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					Nordisk konferanse
Verdiskaping av marine biprodukter etter år 2000
Stjørdal 24. og 25. januar 2001


Biotechnological applications of fish offal in Iceland
Jon Bragi Bjarnason
Science Institute
University of Iceland
Dunhagi 3
IS-107 Reykjavík
ISLAND


Abstract
        Hydrolytic enzymes, especially proteinases, have many uses and potential
applications in industry, medicine and research. Among these are detergent
production, leather processing, chemical modifications and food processing.
Enzymes isolated from cold water marine organisms may prove to be especially
useful for these purposes. The cold-active or psychrophilic enzymes are frequently
more active at low temperatures than their mammalian or bacterial counterparts, a
characteristic which could be beneficial in many industrial processes, as well as
medical, pharmaceutical, hygienic and cosmetic applications.
    A mixture of proteinases, called Cryotin, was prepared by neutral extraction from
Atlantic cod pyloric caeca. The preparation was shown to contain trypsin,
chymotrypsin, elastase and collagenases. Trypsin was purified and resolved into
three differently charged species termed cod trypsin I, II and III, with pI values of 6.6,
6.2, and 5.5 respectively, but a similar molecular mass of 24 kDa. The catalytic
efficiency at 25°C expressed as kcat/Km was 17 times greater for cod trypsin I than
bovine trypsin, when these enzymes were assayed as amidases. Atlantic cod trypsin
demonstrated less resistance to thermal inactivation and treatment in mildly acidic
solutions than bovine trypsin. The amino terminal sequence of cod trypsin enzyme I,
the predominant species from Atlantic cod, showed similarities to other known
trypsins, in particular the porcine and rat trypsins, with 30 identical residues out of
37, bovine trypsin, with 29 residues identical out of 37. Peptide mapping and partial
amino acid sequencing of the three trypsin forms have shown that the cod trypsin
isoenzymes are separate gene products. We have cloned and sequened the cDNA
encoding cod trypsinogen III.
    Two chymotrypsins were purified with isoelectric points of 6.2 and 5.8, but a
similar molecular mass of 26 kDa. It is not clear at this point whether they are
separate gene products. The cod enzymes differed from bovine chymotrypsin in
having more acidic isoelectric points and in being unstable in weakly acidic
solutions. The cod enzymes were found to be more active than bovine chymotrypsin
towards both ester and amide substrates. Cod chymotrypsin retained activity in 50%
(v/v) of various organic solvent solutions. However, it was more thermolabile than
bovine chymotrypsin. We have cloned and sequened the cDNA encoding one of the
cod chymotrypsinogens.
     Elastase from cod has been purified extensively, and has recently been
characterized. At least two distinct collagenases have been isolated from other known
proteolytic activities in the Cryotin mixture. One of these has been highly purified
and is precently being characterized.
     Cryotin, the mixture of proteinases from Atlantic cod, has many potential
applications in industry and medicine, especially in food processing which require
hydrolysis at low temperatures, inactivation under mild conditions or collagen
digestion. It has proven promising in various fish processing applications such as
skinning of fish, removal of membranes and ripening of herring. Cryotin also has
potential as a digestive aid, both for humans and animals, and could be used as an
adjunct in microdiets for fish larvae. Various food processing applications are also
being considered, such as chill-proofing of beer, biscuit manufacture, tenderizing of
meats, hydrolysis of various food proteins such as gelatin, vegetable proteins, and
collagens.
           Precently Cryotin is being prepared on a pilot plant scale for marketing
trials and application tests, both in house and in collaboration with external partners.
Purification of the individual proteinases, trypsin, chymotrypsin, elastase and
collagenase is also being scaled up to allow large scale tests with them to be
conducted.
     Cryotin is now being used in a patented process to prepare high quality all-natural
flavorings for food processing and innovative cooking.
         Penzyme, a pure superactive proteinase from cod, is precently being sold in
Iceland as an enzyme oinment called PENZIM gel or lotion. The PENZIM ointment
is a soothing, moisturizing, cleansing and nourishing skin healing treatment for dry or
chapped skin. It also appears to have very good qualities as a “age-specialist” product
for facial skin, and rejuvenates whole body skin be removing the outermost layer of
dead skin cells. In addition, information suggests that the PENZIM ointment
increases the well being and comfort of people with the following conditions:
Osteoarthritis, rheumatoid arthritis, fibromyalgia
Muscle pains and tendonitis, tenosynovitis, myotenositis
Phlebitis, lymphangitis, varicose veins, hemorrhoids, angiodermatitis
Tennis elbow, golf elbow and various sports related injuries
Swelling and muscle pains due to injuries or accidents, i.e. sprains or broken bones
Wound healing due to burns, cuts or herpes infections
Dry or chapped skin , e.g. due to diabetes
Acne and boils
Psoriasis, eczema and various dermatological conditions, such as contact eczema
Fungal infections of the skin, seborrheic eczema or dermatitis
Warts and cavuses, calluses
Various inflammatory conditions due to injury and itching of the skin, i.e. insect bites




