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ThesisItem Open Access BIOCHEMICAL ASSESSMENT OF HIGH TEMPERATURE AND MUTAGEN EFFECTS ON PSEUDOMONAS SPECIES(Division of Biochemistry, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, 2015) Chalotra, Rashmi; Mallick, S.A.Pseudomonad, are used as potential biocontrol agents against plant diseases for their excretion of cell degrading enzymes (viz. chitinase, lipase, protease) and antibiotics (viz.2,4-DAPG) along with production of plant growth promoter (viz.IAA, GAs). But the survivality and biocontrol activity of the bacteria need to be sustained at high temperature and wide pH range for their wider application. With expectation to further improve in the biocontrol/plant growth promoting activities, Pseudomonas species also need to be mutated by most effective microbial mutagen Ethyl Methane Sulphonate (EMS). Therefore, the present study was carried out with objectives (i)To study the effects of temperature, pH and mutagen on survivality and biochemical characters of Pseudomonas species procured from different crop fields of Jammu region.(ii)To screen out heat resistant Pseudomonas species with good plant growth promoting (PGP) and biocontrol activities.(iii)To develop ethyl methane sulphonate (EMS) mutated Pseudomonas species which will possess maximum biocontrol and plant growth promoting properties. The investigations on the abilities of production of cell degrading enzymes viz. chitinase, lipase, protease , antibiotic 2,4 DAPG and PGPR (IAA and GA) in normal Pseudomonas as well as EMS mutated Species showed that EMS mutation caused the increase in chitinase activity (about 10 folds) and lipase activity (about 2 fold) almost in all Species , on the contrary, the protease activity and 2,4 DAPG production were found to be reduced in all Species except PSM-7 and PSM -10 due to mutation. Highest chitinase production was found both in normal PS-30 and EMS- mutated Species PSM-30 (1.0182 and 1.0395 µmole of N-acetyl glucosamine/ min/mg protein respectively) whereas maximum lipase activity was detected maximum in normal Species PS-11 (2.8491 µmole of p-nitro phenol/min/mg protein) and mutated PSM-18 (3.7550 µmole of p-nitro phenol/min/mg protein) respectively. Due to mutation, the maximum improvement in lipase activity recorded in those Species which were originally poor in lipase production under normal condition. Maximum protease production was determined in PS-11 (0.5411 µmole of tyrosine/min/mg protein) and PSM-15 (0.2667 µmole of tyrosine/min/mg protein) among normal and mutated species respectively. In temperature effect study, all normal Species possessed maximum growth at 350C except PS-11 and PS-30 showed maximum growth at 450C ;however, almost all the mutated Species carried maximum growth at 450C whereas PSM- 11 and PSM-17 which had sustainability up to 550C .Enzymes activities with varying temperature showed the similar activity trend as that of cell growth. On pH study, PS-7 to PS- 17 depicted highest growth at pH 6.5 and PS-18 to PS-30 at pH 8.5; however, all mutated Species showed maximum growth at pH 6.5. Enzymes activities (chitinase, lipase and protease) with varying pH exhibited also the similar trend as that of cell growth. PGPR activity was found to be decreased on mutation. The PS-11 and PS-30, which possessed highest survivality, sustainability in production of in all three enzymes at high temperature and pH 6.5, along with highest production in 2,4-DAPG and PGPR in normal as well as EMS mutated condition, are considered most potential Species for high temperature and pH 6.5. However, on the basis of overall study, the PS-11 is considered to be most efficient Species followed by PS -30(found to perform best at alkaline pH 8.5).ThesisItem Open Access BIOCHEMICAL STUDIES OF MUTAGENIC EFFECT ON AMYLASE ACTIVITY OF BACILLUS ISOLATES(Division of Biochemistry, 2015) Sharma, Neha; Mallick, S.A.Nowadays the new potential of using microorganism as biotechnological sources of