A transglutaminase catalyzing an acyl transfer reaction of a .gamma.-carboxyamide group of a glutamine residue in a peptide or protein chain in the absence of Ca.sup.2+.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a novel transglutaminase and a process for 
producing a protein gelation product using the transglutaminase. 
2. Description of the Background 
Transglutaminases are enzymes which catalyse an acyl transfer reaction of a 
.gamma.-carboxyamide group of a glutamine residue in a peptide chain. 
The transglutaminases form intramolecular or intermolecular 
.epsilon.-(.gamma.-Glu)-Lys cross-linking wherein the .epsilon.-amino 
group of the lysine residue in the protein serves as the acyl receptor. 
When water functions as the acyl receptor, the transglutaminases catalyze 
deamination of glutamine residues to form glutamic acid residues. 
The gelation products of the present invention produced utilizing the novel 
transglutaminases are used as yoghurt, jelly, cheese, gel cosmetics, etc., 
including conventional gel foodstuffs and gel cosmetics. Furthermore, the 
gelation products in accordance with the present invention can be produced 
in a non-heated state and are thermally stable and therefore, can also be 
used over a wide range, such as raw materials for microcapsules, carriers 
for immobilized enzymes, etc. 
Transglutaminases hitherto known are those derived from animals. 
Transglutaminases are widely distributed in, for example, liver of the 
guinea pig [Connellan, et al., Journal of Biological Chemistry, vol. 246, 
No. 4, pages 1093-1098 (1971)] and mammal organs and blood [Folk et al., 
Advances in Enzymology, vol. 38, pages 109-191 (1973) and Folk et al., 
Advances in Protein Chemistry, vol. 31, pages 1-133 (1977)] and, 
characteristics of the enzymes have been investigated. 
Up to now, however, no report has been made on any transglutaminase derived 
from microorganisms. With respect to a process for producing gel products 
of protein using an animal-derived transglutaminase, the present inventors 
have already made investigations resulting in Published Unexamined 
Japanese Patent Application No. 149645/83. 
However, application of the animal-derived transglutaminase to industry, 
particularly, the process for producing protein gelation products involves 
defects as described below. 
It is difficult to obtain animal-derived transglutaminases at low cost and 
in large quantities. Also, there is the restriction that at least 1 unit 
of this expensive enzyme per 1 g of substrate protein and at least 2.0 wt. 
% of a substrate protein concentration are required for gelation. Further, 
the animal-derived transglutaminase is calcium (Ca.sup.2+)dependent so 
that its application is limited. 
Because of the foregoing defects, processes for producing gelation products 
using animal-derived transglutaminases are impractical.

EXAMPLES 
Example 1 
Streptoverticillium mobaraense IFO 13819 was inoculated on 200 ml of medium 
(pH 7) having a medium composition of 0.2% of polypeptone, 0.5% of 
glucose, 0.2% of dipotassium phosphate and 0.1% of magnesium sulfate 
followed by culturing at 30.degree. C. for 48 hours. The obtained seed 
culture solution was added to 20 l (pH 7) composed of 2.0% of polypeptone, 
2.0% of soluble starch "Rastagen" (trademark, manufactured by Nichiden 
Kagaku K.K.), 0.2% of dipotassium phosphate, 0.1% of magnesium sulfate, 
0.2% of yeast extract and 0.05% of defoaming and antifoaming 
polyoxyalkylene glycol "Adekanol" (trademark, manufactured by Asahi Denka 
Kogyo K.K.) followed by culturing at 30.degree. C. for 3 days. After 
filtering, 18.5 l of the culture solution was obtained. The activity was 
0.35 U/ml. 
The culture solution was adjusted with hydrochloric acid to pH of 6.5 and 
passed through a column of methacrylic porous cation-exchange resin 
"Amberlite CG-50" (trademark, manufactured by Rohm & Haas Co., Ltd.), 
which had been previously equilibrated with 0.05M phosphate buffer (pH 
6.5). By this operation, transglutaminase was adsorbed. After washing 
protein impurities out with the buffer, a density gradient of 0.05 to 0.5M 
was prepared by the buffer, through which the system was passed. The 
eluate was fractionated and recovered, and fractions having a high 
specific activity were collected. After the system was diluted to have a 
conductivity of 10 ms or less, it was passed through a column of blue 
Sepharose. By this operation, transglutaminase was adsorbed. After washing 
protein impurities out with 0.05M phosphate buffer (pH 7), a density 
gradient of 0 to 1M was prepared by sodium chloride, through which the 
system was passed. The eluate was recovered and fractions having a high 
specific activity were collected. The fractions were condensed using a UF 
6000 membrane and equilibrated with 0.05M phosphate buffer (pH 7) 
containing 0.5M sodium chloride. 
