(594l) Studies In Fermentative Production and Downstream Processing of Polylysine

Poly-ε-lysine (ε-PL), an unusual naturally occurring homopolyamide of L-lysine having linkage between ε-amino and α-carboxyl groups, is biodegradable, edible and non-toxic towards human and environment. It is preferentially produced by streptomyces species. Potential applications of ε-PL and its derivative have been of multifarious interest in foods, pharmaceuticals and medicine. In spite of commercial production and availability of ε-PL in a few countries, there are knowledge gaps still existing on many aspects of its production and downstream processing. This work was undertaken to look into a few of these aspects.

Initially, various marine strains were screened for the ability to produce ε-PL among which a strain labelled as G 349 was selected for ε-PL production. Biochemical tests and 16S rRNA results showed the ε-PL producer to belong to streptomyces species. Optimization of medium components by classical optimization and with metabolic simulators was carried out. In parallel set of experiments, the production of ε-PL by Streptomyces noursei NRRL 5126 in shake-flask culture was optimized by identifying the most significant medium components which affect ε-PL production by Placket–Burman design and subsequently by a statistical design, viz. evolutionary operation (EVOP) to determine the optimal concentrations of these components. Various metabolic precursors such as amino acids, tricarboxylic acid cycle intermediates and cofactors were investigated for improved production of ε-PL. The marine strain did not produce appreciable amount of ε-PL as compared to standard strain. Hence all further work was done with the standard strain itself.

The logistic and Luedeking-Piret equations have been proposed to describe the time course of ε-PL formation, substrate consumption and cell growth in shake flask level as well as in a fermenter. The growth kinetics of S. noursei NRRL 5126 was investigated under different aeration and agitation combinations in a 5.0 L stirred tank fermenter. Optimized oxygen supply (300 rpm and 2 vvm) in to the stirred tank fermenter shifted mixed growth associated biosynthesis of ε-PL to growth associated biosynthesis. A constant DO at 40% in the growth phase and 20% in the production phase increased the ε-PL yield as well as cell mass to their maximum values of 2 g/l and 20 g/l, respectively. The oxygen transfer rate (OTR), oxygen utilization rate (OUR), and specific oxygen uptake rates (qO₂) in the fermentation broth increased in the growth phase and remained unchanged in the stationary phase.

Use of resting cell cultures to increase the stability and shift in metabolism toward ε-PL biosynthesis was also implied. Optimization of non growth medium to achieve uniform culture density and higher ε-PL titre with minimal degradation was aimed with the help of Box-Behnken design and artificial intelligence. A comparative study of Box-Behnken design and artificial intelligence lead to higher production of ε-PL (1 g/l) during shake flask level.

Purification of ε-PL was done by using various chromatographic techniques such as ion exchange and gel permeation chromatography. Partial characterization of the ε-PL has been done. Further characterization of the same is in progress.

Potential applications of ε-PL to increase the stability of a model enzyme (α-amylase) and to increase the solubility of pharmaceutical drug have been evaluated.