the enzyme solution until the volume ratio between enzyme solution and acetone reached 1:1; 1:2; 1:3; 1:4, and 1:5 (v/v). The solution was left at −20 °C overnight to allow protein precipitation. The precipitates were gathered by centrifugation at 10,000 rpm for 15 min and resuspended in a small volume of sodium-citrate buffer (pH 4.8, 0.05 M) [33]. Cellulase and pectinase activities and protein concentration were measured in the supernatant according to Miller [34] and Lowry et al. [36], respectively, utilizing standard of bovine serum albumin to generate the calibration curve spectrophotometerically at 750 nm (Figure 2). These samples were used for determining the activities of cellulase and pectinase, purification fold, and enzyme-recovery yield [37]. Protein was estimated and suitable precipitants (crude:acetone, 1:4) for characterization were selected. The following equations were used to calculate specific activity, yield, and purification fold of the partially purified enzymes.

$$\mathrm{Specific}\mathrm{activity}\left(\mathrm{U}/\mathrm{mg}\right)=\frac{Total activity}{Total protein}$$

(3)

$$\mathrm{Yield}(\%)=\frac{Total units in partially purified enzyme \times 100\%}{Total units in crude enzyme}$$

(4)

$$\mathrm{Purification}\mathrm{fold}=\frac{Specific activity of partially purified enzyme }{Specific activity of crude enzyme}$$

(5)

2.5. Characterization of Partially Purified Cellulase and Pectinase

The optimum temperature for partially purified cellulase and pectinase activities was determined in the range of 30–90 °C, and the thermal stability was determined after the pre-maintaining of enzyme at each temperature degree for one hour before screening. Ideal pH estimation was carried out at optimum temperature utilizing various buffers with values 3–11, including: glycine-HCl (pH 3.0, 0.05 M); sodium acetate buffer (pH 5.0, 0.05 M); potassium phosphate buffer (pH 7.0, 0.05 M); glycine-NaOH buffer (pH 9.0 and 11.0, 0.05 M) [33], and the pH stability was assessed after preserving with these pH values for 1 h before screening. In addition, cellulase and pectinase activities were valued after maintaining the enzyme with different metal ions (10 mM of K⁺, Mg²⁺, Ba²⁺ and Ni²⁺) for 1 h at optimum temperature and pH, including 70 and 50 °C for cellulase and 50 and 60 °C for pectinase from M. circinelloides and M. hiemalis, respectively, and pH 7.0. Cellulase and pectinase activities were assessed after processing with diverse detergents comprising tween 80 and 20 at concentrations of 1 and 5% v/v, urea (1 and 5% w/v), and Na₂CO₃ (50 and 75 mM), in comparison to control (100% activity) [38,39].

2.6. Data Analysis

All tests and measurements were repeated three times and the values were expressed as the mean ± SD. Significant differences were detected with one-way ANOVA, differences between means were considered using Duncan’s new multiple range test (DMRT) at the 0.05 significance level, using the SPSS software program (version No. 16).

3. Results

3.1. Preliminary and Quantitative Screening of Cellulase and Pectinase Production

The preliminary screening for extracellular fungal cellulase and pectinase revealed that both M. circinelloides and M. hiemalis had a high ability to produce cellulase and pectinase qualitatively on a solid assay medium. They were then quantitatively assayed under SmF and showed high cellulase production (15.70 and 13.85 U/mL, respectively) and pectinase activity (18.21 and 11.98 U/mL, respectively) on tangerine peel as a substrate under static conditions (

Table 2. Cellulase and pectinase production on different substrates under shaking and static conditions.

Substrate	Enzymes	Enzyme Activity (U/mL)
		M. circinelloides		M. hiemalis
		Shaking	Static	Shaking	Static
Pomegranate peel	Cellulase	3.54 ± 0.47 b	3.65 ± 0.34 b	4.53 ± 0.27 b	6.18 ± 0.27 b
	Pectinase	6.01 ± 0.58 B	7.22 ± 0.54 B	7.66 ± 0.41 B	7.06 ± 0.27 B
Tangerine peel	Cellulase	14.67 ± 0.71 a	15.70 ± 0.63 a	13.17 ± 0.36 a	13.85 ± 0.18 a
	Pectinase	12.45 ± 0.65 A	18.21 ± 0.59 A	11.20 ± 0.29 A	11.98 ± 0.24 A
Wheat straw	Cellulase	0.78 ± 0.02 c	0.37 ± 0.04 c	2.66 ± 0.16 c	3.79 ± 0.18 c
	Pectinase	0.99 ± 0.08 C	0.60 ± 0.06 C	1.90 ± 0.20 C	3.51 ± 0.02 C

The data were given as averages of three replicates (mean ± SD). Values followed by the different letters are significantly different at p ˂ 0.05. Small superscripted letters i.e., “^a” were affiliated for cellulase activities on different substrates under shaking and static conditions, while capital superscripted letters i.e., “^A” were affiliated for pectinase activities on different substrates under shaking and static conditions.

).

3.2. Response-Surface Methodology for Optimization of Cellulase and Pectinase Production

Table 1 shows the independent factors with their competent levels employed in the optimization of cellulase and pectinase production, while the BBD of the independent factors along with the predicted as well as experimental values are depicted in

Table 3. Box–Behnken design of optimization variables with experimental and predicted cellulase and pectinase activities of both M. circinelloides and M. hiemalis.

