Industrial enzymes ( naringinase )

Industrial enzymes
Naringinase production
By
Mohammad pooya naghshbandi
Microbial biotechnologist
At
University of Tehran

Contents
• Industrial enzymes
• Classification of enzymes
• Uses of enzymes in industry
• Production of enzymes
Fermentation for enzyme productions :
Semi solid medium
Submerged production
Enzyme Extraction
Packaging and Finishing
Toxicity Testing and Standardization
• IMMOBILIZED BIOCATALYSTS: ENZYMES
Advantages of Immobilized Biocatalysts in General
Methods of Immobilizing Enzymes

• Enzymes are organic compounds which catalyze all the
chemical reactions of living things – plants, animals and
microorganisms. They contain mainly protein; some of
them however contain non-protein components, prosthetic
groups. When excreted or extracted from the producing
organism they are capable of acting independently of their
source. It is this property of independent action which
drew early attention to their industrial use.
modern industrial microbiology and biotechnology . season 11

classification of enzymes
enzyme committee (EC) of the International
Union of Biochemistry and Molecular Biology
(IUBMB) recommended the classification of
enzymes into six groups.
EC 1.11.1.6 = catalase
EC 5.3.1.5 = chimozin

1.Oxidoreductases catalyze a variety of oxidation-reduction reactions.
Common names include dehydrogenase, oxidase, reductase
and catalase.
2.Transferases catalyze transfers of groups (acetyl, methyl, phosphate, etc.).
Common names include acetyltransferase, methylase, protein
kinase, and polymerase. The first three subclasses play major
roles in the regulation of cellular processes.
3.Hydrolases catalyze hydrolysis reactions where a molecule is split into two
or more smaller molecules by the addition of water.
Some examples are: Proteases: Proteases split protein
molecules. They are further classified by their optimum pH as
acid, alkaline or neutral.
4.Lyases catalyze the cleavage of C-C, C-O, C-S and C-N bonds by
means other than hydrolysis or oxidation. Common names
include decarboxylase and aldolase.
5.Isomerases catalyze atomic rearrangements within a molecule. Examples
include rotamase, protein disulfide isomerase (PDI), epimerase
and racemase.
6.Ligases catalyze the reaction which joins two molecules. Examples
include peptide synthase, aminoacyl-tRNA synthetase, DNA
ligase and RNA ligase.

USES OF ENZYMES IN INDUSTRY
• Most of the enzymes used in industry are hydrolases
(i.e., those which hydrolyze large molecules). In particular
amylases, proteases, pectinases, and to a lesser extents
lipases have been most commonly used. Enzymes are
used in a wide range of industries and some uses are
discussed below.

(i) Production of nutritive sweeteners from starch
(ii) Proteolytic enzymes in the detergent industry
(iii) Microbial rennets
(iv) Lactase
(v) The textile industry
(vi) Pectinases for use in fruit juice
(vii) Naringinase
(viii) Enzymes in the baking industry
(ix) Leather baiting
(x)Some medical uses of microbial enzymes

Fermentation for Enzyme Production
1. semi-solid media.
2. submerged fermentation
Most enzyme production is carried out in deep submerged
fermentation; a few are best produced in semi-solid
media.

Semi solid medium
‘Koji’ or ‘moldy bran’
consists of :
moist sterile wheat or rice bran acidified with HCl
mineral salts including trace minerals are added
An inducer is also usually added
10% starch is used for amylase, and gelatin and pectin for
protein and pectinase production respectively.

Submerged production
Most enzyme production is in fact by submerged
cultivation in a deep fermentor
Sometimes some easily metabolizable components of the
medium may repress enzyme production by catabolite
repression. Strong repression is often seen in media
containing glucose. Thus, -amylase synthesis is
repressed by glucose in Bacillus licheniformis and
B.subtilis. Fructose on the other hand represses the
synthesis of the enzyme in B.stearothermophilus.

