I would like to thank everyone who made this thesis and an unforgettable experience possible for me. I would like to express my sincere gratitude to God and my family for their blessings, strength, encouragement and support to complete this work. I would also like to sincerely thank all the members of my doctoral committee, Dr.
I am thankful to Department of Biological Sciences and Bioengineering and Central Instrumentation Facility (CIF), IITG for providing me instruments for my research work. I would also like to thank the present and past Head of the Department of Biosciences and Bioengineering, IIT Guwahati, Prof. I am grateful to all the people I worked with in the laboratory in the Department of Biosciences and Bioengineering for their cooperation and support.
I would like to thank my friends Naveen Kumar, Naresh, Amruta, Bethi, Moushamee, Hasna, Alok and Mounika. I want to receive the support of other teaching and non-teaching staff of Department of Biotechnology, IIT Guwahati.
The presence of 5 mM Ca 2+ increased the activity of CtPME and CtPMEf by 50% and 42%, respectively. The presence of 5 mM EDTA reduced the activity of CtPME and CtPMEf by 40% and 38%, respectively. The melting study of CtPME revealed that it begins to unfold at 68ºC and the protein was complete.
The protein melting peak of CtPME shifted to higher temperature in the presence of Ca2+ ions. Maximum concentration of CtPME and CtPMEf obtained with AIM-LB medium was 150 mg/L and 160 mg/L, respectively. Multiple sequence alignment of CtPME with previously (Uniprot Id: 2ntb and 1qjv) known pectin methylesterase revealed the conserved and semi-conserved amino acid residues (Asp, Asp and Arg).
Comparison of CtPME structure with PME protein from Erwinia chrysanthemi showed conserved catalytic cleft. The ab initio derived dummy atom model of CtPME superimposed well with its modeled structure.
Grinding of jute fibers and bioscrubbing of cotton fabric mixed enzyme treatment showed significant weight loss compared to the respective individual enzyme treatment and similar to that of NaOH treatment. The wettability analysis showed that the cotton fabric treated with CtPME, CtPL1B, mixture of enzymes, and NaOH absorbed a drop of water in 21 s, 18 s, 10 s, and 8 s, respectively. The FESEM analysis of the enzyme-treated cotton fabric showed a smooth surface compared to the control .
ATR-FTIR analysis of enzyme-treated jute or cotton fabric fibers revealed a difference in surface-exposed functional groups compared to controls. Mechanical properties such as Young's modulus and ultimate tensile strength of degummed jute fibers and biodegreased cotton fabrics were investigated by UTM analysis. Degumming of jute fiber with mixed enzymes or biological treatment of cotton fabric gave values of Young's modulus and UTS similar to those of chemical treatment.
The UTM analysis showed that jute fibers treated with the enzyme mixture had a higher tensile strength than the control. To our knowledge, this is the first study on the use of a recombinant pectin methylesterase, CtPME with pectate lyase, CtPL1B for jute fiber degumming and cotton fabric bioscouring.
Chapter 6 describes the isolation of natural pectin, characterization and effect of mixture of pectic oligosaccharides on normal cells (HEK293) and colon cancer cells
All showed reduced proliferation of HT29 cells, but mPOS showed maximum (51%) reduction in proliferation of HT29 cells compared to POS or PP. The microscopic observation of mPOS-treated HT29 cells revealed the reduced connection between the cells and the change in cell morphology from undifferentiated to shrunken sphere shape. Pectin, which is a large polymer compound, is difficult to be absorbed by the digestive tract, and therefore enzymatic degradation facilitates the production of smaller oligosaccharides that can be easily absorbed by the digestive tract, and therefore the pectin oligosaccharides can be prepared from orange peels by using the recombinant CtPME and CtPL1B from Clostridium thermocellum on a large scale.
Therefore, EOP and pectin oligosaccharides can be used for functional food applications that enrich the nutritional values.
