llllllllllllllIllllllllllllllllllllllIllllllllllllllIllllllllllllllllllllll

US005252469A
United States Patent [191 [11] Patent Number: 5,252,469
Andou et al. [45] Date of Patent: Oct. 12, 1993

[54] PROCESS FOR PRODUCING A

TRANSGLUTAMINASE DERIVED FROM OTHER PUBLICATIONS
STREPTOMYCES Goodfellow et al. (1984) The Biology of the Actino
mycetes, pp. 16-17, Acad. Press, N.Y.
[75] Inventors: Hiroyasu Andou; Akin Matsuura; ATCC Catalog of Bacteria & Bacteriophages (1989)
- Susumu Hirose, all of Aichi, Japan 17th Ed., pp. 229, 246-247.
[73] Assignee: Amano Pharmaceutical Co., Ltd., Primary Examiner-Douglas W. Robinson
Aichi, Japan Assistant Examiner-Jon P. Weber
Attorney, Agent, or Firm-Sughrue, Mion, Zinn,
[21] Appl. No.: 750,160 Macpealc 8c Seas '
[22] Filed: Aug. 26, 1991 [57] ABSTRACI
[30] Foreign Application Priority Data A process for producing a transglutaminase which com
prises (l) culturing a microorganism having the identi
Aug. 27, 1990 [JP] Japan ................................ .. 2-226076 t'ying characteristics of a microorganism selected from
the group consisting of Streptomyces sp. No. 83, depos
[51] Int. Cl.5 ...................... .. C12P 21/04; C12N 9/ 10; ited as FERM BP-3505, Streptomyces Iavendulae No.
C12N 9/78 466, deposited as FERM BP-3506, and mutants thereof,
[52] US. Cl. ................................. .. 435/711; 435/193; wherein the microorganism is capable of producing a
435/227; 435/71.1 transglutaminase, wherein the transglutaminase is an
[58] Field of Search ..................... .. 435/227, 193, 71.2 enzyme which catalyzes an acyl transfer reaction of a
[56] References Cited 'y-carboxyamide group of a glutamine residue in a pep
tide or protein chain in the presence or absence of Ca1+
U.S. PATENT DOCUMENTS +and wherein the transglutaminase has an isoelectric
5,055,310 10/1991 Nonaka et a1. ...................... .. 426/46 point of 10 or 6.8, respectively, and is inhibited by Pb
5,156,956 10/1992 Motoki et a1. ................... .. 435/681 ions, and (2) recovering the transglutaminase from the
culture obtained is disclosed.
FOREIGN PATENT DOCUMENTS
0379606 8/1990 European Pat. Off. . 4- Claims, 4 Drawing Sheets
US. Patent Oct. 12, 1993 Sheet 1 of 4 5,252,469

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US. Patent Oct. 12,1993 Sheet 2 of 4 5,252,469

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US. Patent Oct. 12, 1993 Sheet 3 of 4 5,252,469

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US. Patent 0a. 12, 1993 Sheet 4 of 4 5,252,469

FIG]

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TEMPERLTURE(C)
 5,252,469
I 2
due in a peptide or protein chain in the presence and
PROCESS FOR PRODUCING A absence of Ca2+. The present invention is based on
TRANSGLU'IAMINASE DERIVED FROM these ?ndings.
STREPTOMYCES It is therefore one object of the present invention to
provide a process for producing a transglutaminase
FIELD OF THE INVENTION which is free from problems such as quantity of supply,
The present invention relates to a process for produc cost/performance, and ease of puri?cation.
ing a transglutaminase derived from the genus Strepto It is another object of the present invention to pro
myces, and more precisely, to a process for producing a vide a process for producing a transglutaminase which
transglutaminase which comprises culturing a microor has great practicality since no calcium is required for
ganism belonging to the genus Streptomyces, which is the reaction catalyzed by the transglutaminase.
capable of producing a transglutaminase and harvesting The above and other objects, which will become
. the transglutaminase from the culture obtained. more readily apparent hereinafter, have been achieved
BACKGROUND OF THE INVENTION by a process for producing a transglutaminase which
comprises culturing a microorganism belonging to the
Transglutaminases are enzymes which catalyze an genus Streptomyces, which is capable of producing a
acyl transfer reaction of a 'y-carboxyamide group of a transglutaminase and harvesting the transglutaminase
glutamine residue in a peptide or protein chain. from the culture obtained.