1. INTRODUCTION
     Cold-adaptation of ectothermic organisms, such as fishes, involves
compensations in the efficiency of enzyme catalysed reactions, either through
alterations in the catalytic efficiency of the enzymes or through increasing enzyme
concentrations ( Hazel & Prosser 1974; Hochachka & Somero 1985). The
optimisation of an enzyme towards a low temperature environment presumably
involves reducing the rigidity of the enzyme molecule which would lead to a
measurable reduction in stability properties of the enzyme (Ásgeirsson et al. 1989).
Thus, in cold-adapted poikilotherms, natural selection would be expected to favor
enzymes with increased catalytic efficiency at low temperatures, although other
factors, such as structural stability, may restrict the degree of optimisation. We have
been studying digestive enzymes from the Atlantic cod as a possible source of
industrial enzymes with unique and useful properties.
     The present report describes the components of a mixture of proteolytic digestive
enzymes, called Cryotin, which has been prepared by neutral extraction from the
pyloric caeca of Atlantic cod Gadus morhua. This proteinase mixture has many
unique characteristics. The proteinases in the mixture, studied so far, are more active
at low temperatures, when compared to their mammalian counterparts. They are also
thermo-labile as well as acid sensitive. Cryotin has been shown to contain trypsin,
chymotrypsin, elastase and, perhaps most importantly, collagenolytic enzymes, as
well as other proteolytic and peptidolytic activities, but it is practically devoid of
lipase, amylase and nuclease activities.

2. TRYPSIN FROM ATLANTIC COD

    Trypsin was purified from Cryotin with affinity chromatography as previously
described (Ásgeirsson et al., 1989). Trypsin was further resolved into three differently
charged species having pI values of 6.6, 6.2 and 5.5 on a chromatofocusing PBE-94
anion exchange column. All three trypsins were found to have a similar molecular
mass of 24.2 kDa. The amino terminal sequence of cod trypsin enzyme I, the
predominant species from Atlantic cod, showed similarities to other known trypsins,
in particular the porcine and rat trypsins, with 30 identical residues out of 37, bovine
trypsin, with 29 residues identical out of 37, and dogfish trypsin having 26 residues
identical out of 37 (Craik et al. 1984; Walsh 1970; Titani 1975). Peptide mapping and
partial amino acid sequencing of the three trypsin forms have shown that the cod
trypsin isoenzymes are separate gene products (Kristjánsson et al.,1993a). The amino
acid sequence deduced from the trypsinogen III cDNA nucleotide sequence had 58%
amino acid sequence homology with bovine trypsinogen. It contained 222 amino
acids, one less than the bovine analog, with a molecular mass of 23.819 Da
(Gu›mundsdóttir et al., 1993a).

