Ph.D. viva voce examination
Office Biotechnology
Dear all,
There will be a Ph.D. final viva-voce examination as per details given below. All are cordially invited to attend.
|
Name of the Scholar |
S SREEJA |
| Registration Number | BT12D046 |
| Type of Registration | Ph.D. (Regular) |
| Date of Registration | 16.7.2012 |
| Title of the Thesis |
MOLECULAR WEIGHT CONTROL OF HYALURONIC ACID IN RECOMBINANT LACTOCOCCUS LACTIS CULTURES
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| Date, Time & Venue | 1.10.2018, 10.00 am, Biotechnology Seminar Hall |
Guide
Prof Guhan Jayaraman
Abstract:
Molecular weight control of hyaluronic acid in recombinant Lactococcus lactis cultures
Hyaluronic acid (HA) is a bio-polymer with wide applications in the field of medicine and cosmetics. HA of specific molecular weight (MW) is required for specific applications. For specific MW HA production, an understanding of HA MW control by the cell is pivotal. HA MW is affected by numerous factors that could be classified as process factors, genetic factors, metabolic factors and factors intrinsic to the polymerizing enzyme, HA synthase.
In this work, effect of glucose concentration (a process factor) and HA synthase related factors on HA MW in recombinant Lactococcus lactis cultures have been studied. However, before undertaking these investigations, it was necessary to evaluate the accuracy of MW estimations from conventional size exclusion chromatography (SEC), as it was prone to errors due to use of non-HA based SEC calibration and concentration effect. Hence, these two factors were studied and a multivariate correlation to correct for errors due to concentration effect was developed.
The effect of glucose concentration on HA MW during the course of batch fermentation has not been studied previously. It was demonstrated in this work that HA MW decreases by 33-40% during un-aerated batch fermentation due to decreasing glucose concentration. To enable production of specific MW HA around a set MW, a glucostat strategy was developed. It was found that by maintaining constant glucose concentration using glucostat strategy, constant MW HA can be produced during fermentation. However, HA MW was sensitive to glucose concentration only when glucose was the sole limiting nutrient.
Fermentation of Streptococcal species or recombinant organisms expressing Streptococcal HA synthase is the workhorse for industrial HA production. However, the difference in HA MW produced by various Streptococcal HA synthases has not been studied. In this work, we explored the difference in HA MW produced by HA synthases from S. uberis, S. parauberis, S. zooepidemicus, S. pyogenes, which showed 20-30% dissimilarity in their amino acid sequence. The four HA synthases when expressed in L. lactis produced HA MW in a wide range of 0.4-2.2 MDa. To confidently attribute the difference in HA MW observed to difference in HA synthase specific factors, genetic (hasA/hasB mRNA ratio), process (specific growth rate) and metabolic (intracellular availability of monomers) factors were measured and compared. The study revealed that Streptococcal HA synthases produce HA of different MW owing to dissimilarities in their amino acid sequence, which could be exploited for specific MW HA production.
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