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蔗糖酶的提取纯化及蛋白质含量测定研究

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蔗糖酶的提取纯化及蛋白质含量测定研究蔗糖酶的提取纯化及蛋白质含量测定研究 论文 论文题目: 蔗糖酶的提取纯化及蛋白质含量测定研究 作者姓名 周柱林 指导教师 钟莉 学科专业 食品科学与工程1102 所在学院 生物与环境工程学院 提交日期 2013年12月 attempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutandis. Eight, should p...
蔗糖酶的提取纯化及蛋白质含量测定研究
蔗糖酶的提取纯化及蛋白质含量测定研究 论文 论文目: 蔗糖酶的提取纯化及蛋白质含量测定研究 作者姓名 周柱林 指导教师 钟莉 学科专业 食品科学与1102 所在学院 生物与环境工程学院 提交日期 2013年12月 attempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutandis. Eight, should perform in the construction standards and specifications, serial number a 1 GB3323-2005 steel fusion welded butt joints, welding engineering-Ray lighting and quality rating of 2 GB11345-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-2002 industrial pipe welding engineering code for construction and acceptance of field equipment 4 HGJ222-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-2004 petrochemical JB/ T4708-2000 of welding procedure qualification for steel pressure vessels 7 JB/4709-2000 8 JB4730-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 蔗糖酶的提取纯化及蛋白质含量测定研究 周柱林 生物与环境工程学院食品科学与工程1102班 摘要:对啤酒酵母蔗糖酶的相关性质进行研究讨论,实验采用酵母自溶法初步得到粗蔗糖酶,离心除杂质法得到初提取液A,接着制备热提取液B,接着制备乙醇沉淀提取液C,接着采用Q Sepharose-柱层析法得到纯度较高的D液,然后用DNS法、曲线法和分光光度法测定蔗糖酶的活力,用Folin-酚法测定蔗糖酶蛋白质含量及计算比活力,用微量凯氏定氮法测定总蛋白含量,最后用SDS-PAGE法测定蛋白质的相对分子质量,并对其基本性质进行了研究。实验测定结果A、B、C提取液的酶回收率(,)分别为100、128.5、56.6,蛋白回收率(,)分别为100、98.15、4.29,比活力分别为17.4、22.8、230.1,纯化倍数分别为1、1.31、13.22,SDS-PAGE测定酵母蔗糖酶相对分子质量为A:99150、 65660. 45000;B:10140;C:92200、 关键词:蔗糖酶、提取、纯化、酶活力、蛋白质含量、比活力、相对分子质量 1.前言(文献综述): 啤酒酵母也叫营养酵母,可以从其中提取蔗糖酶,蔗糖酶又称为转化酶,属于水解酶类,蔗糖在蔗糖酶的催化下,水解为两种还原糖D一葡萄糖和D一果糖。蔗糖酶在植物的运输贮藏、碳水化合物代谢中发挥主要作用,并在渗透调节、抗逆性生长繁殖、以及信号传导方面也发挥着重要的作用。按水解蔗糖的方式,蔗糖酶可分为从果糖末端切开蔗糖的β-D-呋喃果糖苷酶(β -D-frutofuranosidases,EC 3.2.1.26)和从葡萄糖末端切开蔗糖的α-D-葡萄糖苷酶(α-D-glucosidases,EC 3.2.1.20)。前者存在于酵母中,后者存在于霉菌中。工业上多从酵母中提取。 目前关于酵母中蔗糖酶提取纯化方面的研究较多 , 有SDS抽提法,正交法等等,其中以甲苯自溶法最为常见。本实验采用此自溶法从啤酒酵母中提取蔗糖酶,利用有机溶剂将胞内蔗糖酶释放,经过初提取的离心去杂和等电位沉淀,制得的粗酶经醇沉后,采用Q Sepharose-柱层析法纯化,利用DNS法测定其酶活力,利用Folin-酚法测定蛋白质的含量及比活力,利用微量凯氏定氮法测总蛋白氮,以及用SDS-PAGE测定蛋白质的相对分子质量,并对其基本性质进行了研究,也为酵母蔗糖酶在食品工业中的应用和蔗糖酶基因工程产品的技术开发提供了实验依据。 -2000 8 JB4730-re vessels 7 JB/47092000 of welding procedure qualification for steel pressu-2004 petrochemical JB/ T4708-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-struction and acceptance of field equipment 4 HGJ2222002 industrial pipe welding engineering code for con-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-Ray lighting and quality rating of 2 GB11345-2005 steel fusion welded butt joints, welding engineering-3323andis. Eight, should perform in the construction standards and specifications, serial number a 1 GBattempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutrovisional protective measures have been taken, the responsible unit finedand p . Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the openingusing an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan2000 construction installation engineering construction standard 7, HG ... Pressure gauges, -98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-ion and acceptance specification for 5 GB502752009 mechanical equipment installation engineering construct-1997 steel welded atmospheric pressure vessel 4 GB50231-T47352005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ -GB50128 98 2-ing 1 GB1502000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineer-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T47442 浙江工业大学2013届硕士学位论文 本实验目的在于用自溶法提取酵母蔗糖酶的过程中,测定各步的总蛋白、总活力,并据此计算比活、回收率和纯化倍数。 2.与方法 2.1 材料:啤酒酵母 2.2 试剂与仪器 2.2.1 试剂:醋酸钠;甲苯;95%乙醇;0.5mol/L Tris-HCl pH7.3缓冲液;4mol/L 醋酸;0.05mol/L Tris-HCl pH7.3缓冲液;1mol/L NaCl 的0.05mol/L Tris-HCl pH7.3缓冲液;0.5mol/L NaOH; Q-Sepharose;DNS;葡萄糖标准溶液;0.2mol/L pH4.6 醋酸缓冲液;2mol/L NaOH; 5% 蔗糖;试剂A(碱性铜试剂);试剂B(酚试剂);标准浓度牛血清白蛋白溶液;浓硫酸;硫酸钾3份与硫酸铜1份(质量分数)混合粉末;30%NaOH; 2%硼酸溶液; 混合指示剂; 0.01mol/L HCl标准溶液;30%丙烯酰胺贮液;10%过硫酸铵溶液;分离胶缓冲液贮液;浓缩胶缓冲液贮液; 2*SDS-样品缓冲液;SDS-电极缓冲液贮存液;染色液;脱色液。 2.2.2 仪器:恒温水浴锅,恒温培养箱,高速冷冻离心机 、磁力搅拌器或梯度混合器,搅拌子 层析柱、梯度发生器及搅拌子、紫外分光光度计、点滴板,电炉,恒温水浴锅,分光光度计721分光光度计,刻度吸管0.5ml(*1),2ml*2, 5ml*1, *8),恒温水浴槽,改良式凯氏定氮仪,DYY-60型电泳仪。 试管1.5cm*15cm( 2.3实验过程及方法 2.3.1 蔗糖酶提取及初提纯 a.酵母的自溶 在250ml锥形瓶中加入鲜酵母20g、醋酸钠1.6g,加入1.5ml甲苯用滤布加牛皮纸将瓶口塞住,摇匀10min,放入37?培养箱保温60h,使酵母自溶。 b.制备初提液A 在培养箱中取出装有已自通酵母的锥形瓶,加入10ml蒸馏水,摇匀,倒入塑料离心管中,平衡后用高速冷冻离心机4?、15000r/min离心10min。小心取出离心后中间层的液体,重新倒入离心管中,4?、15000r/min离心10min。仔细倒出上层清液,测出体积为V ,取出3ml保存。 A c.制备热提取液B 预先将水浴加热到50?,将初提液A倒入50ml的锥形瓶中,加4mol/L醋酸3.2ml左右,摇匀,水浴保温20min,在保温过程中不断摇动锥形瓶,取出后迅速在冰浴中冷却,冷却液于4?、15000r/min离心10min。测出上层清液的体积记为V ,取出3ml保存。 B d. 制备乙醇沉淀提取液C 将热提取液B倒入100ml烧杯中,把烧杯放入冰浴中,轻轻搅拌并慢慢加入95%乙醇溶液,体积与热提取液B 相同。整个过程不少于20min,再继续搅拌5min,将烧杯内的液体全部移入离心管中,杯底白色固体保留待用,4?、15000r/min离心10min。仔细地倒掉上层清液,用5ml0.05mol/L attempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutandis. Eight, should perform in the construction standards and specifications, serial number a 1 GB3323-2005 steel fusion welded butt joints, welding engineering-Ray lighting and quality rating of 2 GB11345-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-2002 industrial pipe welding engineering code for construction and acceptance of field equipment 4 HGJ222-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-2004 petrochemical JB/ T4708-2000 of welding procedure qualification for steel pressure vessels 7 JB/4709-2000 8 JB4730-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined Tris-HClpH7.3缓冲液把烧杯中的白色固体溶解,倒入离心管搅拌使离心管内的白色固体溶解,4?、15000r/min离心10min。测量上层清液体积V,全部保存。 C2.3.2 蔗糖酶的纯化 Q Sepharose -柱层析法 A 离子交换柱的填充 B 缓冲液盐度梯度发生器的安装 C 柱的平衡:交换剂成为相应的交换型?在小烧杯中加入15毫升Tris HCl?恒流泵进口插入烧杯液面下?恒流泵出水管一端连接柱子?放松夹子?打开恒流泵?用Tris HCl冲洗?直到缓冲液面与交换剂相切 D 加样及洗穿透峰?夹紧下端夹子, 在烧杯加入15毫升Tris HCl?从上部缓慢加入0.5ml提取液C,放松夹子?样品全部刚好进入交换剂内,相切,夹紧夹子?先打开恒流泵,再放开夹子,?用量筒收集全部流出液体,每管收集3ml?直到液面相切 E 洗脱?将梯度发生器出口与恒流泵相连?恒流泵与柱相连?打开搅拌器,打开连通器旋钮,打开恒流泵?