Fermentation-Based Production and Whole-Cell Immobilization of β-Glucuronidase-Expressing Talaromyces pinophilus Li-93 for Efficient Bioconversion of Glycyrrhizin

Abstract

Glycyrrhizic acid and its derivatives are a crucial class of glycoside terpenoids with significant pharmaceutical and food industry applications. The biotransformation of glycyrrhizin (GL) into glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) and glycyrrhetinic acid (GA) can enhance the production of these valuable compounds. This study aimed to develop strategies to improve the catalytic and operational stability of β-glucuronidase from wild-type Talaromyces pinophilus Li-93, previously known as Penicillium purpurogenum Li-3 (w-PGUS), for efficient GL hydrolysis. Whole cells of T. pinophilus Li-93 expressing w-PGUS were capable of directly converting GL into GAMG. To enhance enzyme stability and reusability, three polymeric supports including, polyurethane foam (PUF), loofah sponge (LS), and polyvinyl chloride (PVC), were evaluated for immobilization of w-PGUS from the fermentation medium. Among these, PUF was the most effective immobilization support, yielding higher immobilization efficiency, GAMG production, and biomass retention. Under optimized conditions (1% PUF, 1.5 g.L⁻¹ w-PGUS inoculum, pH 5.0, 36 °C, 180 rpm), the immobilized w-PGUS produced a final GAMG yield of 3.90 g.L⁻¹, achieving 67.10% immobilization efficiency within 72 h. The PUF-immobilized w-PGUS retained 37.51% of its initial activity after 10 repeated batch reactions, whereas free w-PGUS retained only 6.21%. Additionally, the storage stability of immobilized w-PGUS was significantly higher (40.22%) than that of free w-PGUS (14.74%) after 30 days. Immobilization slightly reduced the initial yield due to mass-transfer limits but enabled much higher cumulative GAMG production through improved stability and reusability.

1. Introduction

Glycyrrhizin (GL), a primary active ingredient of licorice root, consists of a single molecule of glycyrrhetic acid (GA) and two molecules of glucuronic acid linked to the C-3 atom of the aglycon moiety. GL can be hydrolyzed by cells expressing β-glucuronidase into glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) [Figure 1].

The potential applications of GAMG are far beyond GL, as it has been shown to be safer and more effective than its parent compound, and is therefore widely used in the food industry [1,2]. GAMG has a broad spectrum of activities against infection, inflammation, allergy, and cancer [3]. Due to superior physiological effects and a wide range of applications, GAMG is considered a substitute for GL and has more commercial importance than GL.

The biocatalysis of valuable compounds has many advantages over chemical catalysis in terms of physical parameters, specificity, and environmental hazards. The low specificity and yield of GAMG could be improved by biotransforming GL with β-glucuronidase (pgus, EC 3.2.1.31). There are many biological sources of β-glucuronidase, including prokaryotes and eukaryotes, while fungal sources are quite limited. Our research group previously screened a wild-type Penicillium purpurogenum Li-3 (w-PGUS) strain, which has been reclassified recently as Talaromyces pinophilus Li-93 (w-PGUS) and is involved in the direct conversion of GL into GAMG. The w-PGUS carries a unique β-glucuronidase (PGUS, EC 3.2.1.31), and this fungus only grows in a liquid culture medium containing GL, where GL acts as an inducer and sole carbon source [4]. Although the fermentation conditions, including medium composition and physical parameters, had been optimized to enhance the productivity of T. pinophilus and GAMG yield [5], the low enzyme productivity of this strain and the rapid in vitro loss of enzymatic activity were the main limitations to achieving higher GAMG yield.