2.1. Kinetic Properties
    The catalytic efficiency of cod trypsin enzyme I at 25°C, expressed as kcat/Km,
was 17 times greater than that for bovine trypsin when these enzymes were assayed
as amidases using N-benzoyl-L-arginine p-nitroanilide as substrate. This was
revealed as differences in both apparent Km and kcat values. The amidase activity of
the cod trypsin displayed an apparent Km value of 77 µM, approximately eight times
lower than that measured for the bovine enzyme of 650 µM. The turnover number
achieved at 25°C was also greater for the cod enzyme by a factor of two. The esterase
activity of the two enzymes, using p-tosyl-L-arginine methyl ester as substrate, also
displayed dissimilar characteristics with the cod enzyme having a kcat/Km value 2.5
times higher than bovine trypsin. Table 1 summarises the differences in kinetic
properties between cod trypsin, chymotrypsin and elastase as compared with their
mammlian counterparts. It is perhaps of greatest interest that the increased kinetic
efficiency of the Atlantic cod enzymes is maintained at low temperatures.

2.2. Thermal Stability
    Atlantic cod trypsin demonstrated less resistance to thermal inactivation than
bovine trypsin (Ásgeirsson et al., 1989). The highest temperature at which Atlantic
cod trypsin remained fully active for at least 3 minutes was 55°C, as compared to
65°C for bovine trypsin . Half of the initial activity was lost at 52°C and 57°C for
cod and bovine trypsin respectively, inferred from 10 minute incubation experiments
at various temperatures. These data are suggestive of somewhat less structural
stability in cod trypsin which possibly has evolved in response to the need for
optimising kinetic properties at low habitat temperatures. Such structural
destabilisation is not necessarily brought about by fewer covalent links, notably
disulfide bonds, but may rather be due to differences in the weak intramolecular
interactions. The number of hydrophobic interactions expressed in terms of the
average hydrophobicity, are found to be reduced in Atlantic cod trypsin as compared
to bovine trypsin.

2.3. Acid Stability
    Studies on the stability of cod trypsin at various pH values revealed that the
enzyme is unstable in acidic solutions. Bovine trypsin is stable at pH 3.0 at low
temperatures for weeks. The esterase activity of cod trypsin was quite stable in
alkaline medium, but displayed a marked acid lability. Esterase activity was lost
when pH was lowered below pH 5.0, and this effect was apparent after a 30 minute
incubation, but quite pronounced after 18 hours.


3. CHYMOTRYPSIN FROM ATLANTIC COD

    Cod chymotrypsin was isolated on a phenyl-Sepharose column following trypsin
removal with the benzamidine affinity resin. Elastase was eluted from the phenyl-
Sepharose column with a 25 mM tris buffer pH 7.5 containing 10 mM calcium
chloride and 20% (v/v) ethylene glycol, followed by chymotrypsin release from the
column by washing with a 50% (v/v) ethylene glycol solution containing 20 mM
calcium chloride.

3.1. Structural Properties
    Chymotrypsin was further resolved into two differently charged species with
isoelectric points of 6.2 (enzyme A) and 5.8 (enzyme B), but a similar molecular
mass of approximately 26 kDa. However, chymotrypsin B was distinctly larger than
chymotrypsin A (Ásgeirsson & Bjarnason, 1991). The N-teminal sequence of cod
chymotrypsin, enzyme B, was analysed and compared to the amino acid sequence of
bovine chymotrypsin. Only five substitutions were observed in the first 31 amino
acids. Conversion of bovine chymotrypsinogen to the active enzyme involves tryptic
cleavage of the peptide bond between Arg(15) and Ile(16) and subsequent autolytic
removal of the dipeptide Leu(14)-Arg(15). The same modification apparently takes
place during activation of cod chymotrypsinogen, enzyme B, since a gap is observed
in its sequence at this position. Interestindly, the cod chymotrypsins do not show the
B- and C- chain pattern observed for bovine chymotrypsin upon reduction with 2-
mercaptoethanol followed by SDS electrophoresis.
     The amino acid sequence deduced from the cod chymotrypsinogen nucleotide
sequence had a 67% amino acid sequence homology with bovine chymotrypsinogen.
It contained 245 amino acids, as did the bovine enzyme, and yielded a molecular
mass of 26211 Da (Gudmundsdóttir et al., 1993b).
     The cod enzymes differed from bovine chymotrypsin with a pI 8.5 (Laskowski,
1955) in having more acidic isoelectric points and being unstable in weakly acidic
soutions. This is in good agreement with the previously published results of Raae and
Walther (1989). Mammalian chymotrypsins have been found to be very stable in
acidic solutions of pH 3.0 (Wilcox 1970; Bender & Killheffer 1973), while the cod
chymotrypsins displayed marked acid lability at pH values below 5.0.