The obtained condensate was passed through a column of Sephadex G-75 
(manufactured by Pharmacia Fine Chemical Co., Ltd.) which had been 
previously equilibrated with the buffer and the buffer was caused to flow 
therethrough to fractionate the eluate. As a result, the active fraction 
eluted as a single peak. The specific activity was 625 times that of the 
culture filtrate and the recovery rate was 47%. 
Example 2 
Streptoverticillium griseocarneum IFO 12776 was cultured at 30.degree. C. 
for 3 days in a manner similar to Example 1. After filtering, 19 l of the 
culture solution was obtained. The specific activity was 0.28 U/ml. 
The enzyme was purified in a manner similar to Example 1 and a single 
enzyme was obtained by SDS disc electrophoresis. 
Example 3 
Streptoverticillium cinnamoneum sub sp. cinnamoneum IFO 12852 was cultured 
at 30.degree. C. for 3 days in a manner similar to Example 1. After 
filtering, 18.5 l of the culture solution was obtained. The specific 
activity was 0.5 U/ml. 
The enzyme was purified in a manner similar to Example 1 and a single 
enzyme was obtained by SDS disc electrophoresis. 
Example 4 
1) The BTGase (freeze dried product, specific activity of 2.50 Units/mg 
protein) prepared in Example 1 was added to 5 ml of each of 5 and 10 wt. % 
solutions or suspensions of protein foodstuffs prepared by the method 
described in Example 1 of Published Unexamined Japanese Patent Application 
No. 149645/83 or purchased, namely, (1) .alpha..sub.si -casein, (2) 
Na-caseinate, (3) soybean 11S globulin, (4) soybean 7S globulin, (5) 
soybean protein isolate "Ajipron S-2" (manufactured by Ajinomoto Co., 
Inc.), (6) water-extracted soybean protein, (7) acid-precipitated soybean 
protein, (8) soybean protein particles, (9) soybean protein micelles and 
(10) gelatin in 0.02 U per 1 mg of protein followed by shake incubation at 
55.degree. C. for an hour. After allowing to stand at room temperature, 
test tubes with samples were turned upside down, whereby gelation was 
determined by noting whether the samples drifted down or not. The results 
are shown in Table 6. 
2) Rabbit myosin was prepared as follows for use as a substrate for BTGase. 
Following the method of Perry (Perry, S. V. (1955), Methods in Enzymology, 
vol. 2, pp. 582-588, Academic Press, New York), myosin was extracted from 
25 g of rabbit skeletal muscle at 0.degree. C. for 30 minutes in a 3-fold 
amount of 0.45M KCl, 5 mM ATP-MgCl.sub.2 and 50 mM phosphate buffer (pH 6) 
and subsequently collected by dilution precipitation; myosin was dialyzed 
against 0.5M KCl and 20 mM Tris-maleate (pH 7.5) solution and centrifuged 
at 10.sup.5 .times.g for 60 minutes, and the supernatant was used as 
purified myosin. The concentration of protein was 1.5%. BTGase was added 
to the myosin under the same conditions as under 1) above to examine 
gelation ability. The results are shown in Table 6. 
3) Prawn myosin was prepared as follows for use as a substrate for BTGase. 
The shell of a fresh (live) prawn (body length of about 5 cm) was peeled 
off and the prawn flection muscle was taken out. After mincing, the mince 
was washed with ice water and homogenized under cooling in the presence of 
0.1 mM DTT and 0.1 mM PMSF. By centrifugation, actomyosin was extracted 
and separated therefrom. By further ultracentrifugation at 10.sup.5 
.times.g for 60 minutes, actin was removed to give a myosin-rich fraction. 
