Kinetic, Catalytic and Thermodynamic Properties of Immobilized Milk Clotting Enzyme on Activated Chitosan Polymer and its Application in Cheese Making

Milk clotting enzyme (MCE) from Bacillus circulans 25 was immobilized by covalent binding, ionic binding and entrapment methods using various carriers. MCE covalently immobilized on activated chitosan polymer with the bifunctional agent glutaraldehyde (Ch-MCE) exhibited highest immobilization yield (74.6 %). Comparing to the native MCE, Ch-MCE exhibited higher optimum pH, higher optimum reaction temperature, lower activation energy, higher half-life time, lower deactivation rate constant and higher energy for denaturation. After immobilization, maximum reaction rate, Michaelis-Menten constant, specicity constant, turnover number, and catalytic eciency of the enzyme were signicantly changed. Calculated thermodynamic parameters for denaturation (enthalpy, entropy and Gibbs free energy) conrmed that the catalytic properties of MCE were signicantly improved after immobilization. Reusability tests showed that after 7 catalytic cycles, the Ch-MCE retained about 71 % of its activity conrming its suitability for industrial applications.


Introduction
Using rennet enzyme in making cheese is the largest application of enzymes in food processing. Rennin acts in two stages for milk protein coagulation by speci c hydrolysis of peptide bond (Phe 105 -Met 106 ) of κ-casein (da Silva 2017). Recently, there are additional applications of proteases in dairy technology, to accelerate the ripening of cheese and to modify its functional properties (Afroz et al. 2015). Microbial rennin is more acceptable in cheese production as an alternative to chymosin from newborn ruminants due to ethical problems and increased demand for cheese making (da Silva 2017).
Enzymes as bioactive agents have unique characteristics but the effectiveness is limited by their physicochemical properties as stability (Ephrem et al. 2018). The use of soluble enzymes has some drawbacks that increase the consumption of enzymes as instability, easy inactivation, reduction of catalytic stability and di culty of removal from the mixtures. To solve these problems, enzyme immobilization considered an effective technique which not only stabilizes enzymes under operating conditions but also allows easy recovery and reuse (Wehaidy et al. 2018).
Enzymes can be immobilized by adsorption, entrapment, covalent binding and ionic binding methods.
However, immobilization by covalent binding is the most effective procedure in establishing enzymes and inhibiting their leakage due to the formation of strong covalent bond between carrier and enzyme (Eskandarloo and Abbaspourrad 2018). Covalent binding consists of two steps, rst one, activation of functional groups found on carrier surface by a speci c reagent, and the second, adding enzyme to form covalent bond with activated surface of carrier. In the coupling reaction, these activated groups will react with strong electron donating nucleophiles, such as the amino group (NH 2 ) and functional groups of certain amino acids on the surface of most enzymes (such as carboxylic group (COOH) of aspartic acid, amino groups (NH 2 ) of lysine, hydroxyl group (OH) of serine, and sulfhydryl group (SH) of cysteine). Immobilization can be performed using different carriers whose properties play an important role in enzyme behavior. Desired properties of the insoluble carriers include low-cost, non-toxic, high surface area, reusable and good stability (mechanical, chemical and thermal). (Narwal et al. 2016). Since there is no universal carrier suitable for all enzymes and all applications, it is important to examine different carriers using different methods of enzyme immobilization. Various carriers have been used for immobilization e.g. polyacrylic (Esposito et al. 2016), alginate-pectate (Narwal et al. 2016 Chitosan is one of natural polymer (polysaccharides) derived from chitin by deacetylation process and has excellent biocompatibility, no toxicity, cheapness, high mechanical strength, and a susceptibility to chemical modi cations (Cahyaningrum and Sianita 2014; Salazar-Leyva et al. 2017). One common approach for enzyme immobilization on chitosan is through multipoint covalent binding between the functional groups present on the surface of an activated chitosan by cross-linking agents such as glutaldehyde and the surface functional groups of the enzyme protein.
Thermodynamics act as a key tool to understand the thermal deactivation process. Estimation of the thermodynamic parameters of the enzyme as enthalpy (∆H*), entropy (∆S*), and the Gibbs free energy (∆G*) can provide useful information as enzyme behavior, activity and thermostability. The suitability of enzymes for industrial application is judged by their thermodynamic parameters (Zaboli et al. 2019).
The objectives of this study are to promote Bacillus circulans 25 MCE properties by covalent coupling to chitosan after activation and evaluate its catalytic, kinetics and thermodynamic parameters. Finally, it evaluates the reusability of Ch-MCE in cheese making.

