Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 15  |  Issue : 3  |  Page : 158-166

Optimization of the production and characterization of milk-clotting enzyme from Bacillus subtilis isolated from marine sponge


1 Department of Natural and Microbial Products Chemistry, National Research Centre, Giza, Egypt
2 Department of Chemistry, Faculty of Science, Helwan University, Helwan, Egypt

Date of Submission18-Jun-2016
Date of Acceptance20-Jul-2016
Date of Web Publication5-Jan-2017

Correspondence Address:
Hala R Wehaidy
Department of Natural and Microbial Products Chemistry, National Research Centre, Dokki, Giza, 12622
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-4315.197584

Rights and Permissions
  Abstract 

Background and objectives
Milk-clotting enzyme (MCE) has important applications in the dairy industry and in the cheese-manufacturing process. Because of the increase in cheese consumption and the low supply of calf rennets, there is a need for a suitable rennet substitute from an appropriate source. The present investigation aims at microbial production of MCE and medium optimization for maximal enzyme production by the most potent strain. Partial purification and the properties of the partially purified enzyme are also studied.
Materials and methods
In the present study several microorganisms were tested for production of the MCE. MCE/caseinase ratio was investigated and was used as the key parameter for selection of the most potent strain and for medium optimization. Medium optimization experiments were carried out in an attempt to increase the enzyme productivity by the most potent strain. The produced enzyme was partially purified using ammonium sulfate at 50% concentration and the properties of the partially purified enzyme were investigated.
Results and conclusion
Among all the tested bacteria the marine sponge Pseudoceratina arabica isolated bacterium (isolate I) showed the highest milk-clotting activity. This isolate was used for MCE production throughout this study and it was identified as Bacillus subtilis KU710517. Maximum enzyme productivity (581.8 U/ml) and maximum MCE/caseinase ratio were obtained using a medium containing 50 g/l wheat bran with xylose and yeast extract as carbon and nitrogen sources. For the partially purified enzyme, the optimal temperature and pH were 85°C and 5.0, respectively. The enzyme was thermally stable at 45°C and retained 100% activity after 90 min. At 50°C for the same period of time it retained about 82% activity. However, at 60°C, the enzyme lost about 70% of its original activity after 30 min. The activation energy (Ea) of the enzyme was calculated as 6.95 kcal/mol. Km and Vmax values were 4.6 mg/ml and 2933 U/mg protein, respectively.

Keywords: Bacillus subtilis, medium optimization, milk-clotting enzyme


How to cite this article:
Wehaidy HR, Abdel-Naby MA, Shousha WG, El Mallah MI, Shawky MM. Optimization of the production and characterization of milk-clotting enzyme from Bacillus subtilis isolated from marine sponge. Egypt Pharmaceut J 2016;15:158-66

How to cite this URL:
Wehaidy HR, Abdel-Naby MA, Shousha WG, El Mallah MI, Shawky MM. Optimization of the production and characterization of milk-clotting enzyme from Bacillus subtilis isolated from marine sponge. Egypt Pharmaceut J [serial online] 2016 [cited 2020 Nov 29];15:158-66. Available from: http://www.epj.eg.net/text.asp?2016/15/3/158/197584


  Introduction Top


The word ‘rennet’ is applied to the crude enzyme that can coagulate milk either from animal, plant, or microbial source [1]. It is a protease enzyme that is used in cheese manufacture [2]. Milk coagulation occurs in two important phases: first, casein is hydrolyzed in an enzymatic phase and forms paracaseinate. It then forms a curd in the nonenzymatic phase at certain calcium ion concentrations [3]. Calf rennet, which contains chymosin (EC 3.4.23.4), is widely used as an important milk-clotting enzyme (MCE) [4]. Because of the reduced supply of calf rennet and calf diseases like bovine spongiform encephalopathy, there is an increase in the demand for new sources of MCEs to meet the needs of the cheese industry [5],[6]. However, most plant rennets are unsuitable because they give a bitter taste to the cheese [7],[8]. Microbial rennets are more promising enzymes because of its low production costs, greater biochemical diversity, and easier genetic modification [9]. Many fungal and bacterial sources are widely used for cheese production. About 60% of cheese in the USA is manufactured using fungal enzyme sources [10]. Bacillus species are known to produce many extracellular enzymes with a wide range of industrial applications [11].

