3 supplementary data: S2). Depolymerase activity of the Isolate

3 Results:

3.1
Isolation and selection of depolymerase producing bacteria specific for
different strains of K.  pneumoniae

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

Thirty two capsular depolymerase
producing bacteria were isolated from sewage and soil samples. The zone of
clearance was observed on plates containing capsular polysaccharide (CPS)
extracted from different strains of K.
pneumoniae (supplementary data S1). The activity of depolymerase enzyme
obtained from 32 different environmental isolates was checked on the CPS
extracted from 10 different clinical isolates of K. pneumoniae (data not
shown here, supplementary data: S2). Depolymerase activity of the
Isolate No. 30 was observed on CPS of 7 out of the 10 strains tested (70%).

3.2
Quantification of depolymerase

The Isolate
no. 30 that showed maximum extracellular enzyme levels in nutrient broth
supplemented with CPS at 72 hrs (0.9 IU /ml) was selected for further study.

3.3
Identification of the Bacillus siamensis SCVJ30

The morphological and biochemical
characteristics of the selected Isolate SC 30 (SCVJ30) were studied and
according to the Bergey’s
Manual of Systematic Bacteriology, on the basis of biochemical characteristics
the isolate could be placed in the genus ‘Bacillus’. The complete 16S rRNA gene was sequenced and the (1492 bp) analysis clearly confirmed that strain was a
member of the genus Bacillus
and exhibited maximum similarity with the 16S rRNA sequence Bacillus siamensis KCTC 13613 (99.93%
sequence similarity) as shown in figure 1. This sequence was also validated
using Ez-Taxon and showed similarity of 99.93% with Bacillus siamensis KCTC 13613. 
The sequence has been submitted to the NCBI/Nucleotide databases under
accession no MG018338 and named as Bacillus siamensis SCVJ30.

 

 Bacillus
altitudinis 41KF2b (ASJC01000029)

 Bacillus
xiamenensis HYC-10 (AMSH01000114)

 Bacillus
altitudinis strain 41KF2b (NR0423371)

 Bacillus
safensis FO-36b (ASJD01000027)

 Bacillus
zhangzhouensis DW5-4 (JOTP01000061)

 Bacillus
subtilis subsp. subtilis
NCIB 3610 (ABQL01000001)

 Bacillus  siamensis  SCVJ30 (MG018338)

 Bacillus
velezensis CR502 (AY603658)

 Bacillus
siamensis KCTC 13613 (AJVF01000043)

 Bacillus
amyloliquefaciens DSM 7 (FN597644)

 Bacillus
anthracis Ames (AE016879)

 Bacillus cereus strain CCM 2010 (KY6288131)

 Bacillus
cereus ATCC 14579 (AE016877)

 Bacillus
wiedmannii FSL W8-0169 (LOBC01000053)

 Lysinibacillus
fusiformis NBRC 15717 (AB271743)

 Lysinibacillus
xylanilyticus DSM 23493 (LFXJ01000007)

 Lysinibacillus
macroides DSM 54 (LGCI01000008)

 Lysinibacillus
pakistanensis NCCP-54 (AB558495)

 Lysinibacillus macroides strain DSM 54 (KY6436381)

 Lysinibacillus
boronitolerans T-10a (AB199591)

 Lysinibacillus macroides strain DSM 54 (KY6288071)

 Arthrobacter
agilis KCTC 3200 (CLG48653)

71

92

51

56

93

100

57

53

100

49

98

99

100

100

100

45

79

40

0.02

 

Figure
1:
Phylogenetic tree showing the evolutionary relationship among bacterial
isolates drawn in MEGA6 using Kimura 2-parameter method. 16S rRNA gene sequence
accession numbers are given within parentheses. Bootstrap values are given at
nodes. Scale bar 0.02 substitutions per nucleotide position.

 

3.4
Optimization of depolymerase production:

3.4.1 OVAT

An increase in depolymerase activity,
i.e., from0.9 to 1.15 IU/ml, was achieved under following optimized conditions
viz. 0.2mg/ml substrate concentration, 96-hrs incubation time, 37°C temperature,
pH 8.0,  1 % peptone and 75 ml production
volume using OVAT method as given in Table 2.