Enzyme Extraction
The procedures for the extraction of fermentation products described in Chapter
10 are applicable to enzyme extraction. Care is taken to avoid contamination.
In order to limit contamination and degradation of the enzyme the broth is
cooled to about 20°C as soon as the fermentation is over. Stabilizers such as
calcium salts, proteins, sugar, and starch hydrolysates may be added and
destabilizing metals may be removed with EDTA. Antimicrobials if used at all
are those that are normally allowed in food such as benzoates and sorbate.
Most industrial enzymes are extra-cellular in nature. In the case of cell bound
enzymes, the cells are disrupted before centrifugation and/or vacuum filtration.
The extent of the purification after the clarification depends on the purpose for
which the enzyme is to be used. Sometimes enzymes may be precipitated using
a variety of chemicals such as methanol, acetone, ethyl alcohol or ammonium
sulfate. The precipitate may be further purified by dialysis, chromatography,
etc., before being dried in a drum drier or a low temperature vacuum drier
depending on the stability of the enzymes to high temperature. Ultra-filtration
separation technique based on molecular size may be used.

Packaging and Finishing
The packing of enzymes has become extremely important since the
experience of the allergic effect of enzyme dust inhalation by
detergent works. Nowadays, enzymes are packaged preferably in
liquid form but where solids are used, the enzyme is mixed with a
filler and it is now common practice to coat the particles with wax
so that enzyme dusts are not formed.

Toxicity Testing and Standardization
The enzyme preparation should be tested by animal
feeding to show that it is not toxic. This test not only
assays the enzyme itself but any toxic side-product
released by the microorganisms. For a new product
extensive testing should be undertaken, but only spot
checks need to done for a proven non-toxic enzyme in
production. The potency of theenzyme preparation,
based on tests carried out with the substrate should be
determined. The shelf life and conditions of storage for
optimal activity should also be determined.

IMMOBILIZED BIOCATALYSTS: ENZYMES
The major handicap in the traditional use of enzymes is that they are
used but once. This is mainly because the enzymes are unstable in
the soluble form in which they are used and because recovery
would be expensive, even if it were possible.
Interest in immobilized enzymes has grown since the 1960s and
numerous conferences and papers have been held and given on
them.
Imobilized enzymes: An immobilized enzyme may be defined as an
isolated or purified enzyme confined or localized in a defined
volume of space.

Advantages of Immobilized Biocatalysts in General
(i) They can be easily separated from the reaction mixture containing any
residual reactants and reused in subsequent conservations.
(ii) Immobilized enzymes are more stable over broad ranges of pH and
temperature.
(iii) Enzymes are absent in the waste-stream
(iv) Immobilized systems specially lend themselves to continuous
processes.
(v) Reduced costs in industrial production.
(vi) Greater control of the catalytic effect.
(vii) Greater ease of new applications for industrial and medical purposes.
(viii) Immobilized enzymes permit the use of enzymes from organisms
which would not normally be regarded as safe (i.e. non-GRAS).

Methods of Immobilizing Enzymes
The classification method adopted here is the one published in 1995
by the International Union of Pure and Applied Chemistry
(IUPAC)

Immobilization by covalent linkage
This is by far the most widely studied method. The covalent linkage is achieved
between a functional group on the enzyme not essential for catalytic activity
and a reactive group on a solid water-insoluble support. The functional groups
available on enzymes for linkage are amino and carboxyl groups, hydroxyl
groups, imidazole groups, indole groups, phenolic groups and sulphydryl
groups.
Some supports which have been used for immobilization include agarose, celluose,
dextran, chitin, starch, polygalacturonic acid (pectin), polyacrylamide,
polyvivyl alcohol, polystyrene, polyprpylene, polyamino acids, polyamide,
glass and metal aides and bentonite. Many of these are organic, but recently the
use has been advocated of inorganic support on the grounds of reuse of
inorganic materials, non-toxicity, good halflife of enzymes immobilized on
inorganic supports, and the ease with which inorganic materials can be
fashioned to suit any particular enzyme system.