Materials
All the chemical reagents used were of analytical grade. Ceramic, wool and chicken bones were collected from the local market. Milk powder, skim milk powder spray dried (heat treated grade) was made in USA and obtained from the Ministry of Agriculture, Giza. Egypt.

Enzyme production
Milk clotting enzyme from Bacillus circulans 25 has been produced according to the previous work (Ahmed et al. 2018). The medium used for MCE production had the following composition (g/L): lactose 20, yeast extract 1, peptone 1, K 2 HPO 4 2 and MgSO 4 .7H 2 O 0.25. The pH was adjusted to 6.0 prior to sterilization. One mL of cell suspension of 24 h-old slant (OD600 ~ 0.3) was transferred to 50 mL sterile medium in 250-mL Erlenmeyer ask. The asks were incubated at 35°C on a rotary shaker at 180 rpm for Page 4/26 24 h. The broth media after incubation was centrifuged at 6000 x g and 4°C for 15 min and the cell free ltrate was considered as source of crude enzyme.

Milk clotting activity
Milk clotting activity was estimated according to Narwal et al. (2016) method. Enzyme solution (2.5 mL) or certain weight of Ch-MCE was incubated with 10 mL skim milk (12 g dry skim milk/100 mL of 0.01 M CaCl 2 ) at 40°C. The end point is recorded when discrete particles were discernible by stop watch. One unit of the MCE activity (U) was equalized to 10 mL milk clotted within 10 min.

Protein determination
The protein content of the MCE preparation was estimated by the method of Lowry et al. Where: I is the total activity of immobilized enzyme, A is the total activity offered for immobilization and B is the total activity of unbounded enzyme.

Immobilization of MCE by covalent-binding
The beads of chitosan were prepared by shaking 0.4 g chitosan in 5 mL of 0.01 M HCl containing glutaldehyde (GA 2.5 %) at 30°C for 2 h. The beads were precipitated using 0.1 N NaOH. The beads were collected by ltration, washed with distilled H 2 O (to remove the excess GA). Then 5 mL enzyme solution (440U) was mixed with the wet beads by gently shaking. After 2 h at 30°C, the unbounded enzyme was removed by washing with distilled H 2 O until no activity was detected. One gram of other carriers (chitin, wool, chicken bone, As-alumna, ceramic or PVC) was shaken in 25 mL Tris-HCl buffer (0.01 M, pH 6.0) containing 2.5 % GA at 30°C for 2 h. The carriers were ltered off, and washed with distilled H 2 O to remove the excess GA. Then each treated carrier was incubated with Tris HCl buffer (5 mL, 220 U of MCE). After incubation at 30°C for 2 h, the unbounded enzyme was removed by washing with distilled Relative activity (%) = (A1/A2) X 100 (3) Where: A1 is the activity detected under the certain condition and A2 is the activity detected under the optimal condition. The stability to pH was investigated by pre-incubating enzyme samples in 0.01M tris -HCl buffer with pH ranging from 5.0 to 9.0 at 25°C for 1h followed by adjusting the pH to the optimal of each enzyme form. The residual (retained) activity was assayed under the standard conditions and calculated to according to Eq. (4).

