Karam et al.

Microbial Cell Factories (2024) 23:283 Microbial Cell Factories

https://doi.org/10.1186/s12934-024-02521-y

RESEARCH Open Access

Cell immobilization for enhanced milk

clotting enzyme production from Bacillus
amyloliquefacien and cheese quality
Eman A. Karam2, Mohamed E. Hassan1, Nouran A. Elattal1, Amany L. Kansoh2 and Mona A. Esawy1*

Abstract
Background Milk clotting enzymes, essential for milk coagulation in cheese production, are obtained from the
stomach of young ruminants, an expensive and limited source. This study was accomplished by finding a suitable
alternative. Bacterial isolates recovered from honey were screened for milk clotting enzyme activity. and further, by
immobilization of the microorganisms to enhance stability and facilitate their repeated use.
Result The most effective enzyme was produced by a microbe identified as Bacillus amyloliquefaciens based on
16 S rRNA sequencing. The cells were encapsulated in Ca2+ alginate beads. These beads retained complete enzyme
production after being used five times. Glucose and Soybean were selected as the most favorable carbon and
nitrogen sources, respectively. The optimum temperature for activity was 35 ℃ for both free and immobilized cells
but as the temperature was increased to 55 °C and above, the encapsulated form retained more activity than the free
cells. The pH optimum shifted from 6.5 to 7 for the free cells to 7–7.5 for the immobilized cells. The immobilization
process decreased the activation energy for enzyme production and activity, prolonged the enzyme half-life, and
increased the deactivation energy. Enzyme produced by immobilized cells generated a more compact cheese.
Conclusions The finding of this study was to identify a less expensive source of milk-clotting enzymes and
confirm the success of cell immobilization in improving cell rigidity and stability. Also, immobilization of this B.
amyloliquefaciens strain offers an enzyme source of value for industrial production of cheese.
Keywords Bacillus amyloliquefacien, Milk clotting, Cell immobilization, Thermodynamic-cheese

Introduction
Chymosin, the main component of rennet (milk clot-
ting enzyme), is an acid protease produced in the fourth
stomach of young, milk-fed ruminants, such as calves,
lambs and kids. It serves as a milk coagulant in the mak-
ing of cheese by hydrolyzing the kappa-casein chain in
*Correspondence: milk’s casein micelles [1, 2]. The type of milk-clotting
Mona A. Esawy enzyme (MCE), which is essential in the process of milk
mona_esawy@hotmail.com
1
Chemistry of Natural and Microbial Products Department, coagulation and cheese maturation, has a significant
Pharmaceutical Industries and Drug Research Institute, National Research impact on the quality of the cheese. The high cost and
Centre, Dokki, Cairo 12622, Egypt limited availability of rennet from young animals, as well
2
Microbial Chemistry Department, National Research Centre, Dokki, Cairo,
Egypt as its unsuitability for vegetarians, has led to numerous