Further dilution precipitation and ultracentrifugation were repeated to 
give purified prawn myosin. It was found that this purified myosin was 
modified myosin because it had no Ca-ATPase activity and lost the ability 
to bind to actin. To 5 ml of this modified prawn myosin solution having a 
protein concentration of 3.6% (buffer: 0.5M KCl, 5 mM CaCl.sub.2, 25 mM 
Tris-HCl (pH 7.5) and 5 mM DTT) were added 3.6 units of BTGase. By 
immersing in a water bath at 35.degree. C., the reaction was initiated and 
carried out for 35 minutes at maximum. 
The foregoing experimental results of gelation ability are summarized in 
Table 6. 
For purposes of comparison, the results of gelation ability test with 
MTGase are also shown. The amount of MTGase added was 0.1 U per 1 mg of 
substrate protein. 
TABLE 6 
______________________________________ 
Gelation of BTGases of Various Proteins 
(test tube inversion method) 
Protein Foodstuff 
Concentration (%) 
BTGase MTGase 
______________________________________ 
.alpha..sub.S1 -Casein 
5 .smallcircle. 
.smallcircle. 
10 .smallcircle. 
.smallcircle. 
Na-Caseinate 5 .smallcircle. 
.DELTA. 
10 .smallcircle. 
.smallcircle. 
Soybean 11S globulin 
5 .smallcircle. 
.DELTA. 
10 .smallcircle. 
.smallcircle. 
Soybean 7S globulin 
5 .smallcircle. 
x 
10 .smallcircle. 
.smallcircle. 
Ajipron S-2 5 .smallcircle. 
x 
10 .smallcircle. 
.smallcircle. 
Water-extracted 
5 .smallcircle. 
x 
soybean protein 
10 .smallcircle. 
.smallcircle. 
Acid-precipitated 
5 .smallcircle. 
x 
soybean protein 
10 .smallcircle. 
.smallcircle. 
Soybean protein 
5 .smallcircle. 
x 
particles 10 .smallcircle. 
.DELTA. 
Soybean protein 
5 .DELTA. x 
micells 10 .smallcircle. 
.DELTA. 
Gelatin 5 .smallcircle. 
x 
10 .smallcircle. 
.smallcircle. 
Rabbit myosin 
1.5 .smallcircle. 
.smallcircle. 
Prawn myosin 3.6 .smallcircle. 
.smallcircle. 
______________________________________ 
Notes: 
.smallcircle.: gelled 
.DELTA.: weakly gelled 
x: remained as solution 
MTG was reacted at 37.degree. C. for an hour. 
Example 5 
0.1M Tris-HCl buffer (pH 7.6) was added to gelatin (manufactured by Nitta 
Gelatin Co., Ltd.) so as to form 5.10 wt. % solution. Gelatin was 
completely dissolved at 60.degree. C. for 3 minutes and the same BTGase as 
that of Example 4 was added to the solution in 0.02 U/mg protein. After 
thoroughly mixing, the mixture was reacted at 37.degree. C. for an hour 
and then heated in a boiling water bath for 10 minutes. Immediately 
thereafter, the condition was observed. 
For purposes of comparison, the system which was identically treated except 
for adding no BTGase was used as control. The results are shown in Table 
7. 
TABLE 7 
______________________________________ 
-BTG +BTG 
______________________________________ 
5% Gelatin x .smallcircle. 
10% Gelatin x .smallcircle. 
______________________________________ 
Notes: 
x: complete solution 
.smallcircle.: gelled condition (not dissolved even after heating) 
Example 6 
A silk protein aqueous solution was prepared as follows for use as a 
substrate for BTGase. To 100 ml of 9.3M lithium bromide (LiBr) solution 
was added 2.33 g of defatted silk yarn. When the mixture was stirred at 
40.degree. C. overnight, the silk yarn was solubilized. Suction filtration 
of the solution and dialysis to water were carried out to give a crude 
silk protein aqueous solution (approximately 2 wt. %). The same BTGase as 
used in Example 4 was previously charged in test tubes in final 
concentrations of 0.01 U, 0.02 U and 0.04 U/mg protein, respectively, and 
the silk protein aqueous solution was gently added thereto in order to 
avoid gelation due to shaking. For control, a BTGase-free solution was 
also prepared. After allowing each test tube to stand at room temperature 
overnight, the state of the sample in each test tube was observed to give 
the results shown in Table 8. 