Wheat bran acts as a cheap source of energy in the fermentation process for enzyme production. This agroindustrial residue contains many ingredients such as cellulose, crude protein, starch, and trace elements that are used as carbon and nitrogen sources to stimulate the growth of microorganisms and enzyme productivity. Many researchers observed that wheat bran is a potent substrate for enzyme production [12],[13].


  Materials and methods Top


Microorganisms and media

All bacilli used in the screening, except Paenibacillus macerans, were obtained from the Culture Collection of the National Research Centre, Dokki, Cairo, Egypt. However, P. macerans was obtained from the Microbiological Resources Center (Mircen, Faculty of Agriculture, Ain Shams University). The bacterial isolates I, II, III, and IV were isolated from the marine sponge Pseudoceratina arabica. All microorganisms were grown on nutrient agar medium at 37°C. The basal medium used for MCE production contained the following (g/l): wheat bran, 50; yeast extract, 3.0; MgSO4.7H2O, 0.2; K2HPO4, 3.0; and glucose, 4.0. pH was 6.0 before sterilization.

Isolation of marine sponge-associated bacteria

A single colony of P. arabica was collected from Red Sea, Sharm El Sheikh, south of Sinai, Egypt, by scuba diving at a depth of 15–20 m. Sponge specimens were transferred to a sterile plastic bag containing sea water, kept in ice, and transported to the laboratory. Afterwards, the specimens were rinsed several times with sterile sea water and cut into small pieces. The tissues were homogenized in sterile sea water. A volume of 100 μl of 10−1 dilution of the homogenate with sterile sea water was spread onto Zobell marine agar plates supplemented with 50% filtered sea water and cycloheximide (50 μg/ml) as antifungal agent. The plates were incubated at 28°C for 7 days. Colonies were selected on the basis of the morphological features and purified by making streak plates containing sterile Zobell marine agar medium.

Cultivation conditions and enzyme production

One slant was scratched with 10 ml distilled water, and 2 ml of cell suspension was used to inoculate a 250-ml Erlenmeyer flask containing 50 ml of sterile production medium. The flasks were incubated at 37°C on a rotary shaker at 150 rpm for 48 h. The cells were then centrifuged at 3000 rpm for 10 min and the supernatant was collected and assayed for milk-clotting activity (MCA).

Enzyme assays

Assay of milk-clotting activity

MCA was determined as follows: 0.5 ml of the enzyme solution was incubated with 2 ml of reconstituted skimmed milk (12 g dry skim milk/100 ml 0.01 mol/l calcium chloride) at 40°C. MCA was expressed in Soxhlet units, which were calculated using the following equation:



where S is the volume of milk (ml), E is the volume of enzyme (ml), and T is the time necessary for curd fragment formation (s) [14].

Assay of caseinase activity

One milliliter of enzyme was added to 1 ml of 1% casein solution in Tris-HCl buffer (0.2 mol/l, pH 8.5). The reaction mixture was incubated in a water bath at 40°C for 30 min. Then 2 ml of 15% trichloroacetic acid was added and the mixture was centrifuged at 4000 rpm for 10 min. The solubilized proteins in supernatant were measured using the Lowry method at 750 nm [15].

Survey of some bacterial cultures for milk-clotting enzyme production

In this experiment, 10 bacterial cultures were tested for MCE production by submerged fermentation using the basal medium under shaken culture conditions after a 48-h incubation period. The culture filtrates were assayed for both MCE and caseinase activities.

Optimization of some culture conditions for maximization of milk-clotting enzyme productivity by the potent strain

These experiments were undertaken to investigate the effect of different medium compositions on MCE productivity by the potent strain. Thereafter, an optimum culture medium was formulated for the production of MCE.

Effect of wheat bran concentrations

This experiment was undertaken to investigate the effect of wheat bran concentration on the production of MCE. The fermentation was performed for 48 h using different concentrations of wheat bran (10–70 g/l).

