柔性固定化木瓜蛋白酶的制备及在酶解波纹巴非蛤中的应用

在壳聚糖膜的表面接上分子链较长的双醛淀粉作为柔性固定化酶的载体,将木瓜蛋白酶固定在该载体上制成柔性固定化木瓜蛋白酶,并应用于酶解波纹巴非蛤制备小分子肽。通过考察温度、pH 值、加酶量和固定化时间等条件对柔性固定化木瓜蛋白酶的酶活、酶活回收率的影响,确定最优固定化条件。结果表明：双醛淀粉用量0.8mg/g 壳聚糖、柔性固定化酶温度25℃、柔性固定化酶时间20h、加酶量40mg/g 壳聚糖、pH8.0 条件下所得的固定化酶酶活最高,酶活回收率63.35%。将该柔性固定化酶在酶解温度40℃、pH7.0、加酶量1.0%、酶解时间4h条件下制备波纹巴非蛤小分子肽,产率为3.4055%。     

采用双层静电自组装技术制备nPAM-木瓜蛋白酶-CNC催化剂

本研究利用载体材料纤维素纳米晶(CNC)、木瓜蛋白酶(papain,PA)以及非离子型聚丙烯酰胺(non-ionic polyacrylamide,n PAM)的表面电荷特性,制备了新型的固定化木瓜蛋白酶,利用Zeta电位分析,探寻固定化过程中的静电作用机制;并通过傅立叶变换红外光谱(FT-IR)分析固定化PA的组成成分和各物质的相互作用。通过对固定化过程中的各因素进行研究,得到了制备该固定化酶的最优条件,分别为p H 5.0,CNC和PA质量比为36:1(CNC/PA),n PAM(5%质量分数)的添加量为0.3 m L,酶层自组装时间30 min;在此条件下酶活回收率达85.8%。随后,对游离酶和固定化酶的动力学参数研究显示固定化酶的Vmax/Km值(1.40 min-1)明显高于游离酶(0.80 min-1),表明所制备的固定化PA具有更高的催化效率、更广的最适p H范围和温度范围以及操作稳定性,在重复使用5批次之后,相对酶活仍在96.5%以上。可见双层自组装技术是一种进行固定化酶的新途径。     

高碘酸钠氧化法固定化脂肪酶的研究

研究了以醋酸纤维素/聚丙烯复合膜为载体的高碘酸钠氧化法固定化脂肪酶的固定化条件,采用响应面分析法对固定化条件进一步优化,并对固定化酶膜的酶学性质进行了讨论。结果表明,以固定化酶活力为指标,当高碘酸钠浓度为0.15 mol/L,活化为60 min时,浓度为0.013 g/mL的酶液与pH 8.0的磷酸盐缓冲溶液于戊二醛质量分数0.24%,温度4℃下,进行交联反应,反应3.1 h后,获得的固定化酶活最高,为0.52 U/cm2。固定化酶膜的酶学性质为:固定化酶最适温度35℃,最适pH为9.0。     

脂肪酶在磁性纳米粒子上固定化及其酶学性质研究

以Fe_3O_4纳米粒子为载体,碳化二亚胺为交联剂,共价结合制备固定化脂肪酶,探讨脂肪酶固定化影响因素,并对固定化脂肪酶性质进行研究;运用TEM测定其粒径,用FTIR检测脂肪酶—Fe_3O_4磁性纳米粒子耦联。结果表明,脂肪酶固定化适宜条件为:200 mg磁性纳米粒子,加入2 ml 2.5mg/mL脂肪酶磷酸盐缓冲液(0.1mol/L,pH7.5),在4℃超声分散45 min,固定化酶最适pH为7.0,最适温度为45℃,均与游离酶相似;与游离酶相比,该固定化脂肪酶热稳定性明显提高,并具有良好操作和存储稳定性。     