TABLE 8 
______________________________________ 
Sample Condition 
______________________________________ 
Silk protein aqueous solution - BTG 
x 
Silk protein aqueous solution + 
.smallcircle. 
BTG (0.01 U/mg protein) 
Silk protein aqueous solution + 
.smallcircle. 
BTG (0.02 U/mg protein) 
Silk protein aqueous solution + 
.smallcircle. 
BTG (0.04 U/mg protein) 
______________________________________ 
Notes: 
x: Fell down by inverting test tube. Transparent solutionlike state. 
.smallcircle.: Did not fall down even by inverting test tube. Turbid 
gellike state. 
Example 7 
Commercially available milk (crude protein, 2.9%) was condensed to 
approximately 5-fold (crude protein, 14.5%) under reduced pressure. To 1 
liter of the thus obtained condensed milk were added 2 units of BTGase 
shown in Example 4. The mixture was agitated to incubate at 55.degree. C. 
for 30 minutes. The formed gel-like product was heated at 80.degree. to 
90.degree. C. for 20 minutes to inactivate the remaining enzyme. It was 
then cooled to give a pudding-like gel foodstuff. A similar gel-like 
product could be obtained even by adding sugar thereto up to approximately 
10%, if necessary. 
Example 8 
Commercially available milk (crude protein, 2.9%; oils and fats, 32.%; 
moisture content, 89%) was condensed to approximately 5-fold under reduced 
pressure to give condensed milk (approximately 10 liters). To the thus 
obtained condensed milk was added 100 ml of 30% glucono-delta-lactone 
solution. After rapidly mixing them, it was confirmed that the pH was 6.0 
or greater. Then, 100 units of BTGase shown in Example 4 were added. The 
mixture was agitated and settled in an incubator at 45.degree. C. for 45 
minutes to cause gelation. Thereafter the gel was heated to 80.degree. to 
95.degree. C. with caution so that the gel was not broken, whereby 
inactivation of BTGase and decomposition of glucono-delta-lactone to 
gluconic acid were carried out to adjust the pH of the gel to 4 to 5. 
After cooling, the card-like gel was cut into a square of approximately 8 
cm and rendered a salt concentration of approximately 2% by the acid salt 
method. Starter of Penicillium caseicolum was inoculated thereon and 
ripened at 15.degree. C. for 3 weeks at RH of 85% to give cheese. 
In the case of using no glucono-delta-lactone, Lactobacillus acidophillus 
was added and after gelation by BTGase, fermentation was performed at 
40.degree. C. for 2 to 5 hours. Also in this case, similar cheese was 
obtained. 
The cheese obtained by this process can be produced without using expensive 
calf rennet and physical properties thereof were considerably soft 
elasticity, resulting in good quality. 
Example 9 
Condensed milk (1 liter) of Example 8 was cooled to approximately 5.degree. 
C. and a starter composed of Streptococcus thermophillus was added thereto 
and immediately mixed with each other. 1 unit of BTGase as used in Example 
4 was added to the mixture. The system was settled in an incubator at 
35.degree. C. for an hour to cause gelation. Then, the gel temperature was 
raised to 50.degree. C. followed by heating for 40 minutes. After an acid 
was formed by S. thermophillus and flavor was increased, the system was 
further heated at 75.degree. to 85.degree. C. to inactivate BTGase. Upon 
cooling, a yoghurt-like foodstuff having mild acid taste and excellent 
quality was obtained. 
Example 10 
Commercially available soybean milk (manufactured by Meiji Milk Products 
Co., Ltd., Sunglow Soybean Milk, crude protein, 3.1%) was condensed to 
approximately 2.5-fold under reduced pressure and cooled to 20.degree. C. 
or lower to give condensed milk (crude protein, 7.75%). To 1 liter of the 
condensed milk were added 4 units of BTGase shown in Example 4. The 
mixture was charged in a plastic container. After the container was 
covered and sealed, it was heated on a hot water bath of 55.degree. C. for 
30 minutes to cause enzyme reaction and gelation. Thereafter, heating was 
performed using a high frequency dielectric heater (electronic oven, 2450 
MHz, wavelength of 12 cm). Softer bean curd or tofu-like gel free from 
collapse of shape and having good quality was obtained as compared to 
silk-strained tofu or cotton-strained tofu. 