The transglutaminases form intramolecular or inter
molecular e-(y-Glu)-Lys cross-linking wherein the e 20 BRIEF DESCRIPTION OF THE DRAWINGS
amino group of the lysine residue in the protein serves
as the acyl receptor. When water functions as the acyl A more complete appreciation of the invention and
receptor, the transglutaminases catalyze deamination of many of the attendant advantages thereof will be
glutamine residues to form glutamic acid residues. readily obtained as the invention becomes better under
The protein gelation products obtained utilizing the 2 5 stood by reference to the following detailed description
transglutaminases according to the present invention when considered in connection with the accompanying
are used as yoghurt, jelly, cheese, gel cosmetics, etc., drawing, wherein: .
including conventional gel foodstuffs and gel cosmetics. FIGS. 1, 2, 3 and 4 show an optimum pH curv
In the animal-derived transglutaminase, there are optimum temperature curve, pH stability curve and
hitherto known, the transglutaminases which are 30 temperature stability curve of Enzyme No. 83 of the
widely distributed in, for example, liver of the guinea present invention, respectively. FIGS. 5, 6, 7 and 8
pig (Connellan, et al., Journal of Biological Chemistry, show an optimum pH curve, optimum temperature
Vol. 246, No. 4, pages 1093-4098 (1971)) and mammal curve, pH stability curve and temperature stability
organs and blood (Folk et al., Advances in Enzymol curve of Enzyme No. 466 of the present invention,
ogy, Vol. 38, pages 109-191 (1973) and Folk et al., 3 5 respectively.
Advances in Protein Chemistry, Vol. 31, page l-133
(1977)) and characteristics of the enzymes have been DETAILED DESCRIPTION OF THE
investigated. INVENTION
However, application of the animal-derived transg As speci?c examples of the microorganisms belong~
lutaminase to industry, particularly, the process for 40 ing to the genus Streptomyces which can be used in the
producing protein gelation products involves defects as present invention, the following Streptomyces sp. No.
described below. 83 (hereafter, referred to as Strain No. 83) and Strepto
It is difficult to obtain animal~derived transglutami myces Iavendulae No. 466 (hereafter, referred to as
nases at low cost and in large quantities. Also, there is Strain No. 466) which the present inventors isolated
the restriction that at least 1 unit of this expensive en 4 5 from soil samples may be mentioned.
zyme and at least 2.0 wt. % of a substrate protein con The bacteriological characteristics of these new
centration per 1 g of substrate protein are required for strains are set forth below. The experimental proce
gelation. Further, the animal-derived transglutaminase dures used for taxonomical identi?cation were those
is calcium (Ca2+)-dependent so that its application is
limited. 50
described in Experimental Methods for Identi?cation of
Because of the foregoing defects, processes for pro Actinomycetes, Japan Society for Actinomyces Re
ducing gelation products using animal-derived transg search (1985), Classification and Identi?cation of Mi
lutaminases are impractical. croorganisms, Gakkai Shuppan Center (1975), and Ex
Also, in view of the above difficulties of applying perimental Methods for Chemical Classi?cation of Mi
animal-derived transglutaminases in an industrial set 5 5 croorganisms, Gakkai Shuppan Center (1982).
ting, a microorganism-derived transglutaminase which As references for classi?cation and identi?cation,
is produced by the genus Streptoverticillium has previ Bergeys Manual of Determinative Bacteriology 8th
ously been proposed by the present inventors in Euro Ed. (1974), Determinative Bacteriology 7th Ed. and 8th
pean Patent Publication No. 0 379 606Al. Ed., International Journal of Systematic Bacteriology
19, (1969) and 28 (1978), and Experimental Methods for
SUMMARY OF THE INVENTION Identification of Actinomycetes (1985) were used. As to
As a result of farther extensive investigation which colors, .115 Standard Color Cards, Japan Standards
has been made in search of a broad spectrum of micro Association (1972), were referred to.
organisms for use in the production of a transglutami
nase, it has been found that the microoganisms belong III Bacteriological characteristics of Strain No. 83 and
ing to the genus Streptomyces are capable of producing Strain No. 466 a. Morphologic characteristics
transglutaminases which catalyze an acyl transfer reac The morphologic characteristics are shown in Table
tion of the 'y-carboxyamide group of a glutamine resi 1.
