木聚糖酶产生菌的筛选盆鉴定及酶学性质研究

采用富集培养和透明圈平板筛选的方法,从长期堆放青贮饲料的土壤中分离到7株木聚糖酶产生菌。选取透明圈最大的木聚糖酶产生菌X7作为基础菌,对其进行16S rRNA部分序列PCR扩增测定,经基因序列比对(BLAST)同源性分析确定X7为枯草芽孢杆菌(Bacillus subtilis strain GD3b,Genebank编码:HM055602.1)。然后将X7进行发酵产酶并对其酶学性质进行初步研究,结果表明:在温度37℃、摇床转速200 r/min和发酵2~3 d所产的木聚糖酶酶活可达13.47 U/mL;其相应粗酶液酶促反应的最适pH为6,酶促反应的最适温度为55℃。     

枯草芽孢杆菌木聚糖酶Xyn B在毕赤酵母中的表达

编码枯草芽孢杆菌(Bacillus subtilis strain GD3b)木聚糖酶(Xylanse B,Xyn B)基因从重组载体pUC-XynB中切割,并克隆到pPICZa A载体中,经酶切和测序鉴定,成功构建了分泌型毕赤酵母表达载体pPICZa-Xyn B。载体经线性化后转入毕赤酵母X-33,并筛选获得可通过甲醇诱导分泌表达Xyn B重组蛋白的毕赤酵母工程菌X-33/Xyn B。     

木聚糖酶和纤维素酶多酶嵌合体的构建

为探究木聚糖酶和纤维素酶形成的多酶嵌合体降解纤维素的效果,此研究设计了2个含有不同来源锚定域的重组木聚糖酶基因和重组纤维素酶基因,并设计了含有2个不同来源的粘附域和纤维素结合域的脚手架蛋白基因,体外构建了大肠杆菌E.coil BL21(DE3)表达载体,表达纯化了脚手架蛋白、重组木聚糖酶和纤维素酶。同时,体外组装三种元件形成多酶嵌合体,以微晶粉末纤维素为底物检测其降解纤维素的效率。结果显示,纯化所得的脚手架蛋白Scaf、重组木聚糖酶Xyn10B和纤维素酶Cel48F可在体外组装形成多酶嵌合体,该多酶嵌合体酶活(0.107±0.013 U/mL)显著高于游离酶混合物的酶活(0.084±0.001 U/mL)(P0.05)。试验表明木聚糖酶Xyn10B和纤维素酶Cel48F可与脚手架蛋白Scaf体外组装形成多酶嵌合体,并可实现对纤维素的高效降解。     

耐碱性短小芽孢杆菌木聚糖酶在枯草杆菌中的表达及发酵条件优化

研究耐碱性短小芽孢杆菌木聚糖酶在枯草芽孢杆菌中异源表达。从耐碱性木聚糖酶高产短小芽孢杆菌BYG5-20中克隆得到带有自身启动子的木聚糖酶基因xynA,将其构建在大肠杆菌-枯草芽孢杆菌穿梭载体pGJ148中得到重组质粒pGJ148-xynA。采用电转化法将重组质粒pGJ148-xynA转入枯草芽孢杆菌1A747中,得到重组菌B.GJ148-xynA,然后进行诱导表达以及培养基的优化。重组菌GJ148-xynA发酵上清液中木聚糖酶酶活可达93.32IU/ml。耐碱性短小芽孢杆菌木聚糖酶基因xynA可以在枯草芽孢杆菌中实现异源表达,为枯草芽孢杆菌木聚糖酶分泌表达系统的进一步优化奠定了基础。     

4种芽孢杆菌来源木聚糖酶基因在大肠杆菌中的表达及酶活分析

研究旨在比较4种不同来源木聚糖酶基因在大肠杆菌E.coli BL21中表达后的酶活等性质,为生产中筛选高表达量及耐极性木聚糖酶基因提供依据。根据已发表的木聚糖酶基因序列设计引物,分别扩增出浸麻芽孢杆菌(B.macerans)B3、地衣芽孢杆菌(B.licheniformis)B6、蜡样芽孢杆菌(B.cereus)B10和枯草芽孢杆菌(B.subtilis)B12的木聚糖酶基因片段,经克隆表达后获得4种工程菌。经培养和IPTG诱导后进行SDS-PAGE电泳检测及酶特性分析。测序结果表明,4个基因的开放阅读框均为642 bp。其中前3种木聚糖酶基因均为首次报道。4种重组木聚糖酶在55℃下的酶活分别为:36.16、3.85、7.22、98.98 U/ml。4种重组木聚糖酶的最适反应温度均在50~60℃,且枯草芽孢杆菌木聚糖酶最为耐热。结果提示,枯草芽孢杆菌来源的木聚糖酶具有较高的酶活和较好的耐热性。     