Example 11 
In approximately 20 kg of water was immersed 6.5 kg of whole soybeans, in 
which water was thoroughly absorbed at normal temperature overnight to 
swell the soybeans. While adding water thereto, the soybeans were ground 
with a crusher to obtain "go" or a soybean suspension. 25 kg of water was 
added thereto to dilute the go. A small quantity of a defoaming agent was 
added thereto and the mixture was transferred to a boiling vessel. Steam 
was blown into the vessel to heat it. It is preferred that heating 
conditions of elevating to 100.degree. C. over 5 minutes and then keeping 
the temperature for 3 to 5 minutes be adopted. After boiling, tofu lees 
was removed with a tofu lees squeezer to give 30 kg of condensed soy milk 
(crude protein, 7.0%; oil, 8.1% and moisture content, 75%). To the soy 
milk were added 200 units of BTGase shown in Example 4 and the mixture was 
immediately packed in a casing tube (vinylidene chloride-made tube) 
followed by heating at 37.degree. C. for 30 minutes. Then, the tube was 
transferred onto a hot water bath of 90.degree. C. or more and heated (30 
to 60 minutes). Tofu-like gel was obtained in running water. 
Example 12 
Kamaboko or steamed fish paste was prepared by way of experimentation, 
according to a recipe shown in Table 9. Physical properties were measured 
with a rheometer (manufactured by Fudo Chemical Co., Ltd.) and 
organoleptic evaluation (n=10) was performed. The addition amount of 
BTGase was 20 units per 1 g of dry fish paste. The enzyme reaction was 
carried out at 34.degree. C. for 2 hours as in a seating step of 
BTGase-free control. After completion of the reaction, heating was 
performed at 85.degree. C. for 30 minutes to make a product. 
TABLE 9 
______________________________________ 
Control 
BTGase Added 
(%) (%) 
______________________________________ 
Fish paste 66.9 66.9 
grade C 
Potato starch 6.7 6.7 
Sweet sake 2.0 2.0 
Sugar 2.0 2.0 
Table salt 1.7 1.7 
MSG 0.7 0.7 
Water 20.0 19.3 
BTGase 0 0.2 
______________________________________ 
The results of the measurement of physical properties and the organoleptic 
evaluation are shown in Tables 10 and 11, respectively. 
TABLE 10 
______________________________________ 
Breaking Strength (g) 
Strain (%) 
______________________________________ 
Control 454 .+-. 50 44.3 .+-. 2.3 
(BTGase free) 
BTGase 804 .+-. 58 46.3 .+-. 1.3 
______________________________________ 
TABLE 11 
__________________________________________________________________________ 
Texture Profile 
SomeHard toSome 
Veryor lesssay whichor lessVery 
-2-10+1+2 
__________________________________________________________________________ 
1. 2. 3. 4. 
Hardness Crispy texture Collapse Tackiness 
Soft Bad crisp Hard to collapse Tacky 
##STR1## Hard Good crisp Easy to collapse 
on- tacky 
5. 6. 7. 8. 
Elasticity Adhesive- ness Smoothness Texture as a whole 
Non- elastic Easy to adhere to teeth Sandy Not preferred 
##STR2## Elastic Hard to adhere teeth 
Smooth Pre- ferred 
(when control evaluation point was made 
__________________________________________________________________________ 
0) 
As described above, it was found that Kamaboko or fish paste which was 
prepared, by way of experiment, by adding BTGase thereto showed an 
increased breaking strength and provided a preferred texture, as compared 
to the control. 
Example 13 
Sausage was prepared by way of experimentation, according to a recipe shown 
in Table 12. Physical properties were measured with a rheometer 
(manufactured by Yamaden Co., Ltd.) and organoleptic evaluation (n=10) was 
performed. The amount of BTGase added was 1 unit per 1 g of dry pork. The 
enzyme reaction was carried out at 55.degree. C. for 2 hours. After 
completion of the reaction, heating was performed at 80.degree. C. for 30 
minutes to make a product. As a control, BTGase-free product was used. 
