【作者】 胡洪；

【导师】 陈代文； 何军；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2013， 博士

【摘要】 角蛋白酶是一种可降解角蛋白的酶类,在多个行业特别是饲料工业中具有较大的应用前景。但是由于天然菌株产酶水平较低,限制了其应用范围。为了获得高效表达的角蛋白酶(kerA)基因工程菌株,本研究分别构建了kerA基因大肠杆菌和毕赤酵母表达载体,并实现了异源表达；分析角蛋白酶的基因和蛋白结构,利用定点突变技术进行改造,改善重组酶表达水平和热稳定性；系统研究了重组角蛋白酶酶学性质,初步评价其在动物试验中的应用效果。具体研究内容和结果如下：试验一：地衣芽孢杆菌kerA基因的克隆通过PCR以地衣芽孢杆菌DNA为模板扩增出除去信号肽的kerA基因序列。对序列分析显示,该基因长度为1047bp,编码349个氨基酸；与来源于地衣芽孢杆菌PWD-1的kerA基因相比,有5个氨基酸位点发生了改变(Arg-7vs.Glu-7,Asp-121vs. Gly-121, His-136vs. Asn-136, Glu-267vs. Gly-267, Ala-297vs.Val-297),但是仍具有角蛋白酶的三联体催化位点(Asp32, His63和Ser220)。试验二：kerA基因在大肠杆菌中的表达及重组酶酶学性质研究将试验一所得kerA基因克隆进入原核表达载体pET-30a和pET-32a中,转化大肠杆菌BL21得到能够分泌重组角蛋白酶的基因工程菌E. coli BL21-pET-30a-kerA和BL21-pET-32a-kerA,表达的重组蛋白分别命名为EK1和EK2；两种重组酶分子大小分别约为45kDa和55kDa;超声波处理细胞后,破碎液酶活可达18U/mL和13.5U/mL;重组酶EK1和EK2最适反应pH分别为7.5和8.5,最适温度均是50℃；两种重组酶在50-60℃之间热稳定性均较好,在60℃保温30mmin,EK1和EK2相对酶活仍能保持最高酶活的60%以上试验三：kerA基因在毕赤酵母中的表达及重组酶酶学性质研究将除去信号肽和终止密码子的kerA基因克隆进入真核表达载体pPICZaA中,转化毕赤酵母X-33后得到基因重组菌P. pastoris pPICZaA-kerAwt,表达的重组角蛋白酶命名为PK1,其分子大小为39kDa左右,经糖蛋白染色实验证明,重组蛋白属于糖蛋白；甲醇诱导下,重组菌P. pastoris pPICZaA-kerAwt产酶水平最高达195U/mL;重组角蛋白酶PK1最适反应pH和最适反应温度分别为7.5和50℃；该酶在50℃孵育2h后,相对酶活仍能保持最高酶活的50%以上；但是当温度超过60℃时热稳定性较差；就不同底物亲和力而言,重组酶PK1水解羽毛粉(β--角蛋白)的能力要显著强于天青角蛋白(α-角蛋白)。试验四：高效表达角蛋白酶基因的分子设计及其在毕赤酵母中的表达研究利用定点突变技术,通过两种不同的密码子优化策略优化亲本kerA基因,提高重组角蛋白酶的表达水平。优化策略主要包括以下两种方式,一种是将亲本kerA基因的CCG (Pro-3)和GCG (Ala-4)同义突变为CCA和GCT,获得的突变基因为kerAoptil;另外一种是在前者的基础上继续进行突变,将CGC (Arg-322)和CGT(Arg-324)替换为AGA (Arg);将两种优化基因克隆进入pPICZaA载体中,转化P. pastoris X-33得到基因工程菌P. pastoris pPICZaA-kerAopti1和pPICZaA-kerAopti2,表达的重组角蛋白酶分别命名为PK2和PK3；通过实时荧光定量PCR技术检测插入的基因拷贝数和mRNA表达水平,结果表明这两种指标在重组毕赤酵母菌株中均没有显著差异；甲醇诱导下,重组P. pastoris pPICZaA-kerAopti1和pPICZaA-kerAopti2的产酶水平最高分别可达324U/mL和293U/mL,得到的重组蛋白分子大小约为39kDa。试验五：耐热角蛋白酶基因的分子设计及其在毕赤酵母中的表达研究通过定点突变技术,在亲本kerA基因引入两个Cys生成二硫键,提高重组角蛋白酶的热稳定性。利用Disulfide by DesignTM软件,选择将Gln-316和Leu-320突变为Cys,获得突变基因kerAopti3,构建真核表达载体pPICZaA-kerAopti3,电转进入P. pastoris X-33中,得到基因工程菌株P. pastoris pPICZaA-kerAopti3,获得的重组酶命名为PK4,其分子大小约为39kDa,与前期研究一致；甲醇诱导下,重组菌P. pastoris pPICZaA-kerAopti3产酶水平为24U/mL;重组酶PK4最适pH和最适反应温度分别是8.0和50℃；其热稳定性较PK1有所改善,在60℃孵育30min,相对酶活仍有30%以上,而重组酶PK1在60℃保温30min,残余酶活只有13%左右。试验六：重组酶小规模制备及其应用效果评定通过30L发酵罐对基因工程菌P. pastoris pPICZaA-kerAopti1进行小规模诱导产酶,结果表明产酶水平最高可达568U/mL,与摇瓶培养相比提高了150%以上。为了考察重组角蛋白酶对生长育肥猪生长性能和饲粮粗蛋白质消化率的影响,本研究选择60头DLY生长育肥猪(58.1±1.1kg),随机分为5个处理组(n=12),处理1为对照组(CD组),采用玉米豆粕型基础饲粮；处理2-5,均添加5%的羽毛粉等N替代基础饲粮中的豆粕,保证试验饲粮粗蛋白质和赖氨酸含量一致,其中角蛋白酶添加水平分别为0(FD组)、3000U/kg (LFD组)、6000U/kg (MFD组)、9000U/kg (HFD组)。结果表明,与CD组相比,FD组平均日增重(ADG)和饲粮粗蛋白质表观消化率(ADCP)显著降低(P<0.05),随着重组角蛋白酶的添加,LFD、MFD和HFD组的ADG和ADCP也随之增加；试验期间,平均日采食量与耗料增重比在各个组之间没有显著差异(P>0.05),但是FD组的耗料增重比最高。综上结果表明,本研究成功实现了地衣芽孢杆菌kerA基因在大肠杆菌和毕赤酵母中的表达；获得的大肠杆菌基因工程菌株虽然热稳定性较高,但产酶水平较低,不适合于商业化生产；通过密码子优化后,重组菌P. pastoris pPICZaA-kerAoptil产酶水平远远高于大肠杆菌表达系统,是一株较有潜力的生产菌株；经过动物试验初步评定证明重组角蛋白酶可以改善饲粮蛋白质消化率和利用率；对kerA基因进行改造得到耐热重组角蛋白酶,虽然热稳定性有一定提高,但是表达量显著降低。因此,进一步开展角蛋白酶蛋白结构与功能关系的研究十分必要。更多还原