Effect of different carbon sources

For studying the effect of different carbon sources on the production of MCE, glucose from the basal medium was substituted by different carbon sources (i.e. fructose, sucrose, lactose, xylose, arabinose, starch, and cellobiose). The fermentation was performed for 48 h.

Effect of different nitrogen sources

On equivalent nitrogen basis, yeast extract from the basal medium was substituted with different nitrogen sources [i.e. peptone, casein soya bean, NH4Cl, (NH4)2S04, and NaNO3]. The fermentation was also performed for 48 h.

Effect of phosphate concentration

This experiment was undertaken to investigate the effect of phosphate level as K2HPO4 in the culture medium on MCE production. Different concentrations of K2HPO4 ranging from 2.0 to 3.5 g/l were used.

Effect of magnesium sulfate concentration

The present experiment deals with the effect of magnesium sulfate concentration on the production of MCE. In this experiment, magnesium sulfate was added to the culture medium at different concentrations (0.1–0.3 g/l).

Partial purification of the enzyme

This was achieved by precipitation of the concentrated culture filtrate by salting out with ammonium sulfate at 35, 50, 70, and 90% concentrations. The obtained fractions were dried and assayed for enzyme activity.

Properties of the partially purified enzyme

Some properties of the partially purified MCE are studied. These include the effect of reaction temperature, pH, thermal stability, and substrate concentration.

Identification and phylogenetic analysis of sponge-associated strain based on the 16srRNA gene sequence

The gene coding for 16srRNA was amplified by PCR from the isolated genomic DNA using forward (5′-TCACGGAGAGTTTGATCCTG-3′) and reverse (5′-GCGGCTGCTG GCACGTAG TT-3′) primers. Considering the PCR conditions, genomic DNA was initially denatured at 95°C for 3 min, followed by 30 cycles of DNA denaturation at 94°C for 30 s, primer annealing at 48°C for 30 s, and elongation at 68°C for 90 s. Final extension of the amplified products was performed at 68°C for 5 min. The reaction mixture was kept at 4°C [16].


  Results and discussion Top


Survey of some bacterial cultures for milk-clotting enzyme production

After the 48-h incubation period, maximum MCA (564.7 U/ml) and MCA/caseinase ratio (3422.4) were obtained from the bacterial isolate I ([Table 1]). Therefore, this bacterial strain has been used for MCE production in this work.
Table 1: Survey of some microorganisms for milk-clotting activity

Click here to view


Optimization of some cultural conditions for maximization of milk-clotting enzyme productivity

Effect of wheat bran concentrations

Wheat bran has been reported as an ideal medium for microbial protease production [17],[18]. The highest MCE production yield (640 U/ml) was reached at wheat bran concentration of 70 g/l. At higher concentrations, the medium is thicker, which may limit cell growth and affect the enzyme productivity. However, the highest MCA/caseinase ratio (3422.4) was obtained with a wheat bran concentration of 50 g/l ([Figure 1]). Thus, we conclude that this wheat bran concentration is favorable for MCE production. Many authors also used wheat bran as a potent substrate for MCE production by microorganisms [19],[20],[21],[22]. However, some authors reported different optimum concentrations of wheat bran for MCE production from other microorganisms (Paenibacillus spp. BD3526, 30 g/l and Bacillus amyloliquefaciens D4, 180 g/l) [20],[22].
Figure 1: Effect of wheat bran concentrations on milk-clotting activity (MCA)/caseinase ratio.

Click here to view


Effect of different carbon sources

The maximal MCA (581.8 U/ml) was obtained with xylose, arabinose, and cellobiose. However, maximum MCE/caseinase ratio (4216) was obtained with xylose as indicated in [Figure 2]. Thus, xylose proved to be the most favorable carbon source for the production of MCE. This activity is higher than that reported by other authors, who reported other sugars as the best carbon source for MCE production (lactose 58.5 U/ml, glucose 21.5 U/ml) [23],[24],[25].
Figure 2: Effect of carbon sources on milk-clotting activity (MCA)/caseinase ratio.

