【作者】 何军；

【导师】 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 为获得较为优良的木聚糖酶基因工程菌株,本研究以里氏木霉木聚糖酶Ⅱ(Xyn2)的cDNA为目的基因,分别构建了大肠杆菌(Escherichia coli)和毕赤酵母(Pichiapastoris)表达载体,并成功实现了异源表达;在此基础上,为改善重组酶热稳定性,将海栖热袍菌木聚糖酶A(Thermotoga maritima,XynA)的耐热结构域A2融合至Xyn2基因N-末端,实现了杂合基因在毕赤酵母中的分泌表达;系统研究了重组酶酶学性质,并初步评定了该酶在动物生产中的应用效果。主要研究内容及结果如下:1.里氏木霉Xyn2基因的克隆和序列分析利用RT-PCR技术成功从里氏木霉Rut C-30中扩增出Xyn2的cDNA序列,经序列分析证实,该cDNA开放阅读框全长573 bp,编码191个氨基酸;Rut C-30的Xyn2基因序列与另一诱变株VTT-D-79125完全相同;与野生型菌株QM6α相比,两个变异株Xyn2基因内部有两处碱基发生突变,分别是第13位的C变为T,第272的G变为A;这两个位点的突变导致Xyn2第15位氨基酸残基由His突变为Tyr,而91位的Gly则变为Gln;序列分析证实了这两个突变并非来自PCR扩增,而是在菌株的诱变选育过程中形成的。2.里氏木霉Xyn2基因在大肠杆菌中的表达及重组酶酶学性质分析将Xyn2基因分别克隆到E.coli高效表达载体pET28α(+)和pET30α(+)上,转化宿主菌后获得了能够诱导产生重组木聚糖酶的E.coli基因工程菌BL21-pET28α-Xyn2和BL21-pET30α-Xyn2,它们表达产生的重组酶Xyn228和Xyn230分子量分别为25kDa和27 kDa;对细胞破碎液酶活分析表明,其产量分别达到21.9 U/mL和39 U/mL;Xyn228和Xyn230最适反应温度均为50℃,最适反应pH均为5.0;与亲本酶相比,两种重组酶的热稳定性较好,在60℃下保温30 min后,Xyn228和Xyn230相对酶活分别维持在71%和65%。3.里氏木霉Xyn2基因在毕赤酵母中的分泌表达及重组酶酶学性质分析将Xyn2基因克隆到P.pastoris高效表达载体pPICZαA上,转化野生型毕赤酵母X-33后获得了一株基因工程PX-1,在甲醇诱导下,其表达产物PTX2能够有效分泌至培养基中,并且杂蛋白分泌量极少;重组酶PTX2分子量约为21 kDa,与亲本酶一致,在普通摇瓶中诱导,其产酶水平达到了近300 U/mL;PTX2最适反应温度为60℃,最适反应pH为6.0;该酶在50℃下相当稳定,在此温度下保温30 min后,其相对酶活可维持在90%以上;而在60℃下保温30 min后,PTX2相对酶活仅维持在30%左右;PTX2对燕麦木聚糖的亲和力强于桦木木聚糖,其K_m值分别0.9 mg/mL和1.2mg/mL;与亲本酶一样,PTX2不能与纤维素有效结合,但能与木聚糖结合;PTX2为内切型木聚糖酶,当以燕麦木聚糖为底物时,其水解产物主要为木二糖及木二糖以上的木寡糖,另有少量木糖。4.耐热木聚糖酶基因Xyn2-A2的构建及其在毕赤酵母中的表达通过基因全序列合成,将海栖热袍菌XynA的耐热结构域A2融合至Xyn2基因N-末端,成功构建了耐热木聚糖酶基因及其毕赤酵母表达载体pPICZαA-Xyn2-A2,转化毕赤酵母后获得了能够分泌表达耐热木聚糖酶的基因工程菌PXC-1;其重组酶PTXC2分子量大小为27 kDa,在普通摇瓶中进行诱导获得了约90 U/mL的表达水平:PTXC2最适反应温度为65℃,最适反应pH为6.0;该酶耐热性较PTX2有明显改善,在60℃下保温30 min后,PTXC2残余酶活高达74%;与PTX2和亲本酶相似,PTXC2对纤维素几乎没有结合能力,但PTXC2对木聚糖的结合力强于PTX2。5.重组木聚糖酶小规模发酵制备及其应用效果评定利用15 L自动发酵系统对毕赤酵母基因工程菌PX-1进行小规模诱导培养,重组酶PTX2产量提高了近一倍,最高达到了560 U/mL,且纯度较高;在仔猪饲粮中添加液体重组酶PTX2(500 U/kg),仔猪平均日增重提高了16.9%;饲粮粗蛋白、灰分、钙和粗纤维的表观消化率分别提高了1.7%、3.4%、2.5%和2.6%,该结果表明,重组酶PTX2能够部分消除饲粮中木聚糖的抗营养作用,改善动物生产性能。综上所述,本研究构建了木聚糖酶E.coli基因工程菌,其重组酶活性较强,但表达水平偏低;利用毕赤酵母表达系统成功构建了可实现高效分泌表达的基因工程菌PX-1,其产酶量远高于原核表达系统,且重组酶纯度较高,热稳定性较亲本酶也有一定改善;经动物试验初步评定证实,该重组酶具有较好的应用效果;通过对木聚糖酶基因(Xyn2)结构的改造(耐热域融合)可显著改善重组酶热稳定性,但其表达水平偏低。因此如何兼顾重组酶产量与热稳定性是今后仍需要进一步解决的问题。更多还原

【Abstract】 To improve xylanase production, the Xyn2 gene from Trichoderma reesei Rut C-30 was successfully expressed in Escherichia coli and Pichia pastoris, respectively. Meanwhile, a hybrid xylanase gene was constructed by fusion of the thermostabilizing domain （A2） from Thermotoga maritima XynA into the N-terminal region of Xyn2, and subsequently expressed in P. pastoris. Both the native and hybrid enzymes were fully characterized. The main results were listed as following:1. Cloning and sequencing of the Xyn2 geneThe Xyn2 gene encoding the main Trichoderma reesei Rut C-30 endo-β-1, 4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 573-bp open reading frame that encodes a 191-amino-acid propeptide. The cDNA fragment from T. reesei Rut C-30 is 99% identical to that of T. reesei QM6α, differing for only two base pair substitutions. The two substitutions are at nucleotides 43 and 272 of the Rut C-30 Xyn2 sequence, resulting in two amino acids （Try14/Glu91） in the proprotein being different from those in the proprotein （His14/Gly91） encoded by the QM6αXyn2 gene.2. Expression of the Xyn2 gene in Escherichia coliThe Xyn2 gene was cloned into plasmids pET28α（+） and pET30α（+）, and successfully expressed in E. coli BL21 under the control of strong bacteriophage T7 transcription and translation signals. The molecular weight of the