3.2. Kinetic Properties
The kinetic properties of cod chymotryp       sin were compared to those of the
bovine enzyme. The cod enzymes were found to be more active than bovine
chymotrypsin towards both ester and amide substrates. The pseudo second order rate
constant kcat/Km is about 3 to 4 fold higher for cod chymotrypsin than bovine
chymotrypsin when ester hydrolysis is measured using N-benzoyl-L-tyrosine ethyl
ester as substrate at 25°C. When the amide substrate N-benzoyl-L-tyrosine-p-
nitroanilide was employed, the cod chymotrypsin yielded two to four fold higher
values of catalytic efficiency than the bovine enzyme (Table 1). These values
remained consistently higher for the cod enzymes at all temperatures measured,
within the thermal stability of the enzymes. Under the experimental conditions
employed (10 minute incubations), the loss of half-maximal activity occurred at 48°C
for the cod enzyme, compared to 52°C for for the bovine enzyme.

3.3. Stability in Aqueous Solutions and Organic Solvents
     The stability of cod trypsin and chymotrypsin in dilute aqueous solutions was not
maintained over longer storage periods. In the absence of additives the esterase
activity of chymotrypsin was almost completely lost in 3 days, whether at 25°C or
4°C. The addition of calcium chloride proved advantageous in maintaining activity.
Approximately 0.2 M calcium chloride was sufficient to maintain full chymotrypsin
activity for 20 hours at both temperatures. It is not clear whether subsequent loss of
activity may be attributed to autolysis or denaturation. We therefore sought methods
for the preservation of the activity of these enzymes.
     The effects of ethylene glycol on the tolerance of cod chymotrypsin towards
freezing was tested. The esterase activity of the enzyme was well preserved through
three repetitive freezing trials (-26°C) in 25% ethylene glycol, with an activity loss of
only about 20%. If 50% ethylene glycol was used, no activity loss was detected. At
4°C cod chymotrypsin retained full activity in 25% ethylene glycol or glycerol for 20
days.
    The stability of cod chymotrypsin in organic solvents is of interest in relation to
its use in organic synthesis. Stability was measured as residual esterase activity at
25°C after incubation of the enzyme in organic solvents at ratios of 25% and 50%
(v/v) in aqueous buffer for up to 30 days at 4°C. The organic solvents used in the
experiment were dimethyl sulfoxide, dioxane, glycerol, methanol, ethanol, 1,3-
propanediol, acetonitrile and dimethyl formamide. The cod enzyme retained constant
activity for the total duration of the experiment of the 20 days tested in dimethyl
sulfoxide, dioxane and glycerol solutions, and 30 days in the other organic solutions.
The residual activity was approximately the same in all the organic solutions as in
the aqueous buffer standard, except in dioxane, where the activity dropped
immediately to about 20% of the value of the standard, but remained at that level for
the duration of the experiment. This pattern of stability is similar to that observed for
bovine chymotrypsin.


4. ELASTASE FROM ATLANTIC COD

    Elastase from cod has been purified to homogeneity by a phenyl Sepharose
hydrophobic chromatography step followed by gel filtration on Sephacryl S-300 on
which the enzyme is retarded, thus yielding a single band on polyacrylamide gels
(PAGE) indicating a molecular mass for cod elastase of 25 kDa. The purified
elastase gave one band on isoelectric focusing electrophoresis indicative of a single
enzyme species with an isoelectric point higher than 9.3, which is similar to porcine
elastase (Ásgeirsson & Bjarnason, 1993).