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 2 of 13

projects focused on finding other sources of MCE. A were selected and purified by serial subculture and plat-
number of microorganisms have been identified as being ing. Selected bacteria were stored at − 80 °C in the cul-
particularly useful since they grow rapidly, are relatively ture medium with glycerol. The bacterial isolates were
inexpensive to produce and have a diversity of proper- streaked onto agar slants and preserved at 4 °C.
ties desired by cheese makers [3, 4]. Different species of
Bacillus supply some of the most popular distinguish- Screening isolates for milk clotting enzyme production
ing traits. For instance, lipase from Bacillus lichenifor- All pure isolates were inoculated on milk plates to test
mis NCU CS-5 was used to enhance the fatty acid flavor for milk clotting enzyme secretion and incubated at 37 °C
release for low-fat cheeses [5]. Bacillus velezensis DB219, for 28 h. Milk clotting positive strains were determined
Bacillus subtilis PNG27, and Bacillus methanolicus LB-1 by the presence of a clear zone around the colony on the
have been reported as milk clotting producers [6]; [7]; [3]. milk agar plates indicating milk hydrolysis. Colonies hav-
Several studies have used honey as a reservoir for poten- ing a clear zone around them were selected for further
tial probiotic and prebiotic bacteria [8–11]. investigation [16].
Bacillus amyloliquefaciens is an efficient source of milk
clotting enzymes [12]. Immobilization of microbial cells Identification of potent milk clotting producers by DNA
is considered one of the best methods to reduce the eco- and 16 S- rRNA
nomic cost of enzyme production because the cells can Bacterial isolates were grown in Luria-Bertani broth (LB)
be reused several times without losing too much enzyme for 24 h and were harvested by centrifugation at 12,000 g
activity. Moreover, cell immobilization improves enzyme for 5 min. They were washed three times by resuspension
stability, facilitates cell separation from the medium, in 0.85% NaCl and centrifugation. Genomic DNA was
enhances retention of plasmids, increases resistance to extracted using Gene JET Genomic DNA purification kit
toxic contaminants, and promotes secondary metabolite (Thermo Scientific, Lithuania) according to the manufac-
production [13]. Calcium alginate beads are often used turer’s recommendations [17].
for cell immobilization. The beads offer superior biocom- Amplification was done using forward primer 8 F (5′-
patibility, affordability, accessibility, and ease of prepa- CAG GCC TAA CAC ATG CAA GTC-3′) and reverse
ration [14]. Thermodynamic studies help evaluate the primer 1492R (5′-GGG CGG GGT GTACAA GGC-3′).
suitability of an enzyme for a given application. The PCR mixture was carried out in a volume of 50 µl,
This study represents a new attempt to address the containing 22 µl of MQ, 25 µl of DreamTaq Green DNA
clotting problem that has hindered the application of Polymerase (Thermo Fisher Scientific, USA), 1 µl of each
milk clotting in the industrial field. To this end, Bacillus forward and reverse primer (10 µmol/l), and 1 µl of tem-
amyloliquefaciens cells were immobilized on Ca2+ algi- plate. The PCR amplification conditions were 4 min of
nate beads. The cells exhibited high stability and could be preheating at 95 °C, 30 s denaturation at 95 ℃, 45 s of
reused for 5 cycles with no loss of milk clotting enzyme primer annealing at 50 ℃, 1 min extension step at 72 °C,
production. Abiotic production parameters were opti- and post cycling extension of 10 min at 72 ℃ for 35
mized. A thermodynamic study confirmed that immobi- cycles. The reactions were carried out in a thermal cycler
lization decreases the activation energy and increased the (Applied Biosystem Thermal Cycler, USA) [18].
deactivation energy while prolonging the enzyme half-
life. Immobilization of this B. amyloliquefaciens strain Agarose gel preparation
offers an enzyme source of value for industrial produc- Agarose was boiled in 1X TBE buffer. Ethidium bromide
tion of cheese. was added to the melted gel after the temperature cooled
to 55 °C. Melted gel was poured into the mini-gel appa-
Materials and methods ratus tray and the comb was immediately inserted. The
Bacterial sources comb was removed after the gel hardened. Electrophore-
Different honey types were collected from Saudi Arabian ses buffer (1X TBE) covered the gel and 15 µl of dsDNA
Gably honey, Libyan Gably honey and commercial Egyp- was loaded in each well and 3 µl of 2.5 kbp DNA ladder
tian honey made from nectar collected from clover. was added. 16 S rDNA PCR product was extracted from
the gel using Promega Wizard Genomic DNA Purifica-
Isolation of bacterial strains tion Kit [19].
One hundred microliters of each honey sample was
spread on Nutrient agar medium (Peptone, 5.0; Beef Sequence alignment, phylogenetic analysis, and
extract, 3.0; NaCl, 3.0 and agar, 15.0 gm/L) [15]. The bioinformatics analysis
plates were incubated at 37 °C for 24 h or until the bac- Amplified PCR product was purified and sequenced at
terial colonies were grown to sufficient size for colony Macrogen, Inc., Korea. Raw sequencing data was edited
replication (3–5 mm in diameter). Four bacterial colonies (contig and peak chromatogram verification) using

