1

沼津工業高等専門学校研究報告
第 55 号，2021 年 1 月"  B a c illu s s p . J 8 1 3  ) 4 ( 

$":,/"?95*1,#@%*1,#@1<**1@0-!*1@>4=*1@3+;*1@72*1@.#*1@6

&*1@'8*1,2

I m p r o v e m e n t o f S p e c i f i c A c ti v it y o f a n A l k a l i n e C h i t in a s ef r o m A l k a l i p h i l i c B a c i l l u s s p . J 8 1 3 b y Di r e c t e d E v o l u t i o n

SAITO Keisuke*1,#, UNI Fumiya*1,#, WATANABE Toshiki*1, FUKAZAWA Tetsuya*1,
NAGAO Yuri*1, SAMPEI Zenjiro*1, OTAKE Hiroshi*1 YATSUNAMI Rie*1,
FUKUI Toshiaki*1, NAKAMURA Satoshi*1,2Abstract: Chitin is a β-1,4-linked polysaccharide composed of N-acetyl-D-glucosamine units.
Chitinase hydrolyzes β-1,4-glycosidic bonds of chitin. Alkaliphilic Bacillus sp. strain J813
produces a glycoside hydrolase (GH) family 18 alkaline chitinase (ChiJ). To obtain ChiJ mutants
with improved specific activity through directed evolution, random mutation was introduced intochiJ gene by error-prone PCR. Approximately 2,750 clones were screened for chitinase activity
from the random mutation library using high-throughput screening system. Nine mutants withimproved specific activity were obtained. Using soluble chitin as substrate, all ChiJ mutants
showed higher specific activity than wild-type ChiJ. And above all, mutants F272S and F272L
showed the highest activity. Therefore, it was suggested that the amino acid substitution ofPhe272 was particularly important to improve specific activity of ChiJ.

Key Words: Chitinase, GH family 18, Bacillus sp., Directed evolution

=rDqFU[wQ({ĴñFUu:§ŀŎ&Ɣ"6#ż 4767-"&Ï=rDqñ Bacillus sp. J813 ĘFUu( N-=PUr-D-HrJLku

β-1,4 ŵµ"ŒƸ

=rDqFU[wQńŅƅ#!ǂ7!6 [2]Ŀ&Ʃ%ÌŬ"5'ŵµ:¥ĥƔ6ƲŰ FJ813 Ę ńŅ6=rDqFU[wQ (ChiJ) (Ŭƞ¥U[wQ"6FUu(PrtwO&Ġ"ƁĹŉ&ÌƶĥƔƲŰ (GH) d<kqw 18 &ß6ƕÐZm?u&ÒÀ6č¡ņa?CjO"6 ĥ&ǃĴñ"6(CatD)d>et^GUuS?f III ĝZm?u (FnIIID)

0&¡ņ ¿ǃ#7! ƤäFUu'¥ĥ3)FUuŵµZm?u (ChBD)

4Ĝõ76Ɣ&35ńõ6FUuCqKŬ(ǎ¥ŰĐ#(Fig.1) [3]"FnIIID 'ğž(čŕ"6 ChBD!ªƇ¼v©Ū¼vǊ¼%$'ş'ŅěƵ& !Īő({ĴñFUu&ľŌŎ&ŵµ6#"ǁû6 CatD

7!6 [1]FUu'¥ĥƔ(æƳ:ņĂĨ',&36{ĴñFUu'¥ĥƔ:ƪ6# ŕ47!

ŃÈƝƄ:0FU[wQ&36¥ĥƔ1Ɖ9

6 [3,4]

7! 5¥ĥƔ§ŀ'ǎFU[wQ ċ-76

 ďŗš"(ChiJ &36§ŀŎ%FUuCqKŬńŅ& FUu(Ƴñđ z"(ĥ&ĴƔ6 =rDqñ"(¸ù¸ñƪ©&36 ChiJ 'ģĮñ¸y:0{Ĵ#%6 !=rDqñđ z"ǎĮñ:śǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐǐ
*1 ēáěÍÓ

Tokyo Institute of Technology*2 ħĭáěǎťÜƹÓė National Institute of Technology(KOSEN), Numazu CollegeFig. 1 Ï=rDqñ Bacillus sp. J813 ĘŇĒ=rDqF# Equal contribution