4.1. Structural Properties
    The amino terminal sequence of cod elastase showed similarities to other known
elastases. The sequence is identical in 14 out of 20 positions to porcine elastase 1 and
human elastase 1. The cod elastase sequence is also identical in 16 positions out of 20
to porcine elastase 2 and human elastase 2A (Ásgeirsson & Bjarnason, 1993).
Considerable variation is observed in residues 5 to 6, and 8 to 10 among elastases of
both groups 1 and 2. Differences in the N-terminal amino acid sequences which
clearly distinguish type 1 elastases from type 2 elastases are those where the former
has serine instead of tryptophan in position 14 and isoleucine instead of valine in
position 16. The cod elastase was found to contain isoleucine in position 16, a
characteristic of a type 1 elastase, but amino acid residue 14 was identified as
tryptophan, a characteristic of type 2 elastases. Thus, Atlantic cod elastase appears to
have some sequence characteristics in common with both mammalian elastases 1 and
2, as well as hybrid substrate specificity characteristics which includes hydrolysis,
albeit with low activity, at tyrosine and phenylalanine bonds.

4.2. Kinetic Properties
    The Atlantic cod elastase hydrolysed orcein-elastin with twice the specific
activity of porcine elastase at 37°C and with 40% higher specific activity at 0°C. This
further confirms the identity of the cod enzyme as an elastase. Using Succinyl Ala-
Ala-Ala-p-nitroanilide as a substrate for kinetic measurements, Km values were
similar for Atlantic cod elastase and porcine pancreatic elastase 1, the enzyme used
for comparative purposes. The kcat values for the two enzymes differed however,
being about 2 times higher for cod elastase than the porcine enzyme, both at 10°C
and 25°C. The catalytic efficiency, expressed as kcat/Km, determined with this
substrate was therefore more than two-fold higher for cod elastase than for the
porcine enzyme (Table 1).

Table 1
The catalytic efficiency of cod digestive serine proteinases compared to their
mammalian counterparts at two different temperatures. In each case the catalytic
efficiency (kcat/Km) of the cod enzyme was divided by the value obtained for the
mammalian enzyme. The substrate for trypsin was Benzoyl-arginine-p-nitroanilide,
for Chymotrypsin it was Benzoyl-arginine p-nitroanilide and for elastase it was
Succinyl Ala-Ala-Ala-p-nitroanilide.
________________________________________________            ________

   Temperature           Trypsin     Chymotrypsin    Elastase
______________________________________________________     __

        10°C                9.0           2.5                            1.9
        25°C               17.0           2.4                            2.0
________________________________________________                    ________

These results are in good agreement with values of the kinetic parameters for porcine
elastase in the literature and clearly establish the increased catalytic efficiency of the
cod elastase in comparison with the bovine enzyme (Gildberg & Øverbø, 1990).

4.3. Stability Properties
    Cod elastase was stable at pH 5 and above, wheras lowering pH below 5 resulted
in total loss of activity. Even brief titration to acidic pH levels caused total and
irreversible inactivation. This is in clear contrast to porcine pancreatic elastase which
undergoes a reversible conformational change below pH 4.0, and brief titration of this
elastase down to pH 2.6 is fully reversible (Shotton, 1970).
    The cod elastase showed considerably less resistance to thermal inactivation than
the porcine elastase. When the activity of elastase was measured at various
temperatures using Suc-Ala-Ala-Ala-p-nitroanilide as substrate the cod enzyme
reached maximum activity at 40°C, whereas porcine elastase was most active at
50°C. The thermal stability of cod elastase was also investigated more directly by
measuring residual activity at 25°C after preincubating the enzyme at various
temperatures for 10 minutes. The temperature required for half-maximal inactivation
of cod elastase was thus found to be 48°C, but 63°C for porcine elastase, or 15
degrees higher. This manifests a distinct difference in structural stability for the two
related enzymes (Ásgeirsson & Bjarnason, 1993).
4.4. Substrate Specificity
    Specificity studies employing the oxidized insulin B-chain as substrate showed
that substrate specificity of cod elastase was initially restricted to cleavage at the
bond between alanine 14 and leucine 15. Prolonged incubation for up to 24 hours
gave rise to some additional cleavage products which indicated hydrolysis on the C-
terminal side of valine 12, leucine 15, valine 18, phenylalanine 25 and tyrosine 26.
There was no indication that cod elastase was hydrolysing the insulin B-chain at the
C-terminal side of serine 9 or glycine 23 as reported for the porcine enzyme (Sanger
& Tuppy, 1951). Furthermore, the cod enzyme hydrolysed the bond on the C-terminal
side of leucine 15, whereas porcine elastase hydrolysed the bond on the C-terminal
side of leucine 17. However, Atlantic cod elastase did apparently cleave at two
additional sites not hydrolysed by the porcine enzyme, namely phenylalanine 25 and
tyrosine 26, sites more characteristic of the mammalian type 2 elastases. Thus ,
Atlantic cod elastase appears to have some characteristics in common with both
mammalian elastases 1 and 2, in particular substrate specificity which includes
hydrolysis, albeit with low activity, at tyrosine and phenylalanine bonds, and some
hybrid character in the N-terminal amino acid sequence (Ásgeirsson & Bjarnason,
1993).