用20 毫升Tris HCl 缓冲液配制的1摩尔/升 NaCl20 毫升溶液开始梯度洗脱 打开夹子?用量筒收集3 毫升/管,直到全部缓冲液流出 F 离子交换剂的再生烧杯加入15 毫升 NaCl洗脱 ? 不用收集 ? 凝胶冲到小烧杯? 全部凝胶回收 G 测吸光度,酶活力测试,取酶活力最高的2管为柱分离液D。 2.3.3 蔗糖酶活力的测定 2.3.3.1葡萄糖标准曲线制作:烧水,不要太多,进行DNS反应:注意振荡混匀,移液管对应,要及时清洗 试管号 0 1 2 3 4 5 葡萄糖/ml 0 0.8 1.0 1.2 1.4 1.6 蒸馏水/ml 3.0 2.2 2.0 1.8 1.6 1.4 DNS/ml 1.5 1.5 1.5 1.5 1.5 1.5 总体积/ml 4.5 4.5 4.5 4.5 4.5 4.5 A540nm 0.00 0.101 0.261 0.359 0.479 0.574 沸腾后计时。在沸水浴中加热5min。取出后立即用冷水冷却到室温。 2.3.3.2 酶活力测定 A,稀释样品,浓度从低到高,即C?B?A,防止污染。 -92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-struction and acceptance of field equipment 4 HGJ2222002 industrial pipe welding engineering code for con-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-Ray lighting and quality rating of 2 GB11345-2005 steel fusion welded butt joints, welding engineering-3323andis. Eight, should perform in the construction standards and specifications, serial number a 1 GBattempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutrovisional protective measures have been taken, the responsible unit finedand p . Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the openingusing an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan2000 construction installation engineering construction standard 7, HG ... Pressure gauges, -98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-ion and acceptance specification for 5 GB502752009 mechanical equipment installation engineering construct-1997 steel welded atmospheric pressure vessel 4 GB50231-T47352005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ -GB50128 98 2-ing 1 GB1502000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineer-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 8 JB4730-re vessels 7 JB/47092000 of welding procedure qualification for steel pressu-2004 petrochemical JB/ T47084 浙江工业大学2013届硕士学位论文 提取液 A B C D 比例 1:200 1:200 1:200 1:20 移液管原液 0.25ml 0.25ml 0.25ml 0.5ml 定容 50ml容量瓶 50ml容量瓶 50ml容量瓶 10ml移液管 B 水解反应 试管A0 A1 B0 B1 C0 C1 D0 D1 项目 酶液A稀释液 各取4种稀释液2.00 ml,加入8支试管,按项目名称编号 NaOH ml变性 0.5 --- 0.5 --- 0.5 --- 0.5 --- 预热 5%蔗糖试剂瓶,8支试管(放在试管架上),于35? 预热10min 蔗糖ml 各试管加入5%蔗糖 2 ml加入后立即摇匀开始计时,35?准确反应3min NaOH 终止反--- 0.5 --- 0.5 --- 0.5 --- 0.5 应 总体积/ml 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 全部反应后,取出试管架 C 进行DNS反应,测吸光值 试 管A0 A1 B0 B1 C0 C1 D0 D1 号 /ml attempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutandis. Eight, should perform in the construction standards and specifications, serial number a 1 GB3323-2005 steel fusion welded butt joints, welding engineering-Ray lighting and quality rating of 2 GB11345-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-2002 industrial pipe welding engineering code for construction and acceptance of field equipment 4 HGJ222-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-2004 petrochemical JB/ T4708-2000 of welding procedure qualification for steel pressure vessels 7 JB/4709-2000 8 JB4730-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 