Click here to view


Effect of different nitrogen sources

As shown in [Figure 3], maximum MCE production yield (581.8 U/ml) with maximum MCA/caseinase ratio of 4216 was reached with yeast extract. Therefore, the yeast extract that was used in the basal medium as a nitrogen source is used throughout the work for MCE production. This result differs from those of other authors who observed maximum MCE production with other nitrogen sources (beef extract, whey powder, casein) [25],[26],[27]. Other authors observed that the presence of nitrogen sources is not critical for the production of MCE [28]. In general, the effect of nitrogen source on MCE production differs from organism to organism [25].
Figure 3: Effect of nitrogen sources on milk-clotting activity (MCA)/caseinase ratio.

Click here to view


Effect of phosphate concentration

It is well known that phosphate is utilized to form various cellular components [29]. As illustrated in [Figure 4], the highest MCE yield (581.8 U/ml) with an MCA/caseinase activity ratio of 4216 was reached at a phosphate concentration of 3 g/l, which was previously used in the basal medium. This concentration is higher than that investigated by other authors for maximum MCE production (1.3 g/l) [24]. The requirement of phosphorus for protease production was very evident as reported by many researchers [30],[31].
Figure 4: Effect of phosphate concentrations on milk-clotting activity (MCA)/caseinase ratio.

Click here to view


Effect of magnesium sulfate concentration

Magnesium is essential for bacterial growth and cell division [32]. Thus, it was used in many media. The highest MCE yield (600 U/ml) was obtained at low magnesium sulfate concentration (0.1 g/l). Further increase in concentration caused a slight decrease in MCE yield. However, the highest MCA/caseinase ratio (4407.5) was obtained with a magnesium sulfate concentration of 0.25 g/l ([Figure 5]) and hence this concentration was used in this work. From the previous results, the optimum culture medium for MCE production contained the following (g/l): wheat bran, 50; yeast extract, 3.0; MgSO4.7H2O, 0.25; K2HPO4, 3.0; and xylose, 4.0. The pH was 6.0 before sterilization.
Figure 5: Effect of MgSO4 concentrations on milk-clotting activity (MCA)/caseinase ratio.

Click here to view


Partial purification of milk-clotting enzyme

MCE was precipitated with ammonium sulfate and the most active fraction was obtained at 50% concentration. The partially purified enzyme retained a specific activity of 512.8 U/mg protein with 5.7 purification fold ([Table 2]).
Table 2: Fractional precipitation of milk-clotting enzyme with ammonium sulfate

Click here to view


Properties of the partially purified enzyme

Optimum temperature

The effect of the temperature of the reaction MCE was investigated at pH 4.5. The temperature profile of MCE showed that MCA was increased with increase in the reaction temperature up to 85°C. Further increase in reaction temperature resulted in loss in MCA. This may be due to the denaturation of the enzyme above this temperature. This value is higher than those reported by other authors (55 and 60°C) [33],[34]. Other authors also have reported high optimum temperatures for MCE (75°C from Bacillus licheniformis and 85°C from Aloe variegate) [35]. The Arrhenius plot ([Figure 6]) for MCE was linear. The activation energy was calculated as 6.95 kcal/mol from the slope using the following equation:

Figure 6: Arrhenius plot for milk-clotting enzyme (MCE).

Click here to view


where R is the gas constant (R=1.976 cal/mol).

Optimum pH

The effect of the pH of the reaction on the activity of MCE was investigated at pH ranges of 4.0–7.5. MCE was optimally active at pH 5.0 ([Figure 7]). Above this pH the activity decreased gradually, which was due to denaturation of the enzyme. This optimum pH is similar to that reported by other authors [26]. This result is also in agreement with previous findings, in which a reduction in the milk pH resulted in a decrease in the milk-clotting time [36],[37]. Several authors have also reported a decline in MCA at pH near neutrality [38],[39]. The higher activity of the enzyme in acidic pH is highly advantageous, and it can be easily controlled in industrial applications [40].
Figure 7: Effect of pH on milk-clotting enzyme (MCE).

