Soloka Mabika Armel Faly1, Rachel Moyen1,2,  Etienne Nguimbi*1,2, Gabriel Ahombo, Raoul Ampa1,2,  Aimé Christian Kayath1,3, Alain Vouidibio1,2, Cyr Jonas Morabandzaand Simon Charles Kobawila1

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.

Abstract

Two Bacillus strains isolated from Ntobambodi  :Bacillus megaterium (B.me NM 02)  Bacillus licheniformis(B.li NM01),has shown a significant proteolytic activity in the caseinolytic character actively degrading casein.In the present work we set optimization of growth and enzyme production conditions. We therefore cultured strains in LB medium, with time computed the OD value for growth. Using containing-skimmed milk petri dishes, we measure the diameter of the formed halo with sample. Several parameters have been optimized: temperature, pH, various types of media, carbon and nitrogen sources. In both strains, growth is possible from 25 to 60 ° C with an optimum temperature at 37 ° C for B.li and at 35°C for B.me. Enzyme production was observed from 25 to 55 ° C with an optimum temperature of 30 ° C for B.li. Enzyme production was observed from 25 to 50 ° C with an optimum temperature of 35 ° C in B.me. For pH, growth and enzyme production can be at 5, 7 and 9 with an optimum at 7 for both strains. The LB medium is better for growth and enzyme production than TSB for B.li and B.me. Among the carbon sources used, fructose is better for growth after 48 hours of incubation in both strains (B.li: 0,93±0,001, B.me: 0,928±0,002), but for enzyme production fructose remains the best carbon source for  B.li (14,33±1,24) , while starch is the best for B.me (14.66±1,24). Concerning nitrogen sources, in both strains the best source of growth is the yeast extracts (B.li: 0,969±0,015, B.me:0,952±0,01). For enzyme production, the two sources can be used forB.li.(14,333±1,247) but for  B .me (14,333±0,471) only the yeast extracts is the  best  as well as for growth and enzyme production. Furthermore, in both strains the produced enzyme was partially purified by using ammonium sulfate precipitation, and SDS-PAGE has been hold, profiles of specific bands are useful to give more information and differentiate the two strains.

Keywords: Optimization, caseinolytic enzyme, Ntobambodi, Bacillus licheniformis, Bacillus megaterium.

Introduction

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 sales1.[1].

Proteases or proteolytic enzymes find many applications in many domains as:  pharmacy, food, biotechnology, and detergents2,3. Commercially used proteases are produced by bacteria, plants, animals and fungi4,5. 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 of growth. Extracellular protease production is strongly influenced by media composition, the ration of C/N, metabolizable sugars 6 and metallic ions1,7.More over proteases 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 microorganism1. Many studies have been focused on proteases production by microorganisms, these studies have stated strategies of growth and protease production7, 8, 9. Some studies have given information in the relation between growth and proteases 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 beginning 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 proteases production that, depend strictly on each microorganism 13, 14,15.

Caseinolytic proteases P(ClpPs) are complex of  oligoméricproteineswhichcontibute on cellular homeostasy and virulence regulation to bacteria. Whatever most of organismspossesse only one ClpP protein, in some can be encoded two or more ClpP isoforms 16. The caseinolytic protease P(ClpP) is an highly conserved enzyme in bacteria and higher organisms17, 18, 19.

In the republic of Congo fermented manioc leaves are used as a food by Congolese in some area. They are differently called: Ntobamubodi in the Bouenza, Lilleyuka in the Kouilouand  Ntobambodi in the Pool. Microbiological study of the NtobaMbodi has been hold and has shown a diversity of bacteria, among them the species of genus Bacillus20.

Many species of genus Bacillus isolated from NtobaMbodi has been molecularly characterized by their ARNr 16S and ARNr 16S-23S. Many of them have shown proteolyticactivities, as the fibrinolytic enzyme producer Ba NM76 described in 15. A new strain named Lysinibacilluslouembei sp. with a strong proteolytic activity has been isolated from Ntoba Mbodi21.

Two Bacillus strains isolated from Ntobambodi: Bacillus megaterium (B.me NM 02)  Bacilluslicheniformis(B.li NM01), 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 differentiate the strains.

