Membrane Processing of Polygalacturonase from Aspergillus carbonarius

Nakkeeran, E. (2010) Membrane Processing of Polygalacturonase from Aspergillus carbonarius. PhD thesis, University of Mysore.

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Abstract

The investigation was focused on assessing membrane technology for the purification of Aspergillus carbonarius polygalacturonase (PG) produced by submerged (SmF) and solid-state fermentations (SSF) considering its due merits while comparing the performance with alginate affinity purification (AAP) process. Primary assessment of upstream processes revealed that SmF yielded 2.7 fold higher PG production over SSF in terms of starch present in the substrate. Initial attempts on processing SmF culture broth employing a set of microfiltration (MF) and ultrafiltration (UF) membranes showed that diafiltration with 10 kDa UF membrane effectively eliminated carbohydrates that otherwise interfered with the purification process, along with other impurities. Although PG was almost completely retained in the process, there was very little improvement in specific activity. Therefore, further attempts were made to develop an integrated membrane process (IMP) to improve the specific activity by eliminating the contaminant proteins along with other non-protein impurities. The IMP thus developed employing 450 nm MF followed by 50 kDa UF membranes improved the specific activity of SmF-PG to 5590 U/mg protein (4.7 fold) with a greater recovery (76%). However, IMP did not improve the specific activity of SSF-PG due to the presence of other similar molecular mass proteins/enzymes while the PG recovery and carbohydrates elimination were similar to the level achieved processing SmF-PG. SDS-PAGE analysis confirmed the presence of other similar molecular mass contaminant proteins in the SSF culture extract and lesser contaminant proteins in the SmF culture broth, which explained the difference in membrane selectivity. Attempts with AAP revealed its suitability for the purification of PG irrespective of its type of production while displaying better process performance than IMP. In the light of the above, economic analyses were 7 carried out for three potential combinations, SmF-IMP, SmF-AAP and SSFAAP processes for PG production. The analyses showed that the unitary product cost of purified PG by SmF-IMP process was 24-36% lower than SmF-AAP and SSF-AAP processes. Further studies were made with SmF-PG to establish the performance of IMP in a cross-flow system that offers several advantages over stirred membrane cell. Assessing the shear sensitive nature of PG revealed that there is a critical speed of stirring corresponding to a shear stress of 2.1 Pa which should not be exceeded in self-stirred membrane cells. Likewise, peristaltic pump showed its suitability for processing PG over shear intensive gear pump. The specific activity improvement (4.1 fold) attained in cross-flow unit was comparable with the stirred membrane cell (4.7 fold) while achieving greater normalized productivity. In pilot scale system, the specific activity of PG obtained with 200 nm MF followed by 20 kDa UF membranes was comparable with self-stirred system which provided evidence that the performance could be replicated in the scale up. Resistance-in-series model analysis of IMP showed that the fouling resistance was predominant with MF while it was the cake resistance in UF. The efficacy of SmF- and SSF-PG tested for the extraction and clarification of apple juice in terms of sugar yield and clarity suggested the suitability of SmF-PG processed by IMP for formulating as a commercial enzyme. The results obtained in the present study on production of SmF- and SSF-PG had shown that the process selection needs careful considerations backed up with a techno-economic analysis. In the present case, SmF followed by IMP revealed its suitability for the production of purified PG from A. carbonarius in terms of process performance, economics and product application.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	membrane technology, Aspergillus carbonarius polygalacturonase, Downstream processing
Subjects:	600 Technology > 08 Food technology > 16 Nutritive value > 05 Enzymes 600 Technology > 08 Food technology > 05 Processing and Engineering
Divisions:	Food Engineering
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	01 Mar 2011 09:41
Last Modified:	28 Dec 2011 10:21
URI:	http://ir.cftri.res.in/id/eprint/9951

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