Run Order	Variables					Cellulase Activity (U/mL)				Pectinase Activity (U/mL)
						M. circinelloides		M. hiemalis		M. circinelloides		M. hiemalis
	A	B	C	D	E	Exp.	Pred.	Exp.	Pred.	Exp.	Pred.	Exp.	Pred.
1	30	7	5	1	3	20.20	22.28	21.19	22.76	23.67	24.82	18.14	19.80
2	30	9	7	1	3	17.15	18.74	29.86	27.72	19.96	21.94	36.62	35.36
3	30	7	5	5	3	19.30	20.51	18.64	18.46	20.20	21.14	24.74	24.04
4	20	7	7	3	5	27.46	29.96	26.64	25.32	32.99	33.60	35.63	34.70
5	30	7	9	3	1	11.54	14.36	5.77	6.36	8.82	9.90	8.90	7.79
6	20	7	5	3	3	32.66	30.44	17.89	17.89	28.87	28.05	19.05	19.42
7	30	5	7	3	1	14.02	13.13	8.24	8.07	12.94	15.05	14.10	15.32
8	30	7	7	1	5	26.56	27.16	28.87	28.29	31.75	30.80	37.94	37.16
9	30	9	7	5	3	16.08	16.69	14.02	13.76	13.77	14.43	32.58	32.87
10	30	7	7	3	3	13.60	14.53	22.27	23.09	16.08	15.82	29.28	30.13
11	30	7	7	5	1	9.15	8.76	5.52	5.14	8.65	8.78	15.67	17.06
12	40	7	7	3	5	19.05	21.32	29.36	27.35	18.14	20.12	29.61	29.05
13	40	7	7	3	1	8.82	9.18	6.59	5.37	11.21	11.48	11.54	11.52
14	30	7	7	3	3	14.18	14.53	21.85	23.09	16.82	15.82	29.86	30.13
15	20	7	7	3	1	8.57	9.16	9.07	8.54	9.81	8.70	12.37	11.97
16	30	9	7	3	1	7.99	6.21	10.72	11.02	10.22	10.27	21.94	23.24
17	30	7	7	3	3	15.34	14.53	23.92	23.09	15.83	15.82	30.52	30.13
18	30	7	9	3	5	22.84	23.03	22.93	23.60	38.02	39.62	27.55	27.86
19	20	9	7	3	3	20.04	20.63	18.80	20.07	21.03	21.51	36.29	35.89
20	30	7	5	3	5	37.20	34.61	29.44	30.80	29.69	28.04	28.87	29.11
21	20	7	7	1	3	20.95	19.73	18.97	18.98	21.85	21.30	23.92	25.16
22	30	7	7	5	5	21.52	22.51	24.58	23.53	23.26	22.78	28.45	30.26
23	20	7	7	5	3	16.99	17.22	15.67	16.00	14.18	14.69	27.88	27.34
24	30	5	5	3	3	18.97	20.63	16.66	15.30	24.25	25.16	21.03	20.32
25	20	7	9	3	3	16.57	14.49	18.97	19.68	22.43	22.02	22.76	22.62
26	30	7	7	1	1	8.74	7.97	7.83	7.92	11.62	11.26	11.29	10.10
27	40	7	9	3	3	23.50	22.34	12.37	12.26	20.62	21.33	15.25	15.19
28	40	7	7	1	3	15.50	14.84	18.47	19.20	14.84	14.59	23.50	24.26
29	30	9	9	3	3	17.65	18.63	13.19	12.12	24.99	24.37	22.27	22.85
30	40	9	7	3	3	17.07	16.40	20.20	22.26	17.32	15.00	29.05	28.68
31	40	5	7	3	3	14.18	14.54	17.32	17.62	17.07	15.57	28.29	29.12
32	30	9	7	3	5	33.82	31.39	27.63	29.33	33.98	32.40	39.18	39.17
33	30	7	7	3	3	14.51	14.53	22.68	23.09	15.25	15.82	28.04	30.13
34	30	7	5	3	1	10.30	10.35	7.99	9.28	26.39	24.22	10.07	8.90
35	20	5	7	3	3	17.32	18.93	21.44	20.95	18.47	19.76	27.22	28.01
36	30	9	5	3	3	23.67	24.77	33.82	31.95	24.74	26.07	35.05	34.92
37	40	7	5	3	3	15.25	13.96	24.99	24.17	17.73	18.04	20.29	20.74
38	30	7	9	5	3	18.14	16.56	14.92	13.93	18.97	18.20	19.63	18.65
39	30	5	9	3	3	17.65	19.20	25.57	25.01	25.15	24.12	30.02	30.03
40	30	5	7	3	5	22.43	20.90	27.30	28.52	25.98	26.45	39.76	39.66
41	30	7	7	3	3	14.10	14.53	24.74	23.09	14.43	15.82	31.75	30.13
42	30	7	9	1	3	19.38	18.66	16.41	17.16	25.57	25.01	21.44	22.83
43	30	5	7	5	3	16.90	15.03	20.62	22.08	17.89	16.11	31.92	31.68
44	40	7	7	5	3	12.70	13.49	13.60	14.65	9.89	10.70	23.17	22.14
45	30	7	7	3	3	15.42	14.53	23.09	23.09	16.49	15.82	31.34	30.13
46	30	5	7	1	3	17.73	16.83	16.08	15.65	19.54	19.09	30.93	29.13

Exp.: experimental; Pred.: predicted.