 5,252,469
3 4
TABLE 1 ' TABLE l-continued
Morphologic Characteristics Strain No. 83 Strain No. 466 Morphologic Characteristics Strain No. 83 Strain No. 466
SErulated mycelium Position on aerial and on vegetative
Mode of branching monopodial monopodial 5 vegeuflwe myceha
Spore morphology straight (R), ?exuous (F), _ _ myuh?
open loops straight (R) W
(RA) Growth zigzag zigzag
5 "S m
Number 5 50 5- 10 Growth good poor
Spore wall ornamentation smooth smooth 10 Mode of branching radial radial
Shape oblong~oval oblong~oval S?rangiophore
Size, p. 0.8 X 0.8 1.6 0.6 1.0 X Lengm shun hon
_ _ 1-0 _ 1'6 Branching dichotomous
Motility none none
Sporangium none none
Vegetative mycelium 15
Fragmentation not fragmented not fragmented b. Cultural characteristics in terms of growth and color
Sclerotium
The cultural characteristics in terms of growth and
Formation
:lx)(ban'hke " color of Strain No. 83 and Strain No. 466 on various
Sarangjophore _ media (incubated at 28 C. and observed for 30 days) are
set forth in Tables 2 and 3, respectively.
TABLE 2
(Cultural Characteristics of Strain No. 83)
Vegetative Mycelium, Aerial Mycelium, Reverse Color of Diffusible
Medium Growth and Color Growth and Color Vegetative Myeelium Pigment
Sucrose nitrate agar (+) (+) Ivory none
colorless reddish brown
Glycerin asparagine (+ +) (+ +) light yellow none
agar (ISP No. 5) colorless white
Starch inorganic salt (+ +) (+ + +) yellow none
agar (ISP No. 4) reddish brown (5Y 9/4)
(SYR 2/8)
Tyrosine agar (modi?ed (+ +) (+ +) reddish brown none
Okanishi medium) white (2.5YR 6/3)
Yeast molt agar (+ + +) (+ + +) yellow none
(ISP No. 2) colorless Reddish brown (2.5Y 8/6)
(5R 6/3)
Bennets agar (+ +) (+) yellow none
white~reddish (5Y 8/4)
brown
(5R 8/3)
Peptone yeast iron (+ +) (i) yellowish brown none
agar (ISP No. 6) brown (lOYR 6/4)
(2.5Y 4/4)
Peptone yeast (+ + +) (+ + +) yellow none
starch agar colorless reddish brown (2.5Y 8/6)
(5R 6/3)
t: Sparse
+ - + + +: Poor to luxuriant

TABLE 3
(Cultural Characteristics of Strain No. 466)
Vegetative Myceliurn, Aerial Mycelium, Reverse Color of Di?'usible
Medium Growth and Color Growth and Color Vegetative Mycelium Pigment
Sucrose nitrate agar (+) white colorless none
colorless
Glycerin asparagine (+ +) (+) scarlet light
agar (ISP No. 5) scarlet pinkish (2.5K 6/8) yellowish
(2.5K 6/8) white brown
Starch inorganic salt (+ +) (+) yellowish brown none
agar (ISP No. 4) scarlet pinkish (5Y 8/2)
(center white
Tyrosine agar (modi?ed (+ +) (+) brown brown
Okanish medium brown (2.5YR 3/2)
Nutrient agar (+) () brown brown
light brown (2.5YR 4/4)
Yeast molt agar (+ +) (i) (7.5Y 6/5) light
(ISP No. 2) colorless yellowish
brown
Bennet's agar (+ +) (i) none
scarlet
Peptone yeast iron (+ +) () yellowish brown none
agar (ISP No. 6) yellowish brown colorless (10Y 7/4)
(IOY 7/4)
Peptone yeast (+ +) (+) scarlet light
starch agar colorless~ pinkish (7.5Y 5/10) yellowish
 5,252,469
5 6
TABLE 3-continued
(Cultural Characteristics of Strain No. 466)
Vegetative Mycelium, Aerial Mycelium, Reverse Color of Diffusible
Medium Growth and Color Growth and Color Vegetative Mycelium Pigment
scarlet white brown
:t: Sparse
+-+++:Poor\o luxuriant

TABLE S-continued
c. Physiologic characteristics Qridham & Gottlieb agar medium)
Strain No. 83 Strain No. 466
The temperature range for growth and other charac
teristics such as gelatin liquefaction, starch hydrolysis, Starch +++ +++
skin milk coagulation/peptonization, melanoid pigment + + + -- +2 Strongly utilized~utili1ed
15 i: doubtfully utilized
production and hydrogen sul?de production of the -: not utilized
respective strains are shown in Table 4. The carbon
source assimilation spectrum (Pn'dharn & Gottlieb basal Thus, these strains contain diaminopimelic acid in the
agar) of each strain is shown in Table 5. cell wall and LL-diaminopimelic acid intracellularly
d. Chemical characteristics 20 but did not contain. meso-diaminopimelic acid.