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 3 of 13

the Finch T.V 1.4.0 program. Analysis of 16 s rRNA micromole of equivalent L-tyrosine/min under the stan-
sequences of strains was performed using the BLAST (N) dard conditions of the assay.
program of the National Center of Biotechnology Infor-
mation (NCBI) (Rockville Pike, Bethesda MD, USA). Microencapsulation of bacterial cells in Alginate beads
Multiple sequence alignment was done using the Clustal For microencapsulation of bacterial cells, all solutions
W 2.1 program. The phylogenetic trees were constructed and equipment were sterilized at 121 °C for 20 min. Algi-
using the neighbor joining method by MEGA. X. nate beads were prepared according to Bashan [23]. In
brief, 250 mL of bacterial culture was centrifuged, the
Enzyme production supernatant was removed, and the cell pellet was washed
Milk clotting enzyme was produced according to Guleria with saline solution. It was then suspended in 50 mL of
et al. 2016 [12]. The medium used for MCE production a 3% alginate solution and thoroughly mixed. This mix-
had the following composition (g/L): Glucose 10, yeast ture (alginate and cell pellet) was dropped into a sterile
extract 2.5, Casein 5. The pH was adjusted to 7.0 prior to 3% calcium chloride solution using a syringe needle, with
sterilization. One ml of cell suspension from a 24 h-old 10 cm between the needle and the calcium chloride solu-
slant was transferred to 50 ml sterile medium in a 250-ml tion surface. The alginate beads containing bacterial cells
Erlenmeyer ask. The flasks were incubated at 35 °C on a were allowed to harden in a calcium chloride solution for
rotary shaker at 180 rpm for 24 h. After incubation, the 3 h. Beads were collected, washed with sterile water, and
medium was centrifuged at 6000 x g and 4 °C for 10 min stored in saline solution for further investigation [24].
and the cell free filtrate was considered as the source of
crude enzyme. Operational stability of immobilized cells
Operational stability was determined using 1 g of cells
Milk clotting activity immobilized in calcium alginate beads. The immobi-
Milk-clotting activity (MCA) was determined according lized form was incubated with 50 ml of the medium in
to the method of Arima et al. [20] and expressed in terms a 250 ml flask for 24 h at 180 rpm and 35 °C. Then it was
of Soxhlet units (SU). One SU was defined as the amount collected, washed with distilled water, and re-suspended
of enzyme which clotted 1 ml of a solution containing in 50 ml of freshly prepared medium to start a new run.
0.1 g skim milk powder and 0.00111 g calcium chloride The supernatant was assayed for milk clotting activity
in 40 min at 35 °C. In brief, 0.5 ml of material was added after centrifugation. The amount of enzyme that leaked
to a test tube containing 5 ml of reconstituted skim milk out of the beads was determined by dividing the opti-
solution (10 g dry skim milk/100 ml, 0.01 M CaCl2) pre- cal density (OD) of the culture filtrate of the immobi-
incubated at 35 °C for 5 min. The solution was mixed lized cells by the OD of the free cells and subtracting the
well, and the clotting time T (s), measured as the time obtained value from 100.
period from the addition of test material to the first
appearance of clots, was recorded and the clotting activ- Effect of incubation time and temperature on enzyme
ity was calculated using the following formula: Soxhlet production
units (SU) = 2400 × 5 × D/T × 0.5; T = clotting time (s); To study the effect of incubation time on the production
D = Dilution of test material [16]. of milk clotting enzymes from free and immobilized cells,
the BM cultures were incubated for 24 h at 180 rpm and
Protein determination 37 ℃ for different times (6, 12, 24, 36, 48, 50, and 62 h).
The method used to measure protein content was dis- Then the enzyme production is determined according to
closed by Lowry et al. [21]. By deducting the amount of the enzyme activity.”
unbound protein from the protein that was initially intro- The effect of temperature on free and immobilized cells
duced for immobilization, the amount of protein that was was measured by preheating the free and immobilized
immobilized could be estimated. cells at different temperatures (25–60 ℃) for different
time intervals (15,30, 60,90 and 120 min). Then the pre-
Protease activity (PA) heated cells were inoculated in 50 ml of the production
To determine proteolytic activity, the bacterial dilutions medium for 24 h at shaker (180 rpm, 40 ℃). The residual
were streaked on milk agar plates. The plates were incu- activity of enzyme production was assayed under opti-
bated for 24 h at 30 ℃, and strains that produced clear mized conditions. The optimum temperature was taken
zones in the medium were chosen for additional testing. as 100% activity for complete enzyme production and the
The quantitative protease activity was measured accord- relative activity of the produced enzyme at each tempera-
ing to Kembhavi & Kulkami [22]. One unit of protease ture was expressed as a percentage related to the 100%
activity was the amount of enzyme that released one activity.