Immobilization strategies are usually designed to enhance operational and storage stability and to combat perturbations in the physical and chemical environment. Direct immobilization from fermentation media, without affecting growth rate or product yield, is a practical approach to avoid the complex separation and purification processes for enzymes or whole cells [6,7,8]. Different polymer carriers have been employed to immobilize cells or enzymes for stable biotransformation, including natural porous matrices and flexible and rigid synthetic polymeric supports. Natural matrices such as loofah sponge (LS) have been widely used due to their lignocellulosic composition, high porosity, and good biodegradability, which promote microbial adhesion and mass transfer in fermentation processes [9]. Porous rigid synthetic polymers, including polyvinyl chloride (PVC), have also been applied because of their chemical inertness, low porosity, and mechanical stability. While flexible synthetic porous supports such as polyurethane foam (PUF) are among the most widely used carriers owing to their highly interconnected open-cell structure, elasticity, large surface area, and higher mass-transfer properties, which collectively support stable biomass retention and enhanced productivity in repeated biotransformation systems [10]. PUF has several applications in biochemical and biotechnological fields due to its biocompatibility and stability [10,11]. The physical properties of PUF, such as durability, ease of handling, and affordability, make it a remarkable material for immobilizing biomolecules or cells [12]. Moreover, it offers steady mass transfer and minimal mechanical friction, which could help maintain cell vitality and enhance enzyme reactivity [13]. There are many studies on the immobilization of whole cells of different strains of microorganisms in PUF [14,15]. Still, no studies are available on the immobilization of T. pinophilus expressing β-glucuronidase in fermentation media and on its application efficacy for operational and storage stability, which has great potential to biotransform GL directly into GAMG due to its specific mode of action.

In this study, we reported a high immobilization efficiency of w-PGUS in PUF during flask fermentation, achieved by optimizing fermentation parameters. The effects of immobilization on fungal growth and GAMG yield were also controlled to enhance the immobilization efficiency of T. pinophilus in PUF. In addition, the operational and storage stability of PUF-immobilized biocatalyst w-PGUS for the biotransformation of GL into GAMG has been evaluated and compared with that of free w-PGUS for large-scale applications.

2. Materials and Methods

2.1. Chemicals and Reagents

The standard samples of glycyrrhizin (GL) and glycyrrhetic acid (GA) were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO, USA). Standard glycyrrhetic acid monoglucuronide (GAMG) was generously donated by Nanjing University of Technology (Nanjing, China). Polyurethane foam (PUF), loofah sponge (LS), and porous polyvinylchloride (PVC) were purchased from the local market. HPLC-grade methanol was purchased from Sigma-Aldrich (Steinheim, Germany). All other reagents were of analytical grade, and deionized water was purified using a Milli-Q water purification system (Millipore, Bedford, MA, USA). All solutions prepared for HPLC were filtered through a 0.45 μm nylon membrane filter before use.

2.2. Microbial Strain and Fermentation of Glycyrrhizin (GL)

In this study, the microorganism used, Talaromyces pinophilus Li-93 (w-PGUS), was preserved in our biotransformation and microecology lab (Beijing Institute of Technology, Beijing, China). The seed medium consisted of (g.L⁻¹) glucose, 5; NH₄NO₃, 3; KH₂PO₄, 0.8; KCl, 0.5; and MgSO₄, 0.5. A range of GL (C₄₂H₆₅NO₁₆) concentrations (2–8 g.L⁻¹) was evaluated during preliminary optimization with 6 g.L⁻¹ was selected as the optimal concentration for subsequent experiments. The culture medium (g.L⁻¹) comprised C₄₂H₆₅NO₁₆, 6; NH₄NO₃, 3; KH₂PO₄, 0.8; KCl, 0.5; and MgSO₄, 0.5, and was optimized for the maximum growth of w-PGUS [16]. The medium was adjusted to pH 5.0 and sterilized in an autoclave at 121 °C for 20 min before use.

The pure culture of T. pinophilus Li-93 was thawed from a −80 °C frozen stock and transferred to agar medium for pre-culture. The culture (1 mL) was inoculated into a 250 mL flask containing 100 mL of seed medium at 30 °C with agitation at 170 rpm for 72 h. The cells were obtained after centrifugation at 10,000× g at 4 °C and then inoculated into the fermentation media. Each fermentation medium was inoculated with 1 g.L⁻¹ of T. pinophilus into a 1 L flask containing 300 mL of T. pinophilus production medium, in which GL was the sole carbon source and inducer, and cultured at 32 °C and 170 rpm for 72 h.

2.3. Screening of Immobilizing Materials

To evaluate the suitability of different carriers for immobilization of T. pinophilus Li-93, three materials including polyurethane foam (PUF), loofah sponge (LS), and porous polyvinyl chloride (PVC) were investigated. All materials were purchased from the local market in Beijing. Each carrier was prepared, sterilized, and applied under identical fermentation conditions to ensure reliable comparison of immobilization performance.

