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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (3): 37-45    DOI: 10.13523/j.cb.20190306
    
Co-expression of xylanase and mannanase in Pichia pastoris and the enzymatic analyses
Yin YAO,Qi MIN,Hai-rong XIONG,Li ZHANG()
College of Life Science,South-Central University for Nationalities,Wuhan 430074,China
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Abstract  

Xylanase and mannanase are two important hemicellulases and feed enzyme preparations. Construction method of in vitro multimers in Pichia pastoris expression system has been used to construct recombinant plasmid pPICZαA/DSB-ManA which contained both DSB gene and ManA gene. Then, pPICZαA/DSB-ManA was transformed to host cells Pichia pastoris X33 by electroporation to obtain the co-expression recombinant strain X33/DSB-ManA. In shake-flask cultivation, xylanase activity and mannanase activity in the supernatant were 273.6 U/ml and 256.8 U/ml that reached 30.4% and 73.4% activity compared to X33/DSB and X33/ManA, respectively. Properties of the mixed enzyme suggested that the optimal reaction temperature for DSB and ManA is 75℃. Both xylanase and mannanase showed more than 60% activity across temperature ranges 45°C to 75°C. The optimal reaction pH value of DSB and ManA were 6.5 and 6.0, respectively. After incubation 1 hour under the pH3.0 and 40℃ treatment condition, mannanase activity of ManA displayed greater than 80% activity and xylanase activity of DSB revealed more than 50% activity. Meanwhile, DSB and ManA have shown good tolerance to a variety of metal ions and chemical reagents. Both enzyme activities could keep over 60% activity when the treatment concentration of different metal ions and chemical reagents was 1 millimole. Xylanase DSB and mannanase ManA have been co-expressed in Pichia pastoris successfully. The successful expression of the two enzymes of the recombinant strain could offer the theory basis for production and application of compound enzyme in the animal feed.

Key words： Xylanase      Mannanase      Pichia      pastoris      Co-expression      Enzymatic      characteristics     
Received: 06 September 2018      Published: 12 April 2019
ZTFLH:  Q789  
Corresponding Authors: Li ZHANG     E-mail: serena20112014@mail.scuec.edu.cn
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Yin YAO
Qi MIN
Hai-rong XIONG
Li ZHANG
Cite this article:

Yin YAO,Qi MIN,Hai-rong XIONG,Li ZHANG. Co-expression of xylanase and mannanase in Pichia pastoris and the enzymatic analyses. China Biotechnology, 2019, 39(3): 37-45.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190306     OR     https://manu60.magtech.com.cn/biotech/Y2019/V39/I3/37

基因 引物 引物序列(5'→3') 酶切位点
DSB P1 CCGGAATTCTGCGCAACCCCCAACTCGGAG EcoR I
P2 ATAGCGGCCGCTTAGCCCACGTCAGCAACGG Not I
ManA P3 TATGGTACCTCTGGTGGACCAAACTTGTCTG KpnⅠ
P4 CCGTCTAGAAGAGGTACCGATAGCGTTAGTTC XbaⅠ
Table 1 Sequence of PCR amplification primers
Fig.1 Schematic diagram of construction of pPICZαA/DSB-ManA
Fig.2 Electrophoresis analysis of co-expression recombinant plasmid pPICZαA/DSB-ManA (a) Electrophoresis of PCR analysis of DSB and ManA. M: DL5000 DNA marker; 1: ManA; 2: DSB (b) Restriction analysis of recombinant plasmid pPICZαA/DSB and pPICZαA/ManA. M: DL5000 DNA marker; 1: pPICZαA/ManA; 2: pPICZαA/DSB (c) Restriction analysis of pPICZαA/DSB-ManA. M: DL5000 DNA marker; 1: pPICZαA/DSB-ManA
Fig.3 Enzyme activities of X33/DSB and X33/ManA at 144h (a) Xylanase activity of different transmutants of X33/DSB (b) Mannanase activity of different transmutants of X33/ManA
Fig.4 The growth curve(a) and the xylanase activity and mannanase activity curves(b) of X33/DSB-ManA
Fig.5 SDS-PAGE analysis of X33/DSB-ManA fermentation supernatant M: Prestained protein ladder; 1: X33/DSB-ManA fermentation supernatant
Fig.6 Optimum tempature (a) and thermal stability (b) of xylanase (pH 6.5) and mannanase (pH 6.0) of X33/DSB-ManA
Fig.7 Optimum pH (75℃) (a) and pH stability (b) of xylanase and mannanase of X33/DSB-ManA
浓度(mmol/L)

金属离子		 0 0.5 1
Cu2+ 100±1.95 97.22±4.67 83.71±2.81
Fe3+ 100±2.49 105.75±3.16 86.12±6.36
Mn2+ 100±7.52 65.03±1.21 69.79±1.27
Co2+ 100±4.52 104.93±5.30 87.15±3.82
Mg2+ 100±2.93 99.83±4.24 107.56±3.00
Li+ 100±2.85 103.43±3.62 101.33±6.26
Ni2+ 100±4.03 101.38±7.19 102.36±4.31
Ca2+ 100±5.58 121.27±2.82 131.57±4.88
Pb2+ 100±3.94 95.74±2.49 87.14±5.91
SDS 100±4.28 97.74±4.89 99.73±1.85
EDTA 100±2.33 85.06±4.31 90.56±6.99
巯基乙醇 100±2.58 93.29±7.01 87.40±5.55
Table 2 The effects of different chemical reagents and metal ions on the activity of mannanase
浓度(mmol/L)

金属离子		 0 0.5 1
Cu2+ 100±0.26 91.55±0.38 73.18±0.37
Fe3+ 100±0.76 98.29±0.33 97.20±0.14
Mn2+ 100±0.77 64.09±0.54 67.89±0.77
Co2+ 100±0.68 117.13±0.49 96.80±0.74
Mg2+ 100±0.61 90.93±0.75 91.78±1.07
Li+ 100±0.81 97.80±0.54 96.99±0.33
Ni2+ 100±0.45 99.16±0.63 96.69±0.56
Ca2+ 100±0.50 92.79±0.71 96.04±0.35
Pb2+ 100±0.86 92.62±0.77 87.71±1.09
SDS 100±2.22 84.89±0.92 78.51±0.58
EDTA 100±0.50 89.82±1.05 84.04±1.21
巯基乙醇 100±0.61 99.47±2.29 100.49±1.67
Table 3 The effects of different chemical reagents and metal ions on the activity of xylanase
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