一株产凝乳酶解淀粉芽孢杆菌的筛选、鉴定及酶学性质

采用酪蛋白培养基,从甘南牦牛放牧区分离筛选产凝乳酶细菌。通过形态学、生理生化特征和16SrDNA序列同源分析对菌株进行鉴定,并对该菌株所产凝乳酶的特性进行了研究。从牧区采集的56个样品中共筛选得到6株产凝乳酶细菌,复筛得到一株凝乳活力高、蛋白水解力低的菌株GN4.1,经鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),在麸皮培养基中发酵48h,凝乳活力可达1011.56SU/mL,蛋白水解力为14.61U/mL。凝乳酶的最适作用温度为60℃,65℃加热10min后凝乳活力丧失;最适作用pH为5.5,在pH3.5～8.5内稳定性较好。预期该菌株所产凝乳酶有用于乳品加工业的潜力。     

透性化处理对嗜酸乳杆菌细胞亚油酸异构酶表观活力的影响

来源嗜酸乳杆菌的亚油酸异构酶是胞内酶,完整的细胞呈现较低的酶活。研究了乙醇、乙醚、甲苯、溴化十六烷三甲基铵(CTAB)等透性剂对嗜酸乳杆菌细胞亚油酸异构酶活力的影响。结果表明,经过透性化处理的细胞亚油酸异构酶活力显著提高,其中最好的透性剂为CTAB。当CTAB的浓度为1%,30℃保温20～30min,酶活高达405.6U/g,是超声波破碎的细胞表观酶活的5.4倍。透性化细胞中亚油酸异构酶热稳定性提高,60℃保温2h,残余酶活为80%。透性化细胞生物转化共轭亚油酸转化率达84.5%,用透性化细胞可有效转化亚油酸为共轭亚油酸。     

紫色红曲霉FBKL3.0018产酯化酶的酶学性质研究

以从紫色红曲霉FBKL3.0018中固态发酵得到的粗酶制剂为研究对象,对该菌株的酯化酶酶学性质进行了较系统的研究。通过单因素实验确定该酶最适温度为40 ℃,在30 ℃条件下稳定性较好,最适pH值为6.5,在pH5.5～7.5条件下稳定性好。Fe2+、Na+、Mn2+、Mg2+、Li+、Zn2+对酯化酶酶活力有抑制作用,Ca2+在低浓度时有促进作用,高浓度时有抑制作用。K+对酯化酶酶活力有促进作用,并且随着K+浓度的升高,促进作用逐渐增强。吐温-80、十二烷基硫酸钠（SDS）、聚乙二醇6000、阿拉伯胶4种表面活性剂对酯化酶酶活力都有抑制作用。另外,甲醇和乙醇对该酯化酶酶活力有抑制作用,甲酸、乙酸、乳酸对酯化酶酶活力有促进作用。乙酸乙酯在低浓度时对酯化酶酶活力有抑制作用,含量为30%时反而有促进酶活力的作用。酯化酶最大反应速度Vmax为0.016 mol/（L·min）,米氏常数Km为0.015 4 mol/L。     

克鲁维酵母突变株UV-G-40-3菊粉酶性质的研究

研究了克鲁维酵母突变株（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ－ＵＶ－Ｇ－４０－３）所产菊粉酶的分布为胞外酶∶胞壁酶∶胞内酶比是５．７∶１．６∶１。该酶Ｓ／Ｉ为５．３,最适温度为５０℃,最适ｐＨ为４．５,在５０℃以下、ｐＨ４．５～８的范围内比较稳定,４℃贮存稳定性好,１４ｄ后仍保持７６％活力,为外切型菊粉酶,酶解粗菊糖（洋姜提取液）活性为纯菊糖的４倍。     

产酮基还原酶基因工程菌培养温度和转速条件的优化

考察了27 ℃、30 ℃、33 ℃、37 ℃条件下酮基还原酶基因工程菌生长与产酶的影响。结果发现,37 ℃以下随着温度的升高,菌体产酶活性得到提升和提前,37 ℃条件下10 h最大酶活达到了157.5 U/mL。但是所有温度条件下的发酵后期,酶活都出现了下降,发酵后期转速的下降可能是一个原因。因此对37 ℃下不同恒定转速200 r/min、300 r/min、400 r/min、480 r/min的发酵条件进行了实验。结果发现,转速的恒定对于菌体酶活的积累具有重要意义,恒定转速480 r/min条件下酶活逐渐升高,20 h的酶活为217.1 U/mL。此研究确定了酮基还原酶基因工程菌发酵产酶的最佳温度和转速,并对酮基还原酶进一步的补料分批发酵优化具有指导意义。     