2.3.1. Polyurethane Foam (PUF)

PUF was cut into cubes (8 mm × 8 mm × 8 mm) and sequentially washed with 1 M HCl and 1 M NaOH to remove surface impurities. The cubes were then thoroughly rinsed with distilled water and sterilized at 121 °C for 20 min. Sterilized PUF cubes (1% w/v) were added to 300 mL fermentation media in 1 L Erlenmeyer flasks immediately after inoculation with T. pinophilus Li-93. Cultivation was carried out at 32 °C and 170 rpm for 72 h. Immobilization efficiency was determined by measuring the dry cell weight (DCW) attached to the carrier relative to the total biomass, while the concentrations of substrate (GL) and products (GAMG and GA) were determined through HPLC.

2.3.2. Loofah Sponge (LS)

LS pieces (8 mm) were prepared from natural loofah, washed with 1 M HCl and 1 M NaOH to remove lignocellulosic impurities, rinsed with distilled water, and sterilized at 121 °C for 20 min. The sterilized LS (1% w/v) pieces were added to fermentation media under identical conditions as PUF. Immobilization efficiency, GL, GAMG, and GA yields, and biomass retention were quantified following the same analytical procedures.

2.3.3. Porous Polyvinyl Chloride (PVC)

PVC beads (8 mm) were pretreated with ethanol to remove surface contaminants, rinsed with distilled water, and sterilized at 121 °C for 20 min. The sterilized PVC (1% w/v) beads were added to fermentation media under identical conditions as both PUF and LS. Immobilization efficiency, GL, GAMG, and GA yields, and biomass retention were determined using the same protocols.

At the end of cultivation, fermentation broths were centrifuged at 8000 rpm for 10 min at 4 °C to remove free (suspended) cells. Immobilized carriers were recovered by filtration from the fermentation media, washed with distilled water, and lyophilized at −52 °C until a constant weight was achieved to determine the immobilization efficiency. Free biomass was also dried to determine the total dry cell weight (DCW). Aliquots were also taken from the fermentation media at different intervals to determine the concentration of GL, GAMG, and GA through HPLC analysis. The primary objective was to quantify the immobilization yield by determining the fungal biomass successfully attached to the carrier relative to the initial inoculum. Total biomass, including free dry cell weight (fDCW) after centrifugation and immobilized dry cell weight (iDCW), was also determined and compared in the absence of polymer culture media. Immobilization efficiency was calculated using Equation (1) as given below:

$$\mathrm{Immobilizing} \mathrm{efficiency} (\%) = {\displaystyle \frac{\mathrm{weight} \mathrm{of} \mathrm{immobilized} \mathrm{w}\text{-}\mathrm{PGUS}}{\mathrm{Total} \mathrm{biomass} \mathrm{of} \mathrm{w}\text{-}\mathrm{PGUS}}} \times 100$$

(1)

2.4. Determination of Optimal Parameters for PUF Immobilization in Fermentation

The selection of PUF was based on the results of the screening process in Section 2.3. Optimal fermentation parameters for maximizing the immobilization efficiency of T. pinophilus were determined, including PUF dosage, T. pinophilus inoculation concentration, temperature, initial pH, and shaking speed. The GAMG yield, total biomass, and immobilizing efficiency for each parameter were determined. The results were the means of three independent experiments, each with two replicates per condition.

2.5. SEM Analysis

The immobilization and distribution of T. pinophilus on the PUF carrier were examined by scanning electron microscopy (SEM). The immobilized T. pinophilus on PUF carrier was dried in a lyophilizer at a low temperature of −52 °C till a constant weight. Then the specimens were gold-sputtered before SEM (Philips XL30 ESEM, FEI Company, Hillsboro, OR, USA) observation. SEM observations provided better insight into the mycelial adherence to PUF, distribution across the polymer surface, and accumulation pattern during fermentation.