Click here to view


Thermal stability of milk-clotting enzyme

The enzyme solution was heated at different time intervals (15–90 min) at different temperatures (45–60°C) in the absence of substrate. As indicated ([Figure 8]), the enzyme was stable after heat treatment for 90 min at 45°C and retained 100% activity. The enzyme retained about 73 and 47% of its original activity after heating for 15 min at 55 and 60°C, respectively. It was completely deactivated after 90 min of heating at 60°C. These results are better than those reported by other authors, as MCE produced from B. licheniformis retained 73% of its activity when heated for 1 h at 40°C [35]. Also the enzyme produced from Penicillium oxalicum retained only 58% of its activity at the same conditions [34]. However, MCE from B. amyloliquefaciens was completely deactivated when heated at 55°C for 20 min [21].
Figure 8: Thermal stability of milk-clotting enzyme (MCE).

Click here to view


Effect of substrate concentration

MCE activity was assayed at different substrate concentrations (1–5 mg/ml) at optimum assay conditions. The gradual increase in the substrate concentration caused an increase in the enzyme activity. Maximal enzyme activity was attained at substrate concentration of 4 mg/ml. Further increase in substrate concentration did not cause any increase in the enzyme activity. This may be due to saturation of the enzyme active site by the substrate. This result is in agreement with those found by other investigators [34],[41]. From the Lineweaver–Burk plot ([Figure 9]) the Km and Vmax values of MCE were calculated as 4.6 mg/ml and 2933 U/mg protein, respectively.
Figure 9: Lineweaver–Burk plot of milk-clotting enzyme (MCE).

Click here to view


Identification and phylogenetic analysis of sponge-associated strain

A specific band of 1256 bp was observed when resolved on agarose gel. The PCR products were purified with a GeneJET Genomic DNA Purification Kit (Thermo Scientific Company, USA) according to the instruction manual. Partial gene sequences were obtained using the forward primer. The nucleotide sequences were compared with those in the NCBI Gene Bank database (http://www.ncbi.nlm.nkh.gov/) with the basic local alignment search tool (BLAST) algorithm to identify known closely related sequences ([Figure 10]a). The sequence was analyzed with FinchTV software, and thereby a phylogenetic tree was generated by the neighbor-joining algorithm implemented in Geneious R8 software ([Figure 10]b). The results revealed that the aligned sequence had ∼98% identity to Bacillus subtilis strain. The corresponding sequence was submitted in the NCBI Gene Bank under accession number KU710517. Many authors used the genus Bacillus for MCE production [42],[43],[44]. Other authors also used the strain B. subtilis for production of MCE [12],[28],[45],[46],[47],[48].
Figure 10: (a) Nucleotide sequence. (b) Phylogenetic tree for marine sponge bacillus strain.

Click here to view



  Conclusion Top


From the present study, it can be concluded that MCE produced by the marine sponge isolate B. subtilis KU710517 can be considered an alternative to calf rennet in the cheese industry as it has a high MCA/caseinase ratio, which is an important factor in milk clotting. Medium optimization resulted in a high increase in MCE productivity, and MCA/caseinase ratio was increased from 3422 to 4407 after optimization. The partially purified enzyme has high specific activity (512.8 U/mg protein) and high thermal stability at 45 and 50°C, which makes it a promising enzyme in industrial applications. Additional studies must be undertaken in the future to evaluate the enzyme’s safety and benefits in the food industry.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
El-Tanboly ES, El-Hofi M, Youssef BY, El-Desoki W, Ismail A. Utilization of salt whey from Egyptian Ras (Cephalotyre)cheese in microbial milk clotting enzymes production. Acta Sci Pol Technol Aliment 2013; 12:9–19.  Back to cited text no. 1
    
2.
Kumar S, Sharma NS, Saharan MR, Singh R. Extracellular aspartic proteinases from Rhizopus oryzae: purification and characterization. Proc Biochem 2005; 40:1701–1705.  Back to cited text no. 2
    
3.
Fox PF, Guinee TP, Gogan TM, McSweeney PLH. Fundamentals of cheese science (2000).  Back to cited text no. 3
    
4.
Isam AMA, Morishima I, Babiker EE, Mori N. Characterization of partially purified milk-clotting enzyme from Solanum dubium Fresen seeds. Food Chem 2009; 116:395–400.  Back to cited text no. 4
    