. The production of cellulase by M. circinelloides was predicted by the following equation:

Y (U/mL) = 147.0 − 2.858A − 7.08B − 20.72C − 0.38D + 5.22E + 0.01299A² + 0.450 B² + 1.121C² + 0.124D² + 0.395E² − 0.0021AB + 0.3042AC + 0.0144AD − 0.1083AE − 0.294BC − 0.015BD + 1.088BE − 0.021CD − 0.975CE − 0.340DE

(6)

While the production of cellulase by M. hiemalis was fitted by the following equation:

Y (U/mL) = −189.4 + 2.187A + 16.02B + 23.91C + 13.19D + 11.66E − 0.02728A² − 0.034B² − 0.465C² − 0.788D² − 0.929E² + 0.0691AB − 0.1712AC − 0.0196AD + 0.0650AE − 1.846BC − 1.274BD − 0.134BE+ 0.067CD − 0.268CE − 0.124DE

(7)

M. circinelloides pectinase biosynthesis was fitted by the following equation:

Y (U/mL) = 168.2 − 0.245A − 8.11B − 31.47C + 2.49D − 9.01E − 0.00215A² + 0.589B² + 1.689C² − 0.071D² + 0.718E² − 0.0289AB + 0.1165AC + 0.0340AD − 0.2032AE − 0.041BC − 0.284BD − + 0.670BE − 0.196CD + 1.619CE − 0.345DE

(8)

Meanwhile, M. hiemalis pectinase activity was predicted by the following equation:

Y (U/mL) = −228.9 + 3.868A + 2.40B + 38.68C + 12.78D + 20.38E − 0.03621A² + 0.979B² − 1.754C² − 0.447D² − 1.175E² − 0.1039AB − 0.1039AC − 0.0536AD − 0.0650AE − 1.361BC − 0.315BD − 0.526BE − 0.526CD − 0.009CE − 0.866DE

(9)

The highest cellulase activities of both M. circinelloides (37.20 U/mL) and M. hiemalis (33.82 U/mL) were obtained from runs no. 20 and 36, respectively. Run no. 20, for M. circinelloides, consisted of incubation temperature 30 °C, pH value 7, incubation period 5 days, inoculum size 3 mL, and substrate concentration 5 g/100 mL, while run no. 36 for M. hiemalis consisted of incubation temperature 30 °C, pH value 9, incubation period 5 days, inoculum size 3 mL, and substrate concentration 3 g/100 mL. Meanwhile, the highest pectinase production of both M. circinelloides (38.02 U/mL) and M. hiemalis (39.76 U/mL) were obtained from runs no. 18 and 40, respectively. Run no. 18 for M. circinelloides consisted of incubation temperature 30 °C, pH value 7, incubation period 9 days, inoculum size 3 mL, and substrate concentration 5 g/100 mL. In comparison, run no. 40 for M. hiemalis consisted of incubation temperature 30 °C, pH value 5, incubation period 7 days, inoculum size 3 mL, and substrate concentration 5 g/100 mL.

Analysis of variance (ANOVA) for the cellulase and pectinase quadric model of M. circinelloides and M. hiemalis is shown in

Table 4. ANOVA for the experimental results of cellulase biosynthesis by M. circinelloides.

Source	Sum of Squares	Degree of Freedom	Mean of Squares	F-Value	p-Value	Prob > F
Model	1747.96	20	87.40	25.63	0.000	Significant
Linear	1244.08	5	248.82	72.96	0.000
A	74.32	1	74.32	21.79	0.000
B	12.73	1	12.73	3.73	0.065
C	57.29	1	57.29	16.80	0.000
D	14.87	1	14.87	4.36	0.047
E	1084.86	1	1084.86	318.11	0.000
Square	187.20	5	37.44	10.98	0.000
A2	14.73	1	14.73	4.32	0.048
B2	28.31	1	28.31	8.30	0.008
C2	175.35	1	175.35	51.42	0.000
D2	2.14	1	2.14	0.63	0.436
E2	21.82	1	21.82	6.40	0.018
2-Way Interaction	316.68	10	31.67	9.29	0.000
AB	0.01	1	0.01	0.00	0.965
AC	148.07	1	148.07	43.42	0.000
AD	0.33	1	0.33	0.10	0.757
AE	18.76	1	18.76	5.50	0.027
BC	5.53	1	5.53	1.62	0.215
BD	0.02	1	0.02	0.00	0.947
BE	75.75	1	75.75	22.21	0.000
CD	0.03	1	0.03	0.01	0.930
CE	60.78	1	60.78	17.82	0.000
DE	7.41	1	7.41	25.63	0.153
Residual	85.26	25	3.41
Lack of Fit	82.65	20	4.13	7.93	0.150	Not significant
Pure error	2.61	5	0.52
Total	1833.22	45

R²: 0.9535, adjusted R²: 0.9163, and predicted R²: 0.8176.

,

Table 5. ANOVA for the experimental results of cellulase biosynthesis by M. hiemalis.