Immobilization by adsorption
This method is both simple and inexpensive and consists of bringing
an enzyme solution in contact with a water-insoluble solvent
surface and washing off the unadsorbed enzyme. The extent of the
adsorption depends on a number of factors including the nature of
the support, pH, temperature, time, enzyme concentration. In
principle, though not always in practice, adsorption is reversible.
Adsorbents which have been used include alumina, bentonite,
calcium carbonate, calcium phosphate, carbon, cellulose, charcoal,
clay, collagen, diatomaceous earth, glass, ion-exchange resins,
sephadex, and silica gel. Apart from the ease of the operation, the
other advantage is that the enzymes are unlikely to be inactivated
because the system is mild. The disadvantage is that in cases of
weak binding the enzyme may be easily washed away.

Immobilization by micro-encapsulation
Micro-encapsulation consists in packaging the enzyme in tiny usually
spherical capsules ranging from 5-300 in diameter in semi-
permeable (permanent) or liquid (nonpermanent) membranes.
In one method known as the interfacial polymerization technique, the
enzyme solution contains the enzyme as well as one component of
the membrane that will form round the micro-capsule. The emulsion
is stirred vigorously and more of the organic solvent(s) containing
the rest of the capsule-forming reagent is added.
In the second method, the coacervation-dependent method, the added
organic solvents contain all the components of the polymer. In both
cases the enzyme droplets are formed during the vigorous stirring.
The semi-permeable membrane is allowed to harden around the
micro-droplets; the micro-capsules are then washed and then
transferred.

Immobilization by entrapment
In the entrapment of enzymes, no reaction occurs between support
and the enzyme. A cross-linked polymeric network is formed
around the enzyme; alternatively the enzyme is placed in a
polymeric substance and the polymeric chains cross-linked.
Polyacrylamide gels have been widely used for this purpose,
although enzymes do leak through the network in some cases. The
advantages of the entrapping method are: (i) its simplicity, (ii) the
small amount of enzyme used, (iii) the unlikelihood of damage to
the enzyme, (iv) applicability to water insoluble enzymes. The
disadvantages include leakage of enzymes and some chemical and
thermal enzyme damage during gel formation.

Naringinases: occurrence, characteristics, and applications

Introduction to naringinase
Naringinase, an enzyme complex, is commercially
attractive due to its potential usefulness in
pharmaceutical and food industries.
biotransformation of :
steroids
antibiotics
mainly of glycosides hydrolysis
Main application: debittering of citrus juices
Ribeiro, Maria H. "Naringinases: occurrence, characteristics, and applications." Applied
microbiology and biotechnology 90.6 (2011): 1883-1895.

Naringinase, an α-rhamnopyranosidase, expressed α-L-
rhamnosidase (E.C. 3.2.1.40) and β-D-glucosidase
(E.C. 3.2.1.21) activities.
naringinase
α-L-rhamnosidase
β-D-glucosidase
Ribeiro, Maria H. "Naringinases: occurrence, characteristics, and applications." Applied microbiology
and biotechnology 90.6 (2011): 1883-1895.

Naringin : 4′,-5,7′-trihydroxyflavonone-7-
rhamnoglucoside
Prunin : trihydroxyflavonone-7-glucoside
Naringenin : 4′-5,7′-trihydronyflavonone
These molecules have a great potential, especially in
the food and pharmaceutical industries, due to their
recognized antioxidant, anti-inflammatory, anti-
ulcer, and hypocholesterolemic effects, whereas
naringenin has also shown anti-mutagenic and
neuroprotective activities, while prunin has antiviral
activity. Ribeiro, Maria H. "Naringinases: occurrence, characteristics, and
applications." Applied microbiology and biotechnology 90.6 (2011):

Sources and production of naringinase
Naringinase has been reported in the literature
since the earliest of 1938, initially in isolates
from celery seeds (Hall 1938) and later in
grapefruit leaves (Thomas et al. 1958; Ting
1958).
One of the first reports focused on naringinase
production with molds was the work of Kishi,
published in 1955. In this study, 96 strains were
explored and Aspergillus niger was established
as the best producer of naringinase (Kishi 1955).

Ribeiro,MariaH."Naringinases:occurrence,characteristics,andapplications."
Appliedmicrobiologyandbiotechnology90.6(2011):1883-1895.