recombinant protein Xyn228 and Xyn230 were estimated by SDS-PAGE to be 25 kDa and 27 kDa, respectively. The two recombiannt proteins were purified by Ni²⁺-NTA affinity chromatography and enzyme activity assay verified the protein as a xylanase. Both Xyn228 and Xyn230 present a temperature optimum at 50℃and a pH optimum at 6.0. Compared with the native Xyn2, both of them had an improved thermostability and retained more than 60% of its activity after 30 mins incubating at 60℃.3. Expression of the Xyn2 gene in Pichia pastorisThe Xyn2 gene was cloned into the expression vector pPICZαA, and successfully expressed in P. pastoris. The desired P. pastoris strain produced as high as 300 U/mL β-xylanase under the control of the methanol incucible alcohol Oxidase 1 （AOX1） promoter. And the secreted protein PTX2 was estimated by SDS-PAGE as 21 kDa. The activity of PTX2 was highest at 60℃which was 5℃higher than native enzyme. In addition, the recombinant enzyme was active over a broad range of pH 3.0-4.0 with maximal activity at pH 6.0. PTX2 was quite stable at 50℃and reatined more than 90% of its activity after 30 mins incubation at this temperature. However, PTX2 was not stable at 60℃, only 30% activity retained. Using Oat spelts xylan and Birchwood xylan, the determined apparent K_m was 0.9 mg/mL and 1.2 mg/mL, respectively. The enzyme was highly specific towards xylan and analysis of the products from xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products.4. Construction and expression of the thermostable xylanase gene in Pichia pastorisTo improve the thermostability of T. reesei Xyn2, the thermostabilizing domain （A2） of XynA from T. maritima has been engineered into the N-terminal region of the Xyn2 gene. The hybrid gene was successfully expressed in P. pastoris, and a level of 90 U/mL was achieved. The hybrid enzyme PTXC2 produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding enzyme PTX2. The temperature and pH optimum of PTXC2 were at 65℃and 6.0, respectively. The hybrid enzyme was more stable than PTX2 at 60℃, and retained more than 74% of its activity after 30 mins incubation at this temperature. Both the hybrid and native enzyme exhibited a low affinity towards cellulosic substrates. However, the binding capacity of PTXC2 toward oat spelt xylan has been improved. These results also suggested that the N-terminal domain （A2） is responsible for both thermostability and substrate-binding capacity of the T. maritima XynA.5. Production of recombinant xylanase by small-scale fermetation and primary evaluation of its potential for industrial applications.The high-density cultivation of PX-1 was performed in a 15-L automatic fermenter. The highest expression level of 560 U/mL was achieved after 78 h fermentation, which was almost doubled than we achieved in flasks. The culture supernatant was collected and used as a liquid enzyme source. To evaluate its potental for industrial applications, the liquid enzyme （PTX2） was supplemented into a wheat-based diet in weaned piglets. Both the performance and nutrient digestibilities was determined. Results showed that the average body weight gain increased 16.9% when piglets received a diet containing 500 U/kg PTX2 . There also was a positive （0.05<P<0.15） effect on the digestibility values of CP, Ash, Ca, and crude fiber with PTX2 supplemetation. These results suggested that PTX2 has the potential beniefits in the application of animal nutrition and feed industry.更多还原