TABLE 12 
______________________________________ 
Control 
BTGase Added 
(%) (%) 
______________________________________ 
Pork shank 68.4 68.4 
Table salt 1.5 1.5 
Sodium nitrite 0.02 0.02 
Na ascorbate 0.06 0.06 
Sugar 2.1 2.1 
MSG 0.4 0.4 
White pepper 0.3 0.3 
Water 27.22 27.20 
BTGase 0 0.02 
______________________________________ 
The results of the measurement of physical properties and the organoleptic 
evaluation are shown in Tables 13 and 14, respectively. 
TABLE 13 
______________________________________ 
Elasticity Viscosity Coefficient 
(.times. 10.sup.5 dyn.cm.sup.-2) 
(.times. 10.sup.9 dyn.sec.cm.sup.-2) 
______________________________________ 
Control 4.73 1.48 
BTGase Added 
5.83 1.92 
______________________________________ 
TABLE 14 
__________________________________________________________________________ 
Texture Profile 
SomeHard toSome 
Veryor lesssay whichor lessVery 
Item evaluated -2-10+1+2 
__________________________________________________________________________ 
1. 2. 3. 4. 
Hardness Crispy texture Collapse Tackiness 
Soft Bad crisp Hard to collapse Tacky 
##STR3## Hard Good crisp Easy to collapse 
on- tacky 
5. 6. 7. 8. 
Elasticity Adhesive- ness Smoothness Texture as a whole 
Non- elastic Easy to adhere to teeth Sandy Not preferred 
##STR4## Elastic Hard to adhere teeth 
Smooth Pre- ferred 
(when control evaluation point was made 
__________________________________________________________________________ 
0) 
As described above, it was found that sausage which was prepared, by way of 
experimentation, by adding BTGase thereto provided a highly viscoelastic 
and preferable texture because of the gel forming ability of BTGase, as 
compared to the control. 
Example 14 
Whipping cream was prepared by way of experimentation, according to a 
recipe shown in Table 15 and a squeezing property was evaluated. The 
amount of BTGase added was 1 unit per 1 g of dry sodium caseinate. 
Whipping operation was performed using all purpose mixer (manufactured by 
San-Ei Seisakusho K.K.) at 7.degree. to 9.degree. C. As a control, 
BTGase-free product was used. 
TABLE 15 
______________________________________ 
Control 
BTGase Added 
(%) (%) 
______________________________________ 
Palm oil 25.0 25.0 
Sodium caseinate 
5.0 5.0 
Monoglyceride 0.3 0.3 
Water 69.7 69.7 
BTGase -- 0.005 
______________________________________ 
Using each whipping cream, a flower pattern was drawn on a glass plate and 
the condition was observed. With the whipping cream with BTGase added, 
imitation flowers having sharp lines could be drawn. 
Example 15 
Ice cream was prepared by way of experimentation, according to a recipe 
shown in Table 16. Change in shape was observed when allowed to stand at 
room temperature and melt down resistance was evaluated. The amount of 
BTGase added was 25 units per 1 g of dry skim milk and the enzyme reaction 
was carried out at a sterilizing step (68.degree. C., 30 minutes) of ice 
cream mix. After sterilizing, the ice cream mix was aged at 5.degree. C. 
overnight and then frozen at an ice cream temperature of -2.degree. to 
4.degree. C. using an ice freezer (manufactured by Mitsubishi Heavy 
Industries, Ltd.) up to overrun of 90%. After packing in a corn, it was 
hardened at -40.degree. C. to make a product. As a control, ice cream 
prepared by way of experimentation in a similar manner except for adding 
no BTGase was used. 
TABLE 16 
______________________________________ 
Control 
BTGase Added 
(%) (%) 
______________________________________ 
Palm oil 5.0 5.0 
Skim milk 8.0 8.0 
Sugar 13.0 13.0 
Thick malt syrup 
6.0 6.0 
Gujar gum 0.1 0.1 
Carrageenan 0.1 0.1 
Locust bean gum 
0.1 0.1 
Monoglyceride 0.3 0.3 
Vanilla essence 
0.1 0.1 
Water 67.3 67.1 
BTGase -- 0.2 
______________________________________ 
The control collapsed in 15 minutes after allowing to stand at room 
temperature but the ice cream with BTGase added caused no collapse even 
after 30 minutes or longer and provided smooth touch to the tongue as in 
the control. 