透性化植物乳酸杆菌细胞L-阿拉伯糖异构酶表观活力的研究

来源植物乳酸杆菌的L-阿拉伯糖异构酶(-Larabinose isomerase,EC 5.3.1.4.)是一种胞内酶,完整细胞呈现较低的酶活,而经过乙醇、乙醚、甲苯、曲拉通-100和CTAB处理的细胞酶活比完整细胞和超声波破碎的细胞表观酶活力均增加。甲苯透性化细胞优化条件:2%~5%甲苯浓度,15°C保温,作用5 min可得到最大酶活力9.3 U,比超声波破碎的细胞表观酶活力提高5倍。透性化细胞中L-阿拉伯糖异构酶60°C保温2 h,残余酶活80%,比超声波破碎抽提液中的酶对热更为稳定。用透性化细胞作为生物催化剂可有效地转化半乳糖为塔格糖。     

Extraction of Milk Clotting Enzyme from Sodom Apple (Calotropis procera)

A milk clotting enzyme with low proteolytic activity was extracted with ammonium sulfate, at 0.40-0.65 saturation, from sodom apple leaves. The enzyme with apparently a basic isoelectric point was activated by cysteine and was more active at 65°C than at 35°C. Milk clotting activity increased with pH at 65°C, with the enzyme being almost twice as active at pH 6.4 as at pH 5.4-5.7. Storage at 4°C for 15 days resulted in a 30-50% loss in enzyme activity.     

Production of a Thermoresistant Alpha-galactosidase from Thermus sp. Strain T2 for Food Processing

The gene of a thermostable alpha-galactosidase, aglA, from Thermus sp. strain T2 was sequenced, cloned and overexpressed in Escherichia coli (strain MC 2508. The purified enzyme proved to be quite thermostable, retaining high levels of activity even after incubation at 70°C. The optimal T was 65°C at pH 7. The highest activity was achieved at acid pH values, although good activity could be obtained in a broad pH range. Enzyme stability depends on the enzyme concentration at alkaline pH values, suggesting that under these conditions the subunit dissociation may be the first step in the inactivation of the enzyme. However, at pH 5 the enzyme stability becomes independent of the enzyme concentration. The enzyme also exhibited a quite broad specificity, although it shows the best activity with alpha derivatives of galactose, and was able to recognize very different substrates (alpha derivatives of mannose, xylose and maltose, and even beta galactose derivatives). There was no detectable activity against glucose derivatives.     

重组微小毛霉凝乳酶的发酵条件及其酶学性质

通过对重组微小毛霉凝乳酶(recombinant Mucor pusillus rennet,RMPR)发酵条件的研究,确定甲醇诱导体积分数1%、初始OD600nm 0.5、装瓶量100mL/500mL 摇瓶、诱导时间96h 为发酵最适条件。培养液离心取上清用硫酸铵分级沉淀获得粗酶液,并对其酶学性质进行研究,经测定该酶的最适反应温度为60℃,30～45℃酶较稳定,保温90min,剩余凝乳酶活力达86.5%;酶在pH5.5～7.0 范围内,随pH 值升高,凝乳酶活力逐渐降低,pH5.5时凝乳酶活力最高。当Ca2+ 浓度为0.03mol/L 时凝乳酶活力最高,Na+ 浓度对凝乳酶活力没有明显影响,Ni2+ 和Fe2+对酶有抑制作用,Mn2+、K+、Mg2+ 对酶有一定的促进作用。     

Bacillus sphaericus 2297蛋白酶基因sph在毕赤酵母中的表达及重组酶酶学性质

将Bacillus sphaericus 2297蛋白酶基因sph进行毕赤酵母密码子优化后,进行全基因合成,并构建了重组酵母菌P. pastoris X33-ppicZalphA-sph,对重组酶进行酶学性质研究。结果表明,重组酶的最适反应温度和最适反应pH为40 ℃和8.0,且其为20~30 ℃中保温10 h仍有80%以上的酶活力、在pH7.0~9.0条件下孵育24 h,仍能保持60%以上的酶活性。K+、Sr2+对酶活有明显激活作用,而Fe3+、Ba2+对酶活有明显抑制作用;重组蛋白酶SPH在极性常数为0.8~3.1的25%的正丁醇、环己烷、二甲苯中孵育6 d后,仍能保留50%以上的酶活性。本研究为B. phaericus 2297和B. phaericus DS11蛋白酶有机溶剂耐受性机制的研究奠定基础。     