Source	Sum of Squares	Degree of Freedom	Mean of Squares	F-Value	p-Value	Prob > F
Model	2272.99	20	113.65	56.13	0.000	Significant
Linear	1676.41	5	335.28	165.60	0.000
A	1.29	1	1.29	0.64	0.433
B	14.09	1	14.09	6.96	0.014
C	102.55	1	102.55	50.65	0.000
D	56.67	1	56.67	27.99	0.000
E	1501.82	1	1501.82	741.79	0.000
Square	205.80	5	41.16	20.33	0.000
A2	64.93	1	64.93	32.07	0.000
B2	0.16	1	0.16	0.08	0.780
C2	30.24	1	30.24	14.93	0.001
D2	86.81	1	86.81	42.88	0.000
E2	120.61	1	120.61	59.57	0.000
2-Way Interaction	390.78	10	39.08	19.30	0.000
AB	7.64	1	7.64	3.77	0.063
AC	46.89	1	46.89	23.16	0.000
AD	0.61	1	0.61	0.30	0.587
AE	6.75	1	6.75	3.34	0.080
BC	218.07	1	218.07	107.71	0.000
BD	103.80	1	103.80	51.27	0.000
BE	1.15	1	1.15	0.57	0.458
CD	0.29	1	0.29	0.14	0.709
CE	4.60	1	4.60	2.27	0.144
DE	0.98	1	0.98	0.48	0.493
Residual	50.61	25	2.02
Lack of Fit	44.83	20	2.24	1.94	0.239	Not significant
Pure error	5.78	5	1.16
Total	2323.61	45

R²: 0.9782, adjusted R²: 0.9608, and predicted R²: 0.9192.

,

Table 6. ANOVA for the experimental results of pectinase biosynthesis by M. circinelloides.

Source	Sum of Squares	Degree of Freedom	Mean of Squares	F-Value	p-Value	Prob > F
Model	2169.50	20	108.47	48.34	0.000	Significant
Linear	1358.31	5	271.66	121.07	0.000
A	114.58	1	114.58	51.06	0.000
B	1.38	1	1.38	0.62	0.440
C	7.52	1	7.52	3.35	0.079
D	110.20	1	110.20	49.11	0.000
E	1124.62	1	1124.62	501.19	0.000
Square	508.38	5	101.68	45.31	0.000
A2	0.40	1	0.40	0.18	0.675
B2	48.46	1	48.46	21.60	0.000
C2	398.37	1	398.37	177.54	0.000
D2	0.70	1	0.70	0.31	0.581
E2	71.98	1	71.98	32.08	0.000
2-Way Interaction	302.81	10	30.28	13.49	0.000
AB	1.33	1	1.33	0.59	0.448
AC	21.73	1	21.73	9.68	0.005
AD	1.85	1	1.85	0.83	0.372
AE	66.03	1	66.03	29.43	0.000
BC	0.11	1	0.11	0.05	0.827
BD	5.15	1	5.15	2.29	0.142
BE	28.75	1	28.75	12.81	0.001
CD	2.46	1	2.46	1.09	0.305
CE	167.76	1	167.76	74.76	0.000
DE	7.64	1	7.64	3.40	0.077
Residual	56.10	25	2.24
Lack of Fit	52.32	20	2.62	3.46	0.086	Not significant
Pure error	3.78	5	0.76
Total	2225.59	45

R²: 0.9748, adjusted R²: 0.9546, and predicted R²: 0.9035.

and

Table 7. ANOVA for the experimental results of pectinase biosynthesis by M. hiemalis.

Source	Sum of Squares	Degree of Freedom	Mean of Squares	F-Value	p-Value	Prob > F
Model	2938.40	20	146.92	88.77	0.000	Significant
Linear	1720.06	5	344.01	207.85	0.000
A	37.22	1	37.22	22.49	0.000
B	55.19	1	55.19	33.34	0.000
C	5.55	1	5.55	3.35	0.079
D	0.00	1	0.00	0.00	0.962
E	1622.11	1	1622.11	980.06	0.000
Square	962.23	5	192.45	116.27	0.000
A2	114.40	1	114.40	69.12	0.000
B2	133.71	1	133.71	80.79	0.000
C2	429.69	1	429.69	259.61	0.000
D2	27.85	1	27.85	16.83	0.000
E2	192.78	1	192.78	116.48	0.000
2-Way Interaction	256.11	10	25.61	15.47	0.000
AB	17.29	1	17.29	10.45	0.003
AC	19.12	1	19.12	11.55	0.002
AD	4.60	1	4.60	2.78	0.108
AE	6.75	1	6.75	4.08	0.054
BC	118.59	1	118.59	71.65	0.000
BD	6.33	1	6.33	3.83	0.062
BE	17.70	1	17.70	10.70	0.003
CD	17.70	1	17.70	10.70	0.003
CE	0.01	1	0.01	0.00	0.954
DE	48.02	1	48.02	29.01	0.000
Residual	41.38	25	1.66
Lack of Fit	31.96	20	1.60	0.85	0.646	Not significant
Pure error	9.41	5	1.88
Total	2979.78	45

R²: 0.9861, adjusted R²: 0.9750, and predicted R²: 0.9525.

. Model terms with a p-value < 0.05 were deemed significant. The model’s F values for cellulase produced by M. circinelloides and M. hiemalis, (25.63 and 56.13, respectively) and the model’s F values for pectinase produced by the same fungi (48.34 and 88.77, respectively) indicate that the model is significant. Values of “Prob > F” < 0.05 indicated that the model terms are significant. In cellulase activity, A, C, D, E, A², B², C², E², AC, AE, BE, and CE are significant model terms for M. circinelloides; while for M. hiemalis, B, C, D, E, A², C², D², E², AC, BC, and BD are significant model terms. Regarding pectinase biosynthesis, A, D, E, B², C², E², AC, AE, BE, and CE are significant model terms for M. circinelloides; while for M. hiemalis, A, B, E, A², B²,C², D², E², AB, AC, BC, BE, CD, and DE are significant model terms. The “lack of fit F-value” of 7.93 and 1.94 for M. circinelloides and M. hiemalis cellulase, and values of 3.46 and 0.85 for their pectinase, respectively, indicated that the lack of fit is insignificant concerning the pure error. A non-significant lack of fit is proper for the model to be convenient. The multiple correlation coefficient R² = 0.9535 for M. circinelloides and R²= 0.9782 for M. hiemalis cellulase and R² = 0.9748 for M. circinelloides and R² = 0.9861 for M. hiemalis pectinase, nigh to one, indicated preferable interconnection between experimental and predicted values and elucidated the model accuracy with an upgrade response. Regression values were in harmony with adjusted and predicted R².

