5.2 Extracellular Enzymes Quantification
Extracellular enzymes have been qualitatively and quantitatively measured through
various ways ranging from agar plate-based methods to sophisticated advanced
spectrophotometric methods. The enzyme production ability of endophytic
microbes has also been coined for their ability to grow and reproduce in a specific
media. However, a detailed assessment of such abilities has been least known for
endophytic microbes. Overall, the endophytic microbes are isolated from plants
rigorous surface sterilization methods by using tween 80 weak acids, and sterilized
distilled water. Alternatively, the bulk material of plant grown in a sterilized
microbial media is used where endophytic microbes were isolated. Once the pure
culture is maintained, the strains are identified through either 16S rRNA sequencing
or internal transcribed spacer (ITS) region of bacteria and fungi, respectively, using
PCR amplification, sanger sequencing, BLASTn, andfinally a detailed phyloge-
netic analysis. The isolated endophytes are grown in specific growth media to detect
the enzymes producing either on agar plate (initial screening) or detailed spec-
trophotometry methods (UV/VIS orfluorescence). Currently, the advances in
fluorogenic substrate such as 4-methylumbelliferone (MUB) have also been used
for this purpose largely for soil or marine enzyme analysis (Hoppe 1993 ;
Wallenstein et al. 2008 ; Khan et al. 2016 ). However, more sensitive techniques
have to be adopted not only to measure time course estimation and analysis
quantification of these enzymes, but also such studies may be coupled with
molecular and genomic work to validate thefindings and processes (III and Allison
2015 ) (Fig.5.1).
Fig. 5.1 Diagrammatic representation of the isolation, fermentation and quantification of
extracellular enzymes.aGrinding plant,bisolating endophytes,c,dpure culture growth,
dfermentation, e,f, g, hisolation of enzymes, and i quantification through advanced
chromatographic techniques
5 Endophytic Microbes: A Resource... 97