杨桃多酚氧化酶的部分纯化及其特性研究

杨桃多酚氧化酶粗酶液经过DEAE-Toyopearl650M离子交换柱层析,Butyl-Toyopearl650M疏水柱层析后,被纯化了21.6倍,回收率为45.2%。该酶能迅速地催化焦性没食子酸的酶促氧化反应,而对邻苯二酚、间苯二酚、对苯二酚和绿原酸则完全无催化活性。该酶对焦性没食子酸的Km值为7.92mmol/L,其最适pH为8.0,pH稳定性范围在pH4.0-11.0,最适温度为60℃,热稳定性相对较高,在≥90℃加热30min后仍残留约9%的酶活性。该酶的最佳抑制剂是抗坏血酸,其次是NaHSO3和盐酸-L-半胱氨酸,Cu2+、Zn2+、Ca2+等金属离子对该酶也有一定的抑制作用。     

微小毛霉凝乳酶的酶学性质研究

目的：研究微小毛霉(HL-1)凝乳酶的酶学性质。方法：研究了酶的最适温度,酶的pH值稳定性和热稳定性,探讨了金属离子、化学物质和钙离子对酶活力的影响,加酶量对酶反应的影响,测定了酶的蛋白水解活力、相对分子量和酶的Km与Vm值。结果：酶的最适温度为60℃; 最适pH值为5.5;酶在65℃保温5min活力损失95%;钙离子是酶的激活剂,其含量与酶活力正相关,而钾离子则对酶有抑制作用;氯化钠对酶活力的影响不大,但是SDS可导致酶严重失活;加酶量与反应时间成反比;酶的蛋白水解活力为485.3U/g;相对分子质量是42000,Km为0.02380mol/L,Vm为1.1227mg/min。结论：得出了微小毛霉(HL-1)凝乳酶的酶学性质。     

蛹虫草纤溶酶酶学性质的初步研究

采用盐析和离子交换层析法制备蛹虫草纤溶酶粗酶样品,并对其酶学性质进行初步研究。结果显示：该酶最适pH 值和作用温度分别为8.0 和37℃; 在pH6～10.6 范围内较稳定,但对高温较为敏感;EDTA 对蛹虫草纤溶酶具有抑制作用,说明该酶是一种基质金属蛋白酶;金属离子Ca2+、Fe2+ 在低浓度(0.01mmol/L)时对此酶有促进作用,但高浓度时有抑制作用,而NH4+对酶活性影响不大。     

Production of a Thermoresistant Alpha-galactosidase from Thermus sp. Strain T2 for Food Processing

The gene of a thermostable alpha-galactosidase, aglA, from Thermus sp. strain T2 was sequenced, cloned and overexpressed in Escherichia coli (strain MC 2508. The purified enzyme proved to be quite thermostable, retaining high levels of activity even after incubation at 70°C. The optimal T was 65°C at pH 7. The highest activity was achieved at acid pH values, although good activity could be obtained in a broad pH range. Enzyme stability depends on the enzyme concentration at alkaline pH values, suggesting that under these conditions the subunit dissociation may be the first step in the inactivation of the enzyme. However, at pH 5 the enzyme stability becomes independent of the enzyme concentration. The enzyme also exhibited a quite broad specificity, although it shows the best activity with alpha derivatives of galactose, and was able to recognize very different substrates (alpha derivatives of mannose, xylose and maltose, and even beta galactose derivatives). There was no detectable activity against glucose derivatives.     

番石榴多酚氧化酶的部分纯化及其特性研究

番石榴PPO的粗提取液经过80%硫酸铵盐析和DEAE-Toyopearl 650M、CM-Sephadex C-50离子交换柱层析分离后,被纯化了约126倍,回收率为16.13%。该酶迅速地催化焦性没食子酸的酶促氧化反应,而对对苯二酚和绿原酸则完全无催化活性。该酶对焦性没食子酸的Km值为4.6 mmol/L,其最适pH为7.5,pH稳定性范围在pH4.0~11.0,最适温度为50℃,热稳定性相对较高,在≥90℃加热10 min后仍残留约15%的酶活性。该酶的最佳抑制剂是抗坏血酸和NaHSO3,其次是植酸、盐酸-L-半胱氨酸、柠檬酸,Ca2+、Mg2+等金属离子对该PPO也有较强的抑制作用。     