 

Materials and Methods

Bacterial strains and media

Strains used in this study were Bacillus megaterium(B.me NM 02) andBacillus licheniformis(B.li NM 01) available in our laboratory were isolated from Ntobambodi.

The media used were : Luria Bertani ( 0,5%NaCl, 1% Tryptone, 0,5% Extraits de levure ); Tryptone soya broth (TSB); Starch (0, 5%;) Galactose (0, 5% ); Fructose (0, 5%) ; and  mineral solution (MgSO4.5H2O: 0, 06%, KH2PO4: 0, 1%, Cacl2.6H2O: 0.2% , K2HPO4 .0, 2%).

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)12,15.

Enzyme assay

Puri modified techniques9.was used to test caseinolytic enzyme. The 48h culture was collected in 1,5 eppendorf  and centrifuged for 5minutes at 6000rpm in a type microcentrifuge(MICRO STAR 17R from VWR ).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 until à 55-60°C, 10 ml of skimmed milk were added. The mixture were poured in petri dishes. Wholes were made on the plate. 50μl 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 activity12, 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 activity15, 22.

Influence of different kinds of media on the growth and 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, starch 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 I. 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.

 

Table I: different media with different carbon sources

 

Media Carbon sources Nitrogen source Mineral solution
Medium 1 Galactose Yeastextracts MgSO4.5H2O: 0, 06%, KH2PO4 :0,1%,  ,2 Cacl2.6H2O :0.2%, K2HPO.0, 2%
Medium 2 Fructose Yeast extracts MgSO4.5H2O: 0, 06%, KH2PO4 : 0,1%,  Cacl2.6H2O:0.2%, K2HPO.0, 2%
Medium 3 starch Yeast extracts MgSO4.5H2O: 0, 06%, KH2PO4 : 0,1%,  Cacl2.6H2O :0.2%, K2HPO.0, 2%

 

Influence of nitrogen sources on the growth andcaseinolytic 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 II. 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.

Table II: different media with different nitrogen sources

Media Nitrogen sources Carbon source Mineral solution
Medium 1 Yeastextracts starch MgSO4.5H2O: 0, 06%, KH2PO4 : 0,1%,  Cacl2.6H2O :0.2%, K2HPO.0, 2%
Medium 2 Bactopeptone starch MgSO4.5H2O: 0, 06%, KH2PO4 : 0,1%,  Cacl2.6H2O : 0.2%, K2HPO.0, 2%

23, 25,26.

 

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 centrifuge 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 : Tris25 mM, Glycine0.192 M, SDS 0.1% ; Staining solution : Coomassi bleu 2.5 g, methanol 450 ml, glacial acetic acid 100 ml, eau 400 ml, adjusted at 1 l ; Dying solution   : methanol, glacial acetic acid 100 ml, eau 400 ml, adjusted at 1 l.; Protein molecular markers proteins (Protein Page Rulerprestained, Fermentas).

RESULTS

 

Caseinolytic enzyme activity

The Figure1 shows the caseinolyticenzyme activity in three bacillus strains isolated from NtobaMbodi :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.

Fig-1 :   halo of  caseinolytic enzyme activity of   different Bacillus strains isolated from Ntoba Mbodi

B.me : Bacillus megaterium,  B.li : Bacillus licheniformis,   B.amy: Bacillus amyloliquefaciens.

 

 Influence of temperature on growth and caseinolytic enzyme production.

Fig-2shows 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 growth profiles. Growth is possible from 25 to 60°C, with the optimal temperature at 37°C.

Fig-2: growth profiles of Bacillus licheniformis at different.

 DO=Optical Density

 

All growth profiles have the sames phases; exponentially phase, stationary phase and the decreasing phase, but the different amplitudes.

Fig-3showstheinfluence of temperature on caseinolytic enzyme production for  Bacilluslicheniformis. 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 profiles are proportional to increase of the temperature. For the temperatures from 30 to 55°C the amplitude of caseinolytic enzymes production profiles is inversely proportional the increase of temperature. Caseinolytic enzyme production is possible for the temperature from 25 to 55°C, with optimaltemperature of 30°C.

All profiles of caseinolytic enzyme production have the sames phases; latency phase, exponentially phase of enzyme production, stationary phase of enzyme production and the decreasing phase, but the different amplitudes.