industrially relevant enzymes has stimulated renewed interest in the exploration of extra cellular enzymatic activity in several microorganisms. In this study, our purpose was to isolate Bacillus bacteria potenial in amylase production ability, amylase production optimization with variation of pH, High temperature and substrate concentration its improvement through Ethyl Methane Sulphonate (EMS) mutation.Therefore, the present study was carried out with the objectives: (i)To Screen out Bacillus isolates from cereal brans sources having highest amylase activity. (ii)To search for heat resistance strain having highest amylase activity.(iii) To study the effect of Ethyl Methane Sulphonate (EMS) mutagen on amylase activity of isolated strain(s) and to sort out the strain having best performing in α-amylase production /activity. A total of 15 Bacteria were isolated from soil samples collected different mills of Jammu using MYP Agar Base medium and were characterise as Bacillus species through biochemical (positive results for the starch hydrolysis, gelatin liquefaction, simmon’s citrate,casein hydrolysis and H2S production test but showed negative results for urease test), gram and physical tests. Amylase activity was found maximum in the normal strain NBS-9 (2.851µmole/min/mg) and maximum in mutated strain MBS-5 (1.293 µmole/min/mg) at 300C and pH 7.0. In pH effect study, the highest amylase activity was recorded in normal Strains NBS-1 and NBS-10 exhibited maximum amylase activity at pH 6.6 whereas NBS-3 and NBS-8 exhibited maximum amylase activity at pH 5.8 while rest of strain showed maximum activity at pH 7. However,on mutation,MBS-1, MBS-6, MBS-11, MBS-13 and MBS-15 got highest amylase activity at pH 7, while highest amylase activity exhibited in MBS-2, MBS-3, MBS-5 ,MBS-7 strain at pH 6.6 and MBS-4, MBS-8, MBS-9, MBS-10 and MBS-12 at pH 5.8 and MBS-14 at pH 6.2 respectively.So, different isolates indicated different ph for optimal production (most of them in slight acidic range).About temperature, the highest amylase activity was found in normal strains NBS-1, NBS- 4, NBS-6, NBS-7, NBS-8, NBS-10 and NBS-11 at temp 300C and in NBS-2 and NBS-14 at 400C and NBS-3, NBS-5, NBS-9, NBS-12 and NBS-15 at 500C and NBS-14 at 600C.On mutation, the MBS-5, MBS-7, MBS-8, MBS-14 and MBS-15 strains got better amylase activities at 600C.The mutant MBS-7 (1.165 µmole/min/mg protein) was found the best among the lot which possessed maximum amylase activity at 600C. As, Amylase was excreted from different sources i.e. different of Bacillus isolate, it was therefore, important to know Km values of the enzyme obtained from different isolates. Among normal strains, lowest and highest Km value was observed in NBS-15 (0.046 mg/ml) and in NBS-8 (1.060 mg/ml), whereas in mutated strains minimum was observed in MBS-12 (0.036 mg/ml) and maximum in MBS-14 (2.978 mg/ml) . Therefore, NBS-15 and MBS-12 are considered to have best binding capacities with substrate starch in normal and EMS-mutates strains. On the other hand, maximum and minimum Vmax values were seen in normal NBS-9 (6.024 μmol min-1 mg-1 and NBS-2(0.270 μmol min-1 mg-1) whereas highest and lowest Vmax values observed in MBS-12 (125 μmol min-1 mg-1) and in MBS-6 (1.459 μmol min-1 mg-1) respectively. On the basis of overall observation , it is concluded that all the isolates from soil samples are Bacillus species and having significant amylase production abilities and EMS-mutation causes improvement of amylase production abilities in general, especially those are originally ( in normal isolates form) poor in amylase activity. The NBS-13 isolate which possessed highest amylase potential at 600C and at pH 7.0. MBS-11 and MBS-12 which depicted highest amylase values at pH 7.0 and pH 5.8 respectively are considered most efficient amylase producer at corresponding pH.In enzyme quality assessment, NBS-15 and MBS-12 having lowest km values are found best for amylase quality in normal and mutated strains respectively.