The results of analysis for cell wall amino acid com (1) Identi?cation of Strain No. 83
position and intracellular diaminopirnelic acid are Excepting the rare occurrence of open loops (RA),
shown in Table 4. the spore chains are predominantly linear and markably
TABLE 4 25 elongated (arthrospore type chains of 50-80 spores),
Strain No. 83 Strain No. 466 forming a ball-like mass depending on media. The strain
Temperature range for growth 15-37 15-39
produces abundant vegetative hyphae on yeast extract
('C-) containing media (e.g., ISP No. 2 medium), which are
Optimal temperature for 30-34 32-37 characterized by being velvety or downy and azuki-col
growth ('C.) 30 ored (reddish brown). The existence of LL-diaminopi
Hydrolysis of starch melic acid and the absence of meso-diaminopimelic acid
Coagulation of skim milk
Peptonization of skim milk
Production of melanoid
pigment
(Okanishi method)
|+ +a suggest that it belongs to cell wall type I. As to sugars,
galactose and rhamnose were detected but neither arab
inose nor xylose was found. These and other character
35 istics such as fragmentation of hyphae, spore chain
Production of hydrogen sul?de not produced not produced
Liquefaction of gelatin negative negative morphology, Spore motility, aerial mycelium, whorl
Reduction of nitrate (ISP positive negative formation, acid fastness and catalase production sug
No. 6) gested that the strain belongs to the genus.
NaCl tolerance (peptone yeast
extract starch) 40 Streptomyces
4% growth growth
7% no growth no growth Meanwhile, the carbon assimilation spectrum of the
Catalase produced produced strain is relatively narrow, with starch, glucose, salicin,
Cell wall analysis rhamnose and fructose (weak) only being assimilated.
LL-diaminopimelic acid present present The above and other characteristics of the strain sug
meso-DAP absent absent
Total cell sugar
45 gested that it is akin to Streptomyces cinnamonenois,
Rharn, Gal, Rib , Rib
Cell well amino acids Lys, Gly, Asp Lys, Gly, Asp Streptomyces nojiriensis and Streptomyces diastaticus.
Cell wall sugar Rharn, Gal none However, the strain differentiates itself clearly from the
Gram's stain + + above-mentioned species in that it produces melanoid
Acid fastness none none
Dissolution of calcium malate
pigments and does not utilize rhamnose. Therefore, the
++ +
50 strain was identi?ed to belong to a new species of the
Rham: rhamnose
Gal: galactose genus Streptomyces and designated Streptomyces sp.
lib: Riboae No. 83.
Lys: lysine
Gly: glycine (2) Identi?cation of Strain No. 466
Asp; aspartic acid
55 The aerial mycelium is usually so sparse that it looks
TABLE 5
like the vegetative mycelium exposed on the surface. Its
color is scarlet. The colony surface is radial, ?lmy'and
(Pridham 8r Gottlieb agar medium! hard, with hyphae characteristically extending well into
Strain No. 83 Strain No. 466 the agar. As to spore chain morphology, comparatively
Glucose ++ ++ 60 short chains of arthrospores (5-10 spores) are produced
D-Xylose on Pridham-Gottlieb starch medium but spore chains on
L-Arabinose
L-Rhamnose most other media are not uniform, being spherical or
D-Fructose hooked, with the hyphal end being indented in the form
D-Galactose of a hump or branched. As to the cell wall composition,
Raf?nose 65 LL-diaminopimelic acid was detected but meso
D-Mannitol
i-lnositol diaminopimelic acid was absent, indicating that it corre
Salicin sponds to cell wall type I. The ?ndings that the strain
Sucrose utilizes glucose and starch only and tolerates not more
 5,252,469
7 8
than 4% NaCl as well as its straight or digital spore For puri?cation of BTGase from the culture ?ltrate,
morphology, red-series color of aerial mycelium and any method generally used for puri?cation of enzymes
melanoid pigment production suggest that it is a strain can be used.
