唾液酸溶析结晶工艺的研究

大肠杆菌发酵得到聚唾液酸,经酸水解后得到的唾液酸单体纯度较低,需进一步分离和纯化。考察了唾液酸酸水解液的脱色工艺,添加质量浓度为5%的凹土能有效除去水解液中的红色物质。实验中研究了唾液酸单体的溶析结晶工艺,以无水乙醇为主溶剂溶解唾液酸样品,可实现唾液酸与唾液酸二聚体的分离,以乙酸乙酯为析出剂有效地实现了唾液酸单体的结晶析出,当乙醇/乙酸乙酯的比率为1.0时,得到的唾液酸纯度达到90%~92%的质量分数,回收率为55.4%。     

鸡蛋壳膜提取唾液酸过程中蛋白质脱除工艺优化

以鸡蛋壳膜为原料,用乙醇沉淀法脱除蛋白质以提取唾液酸。利用二次回归中心组合设计对影响蛋白质去除率和唾液酸回收率的乙醇体积分数、加热温度、加热时间、pH值4个因素进行回归分析和优化,建立四元二次回归模型。结果表明因素的影响大小顺序为:乙醇体积分数>加热温度>pH值>加热时间,确定最佳工艺参数为:乙醇体积分数30%、加热温度80℃、加热时间1h、pH2。在此条件下,蛋白质去除率为52.3%,唾液酸回收率为82.1%,经验证实验得到实验结果与模型预测值吻合,说明建立的模型确实可行。     

乙醇酸法脱菜籽酚及硫苷的工艺研究及对蛋白品质的影响

研究了乙醇酸法对整粒菜籽多酚及硫苷的脱除工艺,从而对菜籽进行脱毒并提高蛋白品质。采用水媒法对脱酚后的菜籽提油并对水相蛋白提取,对比脱酚前后蛋白质的色泽、纯度和功能特性。结果显示在最佳提取工艺条件(乙醇浓度40%,料液比1:5,pH=5,提取温度80 ℃,反应时间60 min,提取3次)下,多酚提取量为(8.14±0.42) mg/g,硫苷提取量为(32.28±2.32) mg/g,多酚脱除率为51.86%±2.31%,硫苷脱除率为84.66%±0.76%,符合低硫苷菜籽的标准。脱酚使水媒法产出的菜籽蛋白颜色变浅,纯度由57.24%上升到63.63%,持水力、持油力、乳化性、乳化稳定性、起泡性、起泡稳定性分别提高59.31%、26.26%、52.17%、121.29%、60.78%、41.42%,蛋白功能性质得以提升。     

醇洗法脱除玉米胚芽粕中玉米赤霉烯酮的研究

根据玉米赤霉烯酮(ZEN)易溶解于乙醇的原理,利用乙醇对玉米胚芽粕进行浸洗,脱除其中ZEN并提高蛋白质含量和改善感官品质。以玉米胚芽粕为原料,研究乙醇体积分数、醇洗温度、料液比、醇洗时间、醇洗次数对ZEN脱除效果的影响。在单因素试验的基础上,采用正交试验研究确定醇洗法脱除玉米胚芽粕中ZEN的最佳工艺条件为:乙醇体积分数80%,醇洗温度40℃,料液比1∶15,醇洗时间50 min,醇洗次数1次。在最佳工艺条件下,醇洗后玉米胚芽粕中ZEN含量从906.60μg/kg降低至179.21μg/kg,达到国标对饲料粕500μg/kg限量要求,ZEN脱除率达到80%以上,醇洗玉米胚芽粕中蛋白质含量从17.3%提高至21.8%,且色泽、风味等感官品质得到改善。     

基于发酵液特性的聚唾液酸提纯工艺的研究

为了从发酵液中提纯制备聚唾液酸,作者对聚唾液酸特性和发酵液中主要杂质的去除方法进行了研究.结果表明,聚唾液酸具备一定的热稳定性和抗碱性分解能力,并且在一定条件下,可以被乙醇和阳离子表面活性剂氯代十六烷基吡啶(CPC)沉淀.发酵液中的主要杂质可以在一定条件下利用以珍珠岩助滤的方式除去.最终构建了聚唾液酸提纯工艺,利用该工艺得到的产品纯度达到了(98.1±1.6)%,回收率为(56.1±1.7)%.     