2.6. Operational Stability Analysis of PUF-Immobilized w-PGUS

The PUF-immobilized w-PGUS were filtered from fermentation media, washed twice with distilled water to remove unabsorbed cells and then lyophilized till a constant weight. The same concentrations of both immobilized w-PGUS and free w-PGUS (3 g) were added to the 4 mM GL reaction media at pH 5.6, 40 °C, and 150 rpm for 36 h. The catalytic activities of both free and immobilized w-PGUS in each reaction cycle were quantified based on the amount of GAMG formed per cycle as determined by HPLC. The activity in the first cycle was taken as 100%, while the relative activity in subsequent cycles was used to calculate operational stability. After each reaction batch, immobilized w-PGUS were obtained by simple filtration, washed with the buffer, and then introduced again into the freshly prepared GL solutions for the next reaction batch, while free w-PGUS were collected by centrifugation at 8000 rpm at 4 °C, washed with buffer, and then put into the next reaction media under the same set of conditions. The operational stability of both free and immobilized w-PGUS was determined by investigating their catalytic activities in each successive reaction cycle and can be represented as follows:

$$\mathrm{Operational} \mathrm{stability} (\%) = {\displaystyle \frac{\mathrm{w}\text{-}\mathrm{PGUS} \mathrm{activity} \mathrm{in} \mathrm{the} \mathrm{nth} \mathrm{cycle}}{\mathrm{w}\text{-}\mathrm{PGUS} \mathrm{activity} \mathrm{in} \mathrm{the} 1\mathrm{st} \mathrm{cycle}}} \times 100$$

(2)

2.7. Storage Stability Analysis of PUF-Immobilized w-PGUS

Free and PUF-immobilized w-PGUS were stored at 4 °C for specific periods of time (days) and then examined for activity. The storage stability was compared by storage efficiency which was defined as the ratio of free or immobilized w-PGUS after storage to their initial activity.

$$\mathrm{Storage} \mathrm{efficiency} (\%)=\frac{\mathrm{w}\text{-}\mathrm{PGUS} \mathrm{activity} \mathrm{after} \mathrm{storage}}{\mathrm{initial} \mathrm{w}\text{-}\mathrm{PGUS} \mathrm{activity}} \times 100$$

(3)

2.8. Standard Curves of GL, GAMG, and GA

The standard curves for GL, GAMG, and GA were prepared by diluting 3.156 g.L⁻¹, 0.568 g.L⁻¹, and 0.46 g.L⁻¹ of GL, GAMG, and GA, respectively, and then analyzed by high-performance liquid chromatography. Regression analysis was performed to get their respective standard curves, as shown in the Figure 2, Figure 3 and Figure 4 below.

GL, GAMG, and GA regression equations:

GL: y = 8 × 10⁶x + 24,635 (R² = 0.9967)

(4)

GAMG: y = 917,343x + 3037.4 (R² = 0.9997)

(5)

GA: y = 2.8054 × 10⁶x + 6.7022 × 10² (R² = 0.9961)

(6)

Figure 2. Calibration curve of GL concentration versus peak area. The regression equation is y = 8.0 × 10⁶x + 24,635 with R² = 0.9967, indicating excellent linearity across the tested concentration range.

Figure 2. Calibration curve of GL concentration versus peak area. The regression equation is y = 8.0 × 10⁶x + 24,635 with R² = 0.9967, indicating excellent linearity across the tested concentration range.

Figure 3. Calibration curve of GAMG concentration versus peak area. The regression equation is y = 9.17 × 10⁵x + 3037.4 with R² = 0.9997, confirming a very strong correlation between GAMG concentration and measured area.

Figure 3. Calibration curve of GAMG concentration versus peak area. The regression equation is y = 9.17 × 10⁵x + 3037.4 with R² = 0.9997, confirming a very strong correlation between GAMG concentration and measured area.

Figure 4. Calibration curve of GA concentration versus peak area. The regression equation is y = 2.8054 × 10⁶x + 670.22 with R² = 0.9961, demonstrating high linearity suitable for quantitative analysis.

Figure 4. Calibration curve of GA concentration versus peak area. The regression equation is y = 2.8054 × 10⁶x + 670.22 with R² = 0.9961, demonstrating high linearity suitable for quantitative analysis.

2.9. HPLC Analysis

HPLC was used to quantify the substrate and products in the reaction systems (Supplementary File, Figures S1–S15). GL, GAMG, and GA were separated on an octadecylsilane (ODS) column (Shim-pack, VP-ODS, 4.6 × 250 mm, Shimadzu Corporation, Kyoto, Japan) under the following chromatographic conditions: UV detection wavelength 254 nm; flow rate 1.0 mL/min; mobile phase water (pH 2.85 with 0.6% (v/v) acetic acid) and methanol at 19:81 (v/v); and injection volume 10 µL. The retention times of GL, GAMG, and GA were 7.5, 13.5, and 22.7 min, respectively.

