1. Isolation, Production and Characterization of the Crude Pectinase Enzyme Produced by Bacillus sp KCET PEC+ By
M.Naresh kumar. M.Tech
Under the guidance of
Dr.K.Geetha
Professor
Department of Biotechnology
Kamaraj Collage of Engineering and Technology
Virudhunagar.

2. Introduction
The increasing energy demands have focused worldwide attention on the utilization of renewable resources, particularly agricultural and forest residues, the major components of which are cellulose, starch, lignin, xylan and pectin.
Pectinolytic enzymes are widely used in the food industry for juice and wine production.
Pectinases, or pectinolytic enzymes, hydrolyze pectic substances present in the plant cells.

3. Today pectinases are one of the industrially important bacterial enzymes. Pectinases are one of the most widely distributed enzymes in bacteria, fungi and plants.
They have a share of 25% in the global sales of food enzymes.
They are of importance in the applications like fruit juice extraction and its clarification, scouring of cotton, degumming of plant fibers, waste water treatment etc.

4. Pectinase classification
According to the cleavage site, pectinases are divided into three groups:
(1) hydrolases consisting of polygalacturonase, PG (EC ).
(2) lyase/trans-eliminases comprising pectinlyase, PNL (EC ), and pectate lyase, PL (EC ).
(3) pectin esterase, PE (EC ).
Some of the pectinase producing microbes are
Aspergillus carbonarius
Streptomyces lydicus
Aspergillus niger
Bacillus sp.
Penicillium frequentans
Fusarium moliniforme
Aspergillus giganteus.

5. Literature review S .no Litreature Result Year 1.
Isolation, identification and screening of pectinolytic fungi from different soil samples
Isolationand screening of pectinolytic microorganisms.
2012 2.
Production of Bacterial Pectinase(s) from Agro-Industrial Wastes Under Solid State
Fermentation Conditions
Production studies
2008 3.
Production, Characterization and Industrial Application of Microbial Pectinolytic Enzymes
2009 4.
Production of a Novel Pectin Lyase From Bacillus pumilus (P9)
2006
Production and purification of pectinase by soil isolate Penicillium.
Purification studies
2010

6. Objectives
Isolation and screening of pectinase producing organism from fruit market waste.
Assessing the pectinase enzyme productivity by isolate in Yeast Extract Pectin (YEP) medium containing plate.
Production of pectinase using agro-industrial residues in submerged fermentation (SmF).
Effect of initial pH and incubation time of the fermentation medium on pectinase production.
Characterization of crude pectinase produced by isolate.

7. Work plan
Isolation and Screening of bacteria for pectinase production: Assessing the pectinase enzyme productivity of Bacillus sp.PEC+ in YEP containing plate. Effect of initial pH and temperature / incubation time of fermentation medium on pectinase Production. Comparison of pectinase production by using various agro-industrial waste as sole carbon source. Effect of pH, Temperature, and Substrate concentrations on pectinase activity. Effect & Stability of pectinase at different pH and temperature.

8. Materials and Methods

9. Isolation of pectinase producing bacteria:
Sample collection:
Soil samples were collected in plastic bags from fruit market waste dump area from Anna nagar, Madurai district, Tamilnadu, India.
Isolation of pectinase producing bacteria:
One gram of soil sample was added to 100ml of sterile distilled water.
Dilutions of ten folds were made and 1ml aliquots from and dilutions were spread plated on Petri plates containing NB medium with 1.5% pectin.
The plates were incubated at 37˚C and pure colonies were screened for pectinase production. 

10. Screening of pectinase producers:
Isolates were qualitatively confirmed by patching on NB medium and overlayed with iodine – potassium iodide solution.
Colonies with zone of clearance were selected as pectinase producing isolates.
The positive colonies were confirmed for pectinase production by adding 1% hexadecyl trimethyl ammonium bromide (CTAB) and zone of clearance was noted.

11. Production of pectinase using pure pectin as substrate in Smf
The bacterial isolate was grown in YEP medium containing (g/l): peptone, 10; yeast extract, 10; sodium chloride, 5; agar, 10 and pectin, 1%.
The initial pH was adjusted to 7.0 and inoculated with 1% of overnight culture at 37°C.
After every 6 h of incubation, the culture was centrifuged and both supernatant & pellet were analyzed for pectinase presence.

