Membrane Processing of Polygalacturonase from Aspergillus carbonarius

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
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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.