The production rate of GAMG was determined as follows:

GAMG production rate (%) = (P_GAMG/GL₀) × 100

(7)

where GL₀ is the initial concentration of substrate (GL) at time 0 and P_GAMG is the concentration of GAMG at time t.

3. Results and Discussions

3.1. Selection of Immobilizing Material

The selection of polymer carriers for w-PGUS immobilization was primarily based on GAMG yield, total biomass (including free and immobilized dry cell weight (DCW)), and immobilization efficiency. The fermentation media without polymer was taken as a control. The results revealed that PUF was the best carrier for w-PGUS growth, GAMG yield, and immobilization efficiency compared to other carriers [

Table 1. Immobilization efficiency of different polymers in fermentation media of T. pinophilus Li-93 (w-PGUS).

Material	GAMG Yield	Free DCW	Immobilized DCW	Total Biomass	Immobilization Efficiency
	(g.L−1)	(g.L−1)	(g.L−1)	(g.L−1)	(IE %)
Control	4.12 ± 0.08	7.42 ± 0.13	-	7.42 ± 0.13	-
Polyurethane Foam (PUF)	3.52 ± 0.07	2.72 ± 0.11	4.42 ± 0.13	7.13 ± 0.12	62.00
Loofah Sponge (LS)	3.41 ± 0.07	3.04 ± 0.13	3.88 ± 0.14	6.92 ± 0.13	56.06
Porous PVC	3.23 ± 0.08	5.92 ± 0.16	0.56 ± 0.14	6.48 ± 0.15	8.64

All fermentations were performed with a 1% polymer dosage at pH 5.0, 32 °C, and 170 rpm for 72 h with constant inoculum concentrations of w-PGUS. DCW refers to dry cell weight. All experiments were performed in triplicate and reported as mean ± SD. (-) Nill.

]. The average total biomass of w-PGUS without polymer was 7.42 g.L⁻¹ with an average GAMG yield of 4.12 g.L⁻¹. The immobilization efficiencies of PUF, LS, and porous PVC were 62%, 56%, and 8.64% respectively. The GAMG yield decreased in PUF, LS, and PVC to 14.5%, 17.25%, and 21.6%, respectively, compared to without a polymer medium. The total biomass also decreased in PUF, LS, and PVC immobilization media at 4%, 6.73%, and 12.66%, respectively, compared to those without polymer media. This decrease in GAMG yield and biomass could be attributed to a decrease in GL conversion rate, as carriers can increase culture media viscosity and reduce mass-transfer rates by imposing greater resistance [16,17]. The increase in internal mass-transfer resistance negatively affected the efficiency of T. pinophilus, thereby decreasing GAMG yield and total biomass. PUF’s open-cell structure, uniform pores, hydrophilicity, and flexibility enabled dense hyphal penetration, stable biomass retention, and efficient mass transfer. In contrast, LS supported uneven colonization due to its fibrous heterogeneity, whereas PVC’s smooth, hydrophobic, and low-porosity surface limits fungal attachment and growth. These clarifications have strengthened the mechanistic basis for differences in immobilization efficiency and GAMG production.

Based on these results, the PUF carrier had the least effect on T. pinophilus growth rate and the medium showed the lowest resistance to it. GAMG yield and immobilization efficiency of PUF were comparatively much better than those of the other two polymer carriers. Therefore, PUF was selected for further experimentation and trialed for further efficacy analysis.

3.2. PUF Dosage

The effects of PUF dosage on immobilization efficiency, GAMG yield, and total biomass were examined by inoculating fermentation media with varying PUF concentrations. PUF doses including 0.5, 1, 1.5, 2, and 2.5% were added in the fermentation media, and it was found that 1% of PUF dosage exhibited optimum GAMG yield, biomass, and immobilization efficiency [

Table 2. Effect of polyurethane foam (PUF) dosage on the immobilization efficiency, GAMG yield, and total biomass of T. pinophilus Li-93 (w-PGUS) in fermentation media.

