蛋白酶基因改组和表达及其在羽毛粉酶解和肉鸡生产中的应用研究

发布时间：2018-06-24 10:34

本文选题：羽毛粉 + 蛋白酶　；参考：《河南农业大学》2015年博士论文

【摘要】：随着饲料蛋白资源的日益短缺和价格飞速上涨,低质蛋白资源的开发成为研究的热点。羽毛粉蛋白质含量丰富,但其结构稳定,不溶于水,不能被一般的蛋白酶水解,难以被动物消化吸收。本文通过筛选得到对羽毛粉降解效果较好的米曲霉和枯草芽孢杆菌菌株,对两种菌株的中性蛋白酶和碱性蛋白酶基因进行DNA改组和克隆,将改组后的蛋白酶基因成功转入毕赤酵母X33中,并对重组酵母蛋白酶活力和酶学性质进行分析。最后利用重组毕赤酵母发酵羽毛粉并将其应用到肉鸡生产中,对发酵羽毛粉替代鱼粉对肉鸡的饲养效果进行研究,为缓解蛋白资源短缺和提高羽毛粉的饲用价值提供了新的技术途径。主要结果如下:(1)从五种微生物菌株中初筛出产蛋白酶活力较高的米曲霉和枯草芽孢杆菌菌株。通过测定蛋白酶活力和羽毛粉中可溶性蛋白含量,对米曲霉和枯草芽孢杆菌对羽毛粉的降解效果进行研究。结果显示,两种微生物菌株均可对羽毛粉起到降解作用。发酵72 h后,米曲霉产蛋白酶活力达到3256.61 U/g(P0.05),枯草芽孢杆菌产蛋白酶活力达到690.03 U/g(P0.05)。米曲霉发酵羽毛粉48 h时可溶性蛋白含量,由最初的2.21 mg/g提高到362.07 mg/g(P0.05)。(2)使用RT-PCR技术,成功克隆得到米曲霉中性和碱性蛋白酶基因、枯草芽孢杆菌中性和碱性蛋白酶基因,四种蛋白酶基因与NCBI公布的蛋白酶基因同源性均大于99.5%。(3)对克隆得到的四种蛋白酶基因进行DNA改组,将改组蛋白酶基因与pMD19-T质粒连接并转入大肠杆菌中,通过酪蛋白平板筛选,得到三个来自米曲霉的DNA改组中性蛋白酶基因NPGA-S1、NPGA-S2、NPGA-S3和一个来自枯草芽孢杆菌的DNA改组中性蛋白酶基因NPGB-S1。(4)将四种DNA改组蛋白酶基因和两种中性蛋白酶基因电击转入毕赤酵母X33中,通过重组酵母蛋白酶活力测定,筛选出NPGA-S2和NPGA-S3两种改组效果较好的蛋白酶基因。(5)将NPGA-S2和NPGA-S3与四种初始蛋白酶基因一起进行第二轮DNA改组,初筛出一个来自米曲霉的中性蛋白酶DNA改组基因NPGA-S4,将其电击转化至毕赤酵母X33中,通过对三种含米曲霉中性蛋白酶改组基因的重组毕赤酵母产蛋白酶酶活力的测定,最终选出NPGA-S2作为最好的DNA改组蛋白酶基因。与初始蛋白酶基因相比,DNA改组蛋白酶基因在1022 bp处的核苷酸T突变为C,相应的氨基酸由半胱氨酸突变为精氨酸。DNA改组蛋白酶的酶活力比初始蛋白酶活力提高36.4%(P0.05),pH和热稳定性也得到了明显提高,在pH6.5-8.0和30-70℃之间保持稳定(P0.05)。(6)通过鸡强饲试验对发酵羽毛粉蛋白质、钙和磷代谢率进行测定,米曲霉和含蛋白酶基因重组酵母发酵羽毛粉的蛋白质和钙磷表观代谢率显著高于普通羽毛粉和不含蛋白酶基因重组酵母发酵羽毛粉(P0.05)。与普通羽毛粉相比,发酵羽毛粉(含玉米)中各种氨基酸含量均有所提高,含改组蛋白酶基因的重组酵母发酵羽毛粉赖氨酸含量增长接近1倍。(7)用米曲霉和含蛋白酶基因重组酵母发酵羽毛粉替代鱼粉添加到肉鸡日粮中,通过测定黄羽肉鸡生长性能、屠宰性能、肉品质、肠道微生物、血液生化指标和经济效益分析,研究发酵羽毛粉替代鱼粉对肉鸡生产的可行性。饲养试验分为前期(1-28d)和后期(29-56d)两个阶段。前期和后期分别选择250只黄羽肉鸡,分为五个处理,每个处理5个重复,每个重复10只鸡。其中A组为含鱼粉的基础日粮(对照组),B组用等量米曲霉发酵羽毛粉替代基础日粮中的鱼粉,C组用等量不含蛋白酶基因的pGAPZαA重组毕赤酵母发酵羽毛粉替代基础日粮中鱼粉(负对照组),D组用等量含米曲霉蛋白酶基因的pGAPZαA重组毕赤酵母发酵羽毛粉替代基础日粮中鱼粉,E组用等量含米曲霉DNA改组蛋白酶基因(NPGA-S2)的pGAPZαA重组毕赤酵母发酵羽毛粉替代基础日粮中鱼粉。结果表明,饲养试验前期和后期,除负对照组外,添加米曲霉和含蛋白酶基因发酵羽毛粉各组和对照组在日增重、日采食量、饲料转化率和营养物质代谢率方面差异不显著(P0.05)。含DNA改组蛋白酶基因重组酵母发酵羽毛粉组屠宰率、全净膛率和肉品质虽然高于其他各组,但各组之间差异不显著(P0.05)。米曲霉和含蛋白酶基因重组酵母发酵羽毛粉组肠道蛋白酶活力显著高于对照组和负对照组(P0.05)。各组在肠道微生物数量、肠道微生物菌群分布和微生物多样性方面基本一致,其中含DNA改组蛋白酶基因重组酵母发酵羽毛粉组对肠道微生物生长和稳定有促进作用。除对照组外,各组血清中总蛋白、球蛋白和白蛋白差异不显著(P0.05),添加发酵羽毛粉对肉鸡血清酶活和免疫系统无明显影响。另外,经济效益分析也证明,使用发酵羽毛粉替代鱼粉饲喂肉鸡是可行的,为羽毛粉等非常规蛋白质饲料资源的开发和利用奠定基础。
[Abstract]:With the increasing shortage of feed protein resources and the rapid rise in price, the development of low quality protein resources has become a hot spot of research. The protein content of feather powder is rich, but its structure is stable and insoluble in water. It can not be hydrolyzed by the ordinary protease, and it is difficult to be digested and absorbed by animals. This paper has been screened to get better degradation effect of feather powder. The strains of Bacillus subtilis and Bacillus subtilis were reformed and cloned for the neutral protease and alkaline protease genes of two strains. The protease genes were successfully transformed into Pichia pastoris X33, and the recombinant yeast protease activity and the properties of the protease were analyzed. Finally, the featherpowder was fermented by the recombinant Pichia pastoris and applied to the DNA. In the production of broilers, the feeding effect of fermented feather powder instead of fish meal on broilers was studied. A new technical approach was provided to alleviate