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2021-06-18T13:39:05+09:00
2021-06-18T13:39:05+09:00
2021-06-18T13:39:05+09:00
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Ritsumeikan Univ.KO-362
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1 g
10 36 574.5 780 re
f
BT
0 g
/TT0 1 Tf
14.25 0 0 14.25 175.7249 788.25 Tm
(Study on thermostability of thermolysin)Tj
/TT1 1 Tf
12 0 0 12 467.002 759.75 Tm
(Yoshiki Matsumiya)Tj
-35.209 -2.313 Td
(Thermolysin \(TLN, EC3.4.24.27\), which is produced by )Tj
/TT2 1 Tf
22.83 0 Td
(Bacillus thermoproteolyticus )Tj
/TT1 1 Tf
11.832 0 Td
(and )Tj
/TT2 1 Tf
(B)Tj
/TT1 1 Tf
(.)Tj
/TT2 1 Tf
-35.661 -1.5 Td
(stearothermophilus)Tj
/TT1 1 Tf
(, is a thermostable neutral protease binding of 4 calcium ions \(Ca-1, -\
2, -3, -4\) in its)Tj
0 -1.5 TD
(structure. Since TLN shows high stability, the enzyme is widely used for\
the industrial synthesis of an)Tj
T*
(aspartame, and its stability and proteolytic mechanisms have been extens\
ively investigated. Thermal)Tj
T*
(inactivation of TLN at 90 )Tj
/C2_0 1 Tf
10.469 0 Td
<108F>Tj
/TT1 1 Tf
( was mainly caused through an autodegradation. In this study, autodegrad\
ation)Tj
-10.469 -1.5 Td
(and calcium binding of TLN were investigated, and thermostable mutant TL\
Ns were constructed.)Tj
1 -2.563 Td
(Four autodegradation sites in TLN were identified in the presence of Ca)Tj
8.25 0 0 8.25 390.0566 612.75 Tm
(2+)Tj
12 0 0 12 398.8344 611.25 Tm
(. One of the sites Leu)Tj
8.25 0 0 8.25 501.473 612.75 Tm
(155)Tj
12 0 0 12 513.848 611.25 Tm
( was)Tj
-40.112 -1.5 Td
(substituted with Ala, Ser, Phe, and Gly by site-directed mutagenesis. Th\
e thermostability at 80 )Tj
/C2_0 1 Tf
38.048 0 Td
<108F>Tj
/TT1 1 Tf
( increased)Tj
-38.048 -1.562 Td
(in all mutant TLNs compare with WT TLN, and the autodegradation site shi\
fted from the Gly)Tj
8.25 0 0 8.25 482.377 576 Tm
(154)Tj
12 0 0 12 494.752 574.5 Tm
(-Leu)Tj
8.25 0 0 8.25 517.4043 576 Tm
(155)Tj
12 0 0 12 529.7793 574.5 Tm
( to the)Tj
-41.44 -1.562 Td
(Xaa)Tj
8.25 0 0 8.25 51.8184 557.25 Tm
(155)Tj
12 0 0 12 64.1934 555.75 Tm
(-Ile)Tj
8.25 0 0 8.25 80.8457 557.25 Tm
(156)Tj
12 0 0 12 93.2207 555.75 Tm
(. Furthermore, a new autodegradation site, Ile)Tj
8.25 0 0 8.25 312.1445 557.25 Tm
(164)Tj
12 0 0 12 324.5195 555.75 Tm
(-Asp)Tj
8.25 0 0 8.25 347.8516 557.25 Tm
(165)Tj
12 0 0 12 360.2266 555.75 Tm
(, was appeared in all mutant TLNs.)Tj
-26.311 -2.5 Td
(To construct further stabilized mutant TLN, the autodegradation resistan\
t TLN was constructed by)Tj
-1 -1.5 Td
(introducing mutations at the new autodegradation site of L155A. All muta\
nt TLNs showed higher)Tj
0 -1.562 TD
(thermostability than L155A, and the autodegradation at Ala)Tj
8.25 0 0 8.25 319.0879 490.5 Tm
(155)Tj
12 0 0 12 331.4629 489 Tm
(-Ile)Tj
8.25 0 0 8.25 348.1152 490.5 Tm
(156)Tj
12 0 0 12 360.4902 489 Tm
( was suppressed.)Tj
-26.333 -2.5 Td
(Moreover, mutational effects for stability at the autodegradation sites \
of TLN in the conserved region were)Tj
-1 -1.5 Td
(studied. Mutant TLNs in the conserved region only had the proteolytic ac\
tivity in the culture supernatant in)Tj
0 -1.5 TD
(the case of cultivated in LB medium with 5 mM CaCl)Tj
7.5 0 0 7.5 291.1211 423 Tm
(2)Tj
12 0 0 12 294.8711 423 Tm
(. Dialysis study shows that these mutant TLNs require)Tj
-21.864 -1.625 Td
(more calcium-ions than WT TLN. Since the mutational sites were close to \
Ca-1, -2, and -4 binding sites,)Tj
0 -1.562 TD
(these conserved regions seem to play an important role for the binding o\
f Ca)Tj
8.25 0 0 8.25 400.0645 386.25 Tm
(2+)Tj
12 0 0 12 408.8422 384.75 Tm
(.)Tj
-30.362 -2.5 Td
(On the other hand, thermostable mutant TLN, I39F, was obtained by the ra\
ndom mutagenesis treatment.)Tj
-1 -1.562 Td
(Although the mutational site was in the conserved region, mutant TLN bec\
ame more stable without Ca)Tj
8.25 0 0 8.25 526.375 337.5 Tm
(2+)Tj
12 0 0 12 32.5 318 Tm
(comparison with WT and L155A. These results indicate that enhancing calc\
ium binding and changing amino)Tj
0 -1.5 TD
(acids around Ca-3 binding site have a possibility to construct more stab\
le TLN.)Tj
ET
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