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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (10): 30-37    DOI: 10.13523/j.cb.20181004
Orginal Article     
Expression of Chitinase from Trichoderma reesei and Analysis the Composition and Structure of its Hydrolysates
Si-ming JIAO,Gong CHENG,Yu-chen ZHANG,Cui FENG,Li-shi REN,Jian-jun LI,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  

Chitinase encoding gene of Trichoderma reesei was optimized, synthesized and secretorily expressed in Pichia pastoris. The protein concentration of the expressed chitinase reached 0.17mg/ml. The optimum pH and temperature of the chitinase was 5.6 and 65℃, respectively, and enzymatic activity reached 0.52U/ml. The chitinase was continuously thermostable at 50℃. The low deacetylated chitosan was hydrolyzed by this enzyme and the composition and structure of these hydrolysates were analyzed. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS) results showed that these hydrolysates contained at least 41 different kinds of chitooligosaccharides with degree of polymerization of 2~18 and different degree of deacetylation. Nuclear magnetic resonance (NMR) results indicated that, the reducing end of these oligosaccharide fractions were mainly composed of N-Acetylglucosamine while both glucosamine and N-Acetylglucosamine were found in the non-reducing end. These results would provide a reference for study of the relationship between the structure and function of chitooligosaccharides.

Key words： Trichoderma reesei      Chitinase      Pichia pastoris      Chitooligosaccharides      UPLC-QTOF MS      NMR     
Received: 23 June 2018      Published: 09 November 2018
ZTFLH:  Q55  
Corresponding Authors: Yu-guang DU     E-mail: ygdu@ipe.ac.cn
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Si-ming JIAO
Gong CHENG
Yu-chen ZHANG
Cui FENG
Li-shi REN
Jian-jun LI
Yu-guang DU
Cite this article:

Si-ming JIAO,Gong CHENG,Yu-chen ZHANG,Cui FENG,Li-shi REN,Jian-jun LI,Yu-guang DU. Expression of Chitinase from Trichoderma reesei and Analysis the Composition and Structure of its Hydrolysates. China Biotechnology, 2018, 38(10): 30-37.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181004     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I10/30

Fig.1 Electrophoresis map of chitinase gene trchi18 recombinant plasmid restriction enzyme hydrolysates (a) and protein expression products (b) (a) M1: DNA marker; E1: Plasmid trchi18-pGBG1 digested with XhoⅠand NotⅠ; E2: Plasmid trchi18-pGBG1 digested with BglⅡ (b) P1: Expressed protein Trchi18; M2: Protein marker
Fig.2 TIC (a) and mass spectrogram (b) of Trchi18 hydrolysis products
Numbers Retention time (min) Determined m/z Theoretical m/z Predicted components
A1 2.55 447.7 447.2 [A2+Na]+
B1 7.29 586.6 586.2 [D1A2+H]+
C1 9.17 789.8 789.3 [D1A3+H]+
D1 10.99 993.0 992.4 [D1A4+H]+
E1 12.77 747.8 747.3 [D2A2+H]+
F1 14.39 951.0 950.4 [D2A3+H]+
G1 15.62 577.6 577.2 [D2A4+2H]2+
H1 18.65 556.6 556.2 [D3A3+2H]2+
I1 19.45 658.2 657.8 [D3A4+2H]2+
J1 20.27 759.8 759.3 [D3A5+2H]2+
K1 22.26 637.2 636.8 [D4A3+2H]2+
K2 22.26 738.8 738.3 [D4A4+2H]2+
K3 22.26 840.4 839.8 [D4A5+2H]2+
L1 24.92 717.8 717.3 [D5A3+2H]2+
L2 24.92 819.4 818.8 [D5A4+2H]2+
L3 24.92 921.0 920.4 [D5A5+2H]2+
L4 24.92 1 022.6 1 021.9 [D5A6+2H]2+
M1 27.09 798.3 797.8 [D6A3+2H]2+
M2 27.09 900.0 899.4 [D6A4+2H]2+
M3 27.09 1 001.6 1 000.9 [D6A5+2H]2+
M4 27.09 1 103.2 1 102.4 [D6A6+2H]2+
N1 29.90 878.9 878.4 [D7A3+2H]2+
N2 29.90 980.5 979.9 [D7A4+2H]2+
N3 29.90 1 082.2 1 081.4 [D7A5+2H]2+
N4 29.90 1 183.8 1 183.0 [D7A6+2H]2+
N5 29.90 1 285.3 1 284.5 [D7A7+2H]2+
O1 31.55 959.5 958.9 [D8A3+2H]2+
O2 31.55 1 061.1 1 060.4 [D8A4+2H]2+
O3 31.55 1 162.7 1 162.0 [D8A5+2H]2+
O4 31.55 1 264.4 1 263.5 [D8A6+2H]2+
O5 31.55 1 366.0 1 365.1 [D8A7+2H]2+
P1 32.79 761.5 761.0 [D9A4+3H]3+
P2 32.79 829.2 828.7 [D9A5+3H]3+
P3 32.79 896.9 896.4 [D9A6+3H]3+
P4 32.79 964.7 964.1 [D9A7+3H]3+
P5 32.79 1 032.4 1 031.8 [D9A8+3H]3+
P6 32.79 814.8 814.7 [D10A4+3H]3+
P7 32.79 882.9 882.4 [D10A5+3H]3+
P8 32.79 950.7 950.1 [D10A6+3H]3+
P9 32.79 1 018.4 1 017.7 [D10A7+3H]3+
P10 32.79 1 086.1 1 085.4 [D10A8+3H]3+
Table 1 Components identification of Trchi18 hydrolysis products
Fig.3 1H NMR (a) and 13C NMR (b) spectra of Trchi18 hydrolysis products
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