Puri,Munish."Updatesonnaringinase:structuralandbiotechnologicalaspects."

In1964 the studies of Okada et al. (1963) were the basis for
the establishment of a process by Tanabe
Pharmaceuticals Company to produce a preparation of
naringinase from A. niger, marketed as “Kumitanase.”
Later on, Bram and Solomons (1965) studied the effect of
media composition on the production of naringinase with
A. niger, in a 10-L bioreactor, fully baffled, with disc
turbine impellers, and automatic control of temperature
and foam. The best enzyme titers were provided in a
medium containing corn steep liquor–yeast extract.
These authors observed that naringinase formation was
repressed by glucose and stimulated in the presence of
the substrate naringin (Bram and Solomons 1965).

The work of Mateles et al. (1965) showed an
enhanced naringinase production by A. niger
NRRL 72-4 in a culture medium containing
rhamnose. These authors observed that glucose,
lactate, and citrate suppressed the production of
naringinase, as well as sucrose and starch,
although they supported excellent growth.
The abovementioned studies, about the production of
naringinase, were pioneer, and for almost two
decades, no significant developments were
reported.

Shanmugam and Yadav (1995) demonstrated the
extracellular production of naringinase from a
strain of the fungus Rhizopus nigricans. The
culture medium contained sucrose and rice. As
mycelia grew, a decrease in pH was observed.
The higher naringinase activity was observed
nearly 50 h after inoculation. In all reported
fermentation processes, the naringinase was
observed in the extracellular broth.

Zverlov et al. (2000) cloned and expressed with
marked activity the gene of α-L-rhamnosidase
of naringinase in Escherichia coli. This
recombinant naringinase provided an
economical and easily available source of the
enzyme with important impact in the industrial
debittering of citrus juices and in the
pharmaceutical industry.

In order to find new naringinase producers,
Thammawat et al. (2008) isolated 348 fungi from
128 various host samples, collected from 11
different sources in Thailand and China. Forty
fungal isolates were obtained. Secondary screening
was performed measuring the α-L-rhamnosidase
and β-D-glucosidase activities at 40 °C, pH 4.0.
Moreover, from all 40 fungal isolates naringinase
activity was tested at the temperatures of 50, 55,
and 60 °C, at pH 3.0 and 4.0. A. niger BCC 25166
was selected and genetically identified by
Thammawat et al. (2008).

The effect of different physico-chemical parameters,
such as pH, temperature, agitation, and inducer
concentration was evaluated (Puri et al 2010):
The addition of Ca+2 stimulated the naringinase activity,
at optimal pH of 5.5 and 30 °C. A twofold increase in
naringinase production was achieved by the addition of
citrus peel powder to the medium. A large number of
variables were optimized, in a 5-L bioreactor, leading
to significantly improved enzyme production, namely
an increase in sugar concentration (15 g L−1) in the
fermentation medium, further increased naringinase
production (8.9 IU mL−1).

Puri et al. (2010) found the optimum values for the
critical components as follows:
Sucrose : 10.0%
sodium nitrate :10.0%
Naringin : 0.50% (w/v)
pH : 5.6
biomass concentration : 1.58%
These optimal conditions led to a naringinase
production of 8.45 U/mL (Puri et al. 2010).

Activity and characterization of naringinase
Some assay procedures for naringinase activity evaluation
are presently available. These procedures include
spectrophotometric and high-performance liquid
chromatographic (HPLC) determinations.
The use of specific substrates to discriminate between the
two enzymatic activities expressed by naringinase is of
major significance. In fact, both enzymatic activities of
naringinase can be followed and easily measured avoiding
previous subunit protein separation and purification. With
this purpose, p-nitrophenyl α-L-rhamnopyranoside (4-
NRP) and p-nitrophenyl β-D-glucopyranoside (4-NGP)
can be used as specific substrates for α-L-rhamnosidase
and β-Dglucosidase, respectively (Vila-Real et al. 2010b).
Ribeiro,MariaH."Naringinases:occurrence,characteristics,
andapplications."Appliedmicrobiologyandbiotechnology90.6
(2011):1883-1895.