of Streptomyces xanthophaeus or Streptomyces?avotricini For example, there can be used treatment methods
according to Bergeys Manual, 8th Ed, or Streptomyces with an organic solvent such as ethanol, acetone, iso
Iavena'ulae according to the updated edition thereof, viz. propyl alcohol, etc.; salting out using ammonium sul
Bergeys Manual of Systematic Bacteriology (1989). fate, sodium chloride, etc., dialysis, ultra?ltration, ion
Therefore, the strain was identi?ed to belong to the exchange chromatography, adsorption chromatogra
genus Streptomyces and designated Streptamyces laven phy, gel ?ltration, adsorbents, isoeiectric point fraction
dulae No. 466. 10 ation, etc. Further, in the case where the purity of
Incidentally, Streptomyces sp. No. 83 and Streptomy BTGase is increased by the use of these methods in
ces Iavendulae No. 466 have been deposited with the suitable combination, the methods can be performed in
Fermentation Research Institute, Agency of Industrial such a combination. From the enzyme obtained by these
Science and Technology 1-3, Higashi l-chome, Tsuku methods, liquid or solid BTGase can be obtained by
ba-shi, Ibaraki-ken 305, Japan on Aug. 13, 1990 under methods of ultra?ltration condensation, reverse osmosis
the respective deposit number of FERM No. P-11656 condensation, drying under reduced pressure, freeze
(FERM No. BP-3505 under Budapest Treaty) and drying or spray drying, by adding a variety of salts,
FERM No. P~11657 (FERM No. BP-3506 under Buda sugars, proteins, lipids, surfactants, etc., as stabilizers or
pest Treaty), respectively. " without adding them.
For the purposes of the present invention, any micro 20 Measurement of the activity of BTGase is carried out
organism belonging to the genus Streptomyces, which by performing a reaction using benzyloxycarbonyl-L
is capable of producing transglutaminases can be suc glutaminyl glycine and hydroxylamine as substrates in
cessfully employed, preferably Streptomyces sp. No. 83 the absence of Ca2+, forming an iron complex with the
(Strain No. 83) and Streptomyces Iavendulae No. 466 resulting hydroxamic acid in the presence of trichloro
(Strain No. 466) described above. 25
acetic acid, measuring absorption at 525 nm and deter
Methods for culture, puri?cation etc., which com
prise culturing these microorganisms and harvesting mining the amount of hydroxamic acid by a calibration
transglutaminase (hereafter, referred to as BTGase) will curve to calculate the activity. The BTGase activity is
be described below. measured by the method described below, unless other
For the purposes of the present invention, any cultur wise indicated.
ing mode of liquid culture and solid culture can be Measurement of Activity
performed, but from an industrial standpoint, it is ad
vantageous to perform deep aerial spinner culture. As Reagent A
culture sources to be used, there can be used carbon 0.2 M Tris-hydrochloride buffer (pH 6.0)
sources, nitrogen sources, inorganic salts and other 35 0.1 M hydroxylamine
trace nutrient sources conventionally used for culture of - 0.01 M reductive glutathione
microorganisms; in addition thereto, all nutrient sources 0.03 benzyloxycarbonyl-L-glutaminyl glycine
can be employed as long as they are utilizable by mi Reagent B
crooganisms belonging to the genus Streptomyces. As Reagent B is made of equal volumes of the following
the carbon sources for media, there can be used, singly 40 component.
or in combination, glucose, sucrose, rastagen, glycerin, 3 N hydrochloric acid
dextrin, starch, etc. and in addition thereto, fatty acids, 12% trichloroacetic acid
oils and fats, organic acids, etc. As the nitrogen sources, 5% FeCl3.6H2O (dissolved in 0.1 N-HCl)
any of inorganic nitrogen sources and organic nitrogen To 0.05 ml of an enzyme solution is added 0.5 ml of
sources can be used. As the inorganic nitrogen sources, 45 Reagent A and they are mixed with each other. After
there can be used ammonium nitrate, ammonium sul reacting at 37 C. for 10 minutes, Reagent B is added
fate, sodium nitrate, ammonium chloride, etc. thereto to discontinue the reaction and form an Fe com
Further as the organic nitrogen sources there can be plex. Thereafter, absorbance is measured at 525 nm. As
used, for example, powder of soybean, rice, sweet corn a control, absorbance is measured after reacting a previ
and wheat etc., bran and defatted meal, and corn steep 50 ously thermally inactivated enzyme solution in a similar
liquor, peptone, meat extract, casein, amino acids, yeast manner and a difference in absorbance between the
extract, etc. As the inorganic salts and the trace nutrient control and the enzyme solution is measured. Sepa
sources, there can be used salts of phosphoric acid, rately, a calibration curve is prepared using L-glutamic
magnesium, potassium, iron, calcium, zinc, etc., and any acid 'y-monohydroxamic acid instead of the enzyme
other materials that can accelerate growth of the bac solution, and the amount of hydroxamic acid produced
teria or production of BTGase such as vitamins, non is determined by the difference in absorbance described
ionic surfactants, defoaming agents, etc., if necessary. above. An enzyme activity which produces 1 pmol of
Culture may be performed under aerobic conditions at hydroxamic acid per 1 minute is de?ned as 1 unit.