12. Cond.. The pectinase activity was assayed by DNS method.
Pectinase activity was found to be U/ml in supernatant. But, no pectinase activity was found in the pellet.
The supernatant was used as crude enzyme extract for further experiments.

13. Effect of incubation time of fermentation medium on enzyme production
The optimum incubation time for pectinase enzyme production was studied using YEP medium.
After inoculation, 2ml of the medium was immediately collected, subsequent samples were collected at an interval of 6 hrs up to 72 hrs.
The samples were assayed for pectinase activity.

14. Effect of initial pH of the medium on pectinase production
The effect of initial pH of the fermentation medium on pectinase production was studied by preparing the media with different pH in the range of 3 to 9. Temperature was kept constant at 37°C.
After incubation, culture filtrates were assayed for pectinase activity.

15. Effect of agro industrial residues on production of pectinase
The bacterial strain Bacillus sp.PEC+ was grown YEP medium containing 1g each of various substrates (wheat bran, rice bran, sugarcane bagasse and orange peel).
Finally, the culture supernatant was analyzed for pectinase activity using DNS method.

16. CHARACTERIZATION OF CRUDE PECTINASE FROM Bacillus Sp. PEC+

17. Activity & stability of crude pectinase at different temperatures
The effect of temperature on the enzyme activity was determined by performing the standard reaction at different temperature in the range of 30 to 90°C.
Thermostability of pectinase was studied by incubating the enzyme in 0.2M sodium citrate buffer within a temperature range from 30 to 90ºC for 1hr.
Activity & stability of crude pectinase at different pH
The effect of pH on the pectinase activity was determined by performing the enzyme assay with different pH buffers ranging from 3 – 10 pH.
The stability as a function of pH was determined by measuring the residual activity at each pH after 1hr of incubation at room temperature.
The retention of activity was assayed at optimal pH and temperature.

18. Effect of substrate concentration on pectinase activity
Commercial pectin was prepared in different concentrations such as 0.50%, 1.00%, 1.50%, 2.00%, 2.50%, and 3.00% (w/v) and used as substrate for enzyme assay.
Enzyme assay was performed using standard reaction condition.

19. Results and Discussion
Out of the 11 colonies, 6 were selected as positive isolates based on the plate assay involving iodine-potassium iodide solution and CTAB.
Among the six selected positive isolates, 4 were selected based on the size of the zone of clearance formed during the plat assay and designated as C3, C5, C7, C8 .

20. SmF was carried out using YEP medium where isolate C3 showed maximum activity (Table 1). This isolate also showed large zone of clearance in plate assay.
This isolate was grown in submerged state fermentation for quantitative estimation of pectinase enzyme.
The isolate was named as Bacillus sp.PEC+.

21. Iodine-potassium iodide solution overlay plate
assay showing the zone of clearance.
Plate assay using CTAB, showing
zone of clearance.

22. Table 1: Pectinase production by bacterial isolates under Smf condition in YEP medium
Activity (U/ml) C3
0.627 C5
0.255 C7
0.308 C8
0.013
* Here u have given the activity as 0.67 where as in the next slide u have given very high. How did that happen? No correlation……

23. Production of pectinase using pure pectin as substrate by SmF
Maximum enzyme production was observed between 24 to 30th hr in the range of to U/ml respectively and declined subsequently.

24. Effect of initial pH of the medium on pectinase production
Maximal pectinase production ( U/ml) was observed at pH 6.0.
The enzyme activity below and above this optimal pH value was lower.
A decrease in pectinase activity was observed when the pH of the YEP medium was adjusted either at pH 5.0 or pH 9.0.
This decrease in the pectinase production was attributed to decrease in the growth of organism at these pH values .

25. Effect of initial pH on pectinase production
* See the x-axis. pH is 1, 2, 3…. Where as the range used is different????????????

26. Analysis of chemical compositions of various agro industrial residues
Various agro industrial residues were analysed for their chemical composition at Annamalai university, Department of Chemistry, Annamalai Nagar, Tamil Nadu, India.
Chemical analysis revealed that the dry material was composed of the following compositions showed in Table 2:

27. Effect of various substrates on pectinase production
Sugar cane bagasse was found to be the best substrate, among others used, for pectinase production by Bacillus sp.PEC+ (184.1 U/ml).
Other substrates yielded less pectinase activity compared to sugarcane bagasse.

