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Expression, Purification and Enzymatic Activity of α-1,2 Mannosidase I Derived from Trichoderma reesei in Pichia pastoris

Expression, Purification and Enzymatic Activity of α-1,2 Mannosidase I Derived from Trichoderma reesei in Pichia pastoris

Siqiang Li1,2, Tiantian Wang1, Peng Sun1, Airong Gao1, Xin Gong1, Yuanhong Xu2, Baogen Wang2, Jun Wu1* and Bo Liu1*

1Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. 
2School of Biological and Food Processing Engineering, Huanghuai University, 76 Kaiyuan Road, Zhumadian 463000, China.
 
Siqiang Li and Tiantian Wang contributed equally to this article.
 
*      Corresponding author: junwu1969@163.com; liubo7095173@163.com

Fig. 1.

Construction of the expression vector pPIC9-α-M, A) schematic diagram of the expression vector pPIC9-α-M; B) PCR amplification of mds I gene from pAO815α-M, molecular marker (lane 1), mds I gene (lane 2); C) dentification of pPIC9-α-M by enzyme digestion, molecular marker (lane 1), pPIC9-α-M digested by EcoR I and XhoI (lane 2) and circle plasmid pPIC9-α-M as control (lane 3).

Fig. 2.

Expression level analysis of the MDS I in the 5-liter fermentation culture supernatant at different time points by SDS-PAGE, molecular marker (lane 1), 12 h (lane 2), 24 h (lane 3), 48 h (lane 4), 60 h (lane 5), 72 h (lane 6), 84 h (lane 7), 96 h (lane 8), 108 h (lane 9).

Fig. 3.

SDS-PAGE analysis of the purified MDS I, molecular marker (lane 1), 10 µL MDS I from raw liquid (lane 2) and each elution after purification from ammonium sulphate precipitation (lane 3), Phenyl-HP (lane 4), Sephadex G25 (lane 5) was subjected to the 12% SDS-PAGE and stained by Coomassie blue.

Fig. 4.

PMF analysis of the expressive product.

Fig. 5.

Five glycoforms of RNase B and MDS I catalyze reaction.

Fig. 6.
The effect of pH on the activity of MDS I, control (A), pH 3.0 (B), pH 5.0 (C), pH 7.0 (D), pH 9.0 (E) and pH 11.0 (F).
Fig. 8.

The effect of metallic ions on the activity of MDS I, Cu2+ (A), Co2+ (B), Zn2+ (C), Mn2+ (D), control (E), Mg2+ (F) and Ca2+ (G).

Fig. 9.

The influence of pH (A), temperature (B), cation (C) on the reaction of RNase B N-glycans catalyzed by MDS I.

Fig. 7.

The effect of temperature on the activity of MDS I, control (A), 16 ℃ (B), 25 ℃ (C), 37 ℃ (D), 42 ℃ (E) and 60 ℃ (F).

Pakistan Journal of Zoology

February

Pakistan J. Zool., Vol. 56, Iss. 1, pp. 01-501

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