5.
Cavalcanti MT, Teixeira MF, Lima Filho JL, Porto AL. Partial purification of new milk-clotting enzyme produced by Nocardiopsis spp. Bioresour Technol 2004; 93:29–35.  Back to cited text no. 5
    
6.
Roseiro LB, Barbosa MM, Ames J, Wilbey R. Cheese making with vegetable coagulants; the use of Cynara L. for the production of ovine milk cheeses. Int J Dairy Technol 2003; 56:76–85.  Back to cited text no. 6
    
7.
Raposo S, Domingos A. Purification and characterization of milk-clotting aspartic proteinases from Centaurea calcitrapa cell suspension cultures. Process Biochem 2008; 43:139–144.  Back to cited text no. 7
    
8.
Lmdakim MM, Hassan Z, Aween MM, Elshaafi BM, Muhialdin BJ. Milk clotting and proteolytic activity of enzyme preparation from Pediococcus acidilactici SH for dairy products. Afr J Biotech 2015; 14:133–142.  Back to cited text no. 8
    
9.
Ageitos JM, Vallejo JA, Sestelo AB, Poza M, Villa TG. Purification and characterization of a milk-clotting protease from Bacillus licheniformis strain USC13. J Appl Microbiol 2007; 103:2205–2213.  Back to cited text no. 9
    
10.
Yada RY, Nakai S. Use of principal component analysis to study the relationship between physical/chemical properties and the milk clotting to proteolytic activity ratio of some aspartate proteinases. J Agric Food Chem 1986; 34:675–679.  Back to cited text no. 10
    
11.
Wim JQ. Bacterial enzymes. Prokaryotes 2006; 1:777–796.  Back to cited text no. 11
    
12.
Ding ZY, Liu SP, Gu ZH, Zhang L, Zhang KC, Shi GY. Production of milk-clotting enzyme by Bacillus subtilis B1 from wheat bran. Afr J Biotechnol 2011; 10:9370–9378.  Back to cited text no. 12
    
13.
Chandra MS, Viswanath B, Reddy BR. Optimization of extraction of β-endoglucanase from the fermented bran of Aspergillus niger. Indian J Microbiol 2010; 50(Suppl 1):122–126.  Back to cited text no. 13
    
14.
Berridge NJ. Some observations on the determination of the activity of rennet. Analyst (London) 1952; 77:57–62.  Back to cited text no. 14
    
15.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–275.  Back to cited text no. 15
    
16.
Petti CA, Polage CR, Schreckenberger P. The Role of 16S rRNA Gene Sequencing in Identification of Microorganisms Misidentified by Conventional Methods. J Clin Microbiol 2005; 43:6123–6125.  Back to cited text no. 16
    
17.
Dutt K, Gupta P, Saran S, Misra S, Saxena RK. Production of milk-clotting protease from Bacillus subtilis. Appl Biochem Biotechnol 2009; 158:761–772.  Back to cited text no. 17
    
18.
Silva BL, Geraldes FM, Murari CS, Gomes E, Da-Silva R. Production and characterization of a milk-clotting protease produced in submerged fermentation by the thermophilic fungus Thermomucor indicae-seudaticae N31. Appl Biochem Biotechnol 2014; 172:1999–2011.  Back to cited text no. 18
    
19.
Gan BZ, Song X, He XL, Zhang WB, Li F. Separation and identification of a chymosin producing bacterium from soil of yak grazing district in Tianzhu country of Gansu province. Food Sci 2009; 30:158–162.  Back to cited text no. 19
    
20.
He X, Ren F, Guo H, Zhang W, Song X, Gan B. Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4. Korean J Chem Eng 2011; 28:203–208.  Back to cited text no. 20
    
21.
Zhang W, He X, Liu H, Guo H, Ren F, Wen P. Statistical optimization of culture conditions for milk-clotting enzyme production by Bacillus amyloliquefaciens using wheat bran-an agro-industry waste. Indian J Microbiol 2013; 53:492–495.  Back to cited text no. 21
    