 

Table 2:
Optimization of capsular depolymerase by OVAT method

 

Parameter

Range

Optimum range

Optimum activity(IU/ml)

Substrate concentration

0-1 mg/ml

0.2 mg/ml

0.90

Temperature

4-45°C

37°C

0.90

pH

4-9

8

0.97

Incubation time

24-120 hrs

96 hrs

1.05

Production volume

25-100 ml

75 ml

1.08

Nitrogen source

peptone

1.15

 

3.4.2 Screening the significant parameters for
depolymerase production by PB design:

 Out of 11
different factors, 6 factors viz. magnesium sulphate, pH, peptone, ammonium
chloride, incubation time and yeast extract showed positive effects on
production shown in pareto chart (figure 2). Out of these, magnesium sulphate,
pH and peptone had highest influence. For the enhancement of depolymerase yield
CCD was formulated using magnesium sulphate, pH and peptone for further
analysis.

 Figure 2:  Pareto chart showing significant factors
(yellow colored bars) and non-significant factors (blue colored bas).

 

3.4.3 Optimization of depolymerase
production using RSM

 A
CCD was formulated to examine the optimum levels of magnesium sulphate, pH and
peptone showing highest influence in PB analysis (Table 3). By applying
multiple regression analysis on the experimental data, a predictive quadratic
polynomial equation was constructed to illustrate the correlation between
significant factors and depolymerase production as follow:

 

R= +1.89+0.055* A+0.035* B-0.025*
C+0.083* AB+0.12* AC+0.31* BC-0.45* A2-0.38* B2-0.35* C2

 

Figure 3(a-c):  3-D response surface plots showing the effect
of interaction of (a) magnesium sulphate and peptone (b) pH and peptone (c) pH
and magnesium sulphate on capsular depolymerase production from  B. siamensis.

 

Table
3:
Optimization of enzyme production through Response Surface Methodology.

Run Order

Magnesium
Sulphate
(mg)

Peptone
(mg)

pH

Actual
Value (IU/ml)

Predicted
Value (IU/ml)

Residual
(IU/ml)

1

1

7

9

1.92±0.041

1.89

0.031

2

1.5

5

10

0.60±0.017

0.43

0.17

3

1.8409

7

9

0.40±0.016

0.71

-0.31

4

1.5

9

10

1.60±0.037

1.29

0.31

5

1

7

9

1.84±0.045

1.89

-0.049

6

0.159104

7

9

0.81±0.023

0.53

0.28

7

1

7

9

1.90±0.053

1.89

0.011

8

1

7

9

2.20±0.064

1.89

0.31

9

0.5

5

8

0.87±0.032

1.16

-0.29

10

1.5

5

8

0.75±0.053

0.86

-0.11

11

1

7

10.6818

0.59±0.047

0.85

-0.26

12

1

10.3636

9

0.60±0.044

0.87

-0.27

13

0.5

5

10

0.090±0.062

0.24

-0.15

14

1

3.63641

9

0.99±0.073

0.75

0.24

15

1

7

9

1.79±0.049

1.89

-0.099

16

1.5

9

8

0.65±0.028

0.47

0.18

17

1

7

7.31821

1.16±0.069

0.93

0.23

18

0.5

9

8

0.30±0.028

0.44

-0.14

19

1

7

9

1.69±0.075

1.89

-0.20

20

0.5

9

10

0.90±0.051

0.77

0.13

 

Table 4:  Analysis of variance of response surface
methodology for optimization of capsular depolymerase production.

 

ANOVA for Response Surface Quadratic model

Source

Sum of 
Squares

df

Mean Square

F Value

p-value Prob > F

 

Model

6.68

9

0.74

8.36

0.0013

significant

A-magnesium sulphate

0.041

1

0.041

0.46

0.5110

 

B-peptone

0.017

1

0.017

0.19

0.6695

 

C-pH

8.396E-003

1

8.396E-003

0.095

0.7648

 

AB

0.054

1

0.054

0.61

0.4517

 

AC

0.12

1

0.12

1.35

0.2719

 

BC

0.77

1

0.77

8.66

0.0147

 

A2

2.89

1

2.89

32.57

0.0002

 

B2

2.09

1

2.09

23.53

0.0007

 

C2

1.79

1

1.79

20.17

0.0012

 

Residual

0.89

10

0.089

 

 

 

Lack of Fit

0.74

5

0.15

4.93

0.0523

not significant

Pure Error

0.15

5

0.030

 

 

 

Cor Total

7.57

19

 

 

 

 

C.V. %= 27.25       
R2 =0.8827     R2
(pred)=0.2283        R2(Adj)=0.7771

Where R is the depolymerase activity; A,
B, and C were magnesium sulphate, peptone and pH, respectively. The analysis of
variance for the model is summarized in Table 4. The p

x

Hi!
I'm Eileen!

Would you like to get a custom essay? How about receiving a customized one?

Check it out