U[wQ ChiJ 'Zm?uĜõ

2

沼津工業高等専門学校研究報告

第 55 号:ĪpuTlËŌp?epqw:Ĝõ6çƞƣý

:ęƅ37°C " 16 ćƺÄǋ6#"őŎƮÑ2Ǐ1 ƅĘfpOkZ 3)ÄÁ'ōŁƚÝ:Ɖƭî&35¾±ƅ: –20°C " ƮÑŴý ņ×#!(Íƀƅ JM109 Ę:FU[¯ê150 μl 'ĴƅĴİ [50 μg/ml qRUwl0.2%

wQƮÑōŁņ×#!(Íƀƅ BL21(DE3) Ę:ņ

(v/v) Triton X-1005.0 μg/ml DNase I (Sigma)10 mM Tris-pET-CFB (õĻÂ ChiJ :JwZ6ōŁÂfpOHClpH 7.5] &ôĵ37°C " 1 ćƺĴƅåkZ"5pET-21b(+) (Novagen) :ÅŐ#!6 [3]ƭî:Ɖì47yĲ'FU[wQĮñ: 96 Šj?G

Ʀâ'Äǋ&( LB ÄÁ [1.0% (w/vzŔŋ) YqfYu

tfswY:ņ!ĳÔ([Dp?WOG)0.5% ƲĢBFO (Becton Dickinson)
1.0% NaClpH 7.2] :FU[wQƮÑ'ōŁ&(Ɓ¨2Ǐ6 FU[wQ'ūƍƚÝÄÁ ZYM-5052 [5] :ņïƏ&ð!ųĶå 100 ƵńÂ 3)´şËŌÂ ChiJ :JwZ6ōŁÂfpµg/ml '=ucMqu:ı¥OkZ:Ċ6Íƀƅ BL21(DE3) Ę: 5 ml 'Ɓ¨ƚÝÄÁ:¹/ƗǍŨ&ęƅ37°C "xĈÄǋ6#"

2Ǐ2 puTlËŌp?epqwĜũĨ

őŎƮÑ'ōŁƚÝ:Ɖƭî&35¾±ƅ: Bpwftwu PCR &36puTlËŌÝ&(10 mM Tris-HCl ŹƊİ (pH 7.5) &ôĵƢǅĩřŘ

GeneMorph II Random Mutagenesis Kit (Agilent) :ņêåƭî:Ɖì47yĲ:ĸŲŽ÷İ#CqK]GsCUZfp?jw(Cgtu35ƟĸŲŽ÷İ 4'ƵńÂ 3)´şËŌÂ ChiJ 'ūƍBpwftwu PCR ê'°ðĴİ:=EtwOIrǄ

(FYbwr BL-01 (ÙÊűź) *'ƒ»ñ:¡ņ=Ĥī¨&őŎ'Í' DNA āļ:Ir35¾±

d>\W>wGtjYHpd>w&!ƉŵµŹƊİ(10 mM Tris-HCl ŹƊİ (pH 7.5)!Ĵİ&( 100 ËŌƮÑ'Gtw\uH&( Megaprimer PCR of

500 mM CH3COOH Ĵİ:ņì47ň: 10Whole Plasmid Ĩ (MEGAWHOP Ĩ) [6] :ņy"ì

mM Tris-HCl ŹƊİ (pH 7.5) &Û!ƥĔ1':ū

47 DNA āļ:mEfp?jw! pET-CFB :ƍĞ¼#

ƷÂ#!KOD -Plus- (ēĬű) :ņ!L?Gr°ð: SDS-iq=Gqr=kZIrǄĤī¨ (SDS-PAGE) &Ɖ°ðŅĽ&¢ƽƲŰ Dpn I :¥ !ƷÂ DNA :( 12.5% iq=Gqr=kZIr:ņLaemmli 'ĂĨį©êÍƀƅ BL21(DE3) Ę&çƞƣýpuTl[8] &ë!Ɖī¨ê'SubGƞ(GwjMweq

ËŌp?epqw#

q=uYerw R-250 (CBB) ([Dp?WOG) &36ĖƂ:ăÑƶjwDw( SDS-PAGE Molecular Weight2Ǐ3 DNA ÇÅƱƔĔ

Standards, Low Range (Bio-Rad) :ņ

 DNA ÇÅƱ'ƔĔ(ēáěÍÓCwfud<Mq FU[wQĮñ( 1.5 ml j?GtUowe:ņ!ĳÔ

W>PuSwva?Cưƹ&ǈ!ƉSubGƞ'Ôƶ&( Lowry Ĩ [DC Protein Assay(Bio-Rad)] [9] :ņÛĺ(@MƈĲ=reku (»ů2Ǐ4 FU[wQĮñ'ĳÔĨƇ) # ³ĴñÅƞ#!T?FYLu M [70% ſ=PUr©FUu (Íąū©áě35|)] :ņ°ðİ~'Åƞ