Contour plots explained the relationship between parameters and defined each factor’s optimum scale for cellulase (Figure 3a–d and Figure 4a–d) and pectinase (Figure 5a–d and Figure 6a–d) activities by M. circinelloides and M. hiemalis, respectively. The response surface plot constructed any two variables, while other variables were maintained at their optimal level. Contour plots of cellulase and pectinase activities by M. circinelloides revealed significant interactions derived from the analysis of variance and described as significant model terms (Figure 3b,d,g,i and 5b,d,g,i). On the other hand, contour plots of the interactions obtained by ANOVA illustrated model terms significantly influenced cellulase and pectinase production by M. hiemalis (Figure 4b,e,f and 6a,b,e,g,h,j). The remaining interactions insignificantly influenced enzymes production.

3.3. Partial Purification of Cellulase and Pectinase

Extracellular cellulase and pectinase from M. circinelloides and M. hiemalis were partially purified from broth cultures by using different acetone concentrations. The highest cellulase (6.37 and 8.10 U/mL) and pectinase (7.23 and 5.50 U/mL) activities from M. circinelloides and M. hiemalis, respectively, were obtained via the precipitation of crude filtrate with acetone at the ratio 1:4 (

Table 8. Precipitation of cellulase and pectinase produced by M. circinelloides and M. hiemalis using different concentrations of acetone.

Ratio (Crude:Acetone)	Cellulase Activity (U/mL)		Pectinase Activity (U/mL)
	M. circinelloides	M. hiemalis	M. circinelloides	M. hiemalis
1:1	3.11 ± 0.08 d	4.85 ± 0.08 d	4.60 ± 0.24 e	2.87 ± 0.24 e
1:2	3.63 ± 0.10 c	5.36 ± 0.10 c	5.06 ± 0.07 d	3.32 ± 0.07 d
1:3	5.36 ± 0.11 b	7.09 ± 0.11 b	7.08 ± 0.06 b	5.34 ± 0.06 b
1:4	6.37 ± 0.04 a	8.10 ± 0.04 a	7.23 ± 0.05 a	5.50 ± 0.05 a
1:5	5.43 ± 0.10 b	7.05 ± 0.26 b	6.89 ± 0.11 c	5.22 ± 0.07 c

The data were given as averages of three replicates (mean ± SD). Values followed by the different letters are significantly different at p < 0.05.

). Cellulase purification produced 1.73- and 2.03-fold purification, 31.12 and 32.02% cellulase recovery with specific activity of 199.41 and 163.43 U/mg from M. circinelloides and M. hiemalis, respectively, while 1.74- and 1.99-fold purification, 31.26 and 31.51% recovery with specific activity of 216.83 and 215.36 U/mg were obtained from M. circinelloides and M. hiemalis pectinase, respectively (

Table 9. Summary of specific activity, yield and purification fold of cellulase and pectinase produced by M. circinelloides and M. hiemalis.

Purification Steps	Fungal Strain	Enzyme	Total Activity (U/mL)	Total Protein (mg/mL)	Specific Activity (U/mg)	Yield (%)	Purification Fold
Culture supernatant	M. circinelloides	Cellulase	9918.06 ± 63.92	86.05 ± 4.49	115.58 ± 6.33	100.00 ± 0.00	1.00 ± 0.00
		Pectinase	10,740.62 ± 39.60	86.05 ± 4.49	125.17 ± 6.73	100.00 ± 0.00	1.00 ± 0.00
	M. hiemalis	Cellulase	8258.02 ± 91.15	102.39 ± 1.26	80.68 ± 1.86	100.00 ± 0.00	1.00 ± 0.00
		Pectinase	11,063.86 ± 192.37	102.39 ± 1.26	108.07 ± 2.03	100.00 ± 0.00	1.00 ± 0.00
Acetone	M. circinelloides	Cellulase	3087.06 ± 67.34	15.48 ± 0.08	199.41 ± 5.27	31.12 ± 0.61	1.73 ± 0.05
		Pectinase	3357.16 ± 17.87	15.48 ± 0.08	216.83 ± 1.09	31.26 ± 0.22	1.74 ± 0.10
	M. hiemalis	Cellulase	2644.05 ± 27.34	16.19 ± 0.30	163.43 ± 4.34	32.02 ± 0.02	2.03 ± 0.04
		Pectinase	3485.80 ± 57.71	16.19 ± 0.30	215.36 ± 0.70	31.51 ± 0.03	1.99 ± 0.04

The data were given as averages of three replicates (mean ± SD).

).

