Isolating alkaline-adapted bacteria

Three water samples from five different locations of Lake Van, Turkey, (38.520374, 43.314816; 38.309119, 43.040275; 38.560118, 43.282007; 38.427571, 43.258307; 38.422899, 43.285634) were collected and inoculated on nutrient agar (NA), pH 9 at 30 °C. Pure cultures of the isolates were kept at -87 °C in 20% glycerol stocks.

Assessment of cellulase activity among alkaliphilic strains

To assess cellulase activity in isolates, pure cultures were introduced into carboxymethyl cellulose - agar (CMC-A) medium (NA with 10 g/L carboxymethyl cellulose), pH 9, and maintained at 30 °C for 48–72 h. Subsequently, colonies were treated with 0.1% Congo red for 15 min. Following staining, the solution was decanted, and a 1 M NaCl solution was applied for a 30-minute wash cycle. The diameters of cellulolytic zones surrounding the isolates were then gauged and those forming the most expansive zones (Gohel et al. 2014).

Cultivation conditions for cellulase production

The most promising cellulolytic isolates were cultured in 100 mL of carboxymethyl cellulose - broth (CMC-B) composed of 10 g/L CMC, 0.2 g/L MgSO4.7H2O, 5 g/L NaCl, 10 g/L peptone, 2 g/L yeast extract, 1 g/L K2HPO4, and 0.1 g/L CaCl2 (adjusted to pH 9). The medium composition was modified based on Aygan and Arikan (2009). Cultures were incubated at 30 °C and 180 rpm for 5 days, and the activities of the cultures were monitored.

Measurement of cellulase activity

Post-incubation, the cultures were centrifuged at 4 °C and 6000 rpm for 10 min to separate the supernatant, which was used as crude enzyme. Freshly prepared 1% CMC solution in 50 mM Glycine-NaOH buffer at pH 9 (referred to as Buffer A) was used as the substrate solution. Both 0.5 mL substrate solution and 0.5 mL crude enzyme were homogenized and kept in a 35 °C water bath for 30 min. Following incubation, 1 mL of 3,5- Dinitrosalicylic acid (DNS) reagent (Aktas et al. 2023) was added, and the mixture was boiled for 5 min to stop the reaction. After cooling to room temperature, absorbance at 540 nm was measured (Miller 1959), and carboxymethyl cellulase activity was calculated based on the glucose standard graph. One unit of cellulase enzyme activity (1 U) was evaluated as the amount of enzyme that produced 1 µmol glucose in 1 min under the test conditions.

To determine exo-β-1,4-glucanase activity and endo-β-1,4-glucanase activity, 5 mM p-nitrophenyl β-D-cellobioside (pNPC) and 5 mM p-nitrophenyl β-D-glucopyranoside (pNPG) substrate solutions prepared in Buffer A were used, respectively. 100 µL of crude enzyme and 900 µL of respective substrate solutions were mixed and incubated at 35 °C for 30 min. The reactions were terminated by adding 100 µL of Na₂CO₃. Absorbance measurements were performed at 410 nm. As a control, 100 µL Buffer A was used instead of the crude enzyme. Exo-β-1,4-glucanase and β-1,4-glucanase activity calculations were made using a p-nitrophenol standard curve. All the enzymatic experiments were conducted in triplicate.

Identification of cellulase-producing isolates

The bacterial isolate demonstrating the highest cellulase activity, as determined by qualitative and quantitative cellulase activity assays, was selected. The isolate was identified utilizing 16 S rDNA sequence analysis conducted by Macrogen, South Korea and classical identification techniques. The isolate’s morphological characteristics (colony color and shape, cell shape, Gram staining, and KOH test) and biochemical properties (oxidase, amylase, protease, hemolysis, H₂O₂ catalase, urease, lipase, asparaginase, glutaminase, maltose utilization, etc.) were determined. Subsequently, the derived sequence data was deposited into the National Center for Biotechnology Information (NCBI) database, and GenBank accession number was obtained.