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 4 of 13

The activation energy (Ea) was calculated from the T1/2=ln2/kd. The decimal reduction time or the time
slope of the Arrhenius plot [25] of 1000/T versus ln needed to reduce the enzyme value by 90% was calcu-
[enzyme relative activity] (Ea = − slope × R), where R (Gas lated as D-value = ln10/kd.
constant) = 8.314 J. K. mol− 1. The activation energy (Ea) of catalysis for both the
free and immobilized milk clotting enzyme forms was
Effect of carbon and nitrogen sources on enzyme determined from the slope of the Arrhenius plot [log V
production (ln of % residual activity) versus reciprocal of absolute
To evaluate the most appropriate carbon source for temperature in Kelvin (1000/T)], which is calculated as
production of milk clotting enzyme the glucose in the Slope = − Ea /R where R is the gas constant (8.314 mol − 1
medium was replaced with 1% (w/v) of different carbon k − 1). The denaturation energy for the enzyme was deter-
sources (fructose, lactose, mannose, maltose, sucrose, mined from a plot of the log of the denaturation rate
cellulose, and starch). Free and immobilized cultures constants (ln kd) versus the reciprocal of the absolute
were incubated at 35 °C for 24 h. Then milk clotting temperature (K) as follows: Slope = Ed/R.
enzyme activity was determined.
The medium was supplemented with equimolar pH stability
amounts of different organic and inorganic nitrogen The effect of pH on free and immobilized cells was mea-
(casein, peptone, urea, soyabean, NH4Cl, KNO3, NaNO3) sured by subjecting the free and immobilized cells to pHs
and both free and immobilized cultures were incu- ranging from 5 to 9 for time intervals of 15, 30, 45, and
bated at 35 °C for 24 h. Then milk clotting activity was 60 min. Then the treated cells were inoculated in 50 mL
determined. of the production medium for 24 h at 150 rpm, 35ºC, and
the residual activity was assayed under optimized con-
Effect of temperature on enzyme activity ditions. The optimum pH was taken as 100% activity of
The optimal temperature was determined by incubat- enzyme production, and the relative activity at each pH
ing the reaction mixture at different temperatures from was expressed as a percentage of the 100% activity.
25 to 60 °C. The residual activity of enzyme was assayed
under optimized conditions. The optimum temperature Effect of metal ions on production of the milk clotting
was taken as 100% activity the relative activity at each enzyme
temperature was expressed as a percentage related to the Some metal ions (MgSO4.7H2O, NaCl, FeSO4, CaCl2,
100% activity. ZnSO4, MnSO4 and CuSO4) were added separately to the
culture medium at a final concentration of 0.01 M and
Effect of pH on the production of milk clotting enzyme then inoculated by free and immobilized cells incubated
The cell culture for both free and immobilized cells were at 35 ºC for (24 h free cells, 48 h immobilized cells). The
adjusted at pHs ranging from (5–9) in acetate buffer for enzyme production was determined as before.
pH 5–6 or in phosphate buffer for pH 7–9. The BM cul-
tures were incubated at180 rpm and 35 °C for 24 h and Statistical analysis
then assayed for milk clotting production. Data analysis was carried out with MICROSOFT EXCEL
(2007). All data are presented as means ± standard error
Temperature stability of means. The acid and alkali tolerance experiments, the
The effect of temperature on free and immobilized cells bile and pancreatic enzyme tolerance were independently
was measured by preheating the free and immobilized replicated 2 times (n = 2), with 2 measurements per repli-
cells at different temperatures (25–60 ℃) for different cate. The mean of the repeated measurements yielded the
time intervals (15,30, 45 and 60 min). Then the preheated value for each replicate.
cells were inoculated in 50 ml of the production medium
for 24 h at shaker (180 rpm, 35 ℃). The residual activity Result and discussion
was assayed under optimized conditions. The optimum Isolation and identification
temperature was taken as 100% activity of enzyme pro- A preliminary screening of 24 honey isolates revealed
duction and the relative activity at each temperature was variations in the size of the inhibition zones produced on
expressed as a percentage related to the 100% activity. skim milk agar plates, indicative of milk clotting activity.
Isolate NO 2 showed the largest inhibition zone and the
Thermodynamic study highest enzyme activity (Table 1). Of 24 isolates only 14
Thermal inactivation isolates gave a positive result.
The dissociation constant (kd) was estimated by a regres- The isolate was identified as Bacillus amyloliquefaciens
sion plot of relative activity (log) versus time (min). based on 16 S rRNA sequencing. The phylogenetic tree
The half-life (T1/2) for the enzyme was determined by of the isolate (Fig. 1) shows that it belongs to the genus

