JUN 20, 2011, 38(6): 839846

Microbiology China 2011 by Institute of Microbiology, CAS

tongbao@im.ac.cn

Flavobacterium johnsoniae

*
( 250100)

: Flavobacterium johnsoniae ,

, 35 kD ,
50 C, pH 5.0,

: , , , ,

Purification and catalytic properties of a laminarinase from

Flavobacterium johnsoniae
ZHOU Liang ZHU Yong-Tao CHEN Guan-Jun LIU Wei-Feng*
(The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China)

Abstract: Flavobacterium johnsoniae is capable of secreting enzymes which can efficiently hydrolyze
yeast cell wall. Preliminary analysis revealed the presence of glucanase, chintinase and protease activi-
ties in its culture supernatant. A laminarinase was purified from the extracellular components of F.
johnsoniae through several isolation steps including ion exchange, hydrophobic interaction and gel ex-
clusion chromatography. The molecular weight of the purified laminarinase is about 35 kD. The opti-
mum temperature and pH of its catalyzed hydrolysis are 50 C and 5.0, respectively. Laminarin and
laminari-oligosaccharide were hydrolyzed by this laminarinase in an endoglucanase mode with lami-
naritriose as the main product.
Keywords: Flavobacterium johnsoniae, Glucan, Cell wall of yeast, Laminarinase, Purification

Flavobacterium johnsoniae
[1]
, [14],

863 (No. 2006AA10Z342)

* Tel: 86-531-88364324; : weifliu@sdu.edu.cn
2010-12-01; 2011-02-14
 840 2011, Vol.38, No.6

[5]F. johnsoniae TMD-8) Sigma ; (Lichenan)

[6] [711]
, , (Pachyman) Megazyme ;
(Glucan from yeast)
[8,12]
(Laminaribiose)(Laminaritriose)
, F. johnsoniae (Laminaritetraose)(Laminaripentaose)
[23,13]
(Laminarihexaose) ,
-, -1,3- 200810238547.X; CM Sepharose Fast
, Flow, Q Sepharose Fast Flow, Sephacryl S-100HR
[14]
, GE Healthcare ;
[15]
, , ,
: (Olympus),
[16] [13]
Bacillus circulans , (Nikon), (Millipore),
[14]
Micromonospora chalcea (Bio-Rad), TSK-GEL
Bacon 70 Phenyl-5PW (Tosoh), LC-10AD
F. johnsoniae , (Shimadzu), Aminex HPX-42A (Bio-Rad)
, 1.2
; 1.2.1 : 80 C F. john-
, soniae , 30 C200 r/min
[2,13]-, 20 h,
, - 24 h
1.2.2 : 24 h
F. johnsoniae
F. johnsoniae ,
,
,
16 ,
F. johnsoniae

1 ,
, 30 C 36 h,
1.1

1.1.1 : : ATCC17061
1.2.3 :
(Flavobacterium johnsoniae, UW101),
: 5 g/L 50 mmol/L
McBride (CYE):
pH 7.5 Tris-HCl 50 C
Casitone 1%, 0.5%, MgSO47H2O 0.2%,
, DNS [17]1 min
Tris 0.12%, pH 7.5; : Stanier
1 mol 1
, 0.5% (W/V), pH
Bradford [18]
7.5, Stanier : KNO3 0.1%,
K2HPO43H2O 0.1%, MgSO47H2O 0.02%, CaCl2 1.2.4 SDS-PAGE: [19]
0.01%, FeCl36H2O 0.002%, pH 7.5 1.2.5 :
1.1.2 : : (Laminarin) 24 h (2 L),
(Chitin) (CMC) ( 5 kD) 80% (NH 4) 2SO4
(Xylan)(Mannan)(Bed resin ( 5 kD)

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 : Flavobacterium johnsoniae 841

10 mmol/LpH 4.6 , 1.2.8 :

