Production, Partial Purification and Based SDS-PAGE Profiles of Caseinolytic Enzyme in two Bacillus Strains Isolated from Fermented Cassava leaves "Ntoba mbodi" in Congo Brazzaville

1Cellular and Molecular Biology Laboratory, Faculty of Sciences and Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo. 2Unit of Microbiology, Molecular Biology and Bioinformatics, Faculty of Sciences and Techniques, Marien Ngouabi University, Brazzaville, Republic of Congo. 3Institute of Research in Exact and Natural Sciences , Ministry of Research and Technologic innovation, Brazzaville, Republic of Congo.

Enzymes are macromolecules involved in many cellular reactions.Proteases are groups of proteins included in the subclass hydrolases, within the main class enzymes.Serine alkaline proteases (SAP) are one of the most important groups of industrial enzymes.They account for approximately 35% of the total microbial enzyme sales 1 .
Proteases or proteolytic enzymes find many applications in many domains as : pharmacy, food, biotechnology, and detergents 2,3  Commercially used proteases are produced by bacteria, plants , animals and fungi 4,5 .Proteases are produced by many groups of bacteria: Bacillus, Pseudomonas and others.The genus Bacillus is recognize as one of the most important producer of exocellular proteases 1,5 .Proteases production by micro-organisms is a natural phenomenon.Bacillus species produce proteases during the exponential and stationary phases during growth.Extracellular protease production is strongly influenced by media composition, the ration of C/ N, metabolizable sugars 6 and metallic ions 1,7 .More over protease production is also affected by rapidly metabolizable source, like amino acids in the medium and by some physic factors (aeration, inoculum density , pH and temperature).Proteases production by microorganisms is connected to the growth and that is characteristic of each microorganism 1 .Many studies have been focused on proteases production by microorganisms, these studies have stated strategies of growth and protease production 7,8,9 .Some studies have given information in the relation between growth and protease production.Those studies corroborate with the hypothesis that : proteases production is controled by number of mechanisms that occur in the transition between the end of the exponential phase and the begening of the stationary phase 10,11,12 .By any way all studies are strict about the variability and make an amphasis on the optimal conditions of protease production that, depend strictly on each microorganism 13,14,15 .
Caseinolytic proteases P(ClpPs) are complex of oligoméric proteines with contibute on cellular homeostasy and virulence regulation to bacteria.Whatever most of organisms possesse only one ClpP protein, in some can be encoded two or more ClpP isoforms 16 .The caséinolytic protease P(ClpP) is an highly conserved enzyme in bactéria and higher organisms 17,18,19 .
In the republic of Congo fermented manioc leaves are used as a food by Congolese in some area.They are differently called : Ntoba mubodi in the Bouenza, Lilleyuka in the Kouilou and Ntoba mbodi in the Pool.Microbiological study of the Ntoba Mbodi has been hold and has shown a diversity of bacteria, among them the species of genus Bacillus 20 .
Many species of genus Bacillus isolated from Ntoba Mbodi has been molecularly characterized by their ARNr 16S and ARNr 16S-23S .Many of them have shown proteolytic activities, as the fibrinolytic enzyme producer Ba NM76 described in 15 .A new strain named Lysinibacillus louembei sp. with a strong protéolytic activity has been isolated from Ntoba Mbodi. 21.
Two Bacillus strains isolated from Ntoba mbodi : Bacillus megaterium (B.me NM 02) Bacillus licheniformis(B.liNM01), have shown a strong caseinolytic enzyme activity.In the present work we optimize growth and enzyme production conditions (temperature, pH, nitrogen and carbon sources), produce the enzyme.In both strains the produced enzyme was partially purified by using ammonium sulfate precipitation.SDS-PAGE has been hold and profils used to differenciate the strains.

Bacterial strains and media
Strains used in this study were Bacillus megaterium(B.meNM 02) and Bacillus licheniformis (B.li NM 01) available in our laboratory were isolated from Ntoba mbodi.

Culture conditions
250ml Erlenmeyer containing 100ml of LB was inoculated with 1ml of overnight culture and incubated at 37°C for 48 hours.2ml of bacterial suspension were used to compute the optical density (OD) at 600 nm with a type vis spectrophotometer (722 Ningb SI Instrument CO, LTD). 12,15nzyme assay Puri modified techniques [9].wasused to test caseinolytic enzyme.The 48h culture was collected in 1,5 eppendorf and centrifuged for 5minutes at 6000rpm in a type microcentrifuge( ).The supernatant was collected for enzyme activity.
1g of agarose was dissolved in 250 ml erlenmeyer containing 100 ml de PBS at 0,1N, after boiling, we waited untill à 55-60°C,10 ml of skimmed milk were added.The mixture were poured in petri dishes.Wholes were made on the plate.50¼l of sample were added in each whole, and the plate was incubated at 37°C for 12 hours.Caseinolytic activity was measured by lytic area through the diameter of the clear zone 22,23 .

