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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (3): 37-45    DOI: 10.13523/j.cb.20190306
研究报告     
木聚糖酶和甘露聚糖酶在毕赤酵母中的共表达及产酶分析 *
姚银,闵琪,熊海容,张莉()
中南民族大学生命科学学院 武汉 430074
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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摘要：

木聚糖酶和甘露聚糖酶是两种重要的半纤维素酶,也是两种重要的饲用酶制剂,通过毕赤酵母表达系统中的体外串联表达盒构建多拷贝的方法构建了木聚糖酶DSB和甘露聚糖酶ManA共表达重组质粒pPICZαA/DSB-ManA,将该重组质粒电转化至宿主菌毕赤酵母X33中获得共表达两种酶的重组菌X33/ DSB-ManA,实现了两种酶的共分泌表达,经诱导表达后木聚糖酶和甘露聚糖酶的酶活分别为273.6 U/ml和256.8 U/ml,为单独表达重组菌X33/DSB和X33/ManA酶活的30.4%和73.4%。酶学性质的分析显示DSB和ManA的最适反应温度均为75℃,在45℃75℃范围内具有较好的温度稳定性,酶活可保持最高酶活的60%以上;DSB最适pH为6.5,ManA最适pH为6.0,在pH 3.0、40℃条件下,ManA处理1 h能保持最高酶活的80%以上,DSB处理1 h时能保持最高酶活的50%以上;DSB和ManA对多种金属离子和化学试剂(浓度为1 mM)具有较好的耐受性,均可保留60%以上的酶活力。通过单一菌株成功完成了不同酶的共表达,为复合酶饲料添加剂的生产和应用研究提供了一定的理论依据。
关键词： 木聚糖酶;  甘露聚糖酶;  毕赤酵母;  共表达;  酶学特性    
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
收稿日期: 2018-09-06 出版日期: 2019-04-12
ZTFLH:  Q789  
基金资助: * 湖北省技术创新专项重大项目资助项目(2018ABA093)
通讯作者: 张莉     E-mail: serena20112014@mail.scuec.edu.cn
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姚银
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引用本文:

姚银,闵琪,熊海容,张莉. 木聚糖酶和甘露聚糖酶在毕赤酵母中的共表达及产酶分析 *[J]. 中国生物工程杂志, 2019, 39(3): 37-45.

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.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190306    或    https://manu60.magtech.com.cn/biotech/CN/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
表1  PCR扩增引物序列
图1  共表达载体pPICZαA/DSB-ManA的构建示意图
图2  共表达重组质粒pPICZαA/DSB-ManA构建过程中的电泳图
图3  单独表达重组菌发酵144 h产两种酶的酶活力
图4  X33/DSB-ManA发酵过程的生长曲线和产木聚糖酶与甘露聚糖酶的酶活曲线
图5  X33/DSB-ManA发酵上清液的SDS-PAGE分析
图6  X33/DSB-ManA产木聚糖酶(pH6.5)和甘露聚糖酶(pH6.0)的最适温度及热稳定性
图7  X33/DSB-ManA产木聚糖酶和甘露聚糖酶的最适pH(75℃)及pH稳定性
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
表2  不同化学试剂及金属离子对甘露聚糖酶活力的影响
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
表3  不同化学试剂及金属离子对木聚糖酶活力的影响
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