The phylogenetic tree was constructes using the Phylogeny.fr platform (https://www.phylogeny.fr/phylogeny.cgi), which integrates alignment and phylogeny programs. Multiple sequence alignment was carried out using the NCBI MSA Viewer 1.25.0. On the Phylogeny.fr platform, tree construction was completed using the maximum-likelihood method with the default settings (Kuhner and Felsenstein 1994).

Extraction and purification of cellulase

At the end of the 96 h of incubation in CMC-B, bacterial cells were removed by centrifugation at 6000 rpm for 25 min at 4 °C. After centrifugation, ammonium sulfate was gradually added to the supernatant to achieve final concentrations of 20%, 40%, and 60%, respectively, and protein precipitation was carried out. For each concentration, ammonium sulfate was added over 30 min, and the mixture was stirred at 4 °C for a minimum of 4 h. Following this, the solution was centrifuged at 10,000 rpm for 30 min to precipitate the proteins, and the supernatant was separated to continue precipitation at the next ammonium sulfate concentration. The precipitated proteins were dissolved in Buffer A. Ultrafiltration (Millipore, 10 kDa cut-off) was performed at 6000 rpm to remove the remaining ions and salts from the process and to concentrate the protein solution. The obtained samples were loaded onto an anion exchange 16/10 HiPrep Q XL column (Biorad, Biologic LP, USA) equilibrated using Buffer A. The samples were passed through the column at a flow rate of 1 mL/min using a NaCl gradient ranging from 0 to 1 M. Fractions were collected, and cellulase activity measurements were performed on each. Fractions showing high cellulase activity were pooled, concentrated by ultrafiltration, and stored at -20 ºC (Gurkok and Ozdal 2021; Hemsinli and Gurkok 2024) for subsequent enzyme characterization studies.

Investigation into cellulase characteristicsAssessment of protein content and sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiling

The assessment of protein content was carried out using the Lowry method, employing bovine serum albumin (BSA) as the standard for calibration (Lowry et al. 1951). Following quantification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to analyze the molecular weight distribution of the proteins. Samples of crude enzyme extract, ammonium sulfate precipitated protein, and purified cellulase (10 µg each) were loaded onto a 12% polyacrylamide gel and separated using the Bio-Rad Mini-PROTEAN® Tetra Cell system (Laemmli 1970). Post-electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 for protein visualization. A molecular weight marker, Precision Plus Protein™ Unstained Standard (Bio-Rad), with a size range of 250–10 kDa, was used to estimate the molecular weights of the protein bands.

Determining km and vmax values for cellulase

The maximal velocity (Vmax) and the Michaelis-Menten constant (Km) for cellulase were determined using CMC as the substrate within a concentration range of 0.1–3% (Counotte and Prins 1979).

Evaluation of optimal temperature and pH for cellulase activity

To find the optimal temperature for the purified cellulase, enzyme activity assays were conducted across a range of temperatures from 25 to 60 °C, with all other conditions kept constant. Prior to the reaction, enzyme and substrate solutions were kept for 10 min at the specified temperatures.

To determine the optimum pH, the activity assays were conducted in a pH range of 4.0 to 12.0. The used buffers included 50 mM acetic acid-sodium acetate buffer for pH 4.0–5.0, potassium phosphate buffer for pH 6.0, tris-HCl buffer for pH 7.0–8.0, and glycine-NaOH buffer for pH 9.0–12.0. Additionally, the corresponding buffers were used to prepare the substrate solution to maintain the desired pH throughout the assay.

Assessment of enzyme durability across the temperature and pH variations

Temperature stability was assessed by incubating enzyme samples at a temperature range of 25–60 °C for periods of 30, 60, 90, and 120 min. The relative activity of the enzyme was calculated as a percentage of the activity observed in the unincubated control sample, which served as a reference for maximal enzyme activity.

To evaluate pH stability, enzyme solutions were incubated in buffers with a pH range of 6.0 to 12.0 for 1 h at 25 °C. Following this incubation, the relative activity was expressed as a percentage of the activity of the unincubated control sample. The control samples for temperature and pH stability assays were considered to be 100%.