Influence of temperature on the growth and caseinolytic enzyme production
To study the effect of temperature on growth and caceinolytic enzyme production, we made culture on LB medium at different temperatures (25, 30, 35,37, 40 45, 50,55 et 60°C) for 48 h.Each two hours optical density at 600nm and enzyme activity were computed.Testing caseinolytic activity plates were incubated at 37°C, for 12 h.Lytic area through the diameter of the clear zone was measured to show the enzyme activity 12,15,22,23 .

Influence of pH on the growth and caseinolytic enzyme production
To study the effect of pH on growth and caceinolytic enzyme production, we made culture on LB medium at different pH :5, 7, 9 for 48 h.Each two hours optical density at 600nm and enzyme activity were computed.Testing caseinolytic activity plates were incubated at 37°C, for 12 h.Lytic area through the diameter of the clear zone was measured to show the enzyme activity 15,22 .

Influence of different kinds od media on the growth an enzyme production
Two kinds of media were used to study their effects of on growth and caceinolytic enzyme production, we made culture on LB and TSB at pH : 7 for 48 h.Each two hours optical density at 600nm and enzyme activity were computed.Testing caseinolytic activity plates were incubated at 37°C, for 12 h.Lytic area through the diameter of the clear zone was measured to show the enzyme activity 22,23,24 .

Influence of carbon sources on the growth and caseinolytic enzyme production
Three kinds of carbon sources (galactose, Fructose, amidon) were used to study their effects on growth and caceinolytic enzyme production, we made culture on three different media which compositions are given in table 1.Each two hours optical density at 600nm and enzyme activity were computed.Testing caseinolytic activity plates were incubated at 37°C, for 12 h.Lytic area through the diameter of the clear zone was measured to show the enzyme activity 23,25,26 .

Influence of nitrogen sources on the growth and caseinolytic enzyme production
Two kinds of nitrogen sources (yeast extracts, Bactopeptone) were used to study their effects on growth and caceinolytic enzyme production, we made culture on two different media which compositions are given in table 2. Each two hours optical density at 600nm and enzyme activity were computed.Testing caseinolytic activity plates were incubated at 37°C, for 12 h.Lytic area through the diameter of the clear zone was measured to show the enzyme activity 23,23,26 Proteins SDS-PAGE Protein SDS-PAGE has been hold.A 48h culture was centrifuged and the supernatant was precipitated with ammonium sulfate.The centrifugation of the solution was at 4°C, 10000t/ min for 1hour with the centriguge type MICRO STAR 17R from VWR..The precipitate was diluted in PBS and 100 µl was used as a sample The reagents were: Acrylamids 30% / biacrylamids 0.8 % ; Tris-HCL 1.5 M pH 8.8 ; Tris-HCL 0.5 M pH 6.8 ; Electrophoresis buffer : Tris 25 mM, Glycine0.192M, SDS 0.1% ; Staining solution : Coomassi bleu 2.5 g, méthanol 450 ml, glacial acétic acid 100 ml, eau 400 ml, ajusted at 1 l ; Dying solution : méthanol, glacial acétic acid 100 ml, eau 400 ml, ajusted at 1 l.; Protein molecular markers protéines (Protein Page Rulerprestained, Fermentas)

Caseinolytic enzyme activity
The Figure1 shows the caseinolytic activity in three bacillus strains isolated from Ntoba Mbodi : B.me., B.li.,B.amy.The B.amy.is used as a positive control.The halo of B.me is more important than of the positive control.The halo of B.li is as important as af a positive control.The diameter of the halo is the expression of the enzyme production of the strain.E.coli is used as a negative control.