2Ǐ7 ¨¤ÓÔĀ'ŧ' ų Ķ å ( 0.05% (96 Š j ? G t f s w Y ) ƵńÂ3)3)´şËŌÂ ChiJ ūƍ¼:ņųĶå

0.125% (1.5 ml j?GtUowe) #pH 7.537"

0.020.5% 'ÅƞÒÀz1.5 ml j?GtUowe~pH10 ƺ°ð:ƉêÅƞ'¥ĥƔ&!ńƯ

7.537°C " 5 ƺ°ð:ƉÅƞĶå#°ð ČƧåĎŤƶ: Schales ËĨ [7] &!Ôƶ6#&35FU' Hanes-Woolf ftVY [10] &35¨¤ÓÔĀ (kDB[wQĮñ:ŧÛĺ&( N-=PUr-D-HrJLk

qOÔĀ Km3)ƕÐÔĀ kcat) :ŧu:ņ2Ǐ8 ţĜƨnXquH
2Ǐ5 `?OrwfVYOGqw\uHĨ ƵńÂ ChiJ 'ţĜƨnXquH&(Discovery 96 Šj?GtfswY'´@Ar&Ɓ¨ƚÝÄÁ 150 μl

Studio 4.5 ftHpl (BIOVIA) :ņ好アルカリ性 Bacillus sp. J813 株由来アルカリキチナーゼの指向性進化による比活性の向上

3               
3Ǐ1 ǎĮñËŌÂ ChiJ ŅńGtwu'OGqw\uH ƵńÂ ChiJ :JwZ6ōŁÂfpOkZ pET-CFB:ƷÂ#!ņBpwftwu PCR &36 chiJ ƮÑ CatD ǇÃ*'puTlËŌÝ:ƉņCqK]GsCUZfp?jw'Ʊ( Table 1 &śCatD-sp( chiJ ƮÑ CatD ǇÃ' 5’ ĎŤ&=\wr6PuOfp?jw! CatD-ap ( chiJ ƮÑ CatD ǇÃ' 3’ ĎŤ&=\wr6=uUPuOfp?jw"6Bpwftwu PCR 'ŵĕŮ 1.7 kb ' DNA āļ'Éã Śƙ7ì47 DNA āļ: MEGAWHOP Ĩ:ņ! pET-CFB 'úƱ#ŻýêÍƀƅ

BL21(DE3) Ę&çƞƣýpuTlËŌp?epqw#

Fig. 2 ƵńÂ 3)ËŌÂ ChiJ :¹/ĸŲŽ÷İ (A)3)ūƍĞ¼ (B) ' SDS-PAGE
1: ƵńÂ, 2: P254S, 3: N242S, 4: N221I, 5: Q153R/N349D,
6: H281L, 7: F272S, 8: F272L, 9: I69V/P267S, 10:
W302R/D321N, M: jwDw.

Ž÷İ 4'ūƍ:Ɖ´şūƍĞ¼' SDS-PAGE

Table 1 CqK]GsCUZfp?jw'ÇÅƱ'ŵĕ71­xauZ:śĞ¼ ì47# Śƙ7 (Fig. 2B)ê'ĚƖ"("ì47ūƍĞ¼:ņ6##3Ǐ3 ǎĮñËŌÂ ChiJ 'ñƞĚƖ FU[wQĮñĳÔ&Å `?OrwfVYOGqw\ ƵńÂ 3)´şËŌÂ ChiJ 'ūƍĞ¼:ņ³ĴñuHŭ:ņpuTlËŌp?epqw 4ǎĮñËŌÂ

FUu"6T?FYLu M &Û6¥ĥƔĮñ:ĳÔ

ChiJ ŅńGtwu'OGqw\uH:Ɖ 2,749 Ę!'ËŌÂ ChiJ (ƵńÂ ChiJ 35ǎģĮñ'p?epqwĜõGtwu'OGqw\uH'ŵĕ11:ś (Table 2)ľ& F272S

Ę ƵńÂ ChiJ 35ǎĮñ:ś' 11 Ę'ƌ

ChiJ ' 2.2 ~ 2.6 'ģĮñ:ś! 5Phe272 '=Gtwu&¹-76 chiJ ƮÑ&!ÇÅƱ:ĦÔk_ƳŻý

#8!'Ę" 1 ~ 2 Ŧö'=k_ƳŻý Śƙ7

ż 47

-'  2 Ę&!(=k_ƳŻýŦö ´şËŌÂ ChiJ '=k_ƳŻý'§ĕ:35ƘŲ&ƛ(F272S'ƌGtwu#ƴƎ!

