Pectinolytic Bacteria from Soil Samples of Malabar Region. 1. Bergey, D.H., N.R. Krieg, and J.G. Holt, Bergey's manual of systematic bacteriology. 1984 ...
Isolation, Screening and Biochemical Characterization of Pectinolytic Bacteria from Soil Samples of Malabar Region Bijesh Kavuthodi, Vinitha P, Denoj Sebastian Department of Life Sciences, University of Calicut – 673635
RESULTS
INTRODUCTION
Spore Motility Organism Zone dia. Grams (mm) staining staining code
Pectinases constitute a group of enzymes which catalyze
importance in food processing industries, especially for and
clarification
of
fruit
juices.
Other
microorganisms
capable
the
pectinolytic
enzymes
used
for
II
26
+
+
+
Irregular, umbonate, undulate, cream coloured colonies
the basis of zone size ranging from 20mm to 26mm after flooding
III
23
+
+
+
Irregular, umbonate, undulate, cream coloured colonies
the
IV
22
+
-
-
Irregular, umbonate, undulate, cream coloured colonies
plate
with
iodine
solution
(Fig.1).
By
biochemical
Table 1. Microbiological characteres of the selected strains
1
By using these four isolates, pectinase enzyme production
of
producing the enzyme in a cheap rate. At present almost all
Irregular, flat, undulate, large, light brown coloured colonies
sp.(Corynebacterium kutsceri) (Table 1 & 2).
industrial applications of pectinases, it is essential to of
+
III. Bacillus sp. (Bacillus pantotheticus) and IV. Corynebacterium
leather, detergent and paper. In view of the potential source
+
Bacillus sp. (Bacillus coagulans), II. Bacillus sp. (Bacillus subtilis),
pigments from plant materials, textile, pharmaceutical,
new
+
identification, these four microbial strains are identified as; I.
application includes; extraction of oils, flavours and
identify
20
samples. Four isolates with pectinolytic activity were selected on
cell walls. Pectinolytic enzymes having great industrial extraction
I
Thirty six bacterial isolates were screened from the collected
the degradation of pectic polymers present in the plant
Colony characteristics
industrial
applications are produced by fungi. There are a few
was assayed by DNS assay in YEP broth culture medium after
Biochemical tests
24hrs of incubation in 30°C and 150 rpm. The result of this DNS assay was shown in Fig 2.
II
III
IV
Nitrate reduction
+
+
-
-
Urease
-
-
-
-
H2S production
-
-
-
-
Oxidase
+
+
+
+
Starch hydrolysis
+
+
+
+
maximum enzyme activity(0.74 U/mL )was identified using 16S
6.5% NaCl Growth
-
+
-
-
rDNA based molecular technique. Fragment of 16S region
Indole production
-
-
-
-
Methyl red
+
-
-
-
amplified by PCR . A single discrete PCR amplicon band of 1500
Voges proskauer
+
+
-
-
Citrate utilization
+
+
-
-
bp was observed when resolved on agarose gel (Figure 3). In
Glucose
+
+
-
-
molecular identification, the sample strain was most similar to
Lactose
-
-
-
-
Sucrose
+
+
+
-
Mannitol
-
-
-
-
Arabinose
-
-
-
-
reports of pectinase production by bacterial strains. So, the study was presently undertaken which is aimed at screening and isolating pectinolytic bacteria from the soil samples. From thirty six bacterial isolates screened for pectinase production, the bacterium showing maximum enzyme activity was identified using 16S rDNA based
was
Bacillus subtilis (GenBank Accession Number:AB042061.1) based
molecular technique.
Bacterial isolates I
The bacterial isolate II (bacillus sp.) which is showing
2
on nucleotide homology and phylogenetic analysis. The organism also found to produce other exo-pectinase
OBJECTIVES
favourable amounts as follows; polygalacuronase 1.688 U/ml,
Isolation and screening of pectinolytic bacteria from
Pectin lyase 110.2 and pectate lyase 231.0 enzyme units
various samples. • •
( Expressed in units, 0.01 OD change taken as 1 unit of enzyme
Enzymatic assay of the screened isolates.
activity).
Biochemical and molecular characterization of the
CONCLUSION
selected isolate.
MATERIALS AND METHODS
Among thirty six isolated bacterial strains capable of utilize pectin as a soul carbon source, one isolate was selected
• Isolation of pectinase producing bacteria in Yeast
based on the potentiality of enzyme production. By means of
Extract Pectin media.
nucleotide homology and phylogenetic analysis, this isolate
• Screening of enzyme production by iodine solution
was similar to Bacillus subtilis (GenBank Accession Number :
& confirmation by agar well diffusion assay. • Morphological,
Figure 3. Single discrete PCR amplicon band of 1500 bp in agarose gel 1: 16S rDNA amplicon band 2: DNA marker
microbiological
and
AB042061.1). On further analysis, it was revealed that the
biochemical
organism can also able to produce
characteristics of the selected isolates.
Our results indicate the possibility of exo-pectinase
formation, motility, catalase, oxidase, urease, starch tests and IMVIC tests [1,2]. • Colorimetric assay of Pectinase activity by DNS
like
polygalacturonase, pectin lyase and pectate lyase.
This includes; colony morphology, Grams reaction, spore hydrolysis, nitrate reduction, carbohydrate fermentation
exo -pectinases
Fig 1. Zone formation by different bacterial cultures (I, II, III & IV.) showing pectin depolymerisation on YEP media
production by the isolated strain of B.subtilis. Use of cheap Fig 2.Pectinase activity shown by isolated bacterial strains
substrates, media optimization, product enhancement by genetic modification, application etc. are the future prospects of this study.
method [3]. • Culture identification by 16S rDNA based molecular
REFERENCES
technique. DNA isolation, PCR with universal primer (8F and 1492R), DNA sequencing and phylogenetic analysis . • Exo-Pectinase
production
and types of Exo-
1. Bergey, D.H., N.R. Krieg, and J.G. Holt, Bergey's manual of systematic bacteriology. 1984, Baltimore: Williams & Wilkins. 2. Cappuccino, J.G. and N. Sherman, Microbiology, a laboratory manual. 1983, Reading, Mass.: Addison-Wesley. xiii, 466
pectinase activity.
3. Miller, G.L., Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 1959. 31(3): 426-428.
Polygalacturonase assay by DNS, Pectin and pectate lyase
4. Pitt, D., Pectin lyase from Phoma medicaginis var. pinodella. 1988. 161:350-354 .
assay by TBA method [4].
•
enzymes like polygalacturonase, pectin lyase and pectate lyase in
Table 2. Biochemical identification results of the isolated strains