响应面法优化脂肪酶的固定化条件及其水解橄榄油的特性研究

采用Sepharose CL-4B和7种大孔树脂为载体固定Palatase 20000L脂肪酶,对载体进行筛选,以酶活力为指标,采用响应面法优化固定化条件,并考察所制得固定化酶的稳定性和水解橄榄油的酶学性质。结果表明：以大孔树脂HPD-600为载体制得的固定化酶具有较高的活性和良好的稳定性。其最优的固定化条件为：pH3.9,酶与载体比例为9.1mg/g,吸附时间1.8h。在最优条件下制得固定化酶在最适合条件下测得的酶活力达到1440U/g,酶活回收率大于50%。固定化酶最适作用温度为50℃,最适作用pH值为8.0。固定化酶的Km值为0.130g/mL,高于游离酶的Km(0.069g/mL)。固定化酶的热稳定性有一定程度的提高,其重复操作5次后相对酶活力仍保持在58%以上。     

壳聚糖-埃洛石纳米管微球的制备及其对木瓜蛋白酶的固定化

运用交联-吸附法制备壳聚糖-埃洛石纳米管(chitosan-halloysites nanotube,CTS-HNTs)复合微球固定木瓜蛋白酶,并通过傅里叶红外光谱、扫描电子显微镜、荧光标记等方法进行表征。2%CTS+1%HNTs制备的微球对木瓜蛋白酶的固定量最高。固定化条件为木瓜蛋白酶质量浓度1 mg/mL、固定化时间10 h。固定化木瓜蛋白酶最适pH 6.8(游离酶pH 7.2)、最适温度60℃(游离酶50℃),保存30 d该酶相对活性为62%(游离酶27%),使用4次后,木瓜蛋白酶的相对活性仍然保留26.26%。CTS-HNTs微球固定化木瓜蛋白酶耐贮存,操作稳定性强,可以提高酶的利用率,降低酶解的成本,提高生产效率。     

磁性壳聚糖微球固定化脂肪酶研究

以磁性壳聚糖微球为载体,通过戊二醛交联进行脂肪酶固定化,对影响脂肪酶固定化各种因素进行考察,确定最佳条件,并比较游离酶与固定化酶pH和热稳定性。结果表明,固定化适宜条件为:脂肪酶加入量5.0 mg/100 mg载体、温度40℃、时间5 h、pH 8.04、戊二醛浓度10%、最高固载率可达90.56%,酶活4034 U/g载体;与游离酶相比,固定化酶pH和热稳定性都有较宽适用范围。     

大孔树脂D101固定中性脂肪酶及其生物催化应用

以大孔树脂D101固定化中性脂肪酶,研究了固定化条件对酶催化活性的影响,得到了最佳固定化条件:给酶量为90IU/g,固定化温度为35℃,时间为12h,此时固定化酶的活力回收约为60%。固定化酶的半失活温度由游离酶的53℃提高到57℃,最适反应温度和最适pH分别由42℃上升至44℃和由7.5下降到7.3。对固定化中性脂肪酶在生物柴油合成中应用也进行了初步研究。     

双酶水解提取牛蒡菊糖的研究

采用双酶水解法提取牛蒡菊糖。首先从8 种酶中选取3 种对牛蒡菊糖提取率最高的酶,分别为木瓜蛋白酶、植物蛋白酶和酸性蛋白酶,对牛蒡菊糖的提取率分别为8.83%、8.67% 和8.21%。然后对每一种酶采用单因素试验方法研究pH 值、固液比、加酶量、温度以及时间对牛蒡菊糖提取率的影响;再通过3 种酶之间的相互组合试验,选出一组最佳组合为：木瓜蛋白酶+ 植物蛋白酶,其提取率为11.43%。最后采用单因素和正交试验设计方法,研究加酶量、固液比、温度、时间以及pH 值对牛蒡菊糖提取率的影响,得到双酶水解提取牛蒡菊糖的最佳条件组合为：在木瓜蛋白酶加酶量10%、温度50℃、pH7、时间4h、固液比1:15(m/V)的条件下进行酶解,4h 后加入植物蛋白酶,加酶量20%、温度45℃、pH8,时间4h、固液比1:15(m/V),提取液经乙醇沉淀、真空浓缩,得到粗菊糖,菊糖提取率为13.41%,产品中菊糖含量为67.86%,蛋白质含量为1.32%。     

