Market Enzyme Application(s)
Starch ex-amylase conver$iOn of starch into
dextrins
amylo11lucosidase production of fructose-1yrups
glucose isomerase from dextrins
0-amylase conversion of starch into
maltose
Sucrose invertase conversion of sucrose into
glucose and fructose
Dairy 0-galactosidase hydrolysis of lactose,
improvement of Quality and
digestion of products
Beverages pectinases clarification, product and
yield improvement of juices
glucose oxldose removal of oxygen from
+ catalase beverages, removal of glucose
from egg products
Baking ex-amylase bread dough modification,
proteases flour suppfemenration
hemicellulases
am yloglucosidase
Laundry aids alkaline protease presoak and washing,
ex-amylase spot removal
Leather protease bating
Textil.e ex-amylase starch desizlng (e.g. cotton)
The production of pr,oteins in sufficient
amounts is key for theii· study or use as
biotherapeutic agents. Escherichia coli is
the host of choice for recombinant pro-
tein production given its fast growth,
easy manipulation, and cost-effective-
ness. As such, its protein production ca-
pabilities are continuously being im-
proved. Also, the associated tools (such
as plasmids and cultivation conditions)
are subject of ongoing 1·esearch to opti-
mize product yield. In this work, we re-
view the latest advances in recombinant
protein production in E. coli.
The study of proteins or their use in
biotechnological applications often re-
quires their isolation from other cellular
components. Purification can be per-
formed from the natural source of the
protein; however, this approach is usually
cumbersome and inefficient for most of
them. The coding sequence for the pro-
tein of interest can be inserted into an ap-
propriate expression vector and trans-
formed into a prokaryotic host, such as
the bacterium Escherichia coli. Using E.
coli as a microbial cell factory for pro-
ducing recombinant proteins lowers the
costs of production and improves the
yield. Nowadays, many proteins of com-
mercial interest are produced in E. coli.l
In the lab, the recombinant production of
proteins in E. coli is the method of
choice for their structural and functional
study.
In the last decades enzyme separation and purification has become
increasingly important because of the evolving application of enzymes in the
brewing, food, textile, chemical, detergent and pharmaceutical industries.
Together with these developments improved separation techniques were
developed. Production and purification of enzymes are strongly dependent on
the potential market, the proceuing costs, the quality required and the
available technology. Two main product categories can be recognized:
I. Pure preparations, mainly 10 use in -
research ud analytical
applications
2. Crude or partially purified ind us trial enzymes (e.g. proteases and
potysaccharidases). mainly in use in industrial applications.
Most of the enzymes in the first category are purified by clrromatograp-
hic techniques arter a crude isolation, whereas most of the enzymes in categ-
ory two are isolated by precipitation and membrane separation. An important
application area for enzymes is food processing. It can be expected th.at
applications in the food area will require an increasing level of purification in
the future due to trends in this industry and to stricter commodity law
demands.
Recently novel purification techniques have been developed, based on
liquid- liquid extractions and affinity interactions. Although they show con-
siderable potential for the purification of several industrial enzymes they have
n.o t been widely implanted in existing processes yet. The introduction and
application of these techniques i.n the field of (poly)s11ccharidases will be
disclll!sed in this paper.
1.2 (POLY)SACCHARJDE CONVERTING ENZYMES
Among the group of industrially prep:ired bioproducts. enzymes have a
relatively modest position. AS shown in Table I. I 1he market value for bulk
enzymes was estimated to be US S 26S million in 1981. The predominant groups
in this field are those of the proteases and the (poly)saccharide converting
enzymes, which find thei r main applications in the food and beverage industry
Ultrafiltration (UF) is one of I
I - -·
the membrane separation r c.r -••• :r- ""• v_,...
processes and is widely
used for the recovery of I I
I
enzymes (Bohdziewicz, 1
1996). UF separation process is less
inexpensive than other molecules a,nd it also
prevents the loss of enzyme activity. UF is
widely used to purify and concentrate
enzymes, such as proteases.
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streams and bioreactors is widel~
recognized to be economically and
technically challenging. Since the
cost of bio-separation is the main
key, there is an incentive to develop
cost-effective isolation and
purification processes.
Citric acid (CA) has been widely used in c
different industrial sectors, being produced
through fermentation of low-cost feedstocl<.
The development of downstream processes,
easier to operate, environmentally friendly,
and more economic than precipitation, is
certainly a challenge in CA bioproduction.
Large volumes of by-products generated in
precipitation require treatment before
disposal. Adsorption, extraction, and
membrane senaration have been sl1own to
have a lower environmental impact than
precipitation, but the technological maturity
of these methods is still limited. However,
reactive extraction and adsorption l1ave
g1·eat potential for indllstrial applications.
[
• Deep eutectic solvent was first
used as a solvent for ethanol
extraction.
• NRTL binary interaction
parameters for the LLE and VLE
were estimated.
• 93% of water in feedstocl< is
removed during extraction with
no energy demand.
• 99.8% etl1anol is pro duced at a
1
recovery rate of 99.5%.
• The energy demand is reduced
by 19% when compared to the
current processes.
The increasing world deficiency of
protein is becoming a main
problem of humankind. Since the
early fifties, intense efforts have
been made to explore new,
alternate and unconventional
protein. For this reason, in 1996,
new sources ma1
inly yeast, fungi,
bacteria and algae named Single
Cell Protein (SCP) as coined to
describe the protein production
from biomass, originating from
different microbial sources.
Microbial biomass has been
considered an alternative to
conventional sources of food or
feed. Large-scale processes for SCP
production show interesting
features, including:
• The wide variety of
methodologies, raw materials
and microorganisms that can be
used for this purpose
• High efficiency in substrate
conversion
• High productivity, derived from
the fast growth rate of
microorganisms
Substrate
' .
Fcrn1entcr !SCP producing 111icrolx-s) trient supplement
' .
Filtration & Purification
•
Drying
1•
I I
SCP
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