إنتاج وتوصيف أنزيم البروتييز القاعدي من الفطر Metarhizium anisopliae كعامل من عوامل السيطرة الحيوية تجاه عثة الشمع الكبرى Galleria mellonella

Production and Characterization of Alkaline protease from Metarhizium anisopliae as abiological control agent against Greater wax moth Galleria mellonella

This studying consist of  two main axis , the first was carried out to evaluation the efficiency of local isolate of fungus Metarhizium anisopliae  for control Grater wax moth Galleria mellonella L.  by using fungal suspension and secondary metabolites concentrations , and in the second axis the study  was focused on  production and  characterization of  an effective  biological control agent from this fungus . The results showed as later :

1- (A) The different concentration of fungal spore suspension have affected the life stages of G. mellonella . The mortality percentages of eggs was  62.88 % at the concentration of 1×10⁷ spore/ml and decreased to 31.78 % at the concentration of 1×10⁴  spore/ml  . The larval  instars have showed highest mortality rate reached (100 , 96.99 and  90.97  ) %  for 1^st  , 4^th and 7^th larval instars respectively , during 120 h at the concentration of  1×10⁷ spore/ml . The values of  lowest mortality rate when treated with  1×10⁴ spore /ml  during  120 h  were (62.90 , 58.88 , 50.86 ) % for 1^st  , 4^th and 7^th larval instars respectively  .  54.89 %  the highest mortality rate  for the pupae was recorded when treated with  1×⁷10 spore /ml  while  36.85 % were found to be dead when exposed to 1×⁴10 spore /ml. for the adults , and the treatment with highest concentration of spore suspension caused  a high adult mortality, i.e  69.95 %  and  63.94 % for adult males and for females after 168 hours. While the lowest mortality percentages were 41.91 %  and 43.91 % when treated with 1×10 ⁴ spore/ml for the same time .

(B) The relationship between cultural filtrate which contain different secondary metabolites  and  percentage of mortality of  egg ,larvae, pupa  and adult were positive. 60.91 %  of egg dead  at a concentration 100%  , and 26.82 % were dead at the concentration 25% . The full cultural filtrate caused a mortality at  a rate  ( 100  , 97  and 91.99 )  %  from  1^st  , 4^th and 7^th larval instars respectively  during 72 h . Decline to ( 67.96  , 64.96   and 54.95 ) % mortality was found for the same instar respectively  when treated with concentration 25 % at 72 hours . This indicates  that  the secondary metabolites of the fungal were more effective on 1^st larval instars than  the 7^th  instars . that highest mortality was 50.96 % at the concentration 100%  and  32.95 % with concentration 25 %  for pupa  . As for adults the results revealed that the highest mortality was ( 68.90 and 58.98 ) % for male and female respectively at a concentration 100%  while the lowest rate of mortality were (37.98 and 32.97 ) %  for male and female at aconcentration of 25% respectively.

2– Display the isolate of  fungus ability on the production enzymes protease , chitinase and lipase but the optimum production recorded for protease in minimal media supported with casein where the specific activity was 41.12 units / mg protein . The largest inhibitor diameter for resistant organisms was 5.3 mm which taken from destroxins media against S. aureus .

3- The optimal conditions for the enzyme production from the fungus used  in this study were found using medium composed of  Date syrup (DS)  at concentration 0.05 % supplemented with 0.5 % gelatin , at initial pH 5.5 with an inoculum size 16 % , and incubation  at 30 ˚C on a rotary shaker at 100 rpm for 72 h.

4- Protease from culture filtrate of M. anisopliae  , were partially purified by precipitation with 60% ethanol and Ion exchange chromatography using DEAE- Cellulose (Batch wise ) then by gel filtration chromatography using Sephadex G-150 . Fold purification  was 28.726  with 11.97 %  yield for the enzyme at the final step of this purification procedure.

5- The purified Protease  was characterized . The results showed the following :

• The K_m values for protease were (0.178 , 0.179 and 0.182 ) mg/ml , against gelatin , BSA and  casein  as substrates , respectively . While the V_max values were (0.465 , 0.339 and 0. 465) mg/min  for the three substrates  respectively too .

B-  The optimum pH for the Protease  activity was pH 8.0 ,  and it

        was stable in the pH ranged between (7.0 – 9.0)  .

C-The Protease  showed heat stability at (20-30) ˚C for 30 minutes .

D-The efects of protease inhibitors and various metal ions on the

    enzyme activity were determined. Only in the presence of PMSF

    at (5) mM  the enzyme activity was strongly inhibited, indicating that

    protease enzyme belongs to the serine-type peptidase group,  While iron

    chloride (FeCl₂) had stimulated effect on the enzyme activity  .