PUF Dosage	GAMG Yield	Free DCW	Immobilized DCW	Total Biomass	Immobilization Efficiency
(g.L−1)	(g.L−1)	(g.L−1)	(g.L−1)	(g.L−1)	(IE %)
0.5	3.68 ± 0.06	3.52 ± 0.08	3.46 ± 0.10	6.98 ± 0.09	49.50
1.0	3.82 ± 0.08	2.72 ± 0.10	4.42 ± 0.12	7.13 ± 0.11	62.00
1.5	3.28 ± 0.10	2.64 ± 0.13	3.88 ± 0.11	6.52 ± 0.12	59.50
2.0	2.92 ± 0.09	2.63 ± 0.11	3.48 ± 0.12	6.14 ± 0.12	56.66
2.5	2.31 ± 0.12	2.55 ± 0.10	3.21 ± 0.10	5.76 ± 0.10	55.72

All fermentations were performed with 1 g.L⁻¹ inoculum concentrations of w-PGUS at pH 5.0, 32 °C, and 170 rpm for 72 h with different doses of PUF carrier. All experiments were performed in triplicate and reported as mean ± SD.

].

At 0.5% PUF dosage, the GAMG yield was 3.68 g.L⁻¹, which was 3.66% less than the optimal GAMG yield of 3.82 g.L⁻¹ at 1% PUF dosage. Although the single-batch GAMG yield of immobilized T. pinophilus on PUF was slightly lower than that of the free-cell control, the immobilized system exhibited significantly higher operational and storage stability. As a result, the cumulative GAMG production across multiple cycles was substantially higher with the immobilized biocatalyst. At a 1.5% dosage of PUF, GAMG yield and immobilization efficiency decreased 14.14% and 8.55%, respectively, compared to optimal values at 1%. Further increases in PUF dosage from 2 to 2.5% decreased GAMG yield and immobilization efficiency by a significant amount. High carrier concentration was the major rate-limiting factor for the bioconversion of GL, thereby affecting the growth of T. pinophilus and the immobilization efficiency across all materials. However, the effect was most pronounced with PUF, as reported previously [18,19]. This behavior can be attributed to mass-transfer limitations and reduced mixing at higher support dosages. At an optimal dosage of 1% PUF, it exhibited minimal inhibitory effects on fungal growth and was evenly distributed in the fermentation media. It thus produced the maximum GAMG yield, biomass, and immobilization efficiency.

3.3. w-PGUS Inoculation Concentration

The initial inoculation concentration of T. pinophilus for fermentation was determined by inoculating the fermentation media with different concentrations of the w-PGUS seed solution. The results showed that a fungal concentration of 1.5 g.L⁻¹ was the optimal inoculum concentration, producing a GAMG yield of 3.90 g.L⁻¹ and a total biomass of 7.24 g.L⁻¹. Moreover, the immobilization efficiency was 63.81%, and this optimal efficiency was set to 100% to determine relative immobilization efficiency (RIE) at other inoculation levels [Figure 5].

The GAMG yield gradually increased with increasing w-PGUS inoculation concentration, but after an initial cell concentration of 1.5 g.L^-1 w-PGUS, its yield and total biomass decreased as the inoculation became too high, resulting in a negative impact on cell growth and enzymatic activity. The RIE was reduced to 86.85% at 3 g.L⁻¹ inoculation level. High inoculation levels inhibited the growth and enzymatic efficiency of the fermentation system, resulting in lower w-PGUS biomass, as reported by many researchers [19,20].

3.4. Temperature and pH Effects

Temperature has been a key factor in all fermentation media and determines cell yield and biomass by lowering the activation energy and accelerating biotransformation rates. The effect of culture temperature on the immobilization efficiency of w-PGUS was examined at the optimally determined PUF dosage and inoculation concentration, at pH 5.0 and a shaking speed of 170 rpm [Figure 6].

An increase in temperature to 36 °C was observed to positively affect GAMG yield, total biomass, and immobilization efficiency. The increase in biotransformation activity resulted in a maximum yield of GAMG and biomass for w-PGUS. This increase in biomass led to high immobilization efficiency of w-PGUS in PUF. The optimal media temperature was 32 °C, which increased to 36 °C upon addition of PUF to the culture media. Therefore, 36 °C was the optimal temperature for PUF immobilization as it not only increased the GAMG yield (3.90 g.L⁻¹) but also the total biomass of w-PGUS (7.28 g.L⁻¹). The immobilization efficiency of w-PGUS at 36 °C was 64.70%, which was considered 100% RIE. The temperature rise could have resulted from the thermostability of w-PGUS immobilized on the PUF surface or within its pores. Low temperature could be a rate-limiting factor due to the addition of thermostable carriers, which protect cells from temperature perturbations [21,22]. A further increase in temperature to 40 °C reduced the immobilization efficiency (89.14%), resulting in a low yield of GAMG (3.43 g.L⁻¹) and biomass of w-PGUS (6.90 g.L⁻¹).