CM Sepharose Fast Flow [20]
, 00.5 mol/L NaCl , 1 mL/min; , TMD-8
, 10 mmol/LpH 7.5 RID-10A Aminex HPX-42A
PB , Q Sepharose Fast Flow LC-10AD HPLC
, 00.5 mol/L NaCl , 75 C, 0.4 mL/min,
1 mL/min; , 1.2.9 : ,
(NH4)2SO4 1.1 mol/L, 10 mmol/L LCQ Deca XP
PB1.1 mol/L (NH4)2SO4pH 7.0
, 1.10 mol/L (NH4)2SO4
2
, 0.5 mL/min;
, ( 5 kD), 2.1 F. johnsoniae
Sephacryl S-100HR , F. johnsoniae
0.25 mL/min Stanier
1.2.6 pH : pH F. john-
, 50 mmol/L pH 3.08.0 - soniae [2],
, 50 C 10 min; , F. johnsoniae 24 h ,
50 mmol/L pH 6.0 -, ( 1A), F. johnsoniae
30 C70 C 10 min 24 h ,
1.2.7 : 1 ( 1B), F.
(DP26) , johnsoniae
DNS ( 1C 1D),
F. johnsoniae
1 ()
Table 1 Substrate specificity of the purified laminarinase

2.2 F. johnsoniae

Glycosidic bond Ability of F. johnsoniae
Substrate Solubility
style degradation
2 ,
Laminarin + 1,3--D glucan +
Laminaribiose + 1,3--D glucan

Laminaritriose + 1,3--D glucan , ,
Laminaritetraose + 1,3--D glucan + 35 kD ( 3)
Laminaripentaose + 1,3--D glucan + ,
Laminarihexaose + 1,3--D glucan +
,
Pachyman 1,3--D glucan
3 , ,
Glucan from yeast 1,3--D glucan
F. johnsoniae
Yeast cell
Lichenan + 1,3:1,4--D glucan fjoh_4177 16 N
CMC + 1,4--D glucan ( 4)
Chintin 1,4--GlcNAc 143 kD,
Xylan + 1,4--D xylan , ,
Mannan + 1,6--D mannan

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 842 2011, Vol.38, No.6

A B

600 600

C D

1 000 1 000

1
Fig. 1 Morphology of yeast cells in enzyme-producing medium inoculated with F. johnsoniae ( B) or frozen-dried yeast cells
treated with culture supernatant of F. johnsoniae (D)
Note: A is control of B, with no inoculation of F. johnsoniae; C is control of D, treated with heat-inactivated culture supernatant of F. john-
soniae. White arrow: Yeast; Black arrow: F. johnsoniae. Photos A and B were taken under a differential interference contrast microscope;
photos C and D were taken under a phase contrast microscope.

2
Fig. 2 Separation and purification of laminarinase
Note: A: CM Sepharose Fast Flow ion exchange chromatography; B: Q Sepharose Fast Flow ion exchange chromatography; C: TSK-GEL
Phenyl-5PW hydrophobic interaction chromatography; D: Sephacryl S-100HR gel exclusion chromatography.

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 : Flavobacterium johnsoniae 843

2.3 pH

50 C ( 5A) 55 C 60 C ,
; 60 C ,
30% pH 5.0 pH 8.0
, 40%, pH 3.0 ,
( 5B)
2.4

, HPLC
3 SDS-PAGE , -1,3-
Fig. 3 SDS-PAGE analysis of the purified laminarinase
Note: A: Protein marker; B: Laminarinase.

4 Fjoh_4177
Fig. 4 Amino acid sequence of Fjoh_4177 and peptide sequences of the purified laminarinase identified by MS/MS
Note: The MS/MS identified sequences are underlined; The catalytic domain annotated by NCBI is in italic bold.

5 pH-
Fig. 5 Effects of temperature and pH on activities of the purified laminarinase

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 844 2011, Vol.38, No.6

( 1) 1 , , 2.5
4 -1,3- ,
, , 51530 min , HPLC
-1,4- 6A ,

6 HPLC
Fig. 6 HPLC analysis of products from laminarin and laminari-oligosaccharides treated with the purified laminarinase
Note: Controls in A and B: Laminarin treated with heat-inactivated laminarinase; Controls in C, D and E: Laminaritetraose, laminaripentaose
and laminarihexaose treated with heat-inactivated laminarinase, respectively.

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: Flavobacterium johnsoniae 845

, ,
, , F. johnsoniae ,
,
60 min ,
, 41% ( 6B) ,
,
,

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