Optimum temperature and thermal stability
The effect of temperature on the free and Ch-MCE was also determined. The enzyme samples in 0.01M tris -HCl buffer at pH 6.0 and 7.0 (for free and Ch-MCE), respectively were subjected to different temperatures (from 30°C to 100°C). The activation energy (E a ) was estimated from the slope of Arrhenius plot of log the residual enzyme activity (%) against reciprocal of absolute temperature in Kelvin (°K) according to Eq. (5).
Where, E a is the activation energy and R is the gas constant (1.976 Kcal/ mol).
Temperature coe cient value (Q 10 ), the rate of an enzymatic catalysis reaction changes for every 10°C rise in temperature, was calculated as reported by Wehaidy et al. (2018) as Eq. (6). Q 10 = antilog E = (E x 10/RT 2 ) (6) Where E = E a = activation energy For thermal stability, free and Ch-MCE were heated at different temperatures (40-80°C) in the absence of substrate for different time intervals (15-120 min). Every 15 min a sample was removed and the residual activity was estimated under standard assay conditions. The enzyme activity without heating was taken as 100%. Deactivation rate constant (k d ) was determined according to Eq.
The energy for denaturation of enzyme (E ad ) was calculated from Arrhenius plot of (ln k d ) as a function of (1/T) temperature in Kelvin (°K) using the following in Eq. (9).
Effect of substrate concentration Both the free and Ch-MCE activities were assayed with different substrate concentrations ranged from 1 to 12 % (w/v) at optimal assay conditions. Michaelis-Menten constant (K m ) and maximum velocity (V max ) were estimated from Lineweaver and Burke (1934). In addition, the turnover number (k cat ), catalytic e ciency (k cat /K m ), speci city constants (V max /K m ), free energy of substrate binding (∆G* E−S ) and free energy of transition state binding (∆G* E−T ) were estimated according to Abdel-Naby et al. ΔG* E−S (Free energy of substrate binding) = -RT Ln K a , where K a = 1/k m (14) ΔG* E−T (Free energy for transition state formation) = -RT Ln (k cat /K m ) (15) Effect of metal ions The metal ions (ZnSO 4 , CoCl 2 , CaCl 2 , MnSO 4 , CuSO 4 , MgSO 4 , HgCl 2 and NaAsO 2 ) were added individually (10 mM) to the reaction mixture. Both free and Ch-MCE activities were assayed under optimal assay conditions. Suitability of Ch-MCE in the making of cheese (reusability) A weight sample (4 g) of Ch-MCE (wet) was placed in a bag of muslin. The bag was immersed in skim milk solution (10 mL). The mixture was incubated at 85°C until forming the colt. At the end of the reaction, the bag containing the immobilized MCE was removed from the colt, washed with distilled water, and re-suspended in a freshly prepared substrate (10 mL) to start a new run.
Results And Discussion B. circulans 25 MCE was immobilized on various carriers by different methods of immobilization to select the suitable carrier and method. The e ciency of enzyme immobilization was evaluated by different parameters including the residual catalytic activity (RA %), the speci c activity (of the immobilized enzyme), and the immobilization e ciency (IE %). Moreover the immobilization yield (IY %) is the key parameter that it represents the general output of the immobilization process e ciency.

Enzyme immobilization
Immobilization of MCE by covalent-binding Immobilization of MCE by covalent-binding was achieved by cross-linking between the enzyme and activated carriers throughout GA as a spacer group. The reaction happened between the NH 2 groups found in the enzyme protein molecule and the free C = O group located on GA (the cross-linker) forming C = N-bond as reported by Abdella et al. (2020). The amount of MCE used for chitosan was higher than that used for other carriers due to its higher loading e ciency. The data presented in Table 1 Table 3). The results showed that the LE was gradually decreased from 37.2 U/10 mL gel to 18 U/10 mL gel with the gel concentration increase from 2 to 8 %. This probably due to the decrease of the gel porosity with the increase of Na-alginate concentration, and consequently the diffusion limitation was developed. Similar observation was previously reported for entrapped proteases (Abdel-Naby et al. 1998; Lamas et al. 2001).
The pore size of the gel, re ected in the viscosity of the carrier due to the size of the molecule and/or its concentration, can affect the diffusion of substrates or products and limit the reaction rates of the entrapped enzyme. Effect of pH on the free and Ch-MCE activity As illustrated in Fig. 1 the free MCE was optimally active at pH 6.0, however, the Ch-MCE was optimally active at pH 7.0. At higher pH values up to 8.0, the drop in activity was more pronounced with the free than that of the Ch-MCE. These effects may be due to the changes of the ionic microenvironment of the enzyme active site and /or distribution of the surface charges of the carrier after immobilization (Talbert