Moreover, other substrates has been used to follow
the specificity of α-L-rhamnosidase activity
expressed by naringinase, such as disaccharides
and phenol glycosides like methyl 2-O-(alpha-L-
mannopyranosyl)-beta-D-glucoside, methyl 3-O-
(alpha-L-mannopyranosyl)-alpha-D-glucoside,
methyl 5-O-(alpha-L-mannopyranosyl)-beta-D-
glucoside, 6-O-(alpha-L-mannopyranosyl)-D-
galactose, p-nitrophenyl alpha-L-mannoside, and
4-methyl umbelliferone alpha-Lmannoside (Esaki
et al. 1993).

Naringinase activity can also be followed through
rhamnose and glucose determination. To attain
that goal, the 2,4-dinitrosalicylic acid (DNS)
method acid (Miller1959) is one of choice. The
DNS macroassay was modified into a
microassay using a 96-microwell plate,
allowing a higher repeatability, speed, large
sample analysis number, and sample volume
reduction (Nunes et al. 2010, Vila-Real et al.
2010c).

Ribeiro, Maria H. "Naringinases: occurrence, characteristics, and applications." Applied microbiology and
biotechnology 90.6 (2011): 1883-1895.

Applications of naringinase
• Debittering of citrus juices
• Preparation of antibiotics
• Biotransformation of steroids
• Preparation of rhamnose
• Production of prunin
• Production of ginsenosides
• Production of glycolipids

Debittering of citrus juices
• The bitterness of some fruit juices, mainly orange
and grapefruit, is an undesirable quality for the
juices industry. The principal groups of
compounds present in these fruits responsible for
bitterness are flavonoids (e.g., naringin) and
limonoids (e.g., limonin). It had been reported that
when naringin is present in water solutions in
concentrations higher than 20 mg mL−1, the bitter
taste can be detected, although in grapefruit juices,
bitterness is only detectable in concentrations
higher than 300–400 mg mL−1 (Soares and
Hotchkiss 1998).
Ribeiro,MariaH."Naringinases:occurrence,characteristics,and
applications."Appliedmicrobiologyandbiotechnology90.6(2011):
1883-1895.

Mishra and Kar (2003) reduced the bitterness of
grapefruit (Citrus aurantium) juice with
naringinase entrapped in alginate beads, resulting
in 84% naringin hydrolysis. Ribeiro and Ribeiro
(2008) attained a 95% naringin reduction with
naringinase immobilized in k-carrageenan. Olsons
et al.
The enzymatic hydrolysis of naringin combined with
high hydrostatic pressure was carried out in citrus
juices leading to the resolution of one of the major
problems in citrus juice industries, the bitterness
(Ribeiro et al. 2010).
applications."Appliedmicrobiologyandbiotechnology90.6(2011):1883-
1895.

Preparation of antibiotics
Chloropolysporins A, B, and C could be enzymatically
converted to deglycosylated derivatives (Sankyo 1988). A
combination of chloropolysporin C and β-lactam antibiotics
was synergistically effective against methicillinresistant
strains of Staphylococcus. Chloropolysporins havestrong
activity against gram-positive bacteria including methicillin-
resistant Staphylococcus aureus, and these antibiotics also
inhibit the anaerobic gram-positive Enterobacteria. The
deglycosylation of novel glycopeptide antibiotic,
chloropolysporin from Faenia interjecta, was achieved
successfully with rhamnosidase activity of naringinase.The
glycopeptide antibiotic chloropolysporin C is obtained by
the derhamnosylation of chloropolysporin B.