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 5 of 13

Table 1 Screening for milk clotting production example, Zhao et al. [26] detected a novel milk-clotting
Number of strain Diameter of clear zone Activity of enzyme produced by B. amyloliquefaciens GSBa-1. Simi-
(Mm) enzyme
larly, Guleria et al. 2016 [12] isolated a milk-clotting
(SU/ml)
enzyme-producing B. amyloliquefaciens from an apple
1 - -
2 25 2000 ± 086
rhizosphere in the northern Himalayas. Also, He et al.
3 - -
[27], mentioned B. amyloliquefaciens as a milk clotting
4 14 600 ± 066
enzyme producer from the Tibetan Plateau. Additionally,
5 13 400 ± 055 honey was previously mentioned as an efficient reservoir
6 12 343 ± 076 for probiotic bacteria with unique properties [28, 29].
7 13 218 ± 006 These characteristics made B. amyloliquefaciens a prom-
8 - - ising candidate for use in the cheese industry [30].
9 - -
10 - - Cell immobilization and effect on reusability
11 20 1200 ± 086 In this study Bacillus amyloliquefaciens GSBa-1 cells
12 12 240 ± 086 were immobilized in Ca+ 2 alginate beads. As reported by
13 - - many others working with various enzymes, milk clotting
14 32 2400 ± 006 enzyme activity was higher when the enzyme was immo-
15 11 110 ± 077 bilized compared with the free enzyme perhaps because
16 17 750 ± 055 the enzyme is protected from harsh environmental con-
17 12 480 ± 049 ditions. In addition, the alginate matrix has additional
18 15 800 ± 090 advantages of being less expensive, nontoxic, and requir-
19 13 600 ± 048 ing mild conditions for biocatalyst synthesis [31].
20 18 1200 ± 044 The main problem for MCE reusability is the coagu-
21 - - lation around the carrier which may result in blocking
22 - - the active site of the enzyme [32, 33]. In this study, it
23 - - was determined that immobilized cells could be reused
24 - - five times with no loss in activity. Complete milk clot-
ting decreased gradually to 67% of the initial enzyme
Bacillus and is 99.93% homologous to B. amyloliquefa- production after 10 cycles (Fig. 2). This suggests that
ciens strain NBRC15535. the immobilization process improves the stability essen-
Many authors have reported B. amyloliquefaciens tial for cheese manufacture. Data from this study indi-
as an efficient producer of milk clotting enzymes. For cates immobilized Bacillus amyloliquefaciens produced

Fig. 1 Phylogenetic tree based on partial 16 S rDNA sequences, showing the relationship between isolate No (14) Bacillus amyloliquefaciens and other
species belong to the genus bacillus. The tree was constructed using the MEGA11 and neighbor-joining method