a culture temperature within such a range that the bac Enzymological properties of the thus obtained puri
teria grow to product BTGase, preferably at 25 to 35 60 ?ed BTGases, namely, transglutaminase of Streptomy
C. A time period for the culture varies depending upon ces sp No. 83 (named Enzyme No. 83) and transglutami
conditions but the culture may be performed until nase of Streptomyces Iavendulae No. 466 (named En
BTGase is best produced, generally for about 2 to 4 zyme No. 466) are described below.
days. In the case of liquid culture, BTGase is dissolved
in the culture solution and can be harvested from the 65 a) Optimum pH
culture ?ltrate obtained by removing solid contents In the case of using benzyloxycarbonyl-L-glutaminyl
from the culture solution after completion of the cul glycine, and hydroxylamine as substrate, each optimum
ture. pH of Enzyme No. 83 and Enzyme No. 466 lies in a
 5,252,469
9 10
range of approximately 6 to 7 upon the reaction at 37
C. for 10 minutes (shown in FIGS. 1 and 5). g) In?uence of inhibitor
Each inhibitor was added in a concentration of 1 mM.
b) Optimum temperature After allowing the mixture to stand at 25 C. for 30
In the case of using benzyloxycarbonyl-L-glutaminyl minutes, the activity was measured (the results are
glycine and hydroxylamine as substrates, each optimum shown in Table 8). In all BTGases, the activity is inhib
temperature of Enzyme No. 83 and Enzyme No. 466 ited by p-chloromercury benzoic acid (simply referred
lies at about 40~45 C. upon the reaction of pH 7 for to as PCMB), N-ethylmaleimide (simply referred to as
10 minutes (shown in FIGS. 2 and 6). NEM) and monoiodoacetic acid.
c) pH Stability TABLE 8
Inhibitor Enzyme No. 83 Enzyme No. 466
By treatment at 37' C. for 10 minutes, both Enzyme
No. 83 and Enzyme No. 466 are stable at pH of 6 to 9 None 100 100
EDTA 103 108
(shown in FIGS. 3 and 7). PCMB 86 28
15
NEM l0 5
d) Temperature stability Monoiodoicetic acid 92 87
By treatment at pH 7 for 10 minutes, Enzyme No. 83 PMSF 101 103
retains 100 activity at 40 C. and 55% at 50 C.: and
Enzyme No. 466 retains 94% activity at 40' C. and 32% In Table 8, PMSF is the abbreviation for phenylme
20
at 50 C. (shown in FIGS. 4 and 8). thylsulfonyl ?uoride.
e) Substrate speci?city h) isoelectric point
Using each BTGase, reactions of various synthetic According to ampholine isoelectric point electropho
substrates with hydroxylamine were examined. No 25 resis, isoelectric points, pI of Enzyme No. 83 and p1 of
BTGase reacts with the synthetic substrates benzylox Enzyme No. 466 are at approximately 10 and 6.8, re
ycarbonylasparaginyl glycine, benzyloxycarbonyl glu spectively.
tamine and glycylglutaminyl glycine. However, the
reactivity is the highest when the synthetic substrate is i) Molecular weight
benzyloxycarbonylglutaminyl glycine. In this case, the 30 According to SDS disc electrophoresis, molecular
concentration of various synthetic substrates is 5 mM. weight of Enzyme No. 83 and Enzyme No. 466 are
The results are shown in Table 6. In the table, CBZ, approximately 39,000.