苦杏仁醇洗浓缩蛋白制取及脱苷条件研究

以脱皮苦杏仁冷榨饼为原料,采用醇洗工艺制取浓缩蛋白同时脱除苦杏仁苷。通过单因素实验和正交实验得到的最佳工艺条件为:乙醇体积分数75%,提取温度50℃,液料比11∶1,提取时间50 min,提取次数5次。在最佳工艺条件下制取苦杏仁醇洗浓缩蛋白的蛋白质含量为73.61%,苦杏仁苷含量为5.53 mg/kg,苦杏仁苷近乎被完全脱除(脱除率为99.995%)。所得产品颜色洁白、风味清淡略有坚果香味,蛋白质NSI值64.61%,是一种优质的食用蛋白产品。乙醇萃取液即苦杏仁糖蜜中苦杏仁苷含量为3.94%,是提取苦杏仁苷的良好原料。     

紫外光固化硅丙树脂皮革涂饰剂的研究

十二烷基硫酸钠为乳化剂,80℃条件下,m(有机硅预聚体)∶m(丙烯酸酯单体)=4∶50进行共聚,得到有机硅改性丙烯酸树脂乳液。降温至40℃,加入3%光聚合单体和3%光引发剂184(两者用量分别基于丙烯酸酯单体总质量和光聚合单体质量),搅拌0·5h,得到可紫外光固化硅丙树脂乳液。薄膜经过紫外光照射后,断裂伸长率达到367%,抗张强度达到19·3MPa,吸水率为32·2%,玻璃化温度转变点为-45·7℃。     

天然维生素E中PAHs脱除工艺初探

对天然维生素E中PAHs的脱除方法进行了初步探索.考察了脱除溶剂、吸附剂、搅拌时间、搅拌温度、吸附剂加入量以及脱除次数对天然维生素E中PAHs脱除效果的影响,并且考察了吸附剂对天然维生素E含量的影响.实验结果表明,3倍的天然维生素E体积的乙醇溶解天然维生素E(液固比为3 ∶ 1),加入10%的吸附剂2在40℃下搅拌60min对PAHs可以达到较好的脱除效果(对轻质PAHs的脱除率达到98.2%,对重质PAHs的脱除率达到99.6%,对总的PAHs的脱除率达到99.3%),产品中轻质PAHs的残留量为7.5μg/kg,重质PAHs的残留量为6.4μg/kg.而且采用该工艺不影响天然维生素E的含量,脱除PAHs后的天然维生素E具有良好的纯度和收率.     

硅橡胶复合膜用于白酒风味成分渗透汽化分离的实验研究

利用自制PDMS平板复合膜 ,在 30℃、膜下侧压力为 - 0 0 95Mp(6 0mmHg真空度 )的条件下 ,分别对浓度为 5 2°的成品酒和 72°的基酒进行渗透蒸发分离其中的风味物质。实验结果表明新型PDMS复合膜对白酒风味物质具有良好的分离性能 ,原酒中 5种酯类 (乳酸乙酯除外 )和乙缩醛的分离脱除率在 88%以上 ,对高级醇也有良好的分离表现 ,乙醛的脱除率超过 70 % ,在高浓度的乙醇中能保持良好稳定性。在 5 2°成品酒实验中平均总渗透通量达到 2 0 70g/ (m2 ·h) ,平均分离因子 (按乙醇 -水计算 ,下同 )为 4 9;在 72°基酒实验中平均总渗通量为 2 330g/ (m2 ·h) ,平均分离因子为 3 1。     

响应面法优化三氯乙酸脱除滑子菇粗多糖蛋白质的工艺

实验优化了滑子菇粗多糖脱除蛋白质的工艺条件。采用响应曲面法对三氯乙酸从滑子菇多糖中脱除蛋白质的工艺进行研究,在单因素实验基础上,以多糖保留率和蛋白质脱除率为响应值,应用Box-Behnken中心组合实验方法进行三因素三水平的实验设计,通过响应面分析得到优化组合条件。结果表明:最优工艺参数为:料液比为1∶3,脱除时间为50min,三氯乙酸浓度为6%,在此工艺条件下,多糖保留率预测值为75.40%,实测值75.08%;蛋白质脱除率预测值为84.39%,实测值为84.16%,实测结果与预测值符合良好。     