CONTENTS
Cloning, expression and purification of family 8 Carbohydrate Esterase (CE8), Pectin methylesterase (CtPME) and derivatives
Biochemical characterization of CtPME and CtPMEf
SAXS and homology modelling based structure analysis of pectin methylesterase from Clostridium thermocellum ATCC 27405
Green process of degumming of jute fibers and bioscouring of cotton fabric by recombinant pectin methylesterase and pectate
Extraction, characterization and anti-cancer activity of pectic oligosaccharides produced from agro-waste of Orange
- Carbohydrates
- Plant cell wall polysaccharides
- Pectin and its components
- Pectin biosynthesis
- Degree of Esterification (DE)
- High methoxyl (HM) pectins: If more than 50% of the carboxyl groups are methylated the pectins are called high-methyl ester pectins
- Low methoxyl (LM) pectins: If less than 50% of the carboxyl groups are methylated pectins are called low methyl ester (LM) pectins
- Carbohydrate enzymes
- Types of Pectinase
- Carbohydrate Esterases family
- Family 8 Carbohydrate Esterase
- Applications of Pectin polysaccharide
- Applications of microbial pectinases
- The microorganism, Clostridium thermocellum
- Significance and objectives of the present study .1 Significance of the study
- References
- Introduction
- Materials and Methods
- Results and Discussion
- Conclusions
- References
- Introduction
- Materials and Methods .1 Substrates and reagents
- Results and Discussion
- Conclusions
- References
- Introduction
- Materials and Methods
- Conclusions
- References
- Introduction
- Materials and Methods .1 Substrates and chemicals
- Results and Discussion
Polysaccharides are complex macromolecular carbohydrates composed of monosaccharide chains linked together by glycosidic bonds (Berg, 2007). The plant cell wall also contains a smaller amount of structural glycoproteins, hydroxyproline-rich extensions) and phenolic esters (ferulic and coumaric acids) (Matsunaga et al., 2004). Cellulose chains are cross-linked to hemicellulose by pectin with a solid matrix (Taiz et al., 2010). Lignin gives strength to the cell wall and provides resistance to microbial attack (Ralph et al., 2004).
Pectin is present in walls of all higher plants, gymnosperms, pteridophytes and bryophytes (Mohnen et al., 2008). 1.2 (A) Homogalacturonan of pectin molecule and functional groups (B) carboxyl, (C) Ester and (D) Amide in pectin chain (adopted: Ridley et al., 2001). Carbohydrate-active enzymes have been classified into different families based on protein sequence similarity (Cantarel et al., 2009).
1.8 (A) Scanning electron microscope (SEM) images of Clostridium thermocellum, showing normal rod-shaped cells (adapted from Lamed et al., 1987); (B) Transmission electron microscope (TEM) image of cationized ferritin (CF) stained Clostridium thermocellum grown on cellobiose (adapted from Fontes and Gilbert, 2010). The pectin component of the cell wall is believed to play these roles (Ridley et al., 2001). The structural role of pectin in promoting upright plant growth has also been reported (Matsunaga et al., 2004).
More than 80% of the total pectin consists of D-galactopyranosiluronic acid (DGalp A), with the remainder being rhamnose (Rhap), galactose (Galp), xylose (Xylp) and arabinose (Araf) (Ridley et al., 2001). . Glycoside hydrolases and polysaccharide lyases have been classified into different families based on sequence similarity (Lombard et al., 2014). In plants, pectin methylesterase is used for ripening by destabilization in cell wall metabolism (Frenkel et al., 1998).
PME from Aspergillus reptans was also reported to be inhibited by Hg2+ (Arotupin et al., 2008). In cellulosomes with cohesin-dockerin interaction, a number of enzymes organize into a complex (Lamed et al., 1983). The molar residual ellipticity (mre degrees cm2dmol-1) was quantified from the ellipticity values (θ) for the specified range of wavelength (Kelly et al., 2005).
The molecular weight estimation for CtPME was done by the SAXSMoW program (Fischer et al., 2010). Most lipases and esterases contain a catalytic center comprising the Ser-His-Asp triad (Heikinheimo et al., 1999). The RMSD difference between the minimized modeled structure of CtPME and the final developed structure after MD simulation was only 1.202 Å. The MD simulation results showed that the modeled CtPME structure was stable and could be used for further studies.
The production of CtPME (Rajulapati and Goyal, 2017) and CtPL1B (Chakraborty et al., 2015) enzymes was reported earlier.