Influence of température on growth and caseinolytic enzyme production
Fig- 2 shows the influence of temperature on growth profiles for Bacillus licheniformis with the time.For the temperatures from 25 to 40°C, the increase of temperature is proportional with growth profiles.In contrast, after 40°C the increase of temperature become inversely proportional to the Fig- 3 shows the influence of temperature on caseinolytic enzyme production for Bacillus licheniformis.Indeed, the enzyme activity here is used to express the enzyme production with the incubation time.For the temperatures from 25 to 30°C the amplitude of caseinolytic enzyme production profils are proportional to increase of the temperature.For the temperatures from 30 to 55°C the amplitude caseinolytic enzyme production profiles is inversely proportional the increase of temperature.courbes de la production enzymatique augmentent, cela indique que la production enzymatique augmente.Caseinolytic enzyme production is possible for the temperature from 25 to 55°C, with optimal temperature of 30°C.
All profiles of caseinolytic enzyme production have the sames phases; latency phase , exponentially phase of enzyme production, Fig- 4 shows the influence of temperature on growth for Bacillus megaterium.For the temperatures from 25 to 45°C, the increase of temperature is proportional to the increase of the amplitudes of growth profiles.For the temperatures from 40 to 60°C, the increase of temperature is inversely proportional to the amplitudes of growth profiles.The growth of the strain is possible from 25 to 60°C, with the optimal temperature at [ 35°C-37°C] All growth profiles have the sames phases; exponentially phase, stationary phase and the decreasing phase, but the different amplitudes.
Fig- 5 shows the influence of temperature on caseinolytic enzyme production for Bacillus megaterium.For the température from 25 et 35°C, the increase of temperature is proportional to the increase of the amplitude of the enzyme production.For the temperature from 35 to 50°C, caseinolytic enzyme production decrease when the temperature increase.the caseinolytic enzyme production is possible from 25 to 50°C with the optimum at 35°C.
Both profiles have the sames phases which are in correlation with the normal growth profile of bacteria.For both profiles from 0 to 10h , we have the exponentially phase.Stationary phase is from de 10 to 30 hours, for B.li, while from 10 to 34 hours.The decrease phase is from 30 to 48 hours for B.li , from 34 to 48 hours for B.me.
Fig- 7. shows different phases of the caseinolytic enzyme in the two strains.In the both strains , the latency phase is observed between 0 to 2h.For B.licheniformis, the profile comprise: From 2 to 10h,an exponentially phase of enzyme production ; From 10 to 26h, a stationary phase of enzyme production ; From 26 to 30h, a decreasing phase of enzyme production ; From 30 to 48h, a second stationary phase of enzyme production:.For B. megaterium, the profile comprise: From 2 to 8 h, an exponentially phase of enzyme production ; From 8 to 26 h, a stationary phase of enzyme production; From 26 to 48h, a decreasing phase of enzyme production ; From 34 to 48 h, une phase de diminution de production d'enzyme.
Fig- 8 shows how evolve the enzyme production comparing to the growth in B.li.It is shown that the enzyme production is maximal during the stationary phase of the growth.This parallelism is the same for B.me.

Influence of different kinds of media on growth and the enzyme production
Fig- 13 shows for the two strains growth profiles with the same phases.Growth is possible  for the two used media, the LB medium is better than TSB.Fig- 14: shows the sames phases in the two caseinolytic enzyme production profiles.
For LB medium : a latency phase in short and the amplitude of enzyme production is important.For the TSB medium : a latency phase is important and the amplitude of enzyme production is less important than in LB.

Influence of different carbon sources on the growth and caseinolytic enzyme production
In [Table -3] are shown how growth and caseinolytic enzyme production are influenced by different carbon sources in the two studied strains.Bacillus licheniformis and Bacillus megaterium.Sugars are important for growth and enzyme production in the two strains.Fructose is used to be the best carbon source.

Influence of different nitrogen sources on the growth and the caseinolytic enzyme production
Among the two nitrogen sources used, yeast extracts is the best for growth in two strains B. licheniformis and B. megaterium) .For enzyme production both sources can be used in Bacillus licheniformis, in contrast only the yeast extract is efficient in Bacillus megaterium.The results are shown in Table 4 Figure 17 is showing the differentiation of the two bacillus strains by SDS-PAGE , this figure is showing clearly differences between the two strains.