5. COLLAGENASE FROM ATLANTIC COD

     A collagenase preparation, devoid of trypsin, chymotrypsin and elastase, was
obtained by a single DEAE cellulose ion-exchange chromatographic step, following
an initial purification of trypsin on a para-aminobenzamidine Separose-4B affinity
column. The collagenase binds to the ion-exchange column in 25 mM Tris buffer pH
8.5 containing 10 mM calcium chloride, and is eluted of the column with the same
buffer containing 0.1 M sodium chloride. Further purification was achieved by gel-
filtration on an Ultragel AcA-44 gelfiltration column followed by a second DEAE
cellulose ion-exchange column separation step in 25 mM Tris buffer pH 7.5
containing 10 mM calcium chloride yielding collagenase fractions A and B. The
enzyme was eluted from the ion-exchange column at the end of a linear gradient
containing no salt in the beginning and 0.2 M sodium chloride at the end of the
gradient. The enzyme thus isolated is a true collagenase, cleaving native interstitial
collagen at 25°C, whereas trypsin and chymotrypsin do not cleave this substrate, a
type I soluble placental collagen obtained from Sigma (Kristjánsson et al.,1993b).
     Inhibitor studies performed on this collagenase preparation, which is devoid of
trypsin, chymotrypsin and elastase activities, indicate that the enzyme belongs to a
the class of serine proteinases, since it is totally inhibited by soybean trypsin inhibitor
and phenylmethylsulphonyl fluoride (PMSF), and partially inhibited (80%) by the
chymotrypsin inhibitor L-tosylphenylmethyl chloro ketone (TPCK). Percentage
inhibition was obtained by integration of the collagen peaks from densitometry
scanned polyacrylamide gels. Preliminary SDS PAGE suggests that the collagenase
has a molecular mass of approximately 25-30 kDa. These data are in good agreement
with previously reported results on serine collagenases from fiddler crab and catfish
(Grant et al. 1983; Yoshinaka et al.1986). Purification of two cod collagenases to
homogeneity has now been achieved and characterization of these enzymes is
presently under way.