Effect of temperature on the free and Ch-MCE activity
The optimal reaction temperature of the MCE (free and Ch-MCE) was investigated at their optimal pH. As seen in Fig. 3 and Table 4, the free enzyme was optimally active at 75°C however, the Ch-MCE was optimally active at 85°C. Increasing of the optimum temperature for Ch-MCE by 10 degrees is probably a consequence of enhanced thermal stability by immobilization. Covalent binding of enzymes onto chitosan enhanced the optimum temperature and stability of the biocatalysts for thermal inhibition  Thermal stability of free and Ch-MCE Although thermostable enzymes are more suitable for industrial applications than mesophilic enzymes, stability of MCEs for long times at a mild temperatures is very important for their suitability in making cheese. The results in Fig. 5 showed that the immobilization improved the stability of MCE to the thermal inhibition. Thus, after heating for 120 min, the Ch-MCE was stable up to 60°C with 100 % residual activity whereas, the free MCE lost about 90.4 % of its initial activity. In addition, after heat treatment at 70°C for 90 min, the free MCE was MCE Free was completely inhibited however, the Ch-MCE retained 40 % of its initial activity. The increase in enzyme stability after immobilization was possibly related to the higher rigidity of the immobilized form (Yang et al. 2017). In addition, the stability to heat inhibition enhancement after immobilization could be caused by the carrier that protects the enzyme from denaturation by absorbing a great amount of heat (Figueira et al. 2011).
The calculated values of deactivation rate constant (k d ) in Table 4 indicated that the stability of the Ch-MCE to the thermal inhibition was superior to that of the free MCE (the lower k d , the more thermo stable enzyme). For example, k d at 70°C for the free enzyme was 13.6 x 10 − 3 , which was higher by 3-fold than that of the Ch-MCE (4.4 x 10 − 3 ). These results con rm the effectiveness of the MCE immobilization on chitosan for increasing the thermal stability. In addition, half-life (t 1/2 ) of the Ch-MCE at 65, and 75°C were higher by 6 and 5-fold, respectively than that of the free MCE. The binding between the enzyme and the carrier reduces conformational exibility and thermal vibration, thus protecting the immobilized protein from denaturing and unfolding by increasing the temperature (Figueira et al. 2011).
The energy for thermal denaturation (E ad ) for Ch-MCE was greater by 161.39 kJ/ mol than that obtained for free MCE (Fig. 6 and Table 4). This result indicated that immobilized form needed more energy for deactivation compared to the free form. The energy of thermal inactivation is an important factor to judge its thermal stability (Thakrar and Singh 2019).

Effect of substrate concentration
As seen in Lineweaver-Burk plots (Fig. 7) Ch-MCE provided k m 1.3-fold higher and V max 1.3-fold lower than the free MCE. In addition, the V max /K m of Ch-MCE was decreased by 1.6-fold compared to the free MCE (Table 5). This change in enzyme a nity towards its substrate after immobilization probably related    The recorded data in Table 5  Suitability of Ch-MCE in making cheese (reusability) The greatest immobilized enzymes advantage their repeated use which is of practical utility for commercial purposes as it can be easily separated from soluble reactants and products. The Ch-MCE retained about 100 and 90.4 % of the initial activity after being used for 5 and 7 consecutive cycles, respectively. Multipoint covalent binding of enzymes on activated supports promotes a rigidi cation of its structure and reusability (Ahmed et al. 2019). Upon repeated use, gradual decrease in activity was observed ( Fig. 9) which probably due to the enzyme denaturation and loss of the enzyme from the carrier physically. Conversely, Salazar-Leyva et al. (2017) reported that immobilized proteases retained 40 % of its initial activity after the second cycle.

Conclusion
B. circulans 25 milk clotting enzyme was successfully immobilized on activated chitosan (Ch-MCE) with immobilization yield 75% and immobilization e ciency 72%. Based on the results, thermal properties of the Ch-MCE were enhanced. Compared to the free enzyme, Ch-MCE exhibited higher optimum temperature, lower kd (deactivation rate constant) and higher t 1/2 (half-life time). In addition, immobilization improved the quality of MCE by decreasing the activation energy (Ea) by 0.9-fold compared to the free enzyme. Moreover, the E ad (energy for denaturation) of the Ch-MCE was 1.8-fold higher than that of the free enzyme meaning that the immobilization process increases the heat resistance of enzyme (more energy is required to enzyme denaturation). The calculated thermodynamic parameters as enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) demonstrated that covalent binding between enzyme and activated chitosan increased its thermal stability. Also, Ch-MCE showed higher pH stability at different pH values compared with the free enzyme. Ch-MCE is suitable for application in cheese making and can be used successfully for 8 consecutive cycles with 80 % residual activity. It can be concluded that this work helps to overcome the limitations of the reduction in MCE catalytic activity that associated with changes in temperature, pH and inhibitors making it useful in industrial applications and biotechnological process.

Declarations
Authors' contributions Abdel-Fattah; participated in studies. Abdel-Naby; supervised the complete study. Ahmed; performed the research experiments and wrote the manuscript. All authors read and approved the nal manuscript.

Funding
Not applicable.

Availability of data and materials
All data obtained or analyzed during this study are included in this article and available from the corresponding author.  Figure 1 Effect of pH on the activity of free MCE (hollow) and Ch-MCE (solid)