DISCUSSION
The diameter of the halo testifying the caseinolytic enzyme activity is used in this work to express the enzyme production.The Temperature is an important factor for growth and the enzyme production.All growth profiles in the two strains (B.licheniformis and B.megaterium), with the temperature have the same phases, including exponentially phase, stationary phase  and the decreasing phase.Figures 4 and 6.All profiles related to caseinolytic enzyme production, have also the sames phases, including the latence phase, an exponentially enzyme production phase, a stationnary production phase, and the decreasing production phase, Figures 5 and 7.For B.li, the optimal temperature of growth is 37ºC and the optimal temperature of enzyme production is 30ºC.For B.me the optimal temperature of growth is between [35-37] ºC, and the optimal temperature of enzyme production is 35ºC.These results are similar to those already published [15, 12, 27].The growth profiles have not shown a latency phase.The absence of latency phase is related to the used method.The OD measure is done just to hours after the inoculation of an overnight culture.These results are different to those published by [24] which presents a latency phase for different method of inoculation.
The growth and the enzyme production are different phenomens, this is shown on the figure 10.At all temperatures and for the two studied strains, the enzyme production is optimal durind the stationnary phase of the growth, these results are similar to those published in [12, 15, 27].
During the exponnentially phase of growth, bacterial cells are dividing untill the nutriments are still in the medium, when the nutriments goes smaller and smaller in the amount, toxics products accumulate.This brings the changes in the culture conditions.The Figure 11 is illustrating how the pH of the medium has passed from 7 to 9.These results are identical to standard of bacterial culture and those published by [28].
For the two studied strains, the decreasing phases is followed by a stationary phase on which the OD value remained stable , and the caseinolytic enzyme production is still important.This phase is assimilate to the sporulation, these results are in harmony with those related to B. Licheniformis and B. megaterium, which gradually go on sporulation , according to [29 ,30]  All the two strains can be cultured at different pH (5,7 et 9), with the optimum at 7. These results are different than those published by [31], which stipulate that these strains could not growth and can not produce the enzyme when the starting pH of the medium is at 5.
In the two used media, the LB is better rather for growth and fore enzyme production, on the two studied strains.
Among different carbon sources used.The best growth is observed with fructose and galactose which are monosaccharides.For polysacchrides growth and enzyme production are low.
When using the two sources of nitrogen , yeast extracts have been revealed to be better than bactopepton for growth in the two strains (B.li 0,969±0,015 B.me 0,952±0,01).In B.li the two nitrogen sources can be used for enzyme production (Yeast extracts:14,333±1,247, bactopepton: 14,333±1,247), but in B.me only the yeast extract yield the important enzyme production.In deed in our laboratory, we have set the standard of the enzyme production, the reference strains B.amy yield on the fructose medium the standard growth of 0.954±0.001,and at 48h of culture the standard enzyme production is13.333±0.001.It is according to these standard values, the strain is declared a caseinolytic producer (non published results).
We used SDS PAGE to differentiate the two strains through partial purification of protein in the supernatant after culture, and ammonium sulfate precipitation.The Figure 16 is giving the protein profiles of the two studied strains.Specific bands can allow discrimination of the strains.

CONCLUSION
This article have helpful to our laboratory, for we have set new optimal conditions of two new strains in growth and caseinolytic enzyme production.We have discover the specificity of the two strains.Our methods has allowed to discriminate the caseinolytic producers by SDS-PGE.This article has shown the importance of setting standard measure for enzymatic production.In a near future we will do the total purification and sequencing these caseinolytic enzymes.

Fig- 9 :
Fig-9:shows that the pH increase rapidely at the end of the enzyme production phase, from 7 to 9 in the two studied strains.Influence of pH of the medium in the growth and the enzyme production Fig-10 shows the influencce of pH of the medium on growth in the two strains.It is shown that growth is possible at acidic pH.The phases are the same in the two strains.Fig-11 shows the influence of pH on growth in Bacillus megaterium and Bacillus licheniformis.It is shown that growth is possible in basic pH at 9, and the profiles comprises the sames phases.Fig-12 Shpows how caseinolytic enzyme production is possible in different pH of the cultured media The optimum is at pH 7.Influence of different kinds of media on growth and the enzyme productionFig-13shows for the two strains growth profiles with the same phases.Growth is possible

Fig. 14 .Fig. 15 .
Fig. 14.Comparing profiles of caseinolytic enzyme production in two different media for the two studied strains Fig. 15.Proteins profiles on SDS-PAGE of two strains 1: marker in KDa, 2:supernatant after culture of B.li, 3:supernatant after culture of B.Me 4:ammonium sulfate precipitate of B.li supernatant after culture 5: ammonium sulfate precipitate of B.me supernatant after culture Lane 4, two important bands are displayed at 46kDa and 25kDa respectively.Lane 5 one important band is displayed on 56kDa.
. FALY et al.: STUDY OF CASEINOLYTIC ENZYME IN TWO Bacillus STRAINS

Table 1 .
Different media with different carbon sources

Table 2 .
Different media with different nitrogen sources

Table 3 .
Effect of three carbon sources on the growth and the enzyme production

Table 4 .
Effect of two different nitrogen sources on the growth and the caseinolytic enzyme production in B.li and B.me