The pH of the medium has an effective role in the fermentation process. The influence of medium pH on the immobilization efficiency of w-PGUS in PUF carrier was examined within a low acidic profile range (4.4–5.4) at 36 °C and 170 rpm for 72 h [Figure 7].

There was no change in the medium pH with or without the PUF carrier. The maximum RIE was observed at pH 5.0, with results similar at 36 °C, while at a high acidic pH of 4.4, RIE decreased to 89.5% due to a decrease in biomass of w-PGUS (6.80 g.L⁻¹) and GAMG yield (3.40 g.L⁻¹). At a low acidic pH value of 5.4, RIE was 78.65% with a total biomass of w-PGUS 6.72 g.L⁻¹ and GAMG yield of 3.18 g.L⁻¹.

The pH of the fermentation media has a significant effect on biotransformation yield, as it can influence the ionization of the substrate and its binding to enzyme active sites by altering its polarity [23]. GL is a weak tribasic acid, and changes in the ionization of the carboxyl group with pH can influence its binding to the enzyme molecule and, consequently, can affect the course of biotransformation [24]. PUF is a neutral carrier, and its addition to the fermentation medium does not affect its pH; maximum RIE was observed at the same optimal pH.

3.5. Shaking Speed

The effects of shaking speed on the fermentation efficiency of w-PGUS and its immobilization efficiency were determined by maintaining the fermentation flasks at different agitation speeds under previously optimized conditions [Figure 8].

The results revealed that 180 rpm was the optimal shaking speed for immobilization efficiency and biomass of w-PGUS. Almost 67.1% of the w-PGUS immobilized efficiency was achieved, with a maximum biomass of 7.36 g.L⁻¹ and a GAMG yield of 4.08 g.L⁻¹. The optimal shaking speed of the medium was 170 rpm, which increased to 180 rpm with the addition of the PUF carrier, indicating that the mass-transfer rate was the rate-confining factor for the bioconversion of GL into GAMG and the growth of w-PGUS. A further increase in shaking speed at 200 rpm caused a negative impact on GAMG yield (3.22 g.L⁻¹), total biomass (6.9 g.L⁻¹), and RIE (76.50%) of w-PGUS. High shaking speed reduced the immobilization efficiency of w-PGUS by disturbing the absorption rate of w-PGUS to its surface, and it also decreased the mass transfer rate of substrate GL to w-PGUS for its bioconversion into GAMG [25].

3.6. SEM Analysis (SEM-Based Characterization of Immobilized w-PGUS on PUF)

The SEM analysis of the immobilized w-PGUS in PUF is shown in Figure 9.

The w-PGUS absorbed onto the PUF surface, formed a network of mycelia, and were then observed at different surface areas (Figure 9a,b) and magnifications (Figure 9c,d). The interconnected porous architecture of polyurethane foam (PUF) provided a robust three-dimensional scaffold that was highly conducive to the immobilization of whole-cell fungal biomass (w-PGUS). The high porosity and expansive internal surface area of the PUF provided an ideal environment where fungal mycelia could effectively settle via physical absorption and capillary forces. As the w-PGUS biomass grew and expanded its filamentous network (hyphae), these filaments became densely interwoven and mechanically trapped within the complex sponge-like matrix of the foam [26]. The overall surface morphology of PUF showed the stable and gradual growth of T. pinophilus and its strong entrapment in the porosity of PUF. In this study, SEM was employed to evaluate immobilization because it provided direct visualization of fungal adhesion, hyphal penetration, and distribution across carrier surfaces [27,28]. These parameters were directly linked to immobilization efficiency as well as GAMG and GA yields. While XRD can be valuable for determining crystallinity, its scope is limited to material phase analysis and does not provide biological insights into immobilization. As the crystallinity of PUF, LS, and PVC has already been well-documented in the literature [29,30,31], SEM analysis was sufficient to achieve our study’s objectives [32,33]. Our findings demonstrated an inverse relationship between crystallinity and immobilization efficiency: amorphous polyurethane foam (low crystallinity) supported the highest immobilization yield (62%), semi-crystalline loofah sponge showed intermediate efficiency (56%), while highly crystalline PVC exhibited poor efficiency (8.64%).