3.4. Characterization of Partially Purified Cellulase and Pectinase

M. circinelloides partially purified cellulase was highly active at 70 °C (total activity 100%) and decreased gradually at 60–30 °C and 80–90 °C, while the cellulase activity of M. hiemalis was highly active at 50 °C (total activity 100%) and decreased gradually at 40–30 °C and 60–90 °C. The relative cellulase stability from both M. circinelloides and M. hiemalis was high at 30 °C and decreased in the range of 40–90 °C (Figure 7a,b). Partially purified pectinase from M. circinelloides showed the best activity at 50 °C (total activity 100%) and decreased gradually at 40–30 °C and 60–90 °C; meanwhile, the pectinase activity of M. hiemalis was highly active at 60 °C (total activity 100%) and decreased gradually at 50–30 °C and 70–90 °C. The relative pectinase stability from both M. circinelloides and M. hiemalis was high at 30 °C and decreased in the range of 40–90 °C (Figure 7c,d). Furthermore, partially purified cellulase and pectinase from both strains had the highest activity and stability (100%) at pH 7.0, and then, at high or low pH values, the activity and stability of enzymes were reduced (Figure 8a–d).

The cellulase activity of both strains after incubation with 10 mM of K⁺, Mg²⁺, Ba²⁺ and Ni²⁺ was decreased except for K⁺, which increased M. circinelloides partially purified cellulase relative activity and stability by 20.05 and 2.78%, respectively, than control. In contrast, stability decreased except for Ba²⁺ with the M. circinelloides enzyme and K⁺, Mg²⁺, and Ni²⁺ with the M. hiemalis enzyme. Detergents including tween 80 (1 and 5%), tween 20 (1%), and urea (1%), increased M. circinelloides partially purified cellulase relative activity, while a remarkable decrease in relative stability was reported at all concentrations of the tested detergents for enzymes of both strains (

Table 10. Effects of metal ions and detergents on activity and stability of partially purified cellulase from M. circinelloides and M. hiemalis.

Metal Ions and Detergents	Conc.	M. circinelloides		M. hiemalis
		Relative Activity (%)	Relative Stability (%)	Relative Activity (%)	Relative Stability (%)
Control	0	100.00 ± 0.00 e	-	100.00 ± 0.00 a	-
K+	10 mM	120.05 ± 0.69 b	102.78 ± 0.78 A	61.18 ± 0.79 g	94.62 ± 0.76 A
Mg2+	10 mM	60.36 ± 0.69 i	42.83 ± 0.57 I	34.12 ± 0.20 k	73.11 ± 0.39 B
Ba2+	10 mM	30.49 ± 0.21 l	59.83 ± 0.38 H	43.23 ± 0.59 j	35.01 ± 0.73 I
Ni2+	10 mM	41.58 ± 0.24 k	30.64 ± 0.11 J	11.12 ± 0.91 l	50.43 ± 0.92 F
Tween 80	1% (v/v)	105.60 ± 0.81 d	89.41 ± 0.32 C	69.03 ± 0.21 c	58.16 ± 0.93 D
	5% (v/v)	108.43 ± 0.78 c	97.00 ± 0.89 B	68.87 ± 0.25 c,d	49.95 ± 0.75 F
Tween 20	1% (v/v)	106.39 ± 0.24 d	77.17 ± 0.69 F	71.01 ± 0.38 b	42.68 ± 0.83 G
	5% (v/v)	87.46 ± 0.29 g	68.67 ± 0.74 G	64.08 ± 0.35 e	64.29 ± 0.96 C
Urea	1% (w/v)	121.78 ± 0.43 a	88.38 ± 0.38 D	67.97 ± 0.77 d	58.88 ± 0.42 D
	5% (w/v)	91.33 ± 0.35 f	79.55 ± 0.80 E	62.44 ± 0.54 f	56.60 ± 0.99 E
Na2CO3	50 mM	75.25 ± 0.50 h	69.55 ± 0.61 G	56.24 ± 0.54 i	39.66 ± 0.91 H
	75 mM	53.46 ± 0.78 j	42.06 ± 0.76 I	60.14 ± 0.98 h	39.65 ± 0.95 H

The data were given as averages of three replicates (mean ± SD). Values followed by the different letters are significantly different at p ˂ 0.05.

).

After incubation with 10 mM K⁺, Mg²⁺, Ba²⁺, and Ni²⁺, the partially purified pectinase activity of both strains decreased with the exception of K⁺, which increased M. circinelloides pectinase activity by 16.38% compared to the control; meanwhile, stability increased except for K⁺ and Ba²⁺ with M. circinelloides enzyme, and K⁺ and Mg²⁺ with M. hiemalis enzyme. Detergents including tween 80 (1%) increased M. circinelloides pectinase activity by 12.92% compared to the control. In contrast, there was a significant decrease in M. circinelloides and M. hiemalis partially purified pectinase activity with other tested detergents. A fluctuation in significant increase and decrease in pectinase stability of both strains was reported at low and high concentrations of tween 80, tween 20, urea and Na₂CO₃ (

Table 11. Effects of metal ions and detergents on activity and stability of partially purified pectinase from M. circinelloides and M. hiemalis.