+60ƵńÂ 3)´şËŌÂ ChiJ &!³ĴñFê=k_ƳŻýƋś:ËŌÂ ChiJ '·Ŝ#6

Uu¥ĥƔ°ð'¨¤ÓÔĀ:ĳÔ'ŵĕ!

3) F272L)

3Ǐ2 ǎĮñËŌÂ ChiJ 'ōŁ#ūƍ ƵńÂ ChiJ ōŁÂfpOkZ:Ċ6Íƀ
ƅ BL21(DE3) Ę 3)`?OrwfVYO
Gqw\uH&35²ì 9 ş'ǎĮñËŌ
Â ChiJ ŅńGtwu&!Ɓ¨ƚÝÄÁ:ņ!Äǋƅ35ĸŲŽ÷İ:ƛƍì47ĸŲŽ÷İ:Ưđz"SDS-PAGE &CBB ĖƂ:ă´şËŌÂ ChiJ (ƵńÂ ChiJ #¶ĝÍƀƅƅ"³ĴñSubGƞ#!ǎ§ŀńŅ76
# Ć4 #% (Fig. 2A)
 FYbwr BL-01 :ņ=d>\W>w
GtjYHpd>w&35 ´ş ChiJ 'ĸŲ3) F272L (ƵńÂChiJ 'ģĮñ¸y&ÍØ|!6#Table 2 ƵńÂ 3)ËŌÂ ChiJ &36³ņñFUu¥ĥƔ°ð&
6ģĮñ 3)´ş¨¤ÓÔĀ

4沼津工業高等専門学校研究報告

第 55 号'ËŌÂ ChiJ "ƵńÂ ChiJ &ģ! Km 'É¥ .4

Ƴ'Ƥ&(4& 'ƃǌĄ=k_Ƴ (Trp94

7ËŌÂ ChiJ & !(Åƞ#'ƒ»ñ z!6Trp82

# 9  (Table 2)-!'ËŌÂ ChiJ ' kcat'Åƞŵµ*'Ø| ś½76' Phe272 (L(ƵńÂ ChiJ &ģ!ÉÍ!6# 9 eL?Y + 'ĎŤƤ&Ż!60LeL?Y' Km3) kcat 'ÉÍ(ľ&ǎģĮñ:ś+ "'Åƞŵµ:ƌ¦6é£:1#ż 476

F272S3) F272L & !ǉƆ"xĂ"°ð§ŀ:Ƌ kcat/Km &!(F272S3) Phe272)

!Phe272x&};" 574'ġÅ

'=k_Ƴ&Żý76#LeL?

3) F272L &Y + & 6Åƞƒ»ñ&èǆ:| 6# ò7

!ƵńÂ ChiJ ' 2 Şå'¸y Ɛ471''
6ÕƿPhe272 ' Ser % Ala *'=k_ƳŻý&''ËŌÂ ChiJ & !(Í%Ƭ(Ɛ47% 35 Km³ĴñFUu¥ĥƔĮñ ¸yËŌÂ ChiJ &476ÅƞľŌñ'z(°ð§ŀ'Ɠķ 4(Ħ!

!(/8Åƞ#'ƒ»ñ(z!6# 9 ċ-#"(%xĂ"74'=k_ƳŻý&3

5¥ĥƔê'°ðŅĽ1Ŋ-5 4%60LeL?

ÉÍ# 4Åƞƒ»ñ(z#żY +  4Ƨ2 &þ7Ġ'°ð*ŝƉ2%6#1ż 476-Ser 2 Ala ( Phe &ģ+! Ƹ Ŗ (Þ) 0Phe272 ' Ser % Ala *' GH d<kqw 18 &ß6 Bacillus circulans WL-12
ĘŇĒFU[wQ A1 (ChiA1)

3) Serratia marcescens=k_ƳŻý&35LeL?Y + ĎŤ"'ƃǌĄ=k_ƳƸ&36ţǀÖįÎ6#ò76

ŇĒFU[wQ A (ChiA) (ƲŰÅƞƎµ' X ŸŵĉF272S 2 F272A "( kcat 1ÉÍ! 'łŇ#ĜƨƔĔ %7! 5=k_ƳŻý:ÝËŌÂƲ!¥ĥƔê'°ðŅĽ LeL?Y 4Ƨ2 &þŰ'ñƞĚƖ&35Åƞŵµ&ƻ|6LeL?Y ¶Ô763 &%#&¥ !LeL?Y +  4'

