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2       
Materials and methods:

2.1
Bacterial strains and growth conditions

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K.
pneumoniae B5055, used in present study was procured from Dr.
Mathia Trautmann, University of Ulm, Germany and other clinical strains of K.
pneumoniae were procured from Post
Graduate Institute of Medical Education and Research, Chandigarh.
All bacterial isolates were grown in nutrient broth at 37°C and subcultured
periodically.

2.2
Preparation and estimation of substrate

The capsular polysaccharide (CPS)
from different strains of K. pneumoniae was
extracted by method of  Hanlon et al., (2001)  and used as substrate for isolation of
depolymerase producing bacteria. Estimation of CPS was done according to the
method of Dubois (1956).

2.3
Isolation of appropriate depolymerase producing bacteria

Soil and sewage samples from in and
around Chandigarh were screened for the isolation of bacteria capable of producing
depolymerase enzyme specific for capsular polysaccharide of the given strain
following the method of Hoogerheidi (1940).

 

2.4
Assay for depolymerase activity

Depolymerase
activity was determined in supernatant on the basis of release of reducing sugars
following the DNSA method at 560?nm (Miller, 1959).  One unit of enzyme
activity was defined as micromoles of reducing sugars released /ml/min. The
isolate giving the maximum yield and activity was selected for further study.

2.5
Identification of the selected isolate

The identification of the selected bacterial
isolate was done according to
the Bergey’s Manual of Systematic Bacteriology (Claus and
Berkeley, 1986) and 16S rRNA gene
amplification using universal primers.  The bacterial isolate was identified using
nearly complete sequence of amplified 16S rRNA gene on Ez-Taxon and NCBI- BLAST
search servers.  The phylogenetic analysis of bacterial isolate was
performed with all closely related taxas using MEGA6 by Kimura
2-parameter method.

 

2.6 Optimization of depolymerase
production by OVAT

 Capsular
depolymerase production was optimized using OVAT. Various nutritional and
environmental parameters including substrate concentrations (0-1 mg/ml), temperature
(4-45°C), pH (4-9), incubation time
(24-120 h), different nitrogen sources and production volumes (25-100 ml) was
carried out by OVAT method. All the experiments were carried out in
triplicates.

 

2.7
Statistical optimization of depolymerase

 2.7.1 Plackett Burman design

A total of 11
factors based on OVAT and literature search were selected and total 12
experiments was designed using Design Expert10 software (Plackett and Burman,
1946) shown in Table1. The effect of individual factors on enzyme activity was
calculated according to the following equation:

       (?Pi+
– ?Pi-)

Ei =  

                      N

 

Where Ei
is the effect of i parameter, Pi+ and Pi- are responses (depolymerase
activity) of runs at which the parameter was at its high and low level,
respectively and N is the number of runs. Pareto chart showing high influence on
depolymerase production was selected for central composite (CCD) design.

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