22.
Hang F, Liu P, Wang Q, Han J, Wu Z, Gao C et al. High milk-clotting activity expressed by the newly isolated Paenibacillus spp. strain BD3526. Molecules 2016; 21:73.  Back to cited text no. 22
    
23.
Gais S, Fazouane F, Mechakra A. Production of milk clotting protease by Rhizopus stolonifer through optimization of culture conditions. Int J Biol Biomol Agric Food Biotech Eng 2009; 3:340–344.  Back to cited text no. 23
    
24.
Abou Ayana IAA, Ibrahim AE, Saber WIA. Statistical optimization of milk clotting enzyme biosynthesis by Mucor mucedo KP736529 and its further application in cheese production. Int J Dairy Sci 2015; 10:61–76.  Back to cited text no. 24
    
25.
Patil PM, Kulkarni AA, Kininge PT. Production of milk clotting enzyme from Aspergillus oryzae under solid-state fermentation using mixture of wheat bran and rice bran. IJSRP 2012; 2:1–12.  Back to cited text no. 25
    
26.
Foda MS, Moharam ME, Ramadan A, El-Bendary MA. Over production of milk clotting enzyme from Rhizomucor miehei through adjustment of growth under solid state fermentation conditions. Aust J Basic Appl Sci 2012; 6:579–589.  Back to cited text no. 26
    
27.
Yegin S, Fernandes-Lahore M, Guvenc U, Goksungur Y. Production of extracellular aspartic protease in submerged fermentation with Mucor mucedo DSM 803. Afr J Biotechol 2010; 9:6380–6386.  Back to cited text no. 27
    
28.
Shieh CJ, Thi LAP, Shih IL. Milk-clotting enzymes produced by culture of Bacillus subtilis natto. Biochem Eng J 2009; 43:85–91.  Back to cited text no. 28
    
29.
Yoon MY, Yoo YJ, Cadman TW. Phosphate effects in the fermentation of alpha amylase by Bacillus amyloliquefaciens. Biotechnol Lett 1989; 11:57–60.  Back to cited text no. 29
    
30.
Kembhavi AA, Kulkarni A, Pant A. Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM no. 64. Appl Biochem Biotechnol 1993; 38:83–92.  Back to cited text no. 30
    
31.
Ahmed SA. Biochemical studies on microbial proteolytic enzymes. MSc thesis: Cairo University. 1994.  Back to cited text no. 31
    
32.
Webb M. The influence of magnesium on cell division. J Gen Microbiol 1951; 5:480–484.  Back to cited text no. 32
    
33.
El-Bendary MA, Moharam ME, Ali TH. Purification and characterization of milk clotting enzyme produced by Bacillus sphaericus. J Appl Sci Res 2007; 3:695–699.  Back to cited text no. 33
    
34.
Hashem AM. Optimization of milk-clotting enzyme productivity by Penicillium oxalicum. Bioresour Technol 1999; 70:203–207.  Back to cited text no. 34
    
35.
Ahmed SA, Helmy WA. Comparative evaluation of Bacillus licheniformis 5A5 and Aloe variegate milk clotting enzymes. Braz J Chem Eng 2012; 29:69–76.  Back to cited text no. 35
    
36.
Awad S. Effect of sodium chloride and pH on the rennet coagulation and gel firmness. Food Sci Technol 2007; 40:220–224.  Back to cited text no. 36
    
37.
Elmazar MM, El-Sayed ST, Al-Azzouny RA. Screening some local Egyptian seeds extract for milk-clotting activity and physicochemical characterization of Brassica napus seed extract. J Agric Food Tech 2012; 2:28–34.  Back to cited text no. 37
    
38.
Castillo M, Payne FA, Hicks CI, Lopez MB. Predicting cutting and clotting time of coagulating goat’s milk using diffuse reflectance: effect of pH, temperature and enzyme concentration. Int Dairy J 2000; 10:551–562.  Back to cited text no. 38
    
39.
Chazarra S, Sidrach L, López-Molina D, Rodrı́guez-López JN. Characterization of the milk-clotting properties of extracts from artichoke (Cynara scolymus, L.) flowers. Int Dairy J 2007; 17:1393–1400.  Back to cited text no. 39
    
