Alkaline Proteases assay,optimization for parameters.

1. A PROJECT REPORT ON
ISOLATION, IDENTIFICATION AND PRODUCTION OF
ALKALINE PROTEASE ENZYME PRODUCING ORGANISM
FOR THE INDUSTRIALAPPLICATION.
By
Mr.Tejas Vishnu Sonawane
Mr.Girish Sahebrao Sawala
Mr.Yogesh Shivaji Ghayal
Guide : Ms.Nilima Patil mam
(Jai Biotech Industries,Satpur MIDC,Nasik)
Asst.Prof.Mr.C.S Deore Sir (Internal Guide)
MSC II Microbiology
K.A.A.N.M.S College,Satana

2. ABSTRACT
 Alkaline proteases is protein hydrolyzing enzyme which is
most important widely used enzyme.
 Alkaline proteases used in all pharmaceuticals, drug
manufacturing, silver recovery,leather industry, textile industry,
cleaning soultions etc.
 6 alkaline proteases producing Bacillus species isolated from
soil samples.
 The enzyme was partially purified and optimized for different
parmameters like different temprature,pH, effect of metal ion
concentraiton, etc.

3. INTRODUCTION
 Proteases is belongs to Hydrolases class of enzyme. Also known as
proteinases or peptidases are enzymes occurring everywhere in
nature. These carried out proteolysis of proteins.
 It contribute 25% use in industry. Alkaline proteases are particularly
important because they are stable and active under harsh conditions
such as high temperature, pH and presence of surfactants.
 Proteases have optimum pH in the range of 7.0 while alkaline
proteases have pH range 8.0-11.0.
 Alkaline proteases mostly produced from Bacillus spp.
 Eg. B.licheniformis, B.lentus, B.subtilis, B.alcalophilus etc.
 They screened and optimize for different temprature and pH to
determine industraial sutailbility.

4. APPLICATIONS OF PROTEASES :
 Protesases most important group enzyme produced
commercially and are used in the
detergent,brewing,leather, dairy industries.
 It convert biological waste such as
horn,feather,nails,hairs into useful biomass.
 1.Detergent industry
 2.Dairy and food industry
 3.Silver recovery
 4.Leather industry
 5.Medicinal uses
 6.Waste management
 7.Textile industry

5. MATERIALS AND METHOD
 Isolation & Identification of bacteria
 a) Collection of samples :
 1% agitaed soil sample spreaded on skimmed milk agar plate
and incubated at 30°C for 48 hours.
 b) Screening of protease producers
 The isolated colonies were screened for protease production
using skim milk agar plates.
 All the isolates were streaked on to skim milk agar plates and
the plates were incubated for 48 hours at room temperature.
 The clear zones around the colonies were evaluated as protease
producers.

6.  Identification & Characterization of the microorganism using
different tests
 Bacterial isolates those gives zone of clearence showing
maximum enzyme production was proceed for:
 Colony morphology
 Gram statinig
 Strach hydrolysis
 Catalase
 IMViC
 Nitrate reduction
 After that proceed for indentification with the Bergey’s Manual
of Determinative Bacteriology

7. FERMENTATIONS AND CRUDE ENZYME PREPARATION
Loopful growth was innoculated into 10 ml L.B broth and
incubated at 37°C for 24 hours.
After this 100 μl of the bacterial culture was inoculated to 20 ml
of production medium and kept in incubater shaker at 140 rpm,
37°C for 24 hours.
After that enriched broth was centrifuged at 10000 rpm for 10
minutes. Supernatent used as crude enzyme source for enzyme
assay.

8. PROTEASE ASSAY
 Assay method: Kuntiz method
 Substrate: Caesin(pH 9)
 Incubation temprature :37°C
 Time :30 minutes
 Enzyme Substrate reaction stopper: TCA reagent
 Tyrosine realese quantified by measuring abosorbance at 280
nm.

9. PROCESS FOR MAXIMUM ENZYME
PRODUCTION:
 Method: Submerged fermentation
 Carbon source and nitrogen source were optimized
for maximum enzyme production.
 Enzyme production at different time:
 Production media innoculated with 24 Hour
Bacterial strain. Broth incubated at different time
period of 24,48 and 72 Hours. Supernatent used as
crude enzyme source gives different enzyme
activity.