猪繁殖与呼吸综合征病毒ORF7基因的克隆及其在毕赤酵母中表达

用RT-PCR扩增猪繁殖与呼吸综合征病毒(PRRSV)重庆分离株C14-2的ORF7基因(384 bp),构建克隆质粒pMD19-T-ORF7,经EcoR Ⅰ /Not Ⅰ双酶切回收ORF7基因插入酵母表达载体pPIC9K,构建了重组表达质粒pPIC9K-ORF7,进行PCR鉴定和双酶切鉴定.鉴定的pPIC9K-ORF7经Sac Ⅰ线性化后电转化毕赤酵母宿主菌GS115,筛选获得阳性重组菌GS115(pPIC9K-ORF7),再经G-418/YPD筛选获得高拷贝重组菌,重组子经表型鉴定为Mut.重组菌GS115(pPIC9K-ORF7)经甲醇诱导表达,在96 h表达的N蛋白量最大,N蛋白经SDS-PAGE鉴定大小约为15 000;Western blot表明N蛋白能与美洲型PRRSV阳性血清发生特异性反应,具有良好的反应活性.本研究为开展PRRSV ORF7基因在毕赤酵母中表达及应用奠定基础.     

易错PCR技术提高耐碱木聚糖酶基因xylBYG耐热性的研究

为了研究耐碱木聚糖酶基因xylBYG耐热性,试验采用易错PCR技术对耐碱木聚糖酶基因xylBYG进行定向进化,经过2轮易错PCR产生突变基因产物,重组于表达载体pET-32a(+)中,并导入大肠杆菌BL21构建突变体文库。结果表明:经筛选获得了最佳突变菌株37D07,包含4个氨基酸替换,其最适作用温度比野生型提高了13℃,酶活约提高了17%,热稳定性也相应提高。     

变温操作对黑曲霉A-25产木聚糖酶的影响

试验研究不同温度对黑曲霉生物量和木聚糖酶产量的影响,并通过单位质量菌丝的产酶量和酶谱探究木聚糖酶产量提高的原因。结果显示:黑曲霉A-25在33℃培养时获得最大的生物量,而在29℃时获得最高胞外木聚糖酶分泌量。因此,变温操作采用0~48 h在33℃下培养、之后降为29℃,木聚糖酶活力可达721 U/mL,比29℃恒温发酵提高18%,同时发酵周期缩短8 h。进一步研究发现,0~48 h阶段内变温发酵条件下黑曲霉菌丝体生物量明显增加,并且单位质量菌丝体的产酶能力也有不同程度的提高;木聚糖酶的酶谱检测发现,变温发酵促进木聚糖酶XynⅢ的提前表达,同时提高木聚糖酶XynⅠ和木聚糖酶XynⅡ的表达量。     

呼和浩特地区布鲁菌OMP25基因的克隆、序列分析

为了扩增呼和浩特地区布鲁菌OMP25基因的序列,试验参照GenBank上已登录的布鲁菌外膜蛋白OMP25的基因序列设计1对引物,采用聚合酶链式反应(PCR)技术从灭活的羊种布鲁菌中扩增出OMP25基因片段,将其克隆到pMD19-T载体后测序并应用计算机软件进行分析。结果表明:所测定的序列与羊种布鲁菌(B.melitensis)U33003 OMP25基因的核苷酸同源性为99.5%,推导出的氨基酸同源性为98.1%;与绵羊种布鲁菌(B.ovis)U33004 OMP25基因的核苷酸同源性为98.7%,氨基酸同源性为97.0%。     

大肠杆菌O157∶H7独有基因z3276的表达及初步鉴定

经BLAST分析表明,开放阅读框z3276是大肠杆菌O157∶H7独有的遗传标志性基因,编码氨基酸与大肠杆菌Ⅰ型菌毛有较高的同源性,但z3276基因编码蛋白的生物学特性尚不明确。为了明确z3276基因编码蛋白的抗原性,进一步研究其在大肠杆菌O157∶H7致病过程中的作用。采用PCR方法扩增z3276基因,去除其信号肽序列,克隆到原核表达载体pColdⅠ,转化感受态细胞BL21获得阳性重组菌,IPTG诱导表达重组蛋白,以大肠杆菌O157∶H7全菌抗血清为一抗,Western blot方法检测重组z3276蛋白抗原性。结果显示,成功构建重组菌BL21(pColdⅠ-z3276),并获得高效表达;相对分子质量33 000,占菌体总蛋白30%以上,主要以包涵体形式存在于菌体沉淀中;且与兔源全菌多克隆抗血清发生特异性反应,条带明显。     