3.7. Operational Stability of PUF-Immobilized w-PGUS

The major advantage of whole-cell immobilization is the ability to reuse cells for operational stability. The PUF-immobilized T. pinophilus could be easily removed from the fermentation media by filtration. The PUF pieces were washed with distilled water and buffer to leech the unabsorbed cells. The PUF-immobilized T. pinophilus was kept in a lyophilizer for freeze-drying at −52 °C until a constant dry cell weight was achieved. The free T. pinophilus and the freeze-dried PUF-immobilized w-PGUS were added to the GL reaction mixture for operational stability analysis [Figure 10].

The results revealed that PUF-immobilized w-PGUS retained 50.11% of its original activity after eight repeated batches, compared to 10.17% for free w-PGUS. The PUF-immobilized T. pinophilus maintained 37.51% after 10 repeated cycles, while free T. pinophilus exhibited no significant activity (6.21%), which was six times lower than immobilized T. pinophilus. The rapid loss of activity was observed after 10 batches, and, lastly, immobilized PUF retained only 11% of its original activity after 12 cycles.

PUF-immobilized T. pinophilus represented a system in which the cells were absorbed by the polymer’s surface and suspended within the inner core of the pores. The immobilized T. pinophilus was protected by a thermostable, neutral, and non-toxic support, which made the system relatively stable for the efficient biotransformation of GL. The carrier protected T. pinophilus from rapid perturbations in physical parameters, particularly from heat inactivation and trauma to cell membranes caused by external mass stress. The support made T. pinophilus more efficient than free cells due to its protection, regular mass transfer, and a uniform environment, which helped maintain the activity in intracellular β-glucuronidase across repeated batches. The continuous loss or decay in the immobilized T. pinophilus activity could be due to the inactivation of intracellular enzymes or the agglomeration of reaction products inside the cells [34,35].

3.8. Storage Stability of PUF-Immobilized w-PGUS

The storage efficiency of free and PUF-immobilized w-PGUS decreased with the increase in storage time, and the loss of activity was more evident for longer durations. The free and immobilized T. pinophilus lost almost 37.68% and 24.40% of its original activities after 15 days, with decreases reaching up to 85.26% and 59.78%, respectively, after 30 days of storage at 4 °C. The activity retained in PUF-immobilized w-PGUS (40.22%) after 30 days was almost three times higher compared to the free w-PGUS (14.74%). The storage stability of free and immobilized T. pinophilus has been presented in Figure 11 below.

The storage efficiency of free and PUF-immobilized T. pinophilus decreased with the increase in storage time, and the loss of activity was more evident for longer durations of time. The free and immobilized w-PGUS lost almost 37.68% and 24.40% of its original activities after 15 days, which continued to decrease up to 85.26% and 59.78%, respectively, after 30 days of storage at 4 °C. The activity retained in PUF-immobilized w-PGUS (40.22%) after 30 days was almost three times higher compared to the free w-PGUS (14.74%).

The free and immobilized w-PGUS gradually lost enzymatic activity with increasing storage duration, and the loss of activity was found to be proportional to the storage time. The loss of activity in immobilized w-PGUS could be attributed to conformational changes in the enzyme structure during storage. These conformational variations in the intracellular enzyme structure could negatively affect the enzyme efficiency by decreasing its activity. The PUF carrier acted as a physical shield for the fungal cells, maintaining a stable intracellular microenvironment that can protect the internal enzymes from external thermal and physical fluctuations [36,37].

4. Conclusions

The study investigated the direct immobilization efficiency of w-PGUS in PUF under optimized conditions. A maximum immobilization efficiency of 67.1%, corresponding to a GAMG yield of 3.90 g.L⁻¹, was achieved by sustaining both w-PGUS growth and product formation. The PUF-immobilized T. pinophilus showed six times higher operational stability after 10 consecutive batches than free T. pinophilus. The PUF-immobilized w-PGUS also showed almost three times greater storage stability than free w-PGUS. PUF demonstrated higher overall productivity due to greatly enhanced reusability and long-term stability, despite a slight reduction in single-batch GAMG yield.