Biotransformation of steroids
The fenugreek seeds (Trigonella foenum graecum) upon
enzymatic hydrolysis by naringinase produce sapogenins
and diosgenin, precursors of clinically useful steroid drugs.
In this process, the immobilized naringinase and pectinase
could be reused twice without loss of diosgenin yield
(Elujoba and Hardman 1987). α-L-Rhamnosidase expressed
by naringinase can be used in the preparation of many drugs
and drug precursors. α-Lrhamnosidase hydrolyzes the
diosgene (a saponin) to produce α-L-rhamnose and
diosgenin which is used in the synthesis of clinically useful
steroid drugs such as progesterone (Elujoba and Hardman
1987). α-L-Rhamnosidase produced by Curvularia lunata
can remove L-rhamnose from a number of steroidal
saponins (Feng et al. 2007).
applications."Appliedmicrobiologyandbiotechnology90.6(2011):
1883-1895.

Preparation of rhamnose
The α-L-rhamnosidase activity expressed by naringinase
hydrolyzes substrates, as naringin to produce L-rhamnose.
Rhamnose plays the role of chiral intermediate in the
organic synthesis of pharmaceutically important agents
andplant protective agents. The α-L-rhamnosidase cleaves
the L-rhamnose from the glycosides that contain terminal
Lrhamnose. Thus, α-L-rhamnosidase had potential in the
manufacture of L-rhamnose. Daniels et al. (1990) used a
partially purified preparation of naringinase with a high
rhamnosidase activity and low glucosidase activity to
produce rhamnose. Vila-Real et al. (2010a) developed an
easy and cheap method to produce rhamnose inactivating β-
glucosidase expressed by naringinase.

Optimization of process parameters for the production of naringinase
by Aspergillus niger MTCC 1344
Aspergillus niger MTCC 1344 was used to produce
extracellular naringinase in a complex (molasses, yeast
extract and salts) medium. An initial medium pH 4.5 and
cultivation temperature 30 ◦C were optimal for enzyme
production. Among various carbon and organic nitrogen
sources used, molasses and peptone were the most effective
for enzyme yield. The rate of enzyme production was
enhanced when metal ions were added to the medium.
Fermentation conditions are described which produced a
higher rate of enzyme synthesis. An increase in initial sugar
concentration from 6 to 10 g l−1 in the fermentation
medium produced decreased naringinase synthesis while
cell mass growth increased with the increase of sugar
concentration. At a higher sugar level (10 g l−1) the
production of cell mass decreased.
Puri,Munish,AnirbanBanerjee,andU.C.Banerjee."Optimization
ofprocessparametersfortheproductionofnaringinaseby
AspergillusnigerMTCC1344."Processbiochemistry40.1
(2005):195-201.

Production of naringinase:
material and methods
1. Chemicals :
Naringin
Different growth factors
Organic and inorganic nitrogen sources
2. Culture medium and cultivation conditions :
A.niger MTCC 1344
Composition of the medium:
material g/l
NaNO3 2.0
KH2PO4 1.0
KCl 0.5
MgSO4·7H2O 0.5
FeCl3 0.1
naringenin 0.05
Puri, Munish, Anirban Banerjee, and U. C. Banerjee. "Optimization
of process parameters for the production of naringinase by
Aspergillus niger MTCC 1344." Process biochemistry 40.1
(2005): 195-201.

100 ml medium was autoclaved in 500 ml
Erlenmeyer flask for 15 min at 121 ◦C and 15 psi
pressure and the initial pH of the medium was
adjusted to 4.5.
The medium was inoculated with vegetative
mycelia or spore suspension.
Flasks were incubated (28 ◦C, 200 rpm) in a rotary
shaker for 8–10 days.
The salts of different metal ions including Ca2+,
Co2+, Cu2+, Fe3+, Mg2+, Ni2+, Mn2+ and
Zn2+ were used in the culture medium.
Puri,Munish,AnirbanBanerjee,andU.C.Banerjee."Optimizationofprocess
parametersfortheproductionofnaringinasebyAspergillusnigerMTCC
1344."Processbiochemistry40.1(2005):195-201.

Assay methods
Naringinase activity was estimated by
determining residual naringin using the Davis
method [15].
Puri, Munish, Anirban Banerjee, and U. C. Banerjee. "Optimization of process parameters for the production of
naringinase by Aspergillus niger MTCC 1344." Process biochemistry 40.1 (2005): 195-201.

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