不同来源木聚糖酶及其组合对木聚糖水解产物组成、细菌增殖与大肠杆菌黏附性的影响

本试验旨在研究不同来源木聚糖酶及其组合对木聚糖的水解效果,并研究不同木聚糖水解产物对细菌增殖及大肠杆菌对肠道上皮细胞黏附性的影响。试验用木聚糖酶A和木聚糖酶B分别来源于毕赤酵母和米曲霉。采用木聚糖酶A、木聚糖酶B、组合酶1(木聚糖酶A∶木聚糖酶B=3∶7)、组合酶2(木聚糖酶A∶木聚糖酶B=1∶1)、组合酶3(木聚糖酶A∶木聚糖酶B=7∶3)分别水解木聚糖,然后测定木聚糖水解产物对大肠杆菌、枯草芽孢杆菌和乳酸杆菌增殖和大肠杆菌对肠道上皮细胞黏附性的影响。结果表明:1)2种木聚糖酶有一定的组合效应,木聚糖酶A、木聚糖酶B、组合酶1、组合酶2、组合酶3组木二糖和木三糖的总含量分别为95.70%、86.79%、93.11%、94.55%和87.55%,其中木聚糖酶A组木二糖含量最高,组合酶2组木三糖含量最高。2)培养20 h时,5种木聚糖水解产物对大肠杆菌的增殖(以菌液吸光度值表示)没有产生显著影响(P0.05);培养20和30 h时,5种木聚糖水解产物显著促进枯草芽孢杆菌的增殖(P0.05);培养13和17 h时,5种木聚糖水解产物显著促进乳酸杆菌的增殖(P0.05)。3)5种木聚糖水解产物均可以显著降低大肠杆菌对肠道上皮细胞的黏附率(P0.05)。由此可见,通过不同来源木聚糖酶及其组合水解木聚糖,可以产生以木二糖和木三糖为主要组分的水解产物,从而起到促进枯草芽孢杆菌和乳酸杆菌增殖、减少大肠杆菌对肠道上皮细胞黏附的作用。     

饲用木聚糖酶菌株的筛选及鉴定

利用刚果红透明圈法,从采集的土样中筛选分离一株产木聚糖酶的菌株,根据其形态学和生理生化特征,结合16 SrDNA分子水平分析,鉴定该菌为短小芽孢杆菌。摇瓶发酵粗酶液的性质测定表明,该菌株酶活力可达132.53 IU/mL。     

Molecular cloning, nucleotide sequence and characteristics of a xylanase gene (xynA) from Ruminococcus albus 7

A 2.6-kb DNA fragment encoding a xylanase gene ( xyn A) was cloned from the rumen hemicellulolytic bacterium Ruminococcus albus 7. The deduced primary structure of the protein (XynA) was divided into a signal peptide region and 3 domains. Domain A was identified as a family 11 (G) catalytic domain, but one amino acid residue was replaced by another in an active site signature 1 of family 11. Domain B is a stabilizing domain for the catalytic domains of families 10 and 11. Deletion of domain B reduced stability of the xylanase at high temperature and at high and low pH. Domain B may be useful for protein engineering of xylanase. Domain C has sequence similarity to deacetylases and NodB proteins.     

1株产碱性木聚糖酶的芽胞杆菌的分离鉴定及其相关研究

分离到1株产碱性木聚糖酶的革兰氏阳性菌株,通过菌落形态及16srDNA序列同源性分析,确定该菌株为短小芽孢杆菌。生长条件和产酶条件研究结果显示,最适温度和pH分别为50℃和pH8·0。培养基优化试验显示在有机和无机混合氮源条件下(NH4NO30·57%;牛肉膏1%;蛋白胨0·5%;酵母提取物0·5%;木聚糖0·5%)木聚糖酶产量达到最高(180U/mL)。酶学试验表明最适反应条件为50℃,pH8~9;在pH9的条件下,孵育120min时仍具有75%的活力,表明该酶具有较强的碱耐受性。     

MB22木聚糖酶发酵条件的研究

通过因素轮换试验和正交试验,对MB22菌产木聚糖酶的培养基配方和发酵条件进行了研究。结果显示最佳培养基组成是:以玉米芯粉和麸皮为碳源,麸皮120g/l,玉米芯280g/l,(NH4)2SO44g/l,CaCO31.5g/l,Tween80,2.0g/l,MgSO4·7H2O1.5g/l;最佳发酵条件为:起始pH5.0,摇床培养温度30℃,转速160r/min,振荡培养84h。在最佳发酵条件下,MB22菌的最高产酶活力可达898.23U/ml。虽然与一些高产菌株相比该菌株的产酶能力还有待于进一步提高,但该试验结果为进一步进行微生物生产木聚糖酶的研究提供了理论依据。     

Production of large quantities of interferon with calf testicular cells and leucocytes.