Example 16 
A necessary amount of oxhide-derived atelo-collagen powders (manufactured 
by Kouken K.K.) were taken in a test tube and 2 ml of 0.1M Tris-HCl buffer 
(pH 7.5) was added thereto. After maintaining in a water bath at 
55.degree. C. for 15 minutes, the mixture was stirred to prepare 3 to 10% 
atelo-collagen solutions. Before the solutions of high concentration were 
gelled due to cooling, BTGase was added thereto in 0.05 U/mg protein 
followed by incubation at 55.degree. C. for 60 minutes. As a control for 
the all samples, 10% atelo-collagen solution with no BTGase added was also 
incubated likewise. Immediately after completion of the incubation, the 
system was allowed to stand at room temperature for 60 minutes. Then the 
system was further kept in a water bath of 100.degree. C. for 15 minutes 
and then the condition in the test tube was observed. The results are 
shown in Table 17. 
TABLE 17 
______________________________________ 
Immediately 
After After 
After Allowing to 
Heat- 
Completion 
Stand for ing at 
of Incubation 
60 Minutes 100.degree. C. 
______________________________________ 
3% Solution + BTGase 
.smallcircle. 
.smallcircle. 
.smallcircle. 
5% Solution + BTGase 
.smallcircle. 
.smallcircle. 
.smallcircle. 
10% Solution + BTGase 
.smallcircle. 
.smallcircle. 
.smallcircle. 
10% Solution - BTGase 
x .smallcircle. 
x 
______________________________________ 
Key: 
.smallcircle.: gelled 
x: not gelled 
Example 17 
Frozen fresh krill, 1 kg, (manufactured by Taiyo Fishery Co., Ltd.) was 
minced with a frozen cutter and, 30 g of table salt, 100 g of sorbitol 
(manufactured by Ajinomoto, Inc.), 50 g of Shin-Neriaji (manufactured by 
Ajinomoto, Inc.), 40 g of sweet sake and 50 g of potato starch were added 
thereto. Further, 200 units of BTGase dissolved in 300 ml of chilled water 
were added to the mixture followed by kneading with a cutter manufactured 
by Stephan Co., Ltd. for approximately 6 minutes. The temperature 
immediately after the kneading was controlled to 5.degree. to 6.degree. C. 
This krill paste was packed in a casing tube made of vinylidene chloride 
(manufactured by Kureha Chemical Industry Co., Ltd.). After incubating at 
50.degree. C. for an hour, it was heated in a boiling hot water bath for 
25 hours. After heating, it was cooled by running water and physical 
properties thereof were then measured. Namely, the sample was cut into 
pieces having a thickness of 3 cm. Using a spherical plunger having a 
diameter of 7 mm, breaking strength was determined by measuring with a 
rheometer manufactured by Fudo Chemical Industry Co., Ltd. The results are 
shown in Table 18. As a control, a sample was prepared in a similar manner 
except for using BTGase denatured to inactivate by previously heating to 
high temperatures. 
TABLE 18 
______________________________________ 
Breaking Strength (g/cm.sup.2) 
______________________________________ 
Control 286 
BTGase Added 442 
______________________________________ 
That is, it was noted that Kamaboko or krill paste prepared by way of 
experimentation by adding BTGase thereto showed much higher breaking 
strength than the control obtained using the previously inactivated 
BTGase. 
Example 18 
Japanese noodle was prepared in a recipe shown in Table 19. Organoleptic 
evaluation (n=15) and measurement of physical properties were performed. 
TABLE 19 
______________________________________ 
Control 
BTGase Added 
(%) (%) 
______________________________________ 
Strong flour 36.4 36.4 
Weak flour 36.4 36.4 
Table salt 0.5 0.5 
Water 26.7 26.7 
BTGase -- 0.04 
______________________________________ 
The addition of BTGase was 1 U per 1 g of protein. After the enzyme 
reaction was carried out at room temperature for 2 hours, noodles were 
made. The organoleptic evaluation and the measurement of physical 
properties were conducted using Japanese noodles boiled for 12 minutes. 
The length of the noodle used for the measurement of physical properties 
was 7 cm. A tensile test was carried out using a rheometer (manufactured 
by Fudo Chemical Industry Co., Ltd.), and breaking strength and elongation 
to break were measured. The results are shown in Tables 20 and 21. 