A thermostable histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007

A thermostable histamine oxidase (EC 1.4.3.-) was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The enzyme was purified about 715-fold over the cell free extracts with a yield of 55% by ammonium sulfate fractionation and various column chromatographies. The purified enzyme was homogeneous on polyacrylamide gel-electrophoresis (native-PAGE). When the enzyme was kept at 65 degrees C and 70 degrees C for 10 min, the activity was fully stable at 65 degrees C and decreased to 9% of the initial level at 70 degrees C. The enzyme was very thermostable. The optimum pH for histamine oxidase activity was found to be at 9.0, and the enzyme was stable over the pH range of 6 to 9. The purified enzyme showed a single protein band on SDS-PAGE and its molecular mass was estimated to be about 81 kDa. The enzyme showed potent activity toward histamine, whereas it was inactive toward putrescine, cadaverine, spermine, and spermidine. Histamine oxidase was inhibited by N,N-diethyldithiocarbamate (DDTC). The inactive enzyme was restored with Cu2+ to 65% of the initial activity, but Cu+ did not enhance the enzyme activity. It is suggested that Cu2+ is essential for expression of histamine oxidase activity. The enzyme was a copper-containing protein having one atom of copper per mol of the enzyme protein as a result of atomic absorption analysis. The N-terminal amino acid sequence of the purified enzyme was different from that of histamine oxidase from Arthrobacter globiformis IFO12137.     

壳聚糖固定化海藻糖合酶

利用壳聚糖作为载体,将大肠杆菌BL21异源表达且纯化后的海藻糖合酶(TreS),采用吸附法制备成固定化酶。固定化单因素实验结果表明,最适海藻糖合酶与壳聚糖的加入量为32.0 mg/g,最适吸附时间为2.5 h。固定化酶最适反应温度为40 ℃,最适反应pH为6.0,酶活回收率为40.17%,重复使用9次后,固定化酶酶活残留率为64.64%,重复使用性良好。在4~60 ℃范围内放置20 min后,固定化酶的相对酶活均高于87%,与游离酶相比温度稳定性没有明显变化。在pH3.5~8.5范围内放置20 min后,固定化酶的相对酶活均高于60%,酸碱稳定性优于游离酶。反应进程试验结果表明,2 h时海藻糖得率达最大,为61.4%。     

有机改性凹凸棒土交联吸附法固定化磷脂酶A_1研究

以有机硅烷偶联剂KH550改性凹凸棒土作为载体,在单因素试验基础上,研究固定磷脂酶A1最佳工艺条件。优化反应条件为:固定化反应时间5.5 h,固定化反应温度50℃,戊二醛浓度0.4%,体系pH 4.7,最终固定化酶酶活5,100~5,330 U/g。固定磷脂酶A1具有比游离磷脂酶更广温度范围和更宽pH应用范围,固定磷脂酶A1最适pH为5.3,而游离磷脂酶A1为4.8;固定磷脂酶A1在较低和较高pH酶活力均高于游离酶;在58℃处理0.5 h,固定化酶仍能保持高于85%酶活,而游离酶酶活则低于70%。     

枝链霉菌L2001木聚糖酶的化学修饰及其活性中心

分别用多种化学修饰剂,如NBS、WRK对枝链霉菌L2001木聚糖酶进行化学修饰,结果表明,作用于色氨酸的试剂NBS能使酶活明显降低,而作用于谷氨酸、半胱氨酸、赖氨酸、精氨酸、丝氨酸及组氨酸残基的试剂对酶活力影响明显。为了进一步研究色氨酸对酶活力的影响作用,通过测定修饰酶的假一级常数,得到至少有一个色氨酸残基在枝链霉菌L2001木聚糖酶的活性中心,并通过底物对化学修饰的保护试验得到,色氨酸残基处在酶与底物的结合位点。结合荧光光谱再次确定色氨酸处于该酶的活性部位,是保持酶活性的必需基团。     

菠萝皮渣羧甲基纤维素/海藻酸钠复合水凝胶珠固定化菠萝蛋白酶的制备及稳定性研究

本实验以菠萝皮渣羧甲基纤维素、海藻酸钠为原料,制备了菠萝皮渣羧甲基纤维素/海藻酸钠复合水凝胶珠,用于固定化菠萝蛋白酶。采用单因素法分析菠萝皮渣羧甲基纤维素与海藻酸钠的质量比、氯化钙的浓度、菠萝蛋白酶浓度、戊二醛体积分数和交联时间对固定化酶活性的影响。结果表明,固定化酶的优化制备工艺为:菠萝皮渣羧甲基纤维素与海藻酸钠的质量比为2:3,氯化钙的浓度为1.0%,菠萝蛋白酶浓度为2.0 mg/mL,戊二醛体积分数为1.0%,交联时间为60 min。制备的固定化酶比游离酶具有更好的热稳定性,在80℃环境下放置2.0 h后,固定化酶的相对酶活性为35.1%,而游离菠萝蛋白酶在此条件下几乎失活;在pH为11条件下放置24 h后,游离酶的相对酶活性为43.2%,而固定化酶相对酶活性为85.1%,说明固定化酶比游离酶更耐受碱性环境。另外,固定化酶重复使用7次后,相对酶活性为60.5%,说明制备的固定化酶具有较好的重复使用性能。