Gln, Gly and Asn are abbreviations for benzyloxycar Next, properties of BTGases are compared to those
bonyl group, glutaminyl group, glycyl group and as of guinea pig liver-derived transglutaminase. The
paraginyl group, respectively. 35 guinea pig liver-derived transglutaminase was prepared
by the method described in Connellan et al., Journal of
TABLE 6 Biological Chemistry, Vol. 246, No. 4, pp. l093-l098
Substrate Enzyme No. 83 Enzyme No. 466 (1971). In Table 9, comparison in enzymological prop
CBZG1n-Gly 100 100 erties is shown and in?uence of Ca2+ on activity is
CBZ-Gln-Gly-OEt 61 65 shown in Table 10. As will be apparent from Tables 9
CBZGln--GlnGly 49 35 and 10, various differences in enzymological properties
CBZ-Gly-GlnGly-Gly l0 l8
CBZ-Gln 0 0 are noted between transglutaminase of guinea pig liver
CBZAsn--Gly 0 0 (hereafter, referred to as MTGase) which has been
Gly-Gln-Gly 0 0 mainly investigated heretofore and BTGases derived
45 from the genus Streptomyces: in particular, differences
are noted in temperature stability, molecular weight,
t) In?uence of metal ions isoelectric point and substrate speci?city. Further, a
Various metal ions were added to the activity mea difference is noted in that the BTGases of the present
surement system in a concentration of 1 mM (the results invention act in the presence and absence of Ca2+.
are shown in Table 7). In all BTGases, the activity is 50 Therefore, the respective enzymes of the present inven
inhibited by Cu2+, Zn and 1%. tion are different from MTGase in their properties.
TABLE 7 TABLE 9
Enzyme No. 83 Enzyme No. 466
Enzyme Enzyme
No. 83 No. 466 MTGase
Metal Ion (%) (%) 55
Optimum pI-I ca. 6-7 cm 6-7 6
None 100 100 pH Stability 6-9 6-9 67.5
CaCl; 101 99 Optimum temperature ca. ca. 40-45 C. 50-55 C.
BaClz 100 100 40-45 C.
CoClg 100 98
CuClz 89 90
TemErature stability (%)
FeCl; 93 97 40' C. residual rate 111) 94 96
RC1 104 101 50' C. residual rate 55 32 40
MgClg l02 99 Molecular weight ca. 39,030 ca. 39,000 ca. 90,000
Isoelectric point ca. 10 ca. 6.8 4.5
MnClz 102 99
NaCl 102 98 Substrate sEci?city (%)
NiClz 101 99 CBZ-Gln-Gly 100 100 100
PbCl; 59 32 65 CBZGln-G1y--OEt 61 65 122
SrCl; 100 97 CBZ-Gln-Gln-Gly 49 35 288
ZnClz 36 l0 CBZGlyGln--Gly-Gly l0 18 126
CBZGln O 0 0
CBZ-Asn-Gly O 0 0
 5,252,469
11 12
TABLE 9-continued Using a UP 6000 membrane, the culture solution was
desalted and concentrated and the concentrate was
Enzyme Enzyme adjusted to pH 7.0 with hydrochloric acid and passed
No. 83 No. 466 MTGase
Gly-Gln~-Gly 0 0 0
through a column of DEAE-Cellulo?ne (trademark,
manufactured by Seikagaku Kogyo), which had been
previously equilibrated with 0.005 M phosphate buffer
TABLE 10 (pH 7.0). By this operation, transglutaminase was ad
sorbed on the column. The column was ?rst washed
Enzyme No. 83 Enzyme No. 466 MTGase with the same phosphate buffer solution to remove
Metal Ion (%) (%) (%)
None 99 99 ' 0
10 protein impurities and, then, elution was carried out
with the same buffer on a density gradient of 0.005 to
1 mM CaCl; 99 98 39
5 mM QC]; 100 100 100 0.5 M. The eluate was collected in fractions and the
fractions having a high speci?c activity were collected.
Then, using blue Sepharose and CG-SO, the enzyme was
The present invention is now illustrated inv greater puri?ed as in Example 1 to give a pure enzyme giving a
detail with reference to the following Examples, but it single peak in_ SDS disk electrophoresis. The speci?c
should be understood that the present invention is not activity relative to the culture ?ltrate was 169 times and
deemed to be limited thereto. the recovery rate was 37%.