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 6 of 13

Fig. 2 Reusability of the immobilized Bacillus amyloliquefaciens MCA

stable and reusable MCE. Alginate is made up of β-d- immobilized enzyme also had a lower PA in the presence
mannuronate and α-l-guluronate residues, which are of glucose than in the presence of lactose, with MCA to
[1→4], linked. The gradual decrease in production after PA ratios of 9600 and 2307, respectively.
5 cycles could possibly be the result of Ca2+ attaching to Other tested carbon sources had an adverse effect on
various α-guluronate residues, creating a gel network. MCE production, especially cellulose and starch. Starch
Future work will study the possibility of inhibition of the can activate amylase production and cellulose can result
gel network with monovalent metal ions, to extend the in the production of cellulases. Under these circum-
useful lifetime of the beads [34]. stances, milk clotting production will be reduced. Shieh
et al. [35], reported that glucose followed by sucrose were
Factors production of milk clotting enzyme the most effective carbon sources for Bacillus subtilis
Incubation time and temperature, sources of carbon and natto MCE production. However, our results showed that
nitrogen lactose inhibited enzyme activity completely.
The optimal time for milk clotting production (Table 1) Soyabean and peptone were the most effective nitrogen
was obtained after 24 h and 36 h. Below and above this sources for MCE production. The protease activity of the
time slot there was a reduction in enzyme production. free enzyme was 0.272 and 0.26 mg/mL, with MCA to PA
Similar results were reported by Guleria et al. [12], where ratios of 11,029 and 9230, respectively. The immobilized
the highest production of the Bacillus amyloliquefaciens enzyme had a PA of 0.65 and 0.51 mg/g, with MCA to
SP1 milk clotting enzyme activity was obtained after PA ratios of 5882 and 4615, respectively. Therefore, Soy-
24 h. The optimum temperature for the immobilized and bean was selected as the best nitrogen source under the
free MCA production was 35 °C. Below and above this selected conditions.
temperature there was a noticeable drop in MCA pro-
duction (Table 1). Optimum temperature for enzyme activity
Lactose and glucose were the most effective carbon The optimum temperature for the enzyme obtained from
sources for milk-clotting enzyme production, with no the free and immobilized cells was 35 ℃ (Fig. 3a). Also,
noticeable difference between the two (Table 1.). Most the results showed that the activity from the immobilized
proteolytic enzymes cause milk to coagulate, but because enzyme was higher as the temperature increased in com-
the coagulum is continuously digested by proteolysis, sta- parison to the free enzyme. This result is further evidence
ble cheese is rarely produced. The optimal carbon source that cell immobilization tends to protect cells against
should be selected based on the desired proteolytic activ- environmental conditions and gave the enzyme rigidity.
ity (PA). For cheese production, MCE with a high milk
clotting activity to protease activity ratio is desired. In Evaluation of the activation energy for enzyme production
this study, the free enzyme had a lower PA in the pres- and activity
ence of glucose than in the presence of lactose, with Additionally, immobilization reduced the activa-
MCA to PA ratios of 9600 and 2086, respectively. The tion energy (Ea) from 54.96 to 35.99 7 kJ mol− 1 for the

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 7 of 13

Fig. 3 Determination the optimum temperature for both the free and immobilized Bacillus amyloliquefaciens milk clotting (a), Ea for enzyme production
(b), Ea for enzyme activity (c)

enzyme production and from 64.20 to 38.75 7 kJ mol− 1 the energy needed to form the activated complex of
for the enzyme activity (Fig. 3. b, c) and accordingly, sav- enzyme and substrate.
ing energy costs [36]. Wehaidy et al. [37, 38, 39] claimed
the calculated Ea value of Ch-MCE was 1.4-fold lower Optimum pH for enzyme production
than that of the free MCE, indicating that immobilization The pH has an important role in milk clotting activity
increased the enzyme’s catalytic efficiency by reducing (Fig. 4.). The maximum enzyme activity was pH 7 for

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 8 of 13

Fig. 4 Determination the optimum pHs for both the free and immobilized Bacillus amyloliquefaciens milk clotting

and immobilized milk clotting producer cells had 100%

activity below 40 °C. Starting from 45 °C immobilized
cells enzyme was more tolerant to denaturation than free
cells as the incubation time increased. For instance, at
60 °C and one h incubation the free form lost 43% of its
original production while the immobilized form lost 33%
of its original MCE production (Fig. 5a, b). This result
demonstrated the protective effect of alginate immobi-
lization on cell hardness, which acquired enzyme stabil-
ity and tolerance to the severe conditions. Milk clotting
enzyme was incubated for 15 min at temperatures rang-
ing from 30 to 60 °C. With a peak activity at 30 °C, the
relative milk clotting activity decreased as the tempera-
ture rose but as the temperature reach 65 °C, the enzyme
became unstable and milk clotting activity was com-
pletely lost [1].