7!6 [11-13]ChiAÅƞ'ƪ OlwO&ƪƉ63 &%#%$1ż3) ChiA1 'ţĜƨ:ƷÂ#!Ĝũ ChiJ CatD ǇÃ'ţĜƨnXr: Fig. 3&ś ChiA1473  74:Ŷµ6#Phe272 '=k_ƳŻý&35L3) ChiA #'=k_ƳƱœ¶ñ 4ChiJeL?Y +  4'Åƞƪ#¥ĥƔê'°ðŅĽ'þ'LeL?Y&( Tyr17 (5)Trp14 (3)Trp392 (1)

 Ƨ2 &ƪƉ63 &%5ŵĕ#!°ð§ŀ¸yTrp124 (+1)ÒÀ6# üÚ#ģĮñ¸y ƫõ71'#üÚ76³ĴñÅƞ(76!LeL?Y:Ĝõ674'ƃǌĄ=k_øÿƧå ǎƲŰ#'ƊŢŚŀ1ǎ !Ʋ3) Trp245 (+2)Ű'Åƞƒ»ñ 6Şåz!1Ĺ#!ǎŚŀ"Åƞ#'ƊŢ ơ6#ż 476'0Åƞƒ»ñ'z351LeL?Y +  4'Åƞƪ#¥ĥƔ
ê'°ðŅĽþ'§ŀ¸y'èǆ'Ă (6 &Í#!ģĮñ Íã&¸y#ż 473   7 - " &  Beauveria bassiana3 ) Bacillus

licheniformis ŇĒ GH d<kqw 18 FU[wQ'ù¸ñƪ©&36ģĮñ¸y Æº7!6[14,15]74(71~ñƲŰ"5=rDqƲŰ'ģĮñ¸y&ƻ6Æº(ďÆ0!#%6
Fig. 3 ChiJ CatD ǇÃ'ţĜƨnXr T?FYLu M :|Íąū©áě (Ę)

Glu164 (ŷƂ) (ƕÐġÅ, ! Tyr17 (5), Trp14 (3),à¬ƾí®Ê%4)& DNA ÇÅƱƔĔ&«¤

Trp392 (1), Trp124 (+1)

ēáěÍÓCwfud<MqW>PuSwva?Cư

3) Trp245 (+2) (+!Ơ'Åƞŵµ*'Ø|ś½76' Phe272 (LƂ) (´LeL?Y:Ƌ.

ƹ'ŏĝ&óƜ6

好アルカリ性 Bacillus sp. J813 株由来アルカリキチナーゼの指向性進化による比活性の向上

5[8] U. K. Laemmli: Nature, 227 (1970) 680-685.

  [9] O. H. Lowry, N. J. Rosebrough, et al.: J. Biol. Chem., 193[1] ą ď F U u v F Y L u Ó  h w l g w N ,(1951) 265-275.

http://jscc.kenkyuukai.jp/, 2020.10.10 ƼƑ.

[10] S. C. Hanes: Biochem. J., 26 (1932) 1406-1421.

[2] T. Fukazawa, K. Endo, et al.: Chitin and Chitosan in Life[11] T. Watanabe, Y. Ariga, et al.: Biochem. J., 376 (2003)

Science (Eds. T. Uragami, K. Kurita, et al.), pp. 428-429,237-244.

Kodansha (2001).

[12] Y. Papanikolau, G. Prag, et al.: Biochemistry, 40 (2001)[3] Z. Sampei, Y. Nagao, et al.:Nucleic Acids Symp. Ser.,11338-11343.

48 (2004) 167-168.

[13] N. N. Aronson Jr., B. A. Halloran, et al.: Biochem. J.,[4] F. Uni, S. Lee, et al.: Biosci. Biotechnol. Biochem., 76

376 (2003) 87-95.

(2012) 530-535.

[14] Y. Fan, W. Fang, et al.: Appl. Microbiol. Biotechnol., 76[5] F. W. Studier: Protein Expr. Purif., 41 (2005) 207-234.

(2007) 135–139.

[6] K. Miyazaki: Methods Mol. Biol., 231 (2003) 23-28.

[15] C. Songsiriritthigul, P. Pesatcha, et al.: Biotechnol. J.,[7] T. Imoto and K. Yagishita: Agric. Biol. Chem., 35 (1971)

4 (2009) 501–509.

1154-1156.