EXAMPLE 1 As is apparent from above, the microorganism
Streptomyces sp No. 83 was inoculated on 200 ml of derived BTGase of the present invention can be sup
medium (pH 7) having a medium composition of 0.2% 20 plied at low cost and can be easily puri?ed and there
of polypeptone, 0.5% of glucose, 0.2% of dipotassium fore, is highly practical.
phosphate and 0.1% of magnesium sulfate followed by It is also advantageous that by the use of a BTGase'
culturing at 30 C. for 48 hours. The obtained seed according to the present invention, the research and
culture solution was added to 20 l of a medium (pH 7) development on various uses such as a food manufactur
containing 2.0% of polypeptone, 2.0% of rastagen 25 ing or a medical treatment can be accelerated.
(trademark, manufactured by Nichiden Kagaku K.K.), While the invention has been described in detail and
0.2% of dipotassium phosphate, 0.1% of magnesium with reference to speci?c embodiments thereof, it will
sulfate, 0.2% of yeast extract and 0.05% of Adckanol as be apparent to one skilled in the art that various changes
a defoaming agent (trademark, manufactured by Asahi and modi?cations can be made therein without depart
Denka Kogyo K.K.) followed by culturing at 30 C. for ing from the spirit and scope thereof.
3 days. After ?ltering, 18.5 1 of the culture solution was What is claimed is:
obtained. The activity was 0.71 U/ml. 1. A process for producing a transglutaminase com
The culture solution was adjusted with hydrochloric prising the steps of:
acid to pH of 6.5 and passed through a column of CG-50 (l) culturing a microorganism having all of the identi
(trademark, manufactured by Organo Co., Ltd.), which 35 fyingcharacteristics of the microorganism, Strep
had been previously equilibrated with 0.05 M phosphate tomyces sp. No. 83, deposited as FERM BP~3505,
buffer (pl-I 6.5). By this operation, transglutaminase was and mutants thereof, which produces a transg
adsorbed. After washing protein impurities out with the lutaminase; and
same phosphate buffer solution, a density gradient of (2) recovering said transglutaminase from the culture
0.05 to 0.5 M was prepared by the same buffer, through 40 obtained,
which the system was passed. The eluate was fraction wherein said transglutaminase is an enzyme which
ated and recovered, and the fractions having a high catalyzes an acyl transfer reaction of a 'y-carboxya
speci?c activity were collected. After the system was mide group of a glutarnine residue in a peptide or
diluted to have a conductivity of 10 ms or less, it was protein chain in the presence or absence of Ca+ +,
passed through a column of blue Sepharose. By this and wherein said transglutaminase has an isoelec
operation, transglutaminase was adsorbed. After wash tric point of 10, and is inhibited by Pb ions.
ing protein impurities out with 0.05 M phosphate buffer 2. A process for producing a transglutaminase com
(pH 7), a density gradient of 0 to l M was prepared by prising the steps of:
sodium chloride, through which the system was passed. (l) culturing a microorganism having all of the identi
The eluate was recovered and fractions having a high fying characteristics of the microorganism, Strepto
speci?c activity were collected. The fractions were 50
myces Iavendulae No. 466, deposited as FERM
desalted and rechromatographed on a column of CG-50 BP-3506, and mutants thereof, which produces a
to fractionate the eluate. As a result, the active fraction transglutaminase; and
eluted as a single peak. The speci?c activity was 356 (2) recovering said transglutaminase from the culture
times that of the culture ?ltrate and the recovery rate obtained,
was 43%. 55 wherein said transglutaminase is an enzyme which
catalyzes an acyl transfer reaction of a 'y- carboxya
EXAMPLE 2 mide group of a glutamine rersidue in a peptide or
Streptomyces Iavendulae No. 466 was inoculated into protein chain in the presence or absence of Ca+ +,
200 ml of the same preculture medium as used in Exam and wherein said transglutaminase has an isoelec
ple l and cultured at 30 C. for 48 hours. The resulting 60 tric point of 6.8, and is inhibited by Pb ions.
seed culture solution was added to 20] of a medium (pH 3. The process for producing a transglutaminase as
7) containing 2.0% of polypeptone, 2.0% of glycerin, claimed in claim 1, wherein said microorganism is
0.2% of dipotassium phosphate, 0.1% of magnesium Streptomyces sp. No. 83,_deposited as FERM BP-3505.
sulfate, 0.2% of yeast extract and 0.05% of Adekanol, 4. The process for producing a transglutaminase as
which was then cultured at 30 C. for 3 days. The result 65 claimed in claim 1, wherein said microorganism is Strep
ing culture broth was ?ltered to give 18.5 1 of the cul tomyces Iavendulae No. 466, deposited as FERM BP
ture ?ltrate. The activity of this culture solution was 3506.
2.47 U/ml. & Q i t t