10. CHARACTERIZATION OF CRUDE PROTEASE
 a) Effect of pH on enzyme activity
 Effect of pH on alkaline protease activity was determine by
incubating enzyme
 solution with 1% substrate (Casein) prepared in buffers (0.1M)
of different pH such as
 KH2PO4-K2HPO4 (6.0-7.5), Tris –HCl (8.0 -9.0), Glycine-
NaOH (9.0 –13.0) and Na2HPO4-NaOH (11.0-12.0).
.

11. B) EFFECT OF TEMPERATURE ON PROTEASE
ACTIVITY
 To check the optimum temperature of alkaline protease, the
enzyme with substrate
 was incubated at different temperatures (30, 35, 37, 40, 45 and
50°C

12. EFFECT OF METAL IONS ON PROTEASE
ACTIVITY
 Metal ions solutions of below
 Ca2+, Mg2+, Mn2+, Cu2+ and Hg2+
 Metal Salt Concentration : 10 mM.
 1 ml metal solution + 5 ml enzyme solution was
incubatd for 2 Hours and initial and final enzyme
activities were measured.

13. LAB SCALE FERMENTATION
Incubation Temprature: 37°C for 48 hours.
pH : 9
Agitaiton speed : 300 rpm

14. RESULTS
 Isolation of bacteria:
 5 distinct morphologically distinct colonies were isolated
from alkaline Soil sample by serial dilution method was
spred on nutrient agar plate with pH 8,9 and 10. Colonies
were named as I1,I2,I3,E1 and E2.
 Screening for Protease Enzyme
 Isolated 5 strains were streak on skimmed milk agar plate
to check ability of proteases production.only two isolates
gives larger zone of hydrolysis of protein.all isolates
proceeds for futher studies and biochmeical test.

15. Proterolytic activity of Isolates

16. CHARACTERIZATION AND OF PROTEASE
PRODUCING ISOLATES
TABLE 1.

17. TABLE 2.

18. ENZYME ACTIVITY ASSAY

19. PROCESS FOR MAXIMUM ENZYME PRODUCTION
 Protese assay for isolate which gives maximum
enzyme activity
Fig.Enzyme activity of isolates

20. MAXIMUM PROTEASE PRODUCTION IN
DIFFERENT MEDIA
Fig.Enzyme activity in different prodution media

21. ENZYME ACTIVITY AT DIFFERENT TIME
PERIODS:
Fig.Effect of incubation time

22. CHARACTERIZATION OF CRUDE PROTEASE
 Effect of pH on enzyme activity
Fig.Effect of pH

23. EFFECT OF TEMPRATURE ON ENZYME
ACTIVITY
Fig.Effect of Temprature

24. EFFECT OF METAL IONS ON ENZYME
ACTIVITY
Fig.Efffect of metal ions

25. ENZYME ACTIVITY AND GROWTH RATE
 The enzyme activity and growth rate of isolate I2 (Bacillus brevis)
Fig.Graph of enzyme activity V/S growth

26. CONCLUSION :
 Isolated species of Bacillus was identified by morphological
and biochemical testes,
 they were Bacillus insolitus , Bacillus brevis, Bacillus
marinus, Bacillus pasteurii, and
 Bacillus macquariensis respectively.
 1. Alkaline protease produced from isolate I2 i.e. Bacillus
brevis by useing submerged
 fermentation.
 2. The media number 3 (PM3) were the best media for all
Bacillus brevis for Alkaline
 protease production.
 3. Characterization of crude alkaline protease

27.  Optimum enzyme activity at pH 9.
 Optimum enzyme activity at temperature 37oC.
 Enzyme activity rise in the presence of Ca2+, Mg2+,
Mn2+, Cu2+ metal ions and
 decrease in the presence of Hg2+.
 4. In laboratory scale fermenter study shows the
relationship between growth rate V/S
 enzyme activity. The enzyme activity increased as the
incubation times increased.
 Growth rate increased sharply with increasing incubation
times.
 5. Application as detergent of alkaline protease is
successfully carried out