6. CONCLUSIONS

    A mixture of proteinases, called Cryotin, was prepared by neutral extraction from
frozen and homogenized pyloric caeca from Atlantic cod. The preparation was shown
to contain trypsin, chymotrypsin, elastase and collagenases.The catalytic efficiency at
25°C expressed as kcat/Km was 17 times greater for cod trypsin I than bovine
trypsin, when these enzymes were assayed as amidases. Atlantic cod trypsin
demonstrated less resistance to thermal inactivation and treatment in mildly acidic
solutions than bovine trypsin. Two chymotrypsins were purified with isoelectric
points of 6.2 and 5.8, but a similar molecular mass of 26 kDa. The cod enzymes
differed from bovine chymotrypsin in having more acidic isoelectric points and in
being unstable in weakly acidic solutions. The cod enzymes were found to be more
active than bovine chymotrypsin towards both ester and amide substrates. One
elastase has been purified and characterized. It was also found to have a higher
catalytic efficiency and lower thermal stability than its mammalian counterpart,
porcine pancreatic elastase 1. Two Collagenases have also been isolated from other
known proteolytic activities in Cryotin. They are serine proteinases with substrate
specificities similar to trypsin and chymotrypsin.
    Cryotin, the mixture of proteinases from Atlantic cod, has many unique
characteristics for a pancreatic enzyme mixture. It contains practically no lipase,
amylase or nuclease activities, which may be due to proteolytic breakdown of these
enzymes in the initial homogenate. The proteinases in Cryotin have higher catalytic
activities, even at very low temperatures, than comparable mammalian enzymes,
permitting the use of lower amounts of enzyme adjuncts in various processes. They
are more temperature and acid sensitive than enzymes from conventional sources,
allowing the use of milder conditions to destroy residual enzyme activities if needed,
after processing is complete. Finally, Cryotin posesses collagenolytic activities,
lending it the ability to hydrolyse collagens in the native form.
    The cold-active proteinases, purified or in the Cryotin mixture, have many
potential uses in industry, medicine and research, especially in food processing
applications which require hydrolysis at low temperatures, inactivation under mild
conditions or native collagen digestion. It has proven promising in various fish
processing applications such as skinning of fish, removal of membranes and ripening
of herring. Cryotin also has potential as a digestive aid, both for humans and animals.
It is now being tested as an adjunct in microdiets for fish larvae and in the
preparation of fish feed.
    Various food processing applications are also being considered, such as in the
chill-proofing of beer, biscuit manufacture, tenderizing of meats, preparation of
minimally treated fruit and vegetable beverages and hydrolysis of various food
proteins, such as gelatin, vegetable proteins and collagens.
    Precently Cryotin is being prepared on a pilot plant scale for marketing trials and
application tests, both in house and in collaboration with external partners.
Purification of the individual proteinases, trypsin, chymotrypsin, elastase and
collagenase is also being scaled up to allow large scale tests with them to be
conducted.
     Cryotin is now being used in a patented process to prepare high quality all-natural
flavorings for food processing and innovative cooking.
     Penzyme, a pure superactive proteinase from cod, is precently being sold in
Iceland as an enzyme oinment called PENZIM gel or lotion. The PENZIM ointment
is a soothing, moisturizing, cleansing and nourishing skin healing treatment for dry or
chapped skin. It also appears to have very good qualities as a “age-specialist” product
for facial skin, and rejuvenates whole body skin be removing the outermost layer of
dead skin cells. In addition, information suggests that the PENZIM ointment
increases the well being and comfort of people with the following conditions:
Osteoarthritis, rheumatoid arthritis, fibromyalgia
Muscle pains and tendonitis, tenosynovitis, myotenositis
Phlebitis, lymphangitis, varicose veins, hemorrhoids, angiodermatitis
Tennis elbow, golf elbow and various sports related injuries
Swelling and muscle pains due to injuries or accidents, i.e. sprains or broken bones
Wound healing due to burns, cuts or herpes infections
Dry or chapped skin , e.g. due to diabetes
Acne and boils
Psoriasis, eczema and various dermatological conditions, such as contact eczema
Fungal infections of the skin, seborrheic eczema or dermatitis
Warts and cavuses, calluses
Various inflammatory conditions due to injury and itching of the skin, i.e. insect bites
Many of these may have very interesting marketing potentials, such as:
Osteoarthritis,
Various sports related injuries, tennis elbow, golf elbow muscle pains and tendonitis,
Swelling and muscle pains due to injuries or accidents, i.e. sprains or broken bones
Wound healing due to burns, cuts or herpes infections
Dry or chapped skin , e.g. due to diabetes
Acne and boils, in particular teenage pimples
Psoriasis, eczema and various dermatological conditions, such as contact eczema,
child eczema
Fungal infections of the skin
Various inflammatory conditions due to injury and itching of the skin, i.e. insect bites
Thus it appears that PENZIM ointment could compete with and partially replace
products such as Hydrocortisone cremes and skin products containing Salicylates,
Ketoprofens, piroxicam and such.


Acknowledgements:
This work was supported by grants from Nordisk Industrifond, NATO Collaborative
Research Grant, Icelandic Research Council and Icelandic Science Council.