28. Effect of various substrates on pectinase production

29. Effect of partical size of substrate on pectinase production
The effect of particle size of sugar cane bagasse on the production of Pectinase was studied by growing Bacillus sp.PEC+ in four different milled stocks (0.145mm-2.0mm) .
Highest pectinase activity was produced with sugar cane bagasse particle size of 0.5mm (211.6 U/ml).
Generally, smaller substrate particles provide larger surface area for microbial attack and, thus, are a desirable factor.
However, too small a substrate particle may result in substrate agumulation, which may interfere with microbial respiration/ aeration, and therefore result in poor growth.

30. Effect of partical size of substrate on pectinase production:

31. Effect of initial substrate concentration on pectinase production
Various concentrations of sugar cane bagasse ranging from 0.5% to 3% were used in the SmF as sole carbon source.
Maximum pectinase activity (212.3 U/ml) was produced at a concentration of 2.5% (W/V).
Concentration higher than the optimum severely inhibited the pectinase production.

32. Effect of initial concentration of substrate on pectinase production:

33. Effect of temperature on the activity and stability of pectinase produced by Bacillus sp.PEC+
The temperature range was between 30 to 90°C with an interval of 10°C.
The optimum temperature for pectinase activity of Bacillus sp.PEC+ was 80°C after which there was a decline in the enzyme activity.
This clearly showed that pectinase had the maximum activity (170 U/ml) at 80°C.
These results indicate that the enzyme might be a thermostable enzyme.

34. Effect of temperature on Enzyme activity

35. Residual activity of pectinase at different temperature

36. Effect of pH on the activity and stability of pectinase produced by Bacillus sp.PEC+
The range of pH used for the study was from 3 to 10.
The activity was higher at pH 8 (177 U/ml), where as it was very low at acidic pH 3 (136.4 U/ml).
The enzyme was stable at all the pH with highest stability at pH 8.
These results indicate that the enzyme might be alkalophilic in nature.

37. Effect of pH on pectinase activity

38. Pectinase stability at different pH

39. Effect of substrate concentration on pectinase activity
The pectinase showed maximum activity with 0.8 %( w/v) substrate concentration.
And further increase in substrate concentration resulted in decline of the enzyme activity.
The optimum substrate concentration was found to be 0.8% w/v with U/ml pectinase activity.

40. Effect of substrate concentration on pectinase activity

41. CONCLUSION
Maximal pectinase production was observed around pH 6.0 at 30h.
Pectinase production was found to be maximum when sugarcane bagasse was used as sole carbon source.
The optimum pH and temperature for pectinolytic activity was 8.0, indicating that it might be an alkalophilic enzyme.
Additionally pectinase was extremely stable at very high temperature and had wide substrate specificity.
These results indicate that the enzyme might also be thermostable in nature.

42. Reference
Nitinkumar P. Patil and Bhushan L. Chaudhari . (2 1 ) ‘Production and purification of pectinase by soil isolate penicillium sp and search for better agro-residue for its ssf’ , Recent Research in Science and Technology,Vol. 2, No 7,pp
Reda A.Bayoumi,Hesham and M.yassin(2008),”Production of Bacterial Pectinase(s) from Agro-Industrial wastes Under Solid State Fermentation Conditions”,Journal of Applied Sciences Reserch,Vol.4,No.12.
Danielle Biscaro Pedrolli, Alexandre Costa Monteiro, Eleni Gomes and Eleonora Cano Carmona(2009),” Pectin and Pectinases: Production, Characterization and Industrial Application of Microbial Pectinolytic Enzymes”, The Open Biotechnology Journal, Vol 3,pp
Abdul Sattar and Qureshi Muhammad Aqeel Bhutto(2012),” Production of pectinase by Bacillus subtilis EFRL 01 in a date syrup medium “, African Journal of Biotechnology Vol. 11(62), pp
Silva, D.; Da Silva-Martins, E.; Da Silva, R. and Gmes,E. (2002),”Pectinase Production by Penicillium
viridicatum RFC3 by solid states fermentation usingagricultural wastes and agro-industrial by products”.Brazilian Journal of Microbiology 33 (4) : 1 – 10.

43. Thank you