多胺柔性链改性壳聚糖微球固定化木瓜蛋白酶

用反相悬浮交联法制备了单分散壳聚糖微球树脂,以其作为固定化载体基体,经氨基保护、C6羟基环氧化后接枝亲水性多乙烯多胺,制备了粒径为220～300μm、具有较高机械强度的多胺柔性链改性壳聚糖载体。采用该载体对木瓜蛋白酶在pH8.0缓冲液中室温下固定25h,固定化酶表观活力最高可达313U.g-1,活力回收率最高达61.5%,是采用未经多胺分子修饰的壳聚糖微球固定化的2.3倍。该柔性固定化酶的最适温度为65℃,最适pH为8.0,连续使用6～7次后仍保持原来活力的一半。     

Alcoholysis of salicornia oil using free and covalently bound lipase onto chitosan beads

Porcine pancreatic lipase was immobilized on chitosan by covalent binding and retention of its activity was examined. The activities of free and immobilized lipase were determined using olive oil as substrate. The free and immobilized enzymes showed pH 9 as optimum and retained 50% of activity after five cycles. When the substrate concentration was kept constant and enzyme concentration was varied, the K_m and V_max were observed to be 4.0 × 10⁻⁷ and 0.32, and 3.32 × 10⁻⁷ and 0.32, respectively, for free and covalently bound enzyme. This indicates that there is no possibility of conformational change during immobilization. Immobilized enzyme showed improved thermal and storage stability. Alcoholysis of salicornia oil, mediated by free and immobilized lipase, was carried out at 25 °C using methanol in hexane and acetone. Free and immobilized enzyme in hexane produced, respectively, 45% and 55% of fatty acid methyl ester after 12 h.     

固定化脂肪酶性能研究及在洗毛中的应用

利用硅藻土吸附法对解酯假丝酵母发酵产生的脂肪酶进行了固定化,研究了固定化酶和游离酶的酶学性质,以及用固定化脂肪酶对羊毛进行生物酶法洗毛。结果表明:固定化酶比游离酶的最适作用温度提高了5℃,最适作用pH值向碱性方向移动了0.5个单位,热稳定性和pH值稳定性有了大幅度提高。在固定化酶的最适作用pH值下,得到最佳的洗毛条件为:温度45℃、浴比1∶35、时间20 h。     

壳聚糖固定化木瓜蛋白酶提取牛蒡多糖的研究

以壳聚糖为载体、戊二醛为交联剂固定化木瓜蛋白酶,从牛蒡中提取多糖,考察固定化工艺的选择、固定化酶提取牛蒡多糖的优化条件及固定化酶的稳定性。结果表明:壳聚糖固定化木瓜蛋白酶的最佳条件为:壳聚糖浓度2.5%,加酶量0.3g/g载体,时间6h,温度15℃,pH7.5,酶活力回收率38.98%。提取牛蒡多糖的最佳条件为:固液比1:20,pH6.5,温度60℃,时间8h,加酶量1.8g/g载体,多糖提取率11.04%。固定化酶重复使用五次,酶活力仍保持50%以上。     

Improving enzyme immobilization in mesocellular siliceous foams by microwave irradiation

Microwave irradiation was used to immobilize papain and penicillin acylase in mesocellular siliceous foams (MCFs) at low temperature. The maximum loading of papain reached 984.1 mg/g, 1.26 times that obtained using the conventional, non-microwave-assisted method. The half-life (t(0.5)) of papain immobilized in MCFs by microwave irradiation at 80 degrees C was 17 h, 5.21 times that of papain immobilized by conventional means. The activities of papain and penicillin acylase immobilized with the microwave-assisted method were 779.6 U/mg and 141.8 U/mg respectively, 1.86 and 1.39 times of those obtained without microwave immobilization. Using microwave irradiation it only took 140 s for penicillin acylase, an enzyme of large dimensions, to be immobilized in MCFs. In contrast, it took 15 h to do the same using the conventional method. The results showed that microwave irradiation improved the adsorption and immobilization of enzymes in mesocellular siliceous foams.     