冬小麦膨胀素基因EXPB7工程菌构建及纤维素水解作用分析

为了寻找促进纤维素降解的有效方法来提高纤维素酶的降解效率。本研究以东农冬麦1号膨胀素基因EXPB7为供体,构建黑曲霉表达载体,以黑曲霉CICC2462为受体,采用农杆菌介导法遗传转化黑曲霉,构建膨胀素基因黑曲霉工程菌pSZHGS-EXPB7,并对该工程菌发酵液的纤维素水解促进作用进行分析。结果表明,黑曲霉重组膨胀素蛋白在对滤纸处理2 d后崩解效果明显,与出发菌株的分泌蛋白相比,具有一定的促进作用;重组菌株发酵液上清处理滤纸产生的还原糖含量显著高于出发菌株(P<0.05),与仅用酶液处理滤纸相比,能够将纤维素酶的水解效率提高32.9%。     

产广谱细菌素乳酸菌的筛选

从四川传统发酵食品、市售酸奶、自制泡菜、香肠中分离出267株乳酸菌,采用平板挖井法从中筛选了64株对大肠杆菌、金黄色葡萄球菌、藤黄微球菌、铜绿假单胞杆菌、枯草芽孢杆菌有抑制作用的广谱抑菌性乳酸菌菌株,再对这些乳酸菌采用牛津杯抑菌实验,排除酸、过氧化氢干扰后,部分菌株的发酵上清液仍有很强的抑菌作用;进行胰蛋白酶、木瓜蛋白酶处理后,发酵上清液抑菌活性降低,因而确定产生的抑菌物质为蛋白质类细菌素。     

三步沉淀法纯化鸡卵类黏蛋白

鸡卵类黏蛋白(hen’s egg ovomucoid,HOVM)是鸡蛋清中最重要的过敏原蛋白,探讨三步沉淀(一步三氯乙酸沉淀以及两步硫酸铵盐析沉淀)从鸡蛋清中提取并纯化HOVM的工艺条件,并利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及高效液相色谱(high performance liquid chromatography,HPLC)技术检测纯化效果。结果表明:用等体积去离子水稀释的鸡卵清液加入质量分数10%三氯乙酸去除大部分杂蛋白后,所得上清液加入硫酸铵至终饱和度50%,盐析沉淀进一步去除非HOVM的杂蛋白,上清液中最后再追加硫酸铵至终饱和度80%,离心所得沉淀再经过透析冻干即为高纯度的HOVM;HPLC(检测波长220 nm)检测所获HOVM的纯度为99.202%,此工艺条件下得到HOVM的提取率约为27.05%。     

Molecular cloning and characterization of a novel bacteriophage-associated sialidase

Bacteriophage 63D, previously isolated from sewage, is associated with alpha-2,8-linked polysialic acid degrading activity. We cloned a DNA fragment containing the sialidase gene from a 63D phage genomic library and the enzyme was functionally expressed in Escherichia coli. Determination of the nucleotide sequence of the fragment revealed that it contained one open reading frame (ORF) coding for a 108-kDa polypeptide consisting of 984 amino acid residues. The fragment had promoter sequences similar to the E. coli consensus promoters for sigma70. The deduced amino acid sequence of the central region of the ORF showed homology to those of phages K1F (51.6% identity) and PK1E (51.7% identity) endosialidases. Two Asp-box motifs that are widely found in sialidases were conserved. Purification of the soluble enzyme from lysed culture broth of infected E. coli yielded a 90-kDa protein upon SDS polyacrylamide gel electrophoresis, suggesting that the primary translational product is processed to the mature 90-kDa protein. The molecular mass of the enzyme was determined as 360 kDa by gel filtration, indicating that the native enzyme was probably a tetramer of identical 90-kDa subunits.     