Metal Ions and Detergents	Conc.	M. circinelloides		M. hiemalis
		Relative Activity (%)	Relative Stability (%)	Relative Activity (%)	Relative Stability (%)
Control	0	100.00 ± 0.00 c	-	100.00 ± 0.00 a	-
K+	10 mM	116.38 ± 0.68 a	110.11 ± 0.71 A	68.73 ± 0.46 c	68.15 ± 0.75 A
Mg2+	10 mM	98.56 ± 0.78 d	100.10 ± 0.41 C	53.57 ± 0.26 e	53.51 ± 0.44 C
Ba2+	10 mM	61.70 ± 0.57 f	23.28 ± 0.16 I	61.16 ± 0.49 d	67.13 ± 0.94 B
Ni2+	10 mM	26.68 ± 0.35 j	70.47 ± 0.53 G	27.00 ± 0.61 l	46.14 ± 0.72 G
Tween 80	1% (v/v)	112.92 ± 0.87 b	102.08 ± 0.82 B	35.57 ± 0.67 i	52.42 ± 0.41 D
	5% (v/v)	37.82 ± 0.22 i	89.22 ± 0.43 E	51.83 ± 0.24 f	48.55 ± 0.27 F
Tween 20	1% (v/v)	55.80 ± 0.71 g	90.43 ± 0.48 D	41.08 ± 0.42 g	52.07 ± 0.49 D
	5% (v/v)	75.14 ± 0.88 e	85.95 ± 0.59 F	70.61 ± 0.51 b	50.20 ± 0.80 E
Urea	1% (w/v)	62.62 ± 0.64 f	70.44 ± 0.88 G	38.88 ± 0.30 h	50.85 ± 0.74 E
	5% (w/v)	47.27 ± 0.61 h	85.52 ± 0.89 F	30.65 ± 0.49 k	48.51 ± 0.73 F
Na2CO3	50 mM	99.42 ± 0.47 d	58.21 ± 0.82 H	33.83 ± 0.52 j	38.68 ± 0.75 I
	75 mM	56.26 ± 0.41 g	58.13 ± 0.93 H	31.45 ± 0.51 k	40.97 ± 0.55 H

The data were given as averages of three replicates (mean ± SD). Values followed by the different letters are significantly different at p ˂ 0.05.

).

4. Discussion

Fungi are an excellent source for the production of various beneficial enzymes. The lignin present in the polymer matrix in plant cell walls form a hydrophobic network which inhibits the access of microbial enzymes to degrade cell-wall components [40,41]. Therefore, the lowest content of lignin in the citrus peels could enhance microbial-enzyme production due to their tissue structure flexibility, allowing the access of the microorganism to the cellulose, pectin and hemicellulose [18]. In this research, M. circinelloides and M. hiemalis were investigated as potential sources for producing cellulase and pectinase on tangerine peel by submerged fermentation. The type of substrate and the presence of growth factors affect the fungal growth and the biosynthesis of enzymes. Therefore, there is an urgent need to enhance the biosynthesis of the enzymes by optimizing fungal production.

In the current research, M. circinelloides and M. hiemalis afforded high cellulase production (37.20 U/mL and 33.82 U/mL, respectively) and pectinase (38.02 U/mL and 39.76 U/mL, respectively) at the optimum parameters, which consisted of, respectively, incubation temperature 30 and 30 °C, pH value 7 and 9, incubation period 5 and 5 days, inoculum size 3 and 3 mL, and substrate concentration 5 and 3 g/100 mL; while, for pectinase, optimum conditions included incubation temperature 30 and 30 °C, pH value 7 and 5, incubation period 9 and 7 days, inoculum size 3 and 3 mL, and substrate concentration 5 and 5 g/100 mL, respectively, for M. circinelloides and M. hiemalis. The multiple correlation coefficient R² = 0.9535 for M. circinelloides and R² = 0.9782 for M. hiemalis cellulase and R² = 0.9748 for M. circinelloides and R² = 0.9861 for M. hiemalis pectinase, nigh to one, indicated that experimental and predicted values are well-correlated and predicted values elucidated the model accuracy with upgrade response. The low p values, which are attained by the F test, and high R² values indicated that the employed model attained a high significance, and its sufficiency was confirmed [42]. Similarly, Aspergillus niger-ATCC 1640 achieved 0.6045 μmol/mL pectinase production using Citrus macroptera peel (8.4 g/L) in solid-state fermentation. RSM results indicated that the experimental response for pectinase production was convenient with fitted data (R² = 0.9836) [43]. The highest cellulase activity (5.60 IU/mL) was obtained after incubation for 4 days and 5% substrate concentration with pH 5.0 at 30 °C using RSM of Trichoderma viride in SmF of the seed pods of the silk cotton tree [44]. Rhizopus delemar F2 optimal variable values for the maximum production of cellulase (10.40 U/gds) and of pectinase (31.20 U/gds) using solid state fermentation on apple pomace substrate included a moisture ratio of 1:3:5 for 7 days at 30 °C [45].

Conversely, statistical design for the maximum production of pectinase (179.83 U/g in SSF and 1.64 U/mL in SmF) and cellulase (10.81 U/g in SSF and 0.36 U/mL in SmF) by Aspergillus niger NCIM 548 was achieved at optimum conditions in SmF consisting of carbon source concentration 65 g/L, pH 4.6, and time 126 h; while in SSF, moisture content was 65% and pH 4.80 for 156 h [46]. On the other hand, the optimum condition for A. oryzae producing the maximal pectinase (139.56 U/gds) and cellulase (6.01 U/gds) was 67% of moisture content with pH 5.9 at 33 °C, and for 71.8 h of fermentation on soybean residue [47]. Optimum cellulase activity (124.94 U/g) was attained at 1.5% w/v rice straw with pH 7 at 30 °C for 8 days by Aspergillus terreus RS2 [48]. Ramos-Ibarra et al. [18] utilized RSM for a high production of cellulase (1.0 U/g after 24 h) and pectinase (12.3 U/g after 120 h) using Mucor racemosus N9C1 on orange peels by SSF in humidity 70% at 30 °C. They anticipated that increased enzymatic activity reaching its maximum and decreasing at the end of the fermentation period may be attributed to enzyme hydrolysis by proteases.