Fig-3 : Profiles of caseinolytic enzyme production by  Bacillus licheniformis

at different temperatures. AE=enzyme activity=testifying the 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-4 : Growth profiles of  Bacillus megaterium at different temperatures

 

Fig-5 shows the influence of temperature on caseinolytic enzyme production for  Bacillusmegaterium. For the temperature from 25et 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. Thecaseinolytic enzyme production is possible from 25 to 50°C with the optimum at 35°C.

Fig-5: Profiles of caseinolytic enzyme production for Bacillus megaterium at

different temperatures.

 

In Fig-6,both profiles have the same 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-6 : Comparing growth profiles of Bacillus licheniformis (B.li) and  Bacillus megaterium(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  26h, an  exponentially phase of  enzyme production  ;

From 26 to 34 h, a stationary phase of enzyme production;

From 34to  48h, a decreasing  phase of enzyme production ;

Fig-7 :Comparing profiles of caseinolytic enzyme production of   Bacilluslicheniformis (B.li)  and Bacillus megaterium(B.me).

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.

Fig-8: Profiles of growth and enzyme production in  Bacillus licheniformis.

Fig-9: shows that the pH increase rapidly at the end of the enzyme production phase, from 7 to 9 in the two studied strains.

Fig-9: Profiles of growth, enzyme production and pH variation during culture in the two studied strains.

 

Influenceof pH of the medium in the growth and the enzyme production

Fig-10 shows the influence 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 sames in the two strains.

Fig-10 :  Growth profiles of  Bacillus megaterium and B.licheniformis  at pH 5

 

Fig-11 shows the influence of pH on growth in Bacillusmegateriumand  Bacilluslicheniformis. It is shown that growth is possible in basic pH at 9, and the profiles comprise the sames phases.

Fig-11 : Growth profiles at pH 9 in   Bacillus megaterium et B.licheniformis

 

Fig-12 Shows how caseinolytic enzyme production is possible in different pH of the cultured media. The optimum is at pH 7.

Fig-12: Diagrams of caséinolytic enzyme production of B.li and  B.meat different pH.

 

4- 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-13 : Comparing growth profiles in different kinds of media for the two studied strains.

 

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.

Fig-14: Comparing profiles of caseinolytic enzyme production in two different media for the two studied strains.

 

Influence of different carbon sources on the growth and caseinolytic enzyme production.

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

Table III: Effect of three carbon sources on the growth and the enzyme production

  Growth (DO at 600nm) 48h Enzyme production (diameter of halo in mm) 48h
Carbon sources B.li B.me B.li B.me
Galactose 0,903±0,002 0,849±0,001 13,33±1,24 11,33±0,47
Fructose 0,93±0,001 0,928±0,002 14,33±1,24 14,33±1,24
Amidon 0,711±0,001 0,845±0,004 10,00±0,81 16,66±1,24

 

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  Bacillusmegaterium. The results are shown in Table IV

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

 

  Growth (DO at 600nm) 48h Enzyme production (diameter of halo in mm) 48h
Nitrogen sources B.li B.me B.li B.me
Yeast extracts 0,969±0,015 0,952±0,01

 

14,333±1,247 14,333±0,471
Bactopeptone 0.514±0.003 0.739±0.002 14,333±1,247 11.666±0.942

 

7-Differentiation of strains used by SDS-PAGE

Figure 15 is showing the differentiation of the two bacillus strains by SDS-PAGE

Figure 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.

 

 

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 latency phase, an exponentially enzyme production phase, a stationary 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 published15, 12, and 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 phenomena, this is shown on the figure 10. At all temperatures and for the two studied strains, the enzyme production is optimal during the stationary phase of the growth, these results are similar to those published in 12, 15, and 27.

During the exponentially phase of growth, bacterial cells are dividing until 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 illustration 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 toB. Licheniformisand  B. megaterium, which gradually go on sporulation , according to29 ,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 cannot 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 monosaccharaides. For polysaccharides 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 15 is giving the protein profiles of the two studied strains. Specific bands can allow discrimination of the strains.

Conclusion

This article have been helpful for 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.

Acknowledgements: We give thanks to Professor Joseph GomaTchimbakalaScientific Director of for his important help.

 

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