【Abstract】 Keratinases are metallo or serine proteinases which can degrade the insoluble structure forming keratin substrates and have immense potential application in the animal nutrition and feed industry. However, the industrial applications of keratinase are still not extensive because of its low level of expression. To increase its production, the keratinase (kerA) gene from Bacillus licheniformis S90was expressed in Escherichia coli BL21and Pichia pastoris X-33. In addition, the native gene was optimized by site-directed mutagenesis to improve its production and thermal stability, and then expressed in P. pastoris X-33. The biochemical properties of all recombinant keratinases were fully characterized.Experiment1:Cloning of the kerA gene from Bacillus licheniformisThe kerA gene was successfully amplified by PCR from B. licheniformis S90DNA. Sequence analysis revealed that the nucleotide sequence of this gene contains1047bp open reading frame (ORF) encoding349amino acids. As compared to the B. licheniformis PWD-1keratinase, there were only five amino acid mutations in the protein sequence of B. licheniformis S90keratinase (Arg-7vs.Glu-7, Asp-121vs. Gly-121, His-136vs. Asn-136, Glu-267vs. Gly-267, Ala-297vs.Val-297). However, both enzymes share the same triad of catalytic residues including Asp-32, His-63, and Ser-220.Experiment2:Expression of the kerA gene in E. coliThe gene kerA was cloned into two conventional vectors, pET30a and pET32a, and expressed in E. coli BL21. From SDS-PAGE analysis, the recombinant keratinases were45and55kDa. They had different optimal pH values (7.5and8.5) but the same optimum temperature of50℃. Both of them had improved thermal stability and kept approx.60%of its max activity after30min at60℃.Experiment3:Expression of the kerA gene in P. pastoris X-33The gene kerA was cloned into plasmid pPICZaA and expressed in P. pastoris X-33under the control of AOX1promoter. The highest keratinase activity produced by P. pastoris pPICZaA-kerAwt was195U/mL. The keratinase secreted by recombinant P. pastoris was named PK1. The molecular weight of PK1was approx.39kDa. The enzyme PK1was active at pH values from7-9and had optimum pH of7.5. It was stable at moderate temperature and had optimum temperature at50℃. The PK1is much more stable at optimum temperature (50℃), remained approx.50%of the maximum activity at this temperature for2h. However, it was rapidly inactivated at higher temperatures60-70℃for30min. When the substrate chicken feather (β-keratin) meal was used, the yield of enzymatic hydrolysis was significantly higher than the substrate keratin azure (a-keratin).Experiment4:Expression of the high-yield kerA gene in P. pastoris X-33The main kerA gene was optimized by two codon optimization strategies and expressed in P. pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The obtained recombinant keratinase was named PK2and PK3respectively. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in P. pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZaA-kerAoptil and pPICZaA-kerAopti2was324U/mL and293U/mL respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. And the secreted recombinant protein was determined by SDS-PAGE was approx.39kDa.Experiment5:Expression of the thermostable kerA gene in P. pastoris X-33Improvement of thermal stability of kerA was tried by engineering a de novo designed disulfide bridge. Disulfide design was performed firstly using Disulfide by DesignTM program. In the present study, only one disulfide bond (Gln320and Leu316) was selected, and the respective amino acids were substituted with Cys residues. The newly designed gene which was named kerAopti3expressed in Pichia pastoris. The highest keratinase activity produced by P. pastoris pPICZaA-kerAwt was24U/mL. The obtained recombinant keratinase was named PK4. The molecular weight of PK4was approx.39kDa. The PK4was optimally active at pH8.0and50℃. The PK4produced clearly improved the thermal stability. It was more stable than PK1at60-70℃.Experiment6:Production of recombinant keratinase by small-scale fermentation and its effect on growth performance and apparent crude protein digestibility in finisher pigsThe high density cultivation of Pichia pastoris pPICZaA-kerAopti1was performed in a30-L fermenter. The highest expression level of568U/mL was achieved after72h fermentation. Then, the recombinant keratinase was added to a feather meal dietary to detect its effect on growth performance and crude protein digestibility in finisher pigs. Sixty Duroc×Landrace×Yorkshire finisher pigs (58.1±1.1kg of initial body weight) were randomly allotted to five groups (n=12). The dietary groups include:(1) basal diet without feather meal and keratinase supplementation (CD);(2-5) feather meal dietary group (5%of feather meal inclusion) with keratinase supplementation at a final concentration of0U/kg (FD group)、3000U/kg (LFD group)、6000U/kg (MFD group)、9000U/kg (HFD group). The results indicated that the average daily body-weight gain (ADG) and the apparent digestibility of crude protein (ADCP) were significantly reduced for FD group compared to CD group (P<0.05), but the ADG and ADCP were increased by FD group keratinase supplementation. In addition, no significant effect of keratinase treatment on feed intake and efficiency.In summary, the broad the pH profile, substrate specificity and expression level make the recombinant keratinase from P. pastoris pPICZaA-kerAopti1a suitable applicant for various industrial applications.更多还原