Interferon (IF) was induced in calf testicle monolayer cultures and leucocycte suspension cultures with Semliki forest virus and Newcastle disease virus to evaluate the potency of those cells for producing IF in large quantities for clinical experiments. Interferon activity was measured by an RNA-inhibition test using Semliki forest virus as challenge. To produce 10(6) units of IF, leucocytes from 67-5 litres of blood or 7-2 X 10(9) testicle cells were needed.     

两种木聚糖酶对黄羽肉鸡生长性能和养分代谢率的影响

试验旨在探讨不同来源木聚糖酶对黄羽肉鸡生长性能的影响。将1日龄健康黄羽肉鸡540羽,随机分成3组,每组6个重复,每重复30羽,试验期42 d。对照组饲喂小麦-玉米-豆粕型基础日粮,试验A、B组分别饲喂基础日粮中添加200 g/t木霉产木聚糖酶和200 g/t细菌产木聚糖酶的试验日粮,木聚糖酶酶活均为30 000 IU/g。结果表明,与对照组相比,试验A、B组的平均日增重和粗脂肪、粗纤维、钙、磷的表观代谢率及日粮表观代谢能均显著提高（P < 0.05）,料重比显著降低（P < 0.05）,粗蛋白质表观代谢率分别提高了5.90%（P < 0.05）和3.54%（P > 0.05）。试验A、B组之间各测定指标没有显著差异（P > 0.05） 。上述结果表明日粮中添加200 g/t木霉产木聚糖酶或细菌产木聚糖酶均能提高养分代谢率,改善肉鸡生长性能,木霉产木聚糖酶的效果稍好于细菌产木聚糖酶。     

Effects of Two Xylanases on Growth Performance and Nutrient Metabolizability of Yellow-feather Broilers

The experiment was aimed to investigate the effects of xylanases from different sources on growth performance and nutrition metabolizability of Yellow-feather broilers.540 of healthy 1-day-old Yellow-feather broilers were randomly divided into three groups with 6 replicates per group and 30 broilers per replicate,and the trial period was 42 d.The control group was fed a wheat-corn-soybean meal diet,and groups A and B fed the basal diet supplemented with 200 g/t Trichoderma xylanase (30 000 IU/g) and bacterial xylanase (30 000 IU/g), respectively.The results showed that,compared with the control group,the average daily gain,apparent metabolizability of crude fat,crude fiber,calcium and phosphorus,and apparent metabolizable energy were significantly higher (P < 0.05),the F/G were significantly decreased (P < 0.05) in both groups A and B,their apparent metabolizabilities of crude protein increased by 5.90% (P < 0.05) and 3.54% (P > 0.05), respectively.There were no significant differences for all the determined indexes between groups A and B (P > 0.05).The supplementation of Trichoderma xylanase or bacterial xylanase at 200 g/t could improve nutrient metabolizability,thus improve growth performance of broilers,while the effects of Trichoderma xylanase were better than that of bacterial xylanase.     

Bacterial responses to different dietary cereal types and xylanase supplementation in the intestine of broiler chicken.

Several studies were carried out to investigate the influence of dietary cereals differing in soluble non starch polysaccharides (NSP) content and a xylanase preparation on selected bacterial parameters in the small intestine of broiler chicken. Compared to a maize diet colony forming units (CFU) of mucosa associated bacteria were higher in a wheat/rye diet, most notably for enterobacteria and enterococci. Xylanase supplementation to the wheat/rye diet generally led to lower CFU, especially in the first week of life. However, xylanase supplementation also displayed higher in vitro growth potentials for enterobacteria and enterococci. Bacterial growth of luminal samples in minimal media supplemented with selected NSP showed that the wheat/rye diet enhanced bacterial capacities to utilize NSP only in ileal samples. The xylanase application generally shifted respective maximum growth to the proximal part of the small intestine. The presence of soluble NSP from wheat or rye in the diet per se did not enhance bacterial NSP hydrolyzing enzyme activities in the small intestine, but xylanase supplementation resulted in higher 1,3-1,4-beta-glucanase activity. Compared to a maize diet the activity of bacterial bile salt hydrolases in samples of the small intestine was not increased due to inclusion of wheat/rye or triticale to the diet. However, xylanase supplementation led to a reduction with a corresponding increase of lipase activity. It was concluded that dietary cereals producing high intestinal viscosities lead to increased overall bacterial activity in the small intestine. The supplementation of a xylanase to cereal based diets producing high intestinal viscosity, changes composition and metabolic potential of bacterial populations and may specifically influence fat absorption in young animals.