In the present investigation, M. circinelloides and M. hiemalis partially purified cellulase and pectinase showed 6.37 and 8.10 U/mL and 7.23 and 5.50 U/mL activities, respectively, and cellulase 1.73- and 2.03-fold purification, 31.12 and 32.02% cellulase recovery with specific activity of 199.41 and 163.43 U/mg; while 1.74- and 1.99-fold purification, 31.26 and 31.51% recovery with specific activity of 216.83 and 215.36 U/mg, respectively, were obtained. Our results were in agreement with Almowallad et al. [25], who utilized Aspergillus niger AUMC 4156, Penicillium oxalicum AUMC 4153, and Paecilomyces variotii AUMC 4149 on orange peel (3% w/v) by SmF and obtained pectinase activity in static (52.22, 14.06 and 49.26%) and shaken cultures (48.89, 2.94, 50.00%), respectively. Orange peel as a sole carbon source afforded the highest protein content in filtrates with all tested fungal strains in stirred (2.57, 3.75, and 3.40 mg/mL) and static cultures (4.74, 4.45, and 4.98 mg/mL), respectively. Statistical-derived optimum conditions for crude cellulase produced by the SmF of A. niger using A. hypogaea shells as a carbon source involved 120 h incubation with pH 4 at 40 °C, along with of 13 × 10⁵ CFU/mL inoculum size, while purified cellulase resulted in a 68.12-fold purification with yield 3.87% and specific activity of 484.3 U/mg [23].

Partially purified cellulase and pectinase from M. circinelloides and M. hiemalis demonstrated the highest activity at neutral pH, and 70 and 50 °C, for cellulase and 50 and 60 °C, for pectinase, respectively. Thakur et al. [13] highlighted that each enzymatic application requires unique properties with respect to specificity, stability, temperature, and pH dependence. High temperature increases the solubility of reactants and products by decreasing viscosities, resulting in faster hydrolysis [49], and longer active life under high temperatures would make enzymes favorable for efficient biomass conversion. Therefore, thermo-stability is the most significant property for the enzyme used under extreme bioprocessing conditions to be efficient [50]. Optimally, purified pectinase from Rhizomucor pusillus was active at 55 °C and pH 5.0, and showed stability up to 50 °C and a pH range between 4.0 and 5.0 for 120 min incubation, while the stability decreased rapidly over pH 5.0 and 60 °C [51]. Aspergillus sp. Gm showed the highest pectinase production by SmF using 1% pectin at 30 °C for 48 h; meanwhile, the purified pectinase activity optimum temperature was 30 °C, 75.4 U/mL; pH was 5.8, 72.3 U/mL; and substrate concentration 0.5%, 112.0 U/mL, and enzyme thermo-stability decreased 50% within 10 min incubation at 60 °C [52].

Our results revealed a decrease in partially purified pectinase activity of both strains after incubation with 10 mM K⁺, Mg²⁺, Ba²⁺, and Ni²⁺, while 10 mM K⁺ increased M. circinelloides pectinase activity by 16.38%. In contrast, notable pectinase stability increased with Mg²⁺ and Ni²⁺ for M. circinelloides enzyme, and with Ba²⁺, and Ni²⁺ for M. hiemalis enzyme. Thakur et al. [13] tested phenolic acids (0.05 mM), metal ions (Mn²⁺, Co²⁺, Mg²⁺, Fe³⁺, Al³⁺, Hg²⁺, and Cu²⁺), and thiols, and found that they exerted an inhibitory impact on the polygalacturonase from Mucor circinelloides ITCC 6025. They suggested that the enzyme did not need any metal ions for its activity expression.

5. Conclusions

The present investigation utilized the response-surface methodology via the Box–Behnken design to improve cellulase and pectinase production by M. circinelloides and M. hiemalis strains. The experimental results are consistent with predicted responses. The produced enzymes were partially purified and characterized. The optimum parameters for cellulase production by M. circinelloides were incubation temperature 30 °C, pH value 7, incubation period 5 days, inoculum size 3 mL, and substrate concentration 5 g/100 mL, and for pectinase production were incubation temperature 30 °C, pH value 7, incubation period 9 days, inoculum size 3 mL, and substrate concentration 5 g/100 mL. For M. hiemalis, the optimum parameters for cellulase production were incubation temperature 30 °C, pH value 9, incubation period 5 days, inoculum size 3 mL, and substrate concentration 3 g/100 mL, and for pectinase production were incubation temperature 30 °C, pH value 5, incubation period 7 days, inoculum size 3 mL, and substrate concentration 5 g/100 mL. The influence of single, interaction and quadratic factors on cellulase and pectinase production was investigated using non-linear regression equations with significant R² and p values. The partial purification of M. circinelloides and M. hiemalis cellulase produced 1.73- and 2.03-fold purification with 31.12 and 32.02% recovery, respectively. Meanwhile, 1.74- and 1.99-fold purification with 31.26 and 31.51% recovery were obtained from M. circinelloides and M. hiemalis pectinase, respectively. A significant increase and decrease in the activity and stability of M. circinelloides and M. hiemalis partially purified enzymes was reported after incubation with different concentrations of metal ions and detergents. The response-surface methodology was effective and satisfactory, and investigated many factors simultaneously. More research is needed to scale up enzymes production for a wide range of applications.