Thermodynamic study
The deactivation rate constant was determined as shown
in Fig. 6a, b for the Bacillus amyloliquefacien milk clot-
ting produced from the free and immobilized cells, aim-
ing to evaluate Ed, t1/2, and D values. These parameters
Fig. 5 Thermal stability for the free MCA (a) and the immobilized MCA (b)
gave a clear picture of the extent to which cell immobili-
zation succeeded. The data demonstrated that first-order
the free enzyme and 7-7.5 for the enzyme obtained from kinetics was present in the linear log relative activity
the immobilized cells. A similar result was obtained by versus time plots. Also, the determination of the dena-
Esawy & Blanc [32, 33] who mentioned that the optimum turation energy (Ed) demonstrated the vital role of cells
activity of free and immobilized Bacillus licheniformis immobilization in enzyme stability, where the deactiva-
5A1 MCA was obtained at pH 7. This result indicated tion energy of the milk clotting produced from the immo-
that the cell immobilization process protected the cells bilized cells increased about 2-fold (from 25.62 kJ mol − 1
from different environmental factors affecting the cell to 55.19 kJ mol − 1 (Fig. 7) compared with the free cells.
vulnerabilities. This indicates that the immobilized enzyme required
additional energy to denature, which was a positive indi-
Thermal stability cation of an improvement in thermal stability [40]. A low
One of the most critical application requirements was the Ed hinders application of the enzyme in the industrial
temperature stability of the immobilized cells. The free field [41]. The t1/2 of the free and immobilized forms were

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 9 of 13

Fig. 6 Deactivation rate constant for the free MCA (a) and the immobilized MCA (b)

Fig. 7 Arrhenius plot to calculate activation energy for denaturation (Ed) for the free and immobilized MCA

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 10 of 13

Table 2 Effect of different parameters on milk clotting production

Incubation time (h) 6 12 24 36 48 50 62 -
Activity (US) Free enzyme 400 ± 026 1200 ± 022 2400 ± 043 2400 ± 023 1750 ± 055 1000 ± 065 300 ± 045 -
Activity (US) Imm enzyme 300 ± 007 1000 ± 065 2400 ± 046 2000 ± 096 1750 ± 034 1200 ± 081 800 ± 082 -
Temperature °C 25 30 35 40 45 50 55 60
Activity (US) Free enzyme 400 ± 086 1200 ± 086 2400 ± 086 2400 ± 086 2000 ± 086 1750 ± 086 1200 ± 086 800 ± 086
Activity (US) Imm enzyme 300 ± 086 1500 ± 010 2400 ± 20 1714 ± 090 1500 ± 20 1000 ± 099 800 ± 076 400 ± 086
Carbon sources (1%) Lactose Glucose Fructose Mannose Maltose Sucrose Starch Cellulose
Activity (US) Free enzyme 2400 ± 190 2400 ± 310 2000 ± 086 2181 ± 097 2000 ± 099 2000 ± 200 1714 ± 180 1500 ± 190
Activity (US) Imm enzyme 2400 ± 20 2400 ± 099 1714 ± 190 1500 ± 200 2181 ± 240 2000 ± 220 1500 ± 190 1333 ± 086
Nitrogen source (equimolar amount) peptone Casein (control) Gelatin Na NO3 Urea Soya been KNO3 Na NO3
Activity (US) Free enzyme 3000 ± 099 2400 ± 088 2181 ± 099 800 ± 190 1741 ± 160 3000 ± 320 1500 ± 086 2000 ± 086
Activity (US) Imm enzyme 2500 ± 220 2400 ± 080 1500 ± 155 800 ± 086 1714 ± 077 3000 ± 090 1500 ± 186 2000 ± 069

Figs. 8 The picture of the cheese formed by the CF of the free (a) and immobilized enzymes (b) at the optimum conditions for enzyme activity

