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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (9): 19-26    DOI: 10.13523/j.cb.20180903
Orginal Article     
Preparation and Composition Analysis of Chitooligosaccharides with Low Degree of Deacetylation by Hydrolysis of Bacillus subtilis Chitosanase
Gong CHENG,Si-ming JIAO,Li-shi REN,Cui FENG,Yu-guang DU()
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
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Abstract  

Chitosanase encoding gene of Bacillus subtilis 168 was optimized, synthesized and secretorily expressed in Pichia pastoris. The protein concentration of the expressed product reached 0.30mg/ml. The optimum pH and temperature of the expressed chitosanase was 5.6 and 55℃, respectively, and enzymatic activity reached 84.54U/ml. The chitosanase was continuously thermostable at 50℃. The low deacetylated chitosan was hydrolyzed by this enzyme and the composition of these products were analyzed through utra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). The results showed that these hydrolysates contained at least 37 different kinds of chitooligosaccharides with degree of polymerization of 2-18 and different degree of deacetylation. In summary, chitooligosaccharides with low degree of deacetylation were prepared through Bacillus subtilis 168 chitosanase expressed in Pichia pastoris and its composition analyzed, which can provide a reference for the study of the relationship between the structure and function of chitooligosaccharides.

Key words： Bacillus subtilis      Chitosanase      Pichia pastoris      Chitooligosaccharides      Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry     
Received: 17 May 2018      Published: 12 October 2018
Corresponding Authors: Yu-guang DU     E-mail: ygdu@ipe.ac.cn
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Gong CHENG
Si-ming JIAO
Li-shi REN
Cui FENG
Yu-guang DU
Cite this article:

Gong CHENG,Si-ming JIAO,Li-shi REN,Cui FENG,Yu-guang DU. Preparation and Composition Analysis of Chitooligosaccharides with Low Degree of Deacetylation by Hydrolysis of Bacillus subtilis Chitosanase. China Biotechnology, 2018, 38(9): 19-26.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180903     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I9/19

Fig.1 Electrophoresis map of chitosanase gene bscsn recombinant plasmid restriction enzyme hydrolysates (a) and protein expression products (b)(a) M1: DNA marker; E1: Plasmid pGBG1-bscsn digested with XhoⅠand NotⅠ; E2: Plasmid pGBG1-bscsn digested with BglⅡ (b) P1: Protein marker; M2: Expressed protein BSCSN
Fig.2 Effect of pH (a) and temperature (b) on the activity of chitosanase BSCSN and its thermostability (c)
Fig.3 TIC(a) and mass spectrogram(b) of BSCSN hydrolysis products
Numbers Retention time
(min)		 Determined
(m/z)		 Theoretical
(m/z)		 Predicted
components		 DP DDA(%)
A1 9.65 341.4 341.1 [D2+H]+ 2 100
A2 9.65 544.6 544.2 [A1D2+H]+ 3 67
B1 15.31 502.6 502.2 [D3+H]+ 3 100
C1 17.30 705.8 705.3 [A1D3+H]+ 4 75
C2 17.30 909.0 908.4 [A2D3+H]+ 5 60
D1 20.76 663.7 663.3 [D4+H]+ 4 100
D2 20.76 866.9 866.4 [A1D4+H]+ 5 80
D3 20.76 1070.1 1069.4 [A2D4+H]+ 6 67
Numbers Retention time
(min)		 Determined
(m/z)		 Theoretical
(m/z)		 Predicted
components		 DP DDA(%)
E1 24.89 514.6 514.2 [A1D5+2H]2+ 6 83
E2 24.89 616.2 615.8 [A2D5+2H]2+ 7 71
E3 24.89 717.8 717.3 [A3D5+2H]2+ 8 63
E4 24.89 819.4 818.8 [A4D5+2H]2+ 9 56
F1 27.91 1189.3 1188.5 [A1D6+H]+ 7 86
F2 27.91 696.8 696.3 [A2D6+2H]2+ 8 75
F3 27.91 798.4 797.8 [A3D6+2H]2+ 9 67
F4 27.91 900.0 899.4 [A4D6+2H]2+ 10 60
F5 27.91 1001.6 1000.9 [A5D6+2H]2+ 11 55
F6 27.91 1103.2 1102.4 [A6D6+2H]2+ 12 50
G1 30.08 777.4 776.8 [A2D7+2H]2+ 9 78
G2 30.08 879.0 878.4 [A3D7+2H]2+ 10 70
G3 30.08 980.6 979.9 [A4D7+2H]2+ 11 64
G4 30.08 1082.1 1081.4 [A5D7+2H]2+ 12 58
G5 30.08 1183.8 1183.0 [A6D7+2H]2+ 13 54
G6 30.08 1285.4 1284.5 [A7D7+2H]2+ 14 50
G7 30.08 1387.0 1386.1 [A8D7+2H]2+ 15 47
H1 31.56 640.0 639.6 [A3D8+3H]3+ 11 73
H2 31.56 707.8 707.3 [A4D8+3H]3+ 12 67
H3 31.56 775.5 775.0 [A5D8+3H]3+ 13 62
H4 31.56 843.3 842.7 [A6D8+3H]3+ 14 57
H5 31.56 911.0 910.4 [A7D8+3H]3+ 15 53
H6 31.56 978.7 978.1 [A8D8+3H]3+ 16 50
I1 32.69 761.5 761.0 [A4D9+3H]3+ 13 69
I2 32.69 829.3 828.7 [A5D9+3H]3+ 14 64
I3 32.69 897.0 896.4 [A6D9+3H]3+ 15 60
I4 32.69 964.7 964.1 [A7D9+3H]3+ 16 56
I5 32.69 1032.4 1031.8 [A8D9+3H]3+ 17 53
I6 32.69 1100.1 1099.4 [A9D9+3H]3+ 18 50
Table 1 Component identification of BSCSN hydrolysis products
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