7. REFERENCES
Ásgeirsson, B., Fox, J.W. & Bjarnason, J.B. (1989). Purification and
     characterization of trypsin from the poikilotherm Gadus morhua European
Journal of Biochemistry., 180, 85-94.
Ásgeirsson, B., & Bjarnason, J.B. (1991). Structural and kinetic properties           of
chymotrypsin from Atlantic cod (Gadus morhua). Comparison           with         bovine
chymotrypsin. Comparative Biochemistry and Physiology. 99B, 327-335.
Ásgeirsson, B., & Bjarnason, J.B. (1992). Properties of elastase from Atlantic cod.
Biochem.Biophys. Acta Sumitted for publication.
Bender, M.L. & Kilhleffer, J.V. (1973). Chymotrypsins Pp. 149-199 in            CRC,
Critical reviews in biochemistry.
Craik, C.S., Choo, Q.-L., Swift G.H., Quinto, C., MacDonald, R.J. & Rutter, W.J.
(1984). Structure of two related rat pancreatic genes. Journal of Biological
Chemistry 259, 14255-14264.
Gildberg, A. & Øverbø, K. (1990). Purification and characterization of pancreatic
elastase from Atlantic cod (Gadus morhua). Comparative Biochemistry and
Physiology. 97B, 775-782.
Grant, G.A., Sacchettini, J.C. & Welgus H.G. (1983). A collagenolytic          serine
protease with trypsin-like specificity from the fiddler crab Uca pugilator.
Biochemistry 22, 354-358.
Gudmundsdóttir, A., Gudmundsdóttir, E., Óskarsson, S., Bjarnason, J.B., Eakin, A.
& Craik, C.S. (1993). European Journal of Biochemistry., 217, 1091-1097.
Gudmundsdóttir, A., Óskarsson, S., Bjarnason, J.B., Eakin, A. & Craik, C.S.(1994).
Biocim . Biophys Acta, 1219, 211-214.
Hazel, J.R. & Prosser, C.L. (1974). Molecular mechanism of temperature
     compsentation in poikilotherms. Physiological reviews 54, 620-677.
Hochachka, P.W. & Somero, G.N. (1985). Biochemical Adaptation,             Princeton
University Press, Princeton, New Jersey.
Kristjánsson M.M., Gudmundsdóttir, S., Fox, J.W. & Bjarnason, J.B.(1993b). Comp.
Biochem. Physiol. 110B, No. 4, pp. 707-717.
Laskowski, M. (1955). Chymotrypsinogens and chymotrypsins.
 In: Methods in Enzymology, (Eds) Colowick, S.P. & Kaplan, N.O.            Vol.II.pp.8-
26. Academic Press Inc., New York.
Raae, A.J. & Walther, B.T. (1989). Purfication and characterization of chymotrypsin,
trypsin and elastase from cod. Comparative Biochemistry and Physiology 93B:317-
324.
Sanger, F. & Tuppy, H. (1951). The amino acid sequence in the            phenylalanyl
chain of insulin. Biochem. J. 49, 481-490.
Shotton, D.M. (1970). Elastase. In: Methods in Enzymology. (Eds)           Colowick,
S.P. & Kaplan, N.O. Vol. 19 pp. 113-140 Academic Press              Inc., New York.
Tani, T., Ohsumi, J., Mita, K. & Ikeda, S. (1982)Identification of a novel class      of
elastase isoenzyme, Human pancreatic elastase III, by               cDNA            and
     genome gene cloning. J. Biol. Chem. 263, 1231-1239.
Titani, K., Ericsson, L.H., Neurath, H. & Walsh, K.A. (1975). Amino acid sequence
of dogfish trypsin. - Biochemistry 14, 1358-1366.
Walsh, K.A. (1970). Trypsinogens and trypsins. - In Methods in           Enzymology
(Eds) Perlman, G.E. & Lorland, L. Vol. 19. pp.41-63. Academic Press Inc., New
York.
Wilcox, P.E. (1970). Chymotrypsinogens - Chymotrypsins.In        Methods           in
    Enzymology (Eds) Perlman G.E.& Lorland, L., Vol.19. pp. 64-108. Academic
Press Inc., New York.
Yoshinaka, R., Sato, M., Itoko, M., Yamashita, M. & Ikeda, S. (1985). Purification
and characterization of a collagenolytic serine   proteinase from the catfish
pancreas. - Journal of Biochemistry 99, 459-467.

				
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