透明质酸发酵液的絮凝预处理研究

通过测定过滤滤饼常数、透光率及处理前后透明质酸浓度 (CHA)的变化 ,分析讨论了用絮凝法预处理透明质酸发酵液的合理性。实践证明 :透明质酸发酵液经絮凝预处理后 ,不仅可以大大改善发酵液的固液分离效果 ,同时其滤清液的纯度亦有一定幅度的提高 ,在超滤过程中污染程度明显减少 ,渗透通量增加     

液液萃取-气相色谱法快速测定发酵液中的己酸含量

对液液萃取-气相色谱法测定发酵液中己酸的方法进行研究。样品经硫酸酸化后采用萃取剂萃取,上层液再经微孔滤膜过滤。结果表明,乙酸乙酯为最佳萃取剂,发酵液样品最佳pH值为2。处理后样品直接进入白酒分析专用色谱柱(型号FFAP),火焰离子化检测仪(flame ionization detector,FID)检测,进样量0.6μL,以2-乙基正丁酸为内标定量。该方法加标回收效率98.19%~104.43%,相对标准偏差2.19%~3.51%,精密度3.46%,该方法精确度高、准确、可靠,可用于发酵液中己酸含量的快速定量检测。     

Production of recombinant human antithrombin by Pichia pastoris

This paper deals with the production of recombinant human antithrombin (rAT) by the methylotrophic yeast Pichia pastoris. In preliminary methanol-limited fed-batch fermentation, the rAT concentration reached 324 mg/l at 192 h of cultivation, but the specific heparin cofactor (HC) activity of rAT in the culture supernatant was 10% of that of plasma-derived antithrombin (pAT). To improve the specific HC activity of rAT, effort was first focused on the optimization of culture pH and media composition, resulting in protection of rAT against pH-dependent instability and proteolytic degradation. However, even in the optimized methanol-limited fed-batch fermentation, the specific HC activity of rAT in the culture supernatant was still 20% that of pAT. To investigate the unknown mechanisms involved in the decreased specific HC activity of rAT, the culture supernatant of mock-transfected cells was prepared by methanol-limited fed-batch fermentation. When pAT was added to this supernatant, a rapid decrease in HC activity was observed; the residual HC activity was 26% after 24 h of incubation at 25 degrees C. The loss of pAT activity was prevented by addition of a formaldehyde scavenger, amino urea, to the supernatant. In addition, alcohol oxidase activity was observed in the supernatant, resulting in the accumulation of formaldehyde in the culture broth. These results suggest that the formaldehyde produced by methanol oxidation in the culture broth of P. pastoris might decrease the HC activity of rAT during fermentation. Replacing the methanol with glycerol as the carbon source improved the specific HC activity of rAT from 20% to above 40% of that of pAT. In the glycerol-limited fed-batch fermentation, rAT is expressed at 100 mg/l under the control of the truncated mutated AOX2 promoter.     

人白介素2-人血清白蛋白融合蛋白的纯化及活性鉴定

采用课题组已构建的携带人白介素2-人血清白蛋白融合蛋白(rhIL-2-HSA)基因的重组毕赤酵母Pichia pastoris GS115进行rhIL-2-HSA的分离纯化研究。发酵上清液经超滤浓缩、Blue Sepharose亲和层析、Octyl Sepharose疏水层析和DEAE Sepharose离子交换层析纯化获得较高纯度的目的产物,SDS-PAGE检测为单一条带。Western Blot分析结果显示,目的产物同时具有HSA和IL-2的抗原性,为HSA和IL-2的融合分子。经CTLL-2/MTT细胞增殖法测定,纯化的融合蛋白的生物学活性为1.147×107 IU/mg。     

Production and optimization of a kiwi pectin methylesterase inhibitor in Pichia pastoris GS115

Gene sequence coding of a kiwi pectin methylesterase inhibitor was optimized, synthesized, and expressed in Pichia pastoris GS115 based on P. pastoris preferred codon usage. The expression level of the recombinant protein (kwPMEI) increased by 89.74% after codon optimization. Expression conditions of recombinant strains were optimized. The highest production of kwPMEI was achieved using 0.8% sorbitol (added every 24 h), 0.05% oleic acid (added at the beginning of induction), and 0.5% methanol (added every 12 h). kwPMEI was purified using Ni²⁺ chelating affinity chromatography and 17 mg of the protein was harvested from 60 mL of a culture supernatant. Activity analysis showed that kwPMEI efficiently inhibited the activity of different plant PMEs. High expression levels and purification of kwPMEI will promote applications in fruit and vegetable juices.