TABLE 20 
__________________________________________________________________________ 
Texture Profile 
(evaluation point of the sample added with BTGase when the control was 
made 0) 
SomeHard toSome 
Veryor lesssay whichor lessVery 
Item evaluated -2-10+1+2 
__________________________________________________________________________ 
1. 2. 3. 4. 
Hardness Crispy texture Collapse Tackiness 
Soft Bad crisp Hard to collapse Tacky 
##STR5## Hard Good crisp Easy to collapse 
on- tacky 
5. 6. 7. 8. 
Elasticity Adhesive- ness Smoothness Texture as a whole 
Non- elastic Easy to adhere to teeth Sandy Not preferred 
##STR6## Elastic Hard to adhere teeth 
Smooth Pre- ferred 
__________________________________________________________________________ 
TABLE 21 
______________________________________ 
Breaking 
Strength (g) 
Elongation (%) 
______________________________________ 
Control 706 .+-. 23 
64 .+-. 5 
(BTGase free) 
BTGase 885 .+-. 48** 
51 .+-. 13 
______________________________________ 
n = 10 **Significant difference was noted with a significance level of 1% 
 
The results of the organoleptic evaluation were consistent with those of 
the measurement of physical properties, and it was found that by adding 
BTGase, cross-linking structure between gluten molecules was formed and 
noodles having a firm texture similar to special Japanese noodles of the 
someday district. 
Example 19 
Spaghetti was prepared according to a recipe shown in Table 22. 
Organoleptic evaluation (n=15) and measurement of physical properties were 
performed. 
TABLE 22 
______________________________________ 
Control 
BTGase Added 
(%) (%) 
______________________________________ 
Strong flour 73.7 73.7 
Table salt 0.6 0.6 
Water 25.7 25.7 
BTGase -- 0.04 
______________________________________ 
The addition of BTGase was 1 U per 1 g of protein. After the enzyme 
reaction was carried out at room temperature for 2 hours, spaghetti was 
made with a pasta machine (manufactured by Lucky Coffee Maker Co., Ltd.). 
The organoleptic evaluation and the measurement of physical properties 
were conducted using spaghetti boiled for 5 minutes and 30 seconds. The 
length of the spaghetti used for the measurement of physical properties 
was 7 cm. A tensile test was carried out using a rheometer (manufactured 
by Fudo Chemical Industry Co., Ltd.) and, breaking strength and elongation 
to break were measured. The results are shown in Tables 23 and 24. 
TABLE 23 
__________________________________________________________________________ 
Texture Profile 
(evaluation point of the sample added with BTGase when the control was 
made 0) 
SomeHard toSome 
Veryor lesssay whichor lessVery 
Item evaluated -2-10+1+2 
__________________________________________________________________________ 
1. 2. 3. 4. 
Hardness Crispy texture Collapse Tackiness 
Soft Bad crisp Hard to collapse Tacky 
##STR7## Hard Good crisp Easy to collapse 
on- tacky 
5. 6. 7. 8. 
Elasticity Adhesive- ness Smoothness Texture as a whole 
Non- elastic Easy to adhere to teeth Sandy Not preferred 
##STR8## Elastic Hard to adhere teeth 
Smooth Pre- ferred 
__________________________________________________________________________ 
TABLE 24 
______________________________________ 
Breaking 
Strength (g) 
Elongation (%) 
______________________________________ 
Control 29 .+-. 2 77 .+-. 7 
BTGase 27 .+-. 1 54 .+-. 9** 
______________________________________ 
n = 10 **Significant difference was noted with significance level of 1%. 
Even though BTGase acted on spaghetti, no significant change occurred in 
the texture as shown in Table 23 but powders mixed at the preparation step 
were light and operability was greatly improved in that feeding into a 
screw was smooth and heat generation in a cylinder was minimized, etc. 
Effects of the Invention 
Microorganism-derived BTGase of the present invention can be supplied at 
low cost and can be easily purified and therefore, is highly practical. 
It is also advantageous that by the use of a BTGase according to this 
invention, gelation products having excellent quality can be produced 
using a very low enzyme (BTGase) concentration and in a very low substrate 
concentration in the absence of calcium or even in the presence of 
calcium. 
Obviously, numerous modifications and variations of the present invention 
are possible in light of the above teachings. It is therefore to be 
understood that within the scope of the appended claims, the invention may 
be practiced otherwise than as specifically described herein.