determined at 50, 55, 60 ℃. The t1/2 of the enzyme pro- lost only 15% after one hour (Table 2). This result con-
duced from the free cells was 238, 182, 133, minutes and cluded that the cells tolerance and stability increased
immobilized enzyme was 216, 192, and 164 min respec- after the cell immobilization. Since it shielded the cells
tively. The D values for free enzyme were 793, 605, and from toxic materials and harsh environments caused by
442 min. For enzyme produced from immobilized cells, pH extremes and accordingly protected the produced
the D values were 718, 638, and 547 min. The previ- enzyme to some extent. According to Pervez et al. [43],
ous results indicate the success of the cell immobiliza- immobilization reduces the inhibition of enzymes either
tion process in increasing the cell’s rigidity and stability, by stabilizing the structure of the enzyme or by removing
allowing the immobilized cells to be reused several times the inhibitor.
without enzyme loss. Figure 8a, b show examples of cheese formation using
the culture filtrate of the free and immobilized cells
pH stability (Fig. 8b) under the optimum conditions for enzyme activ-
The free and immobilized cells was incubated at pHs ity. It was noticed that the cheese was more compact
5–9 for different time intervals. Both the free and immo- when using the culture filtrate of the immobilized cells
bilized forms showed complete stability up to one hour compared with the culture filtrate of the free cells. This
at pHs 5–7. In industrial applications, there is a press- could be due to the fact that the milk clotting resulting
ing need for pH stability in this range [42]. At pH 8, the from the immobilized cells was more efficient than the
enzyme produced from the free cells gradually lost up free form. Further study will be done to check the cheese
to 73% of its original production after one hour. On the texture, taste, and quality.
other hand, the enzyme produced from the immobilized
cells kept its full production for 45 min at pH 8 and only Effect of different metal ions
lost 5% of its original production after one hour. At pH As seen in Table 4, all tested metals had adverse effects
9, the role of the cells immobilization process in enzyme on MCE production on both forms except NaCl and
protection was clearer, since the free enzyme lost 35% of MnSO4. It was shown that NaCl and MnSO4 increased
its original production while the immobilized enzyme the production of the free forms by about 15.8% and

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 11 of 13

Table 3 Effect of pH stability on the activity of milk clotting enzyme for free and immobilized cell
pH Milk clotting activity (MCA)(%)
Free cell Immobilized cell
Time 15 30 45 60 15 30 45 60
min min min min min min min min
5 100 ± 006 100 ± 003 100 ± 055 100 ± 009 100 ± 007 100 ± 086 100 ± 001 100 ± 000
6 100 ± 000 100 ± 006 100 ± 003 100 ± 000 100 ± 001 100 ± 001 100 ± 002 100 ± 001
7 100 ± 086 100 ± 066 100 ± 086 100 ± 086 100 ± 086 100 ± 086 100 ± 086 100 ± 086
8 100 ± 088 95 ± 056 86 ± 066 73 ± 007 100 ± 086 100 ± 086 100 ± 086 95 ± 086
9 100 ± 004 86 ± 006 70 ± 080 65 ± 056 100 ± 022 100 ± 001 90 ± 006 86 ± 005

Table 4 Effect of different metal ions on Bacillus amyloliquefaciens milk clotting

in Performed the experiments, Analyzed and interpreted the data, review

9.4% respectively. For immobilized enzyme, the metals & editing, and A. L. K. Share in Conceptualization, and M A. E. Supervision,
Conceptualization, Writing, original draft, review & editing. All authors have
increased enzyme production by 19.9% and 19.3% cor- read and approved the final manuscript.
respondingly. The activation of enzyme production by
NaCl could reflect the fact that the microorganisms were Funding
No funding information avalaible.
collected from honey, an osmophilic medium [44]. Fur- Open access funding provided by The Science, Technology & Innovation
ther, cell immobilization shortens the non-productive Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
development phase, it enables more efficient operation. It (EKB).
has been suggested that immobilized cells with high cell Data availability
densities increase product yield [45]. Pushkarev et al. [46] The authors confirm that the data supporting the findings of this study are
demonstrated that the milk-clotting activity of a mutant available within the article.
version of recombinant reindeer (Rangifer tarandus)
chymosin was stimulated by Mg2+, Mn2+, and Ca2+. The Declarations
result deduced that Bacillus amyloliquefaciens milk clot- Ethics approval and consent to participate
ting was Ca2+-independent. This article contains no studies with human or animal subjects conducted by
any of the authors. Consent for publication All authors approve for publication.
Acknowledgements
This work was supported by National Research Center, Chemistry of Natural Competing interests
and Microbial Products Department. We thank Joan Combie, Montana The authors declare no competing interests.
Biopolymers Inc., for help translating this paper.
Received: 5 May 2024 / Accepted: 3 September 2024
Author contributions
E.A.K. Performed the experiments; Analyzed and interpreted the data, review
& editing, and M. E. H. Investigation and Methodology, and N. A. E. Share

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Karam et al. Microbial Cell Factories (2024) 23:283 Page 12 of 13

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