40.
El-Sayed ST, Elmazar MM, Al-Azzouny RA. Purification and characterization of a novel milk-clotting enzyme from Brassica napus seeds. Aust J Basic Appl Sci 2013; 7:482–493.  Back to cited text no. 40
    
41.
Wahba A, El-Abbassy F, El-Shafei H, Awad S. Effect of some factors on the activity of milk clotting enzymes. Egypt J Food Sci 1995; 23:27–35.  Back to cited text no. 41
    
42.
An Z, He X, Gao W, Zhao W, Zhang W. Characteristics of miniature Cheddar-type cheese made by microbial rennet from Bacillus amyloliquefaciens: a comparison with commercial calf rennet. J Food Sci 2014; 79:214–221.  Back to cited text no. 42
    
43.
Lemes AC, Pavon Y, Lazzaroni S, Rozycki S, Brandelli A, Kalil SJ. A new milk-clotting enzyme produced by Bacillus sp. P45 applied in cream cheese development. Food Sci Technol 2016; 66:217–224.  Back to cited text no. 43
    
44.
Ahmed SA, Wehaidy HR, Ibrahim OA, Abd El Ghani S, El-Hofi MA. Novel milk-clotting enzyme from Bacillus stearothermophilus as a coagulant in UF-white soft cheese. Biocatalys Agri Biotechnol 2016; 7:241–249.  Back to cited text no. 44
    
45.
Talantikite-Kellil S, Nouani A, Saunier M, Gillmann L, Brahmi A, Fazouane F. Isolation and identification of bacterial strain I33M producing milk-clotting enzyme: optimization of culture parameters using response surface. Afr J Biotechnol 2012; 11:16594–16606.  Back to cited text no. 45
    
46.
Narwal RK, Bhushan B, Pal A, Panwar A, Malhotra S. Purification, physico-chemico-kinetic characterization and thermal inactivation thermodynamics of milk clotting enzyme from Bacillus subtilis MTCC 10422. Food Sci Technol 2016; 65:652–660.  Back to cited text no. 46
    
47.
Wu FC, Chang CW, Shih IL. Optimization of the production and characterization of milk clotting enzymes by Bacillus subtilis natto. Springerplus 2013; 2:33.  Back to cited text no. 47
    
48.
Li Y, Liang S, Zhi D, Chen P, Su F, Li H. Purification and characterization of Bacillus subtilis milk-clotting enzyme from Tibet Plateau and its potential use in yak dairy industry. Eur Food Res Technol 2012; 234:733–741.  Back to cited text no. 48
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2]


This article has been cited by
1 Application of Milk-Clotting Protease from Aspergillus oryzae DRDFS13 MN726447 and Bacillus subtilis SMDFS 2B MN715837 for Danbo Cheese Production
Jermen Mamo,Paulos Getachew,Mbugua Samuel Kuria,Fassil Assefa
Journal of Food Quality. 2020; 2020: 1
[Pubmed] | [DOI]
2 Statistical optimization of B. subtilis MK775302 milk clotting enzyme production using agro-industrial residues, enzyme characterization and application in cheese manufacture
Hala R. Wehaidy,Walaa A. Abdel Wahab,Adel M.M. Kholif,Mostafa Elaaser,Wafaa K. Bahgaat,Mohamed A. Abdel-Naby
Biocatalysis and Agricultural Biotechnology. 2020; 25: 101589
[Pubmed] | [DOI]
3 Improving the catalytic, kinetic and thermodynamic properties of Bacillus subtilis KU710517 milk clotting enzyme via conjugation with polyethylene glycol
Hala Refaat Wehaidy,Mohamed Ahmed Abdel-Naby,Wafaa Ghoneim Shousha,Mohammed I.Y. Elmallah,Michael Mounir Shawky
International Journal of Biological Macromolecules. 2018; 111: 296
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and me...
Results and disc...
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed1843    
    Printed88    
    Emailed0    
    PDF Downloaded195    
    Comments [Add]    
    Cited by others 3    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]