the shortage of protein resources and improve the feeding value of feather powder. The main results are as follows: (1) from the first screening of five strains of microorganism, Aspergillus oryzae and Bacillus subtilis producing high activity of protease have been screened. Determination of protease activity and soluble protein content in feather powder, the degradation effect of Aspergillus oryzae and Bacillus subtilis on feather powder was studied. The results showed that two microbial strains could degrade feather powder. After 72 h fermentation, the protease activity of Aspergillus oryzae reached 3256.61 U/g (P0.05), Bacillus subtilis producing protease The activity reached 690.03 U/g (P0.05). The soluble protein content of Aspergillus oryzae Fermented feathers 48 h increased from 2.21 mg/g to 362.07 mg/g (P0.05). (2) RT-PCR technology was used to clone the neutral and alkaline protease genes of Aspergillus oryzae, Bacillus subtilis neutral and alkaline protease genes, and four protease genes published with NCBI. The homology of the protease gene is greater than 99.5%. (3) to reorganize the four protease genes obtained from the cloned gene. The protease gene is reorganized by DNA. The protease gene is connected to the pMD19-T plasmid and transferred into the Escherichia coli. By screening the casein plate, three DNA reformed neutral egg white enzyme genes from Aspergillus oryzae, NPGA-S1, NPGA-S2, NPGA-S3, and one from the blight are obtained. The DNA modified neutral protease gene NPGB-S1. (4) of Bacillus buds (4) converted four DNA protease genes and two neutral protease genes into Pichia pastoris X33. Through recombinant yeast protease activity determination, two protease genes of NPGA-S2 and NPGA-S3 were screened out. (5) NPGA-S2 and NPGA-S3 and four species were first introduced. The initial protease gene was reorganized in second rounds of DNA. A neutral protease DNA restructure gene NPGA-S4 from Aspergillus oryzae was screened, and its electric shock was converted to Pichia pastoris X33. The activity of protease production of the recombinant Pichia pastoris containing three species of Aspergillus oryzae neutral protease was determined, and NPGA-S2 was selected as the best. DNA reformed protease gene. Compared with the initial protease gene, the nucleotide T of the DNA reformed protease gene at 1022 BP mutation was C. The corresponding amino acids increased by 36.4% (P0.05), and pH and thermal stability were significantly increased from the cysteine to the arginine.DNA reformed protease, and the pH and thermal stability were also significantly increased in pH6.5-8.0. Stability (P0.05) was maintained between 30-70 and 6. (6) the protein, calcium and phosphorus metabolism of fermented feather powder was measured by the strong feeding test of chicken. The apparent metabolic rate of protein and calcium phosphorus of Aspergillus oryzae and protease gene recombinant yeast feathers was significantly higher than that of ordinary feather powder and recombinant yeast fermented feathery powder without protease gene (P0.05). Compared with ordinary feather powder, all kinds of amino acids in fermented feather powder (corn) were improved, and the content of lysine content of the recombinant yeast fermented feathery powder containing the reorganized protease gene increased nearly 1 times. (7) using Aspergillus oryzae and protease gene recombinant yeast fermented feathery feathers added to the broiler diet by measuring Huang Yurou. The growth performance, slaughter performance, meat quality, intestinal microorganism, blood biochemical indexes and economic benefits were analyzed. The feasibility of fermented feather meal instead of fish meal on broiler production was studied. The feeding experiment was divided into two stages (1-28d) and later stage (29-56d). In the early and late stages, 250 yellow feathered broilers were divided into five treatments, and 5 were treated with each treatment. Repeat, 10 chickens were repeated each, of which group A was a basal diet containing fish meal (control group), and the B group used Aspergillus oryzae Fermented feathers to replace fish meal in basal diet, and group C with pGAPZ alpha A recombinant Pichia pastoris fermented feathery powder with equal amount of non protease gene instead of fish meal in basal diet (negative control group), and D group with Aspergillus oryzae in the same amount. The gene pGAPZ alpha A recombinant Pichia pastoris fermented feathers to replace the fish meal in the basal diet, and the E group used the pGAPZ alpha A recombinant Pichia pastoris feathers from the same amount of Aspergillus oryzae DNA to replace the fish meal in the basal diet with the same amount of Aspergillus oryzae DNA (NPGA-S2). The results showed that the early and later period of the feeding test, except for the negative control group, added Aspergillus oryzae and protease. There was no significant difference in daily weight gain, daily feed intake, feed conversion rate and nutrient metabolism rate (P0.05) in each group and control group (P0.05), but the total net rate and meat quality were higher than those of other groups, but the difference was not significant (P0.05), Aspergillus oryzae. The protease activity of the enteric fermented feather meal group containing the protease gene was significantly higher than that of the control group and the negative control group (P0.05). The number of intestinal microbes, the distribution of intestinal microflora and the microbial diversity in each group were basically the same, including the DNA reorganized protease gene regrouping yeast fermented feather powder group to the intestinal microorganism growth. In addition to the control group, there was no significant difference in total protein, globulin and albumin in the serum (P0.05), but the addition of fermented feather powder had no obvious effect on the serum enzyme activity and immune system of broilers. In addition, the analysis of economic benefits also proved that it was feasible to feed the chicken with fermented feather powder instead of fish meal, and the feather powder was very important. Laying the foundation for the development and utilization of protein feed resources.
【学位授予单位】：河南农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S831

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