聚丙烯二氧化钛负载膜固定化脂肪酶的研究

利用二氧化钛的吸附性,将脂肪酶固定于聚丙烯二氧化钛负载膜上,研究固定化酶的水解特性。结果表明,二氧化钛负载膜上酶的最大负载量为3 0U/cm2 ,固定化酶的最适反应温度为30℃,与自由酶最适温度相比降低了6℃;固定化酶的最适pH为8 7,与原酶最适pH相比,向碱性一侧偏移了0 9个单位;在对大豆油水解时,固定化酶的催化效果比自由酶提高了2 5倍     

面包酵母FX-2酶联辅助自溶工艺研究

该文主要研究了外源酶添加对面包酵母（Saccharomyces cerevisiae）FX-2自溶过程的影响。 首先通过单一对照实验,发现甘露 聚糖酶、木瓜蛋白酶和β-葡聚糖酶能显著提高酵母自溶效率。 其次,以上述三种酶为对象,考察了酶添加量、pH和温度对面包酵母酶 联辅助自溶过程的影响,在单因素试验的基础上,采用正交试验设计分别确定了各酶的最佳助溶条件。 最后,进一步比较验证了单 酶、双酶和多酶联用的助溶能力,并获得最优的酶法辅助自溶工艺：13%酵母乳调pH至5.5,添加0.6‰木瓜蛋白酶,于57.5 ℃条件下助 溶30 min后,调节温度为55 ℃、pH为6.0、添加0.2‰ β-葡聚糖酶,助溶至10 h。 该条件下,细胞死亡率、酵母乳离心上清液干物质含量及 细胞壁水解度分别达100%、9.40%和54.41%,辅助自溶效果好。     

Activity of diastase α-amylase immobilized on polyanilines (PANIs)

Starch hydrolysing enzyme α-amylase (EC 3.2.1.1) was immobilized by physical adsorption and covalent binding onto chemically synthesised polymer, polyanilines (PANIs) in two different forms, emeraldine salt and emeraldine base powder. The immobilization efficiency was affected by the pH of the immobilization medium, contact time and amount of enzyme. The kinetic parameters, reusability and storage stability of the enzyme were studied under free and immobilized condition at optimum pH and temperature. The immobilized enzyme showed enhanced storage stability over a period of 6 months, while free enzyme in solution lost all of its activity within a period of 8 days. Reusability of the enzyme was improved by immobilization. The K_m values for starch hydrolysis were found to be high for the immobilized enzymes.     

棉织物上SESA活化法固定化脂肪酶工艺的研究

研究了对-β-硫酸酯乙砜基苯胺活化法在棉织物上固定化脂肪酶的工艺条件,并考察了固定化酶的最适作用温度和pH及间歇操作稳定性。最佳固定化条件为醚化pH为10.0,偶联pH为7.0,脂肪酶的浓度为6mg/ml,偶联时间为12h,所得固定化脂肪酶的最大活力为35U/g(棉)·min。固定化酶的最适作用温度为35℃,最适pH为8.0,半衰期为6d。     

Immobilized Condensed Tannins and Their Interaction with Proteins

Condensed tannins from grapes immobilized on Sepharose 4B were found to have protein binding properties similar to tannins in free solution. Proteins bound to the gel at pH values below their individual isoelectric points and were eluted when the pH was increased. A good correlation was obtained between the isoelectric point and the elution pH for the following enzymes and proteins: alkaline phosphatase, ovalbumin, β-lactoglobulin, bovine serum albumin, α-amylase,γ-globulin, and cytochrome c. Trypsin represented an exception to the rule, α-Amylase retained enzymatic activity after elution from the gel.     

Preparation and Properties of Rhizopus oryzae Lipase Immobilized Using an Adsorption-Crosslinking Method

The aim was to develop an enzyme immobilization method for Rhizopus oryzae lipase to improve its acidolysis activity and stability. Lipase was adsorbed into NKA-9 resin and then crosslinked with glutaraldehyde as a crosslinker. The optimum conditions obtained using the response surface methodology were as follows: 34.6 mg lipase/100 mg support, 4.1 h of adsorption time, 45°C, pH 6.8, 0.5% glutaraldehyde concentration, and 2.5 h of crosslinking time. The acidolysis activity of the immobilized lipase was 31.78%, the free lipase activity was only 11.02%. The immobilized lipase showed better performance, such as higher acidolysis activity, better pH tolerance and temperature stability, enhanced storage stability, and improved reusability.