Assessing the effects of metal ions, inhibitors, and solvents on cellulase activity

To examine the impact of metal ions, 5 mM and 10 mM solutions of various metal ions (AgNO₃, BaCl₂, CaCl₂, Cd(NO₃)₂, CoCl₂, CuSO₄, FeCl₂, HgCl₂, MgCl₂, MnCl₂, NiCl₂, and ZnCl₂) were prepared. Additionally, to investigate the effects of inhibitors, 20% solutions of various inhibitors such as EDTA (ethylenediaminetetraacetic acid), Triton X-100, H₂O₂, SDS, Tween 80, and Tween 20) were used. For evaluating the effects of solvents, 20% solutions of ethanol, DMSO (dimethyl sulfoxide), glycerol, acetone, and isopropanol were prepared. All solutions were dissolved in Buffer A. The enzyme and test solutions were incubated for 1 h at room temperature in a 1:1 ratio. Enzyme activity in the presence of each test solution was compared to that of a control sample, which was incubated under identical conditions without any test solutions, to calculate the relative activity.

Assessment of cellulase activity and stability in the presence of commercial detergents

To assess the impacts of commercial detergents on cellulase activity, standard activity assays were performed with the addition of 1% liquid soap, dishwashing detergent, liquid laundry detergent, and powdered laundry detergent. To evaluate the impacts of these detergents on cellulase stability, the enzyme was mixed with the detergent solutions (1% liquid soap, dishwashing detergent, liquid laundry detergent, and powdered laundry detergent in Buffer A) at a 1:1 ratio. The mixtures were incubated at room temperature for 1 h. Following incubation, cellulase activity was measured under standard conditions. The relative activity was then calculated by comparing it to a control sample, which was incubated under the same conditions but without detergents.

Biotechnological applications of cellulaseAssessment of cellulase for surface fuzz removal from cotton fabrics

The depilling effect of cellulase on cotton fabric surfaces was evaluated by treating cotton fabric samples with cellulase-rich supernatant obtained from the bacterial culture grown in CMC-broth medium for 96 h. Fabric pieces of approximately 2–2.5 cm were immersed in 25 mL of enzyme solution, and the samples were subjected to shaking (100 rpm) to ensure uniform exposure for varying time intervals (30, 60, 90, and 120 min). After each incubation period, the treated fabric pieces were thoroughly rinsed with deionized water to remove any residual enzyme. The rinsed fabrics were subsequently dried at 60 °C for 1 h. The extent of fuzz removal from the fabric surface was evaluated through visual inspection, providing qualitative insights into the efficiency of cellulase in reducing pilling (Duran et al. 2008).

Exploring the potential of cellulase in textile dye removal: a case study with indigo

To evaluate cellulase’s ability to degrade fabric dyes, indigo dye was used as the dyeing agent. Indigo, which is insoluble in water, was converted to its soluble form, leuco-indigo, under alkaline conditions (Pathak and Madamwar 2010). Soluble leuco-indigo was prepared by reducing indigo dye powder (5 g/L) with NaOH (2 g/L) and Na2S2O4 (2 g/L). Fabric pieces, measuring 2–2.5 cm, were added to the dye solution. Dyeing was performed at room temperature for 1 h. After dyeing, the samples were air-oxidized, rinsed, and air-dried. Fabric pieces were immersed in 5 mL of enzyme solution for 1, 3, 5, 15, and 20 h. After incubation, the fabric pieces were washed with distilled water to remove any residual dye. The cleaned fabrics were dried in an oven at 60 °C for 1 h, and the dye concentration levels on the fabric were visually inspected (Tayade and Adivarekar 2014).

Statistical data analysis

All experiments were carried out in triplicate, and the resulting data were analyzed using Microsoft Excel 2016 (Microsoft Corporation). The data are reported as mean values with standard deviations. Statistical significance was assessed at a 95% confidence level, with p-values less than 0.05 (p < 0.05) considered significant.