反0.1 0.1 0.1 0.1 0.15 0.15 0.3 0.3 应 液V 从上次水解反应液中分别取出V测,用ABCD移液管,先取对照 测 蒸 馏2.9 2.9 2.9 2.9 2.85 2.85 2.7 2.7 水 DNS 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 总 体4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 积 A 0.00 0.173 0.00 0.257 0.00 0.698 0.00 0.662 540nm 2.3.4 蔗糖酶蛋白质含量测定及比活力计算 2.3.4.1蛋白质含量标准曲线绘制 :Folin-酚反应 试管号 0 1 2 3 4 5 标准牛血清蛋白液0 0.2 0.4 0.6 0.8 1.0 /ml 蒸馏水/ml 4.5 4.3 4.1 3.9 3.7 3.5 试剂A/ml 1.0 1.0 1.0 1.0 1.0 1.0 第一试剂B/ml 0.3 0.3 0.3 0.3 0.3 0.3 次 第二0.2 0.2 0.2 0.2 0.2 0.2 次 总体积/ml 6.0 6.0 6.0 6.0 6.0 6.0 0.70A660 0 0.181 0.323 0.457 0.576 3 2.3.4.2 未知蛋白质浓度测定 -2004 petrochemical JB/ T4708-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-struction and acceptance of field equipment 4 HGJ2222002 industrial pipe welding engineering code for con-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-Ray lighting and quality rating of 2 GB11345-2005 steel fusion welded butt joints, welding engineering-3323andis. Eight, should perform in the construction standards and specifications, serial number a 1 GBattempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutrovisional protective measures have been taken, the responsible unit finedand p . Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the openingusing an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan2000 construction installation engineering construction standard 7, HG ... Pressure gauges, -98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-ion and acceptance specification for 5 GB502752009 mechanical equipment installation engineering construct-1997 steel welded atmospheric pressure vessel 4 GB50231-T47352005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ -GB50128 98 2-ing 1 GB1502000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineer-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 8 JB4730-re vessels 7 JB/47092000 of welding procedure qualification for steel pressu6 浙江工业大学2013届硕士学位论文 A 样品稀释 A B C D 稀释比例 1:100 1:20 1:1 1:100 0.5ml0.5ml原0.5ml原原液:液 +9.5 稀释方法 液:50 ml不稀释 50 mlml 移液管容量瓶 容量瓶 量取 B 测样品蛋白含量 试剂量/ml所加试剂 管号 A0 A1 B1 C0 C1 D0 D1 样品稀释液/ml移液0 0.5 0.5 1/20 Tris 0.5 Tris 3 管:酶液 0.5 ml 3ml 水 / ml 4.5 4.0 4.0 4.0 4.0 1.5 1.5 试剂A 1.0 1.0 1.0 1.0 1.0 1.0 1.0 试剂B 第一次 0.3 0.3 0.3 0.3 0.3 0.3 0.3 第二次 0.2 0.2 0.2 0.2 0.2 0.2 0.2 总体积/ml 6.0 6.0 6.0 6.0 6.0 6.0 6.0 OD660nm/A O O.625 0.355 0 0.508 0 0.193 2.3.5 微量凯氏定氮法测总蛋白氮 将0.04ml B样液与3ml浓硫酸、20mg硫酸钾-硫酸铜混合物加热消化。洗涤微 量凯氏定氮仪时,用装有10ml 2%硼酸溶液和4滴混合指示剂的锥形瓶检测,变 绿则重新洗涤。将消化液定容至50ml,取5ml于反应室,同时加入10ml30% NaOH, 用少量水洗涤漏斗后水封。等到颜色变绿后计时3min,再蒸馏1min,滴定。六 次滴定消耗HCl的体积(ml)分别为:0.48、0.55、0.52、0.45、0.50、0.50 。 2.3.6 SDS-聚丙烯酰胺凝胶电泳测定蛋白质的相对分子质量 attempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutandis. Eight, should perform in the construction standards and specifications, serial number a 1 GB3323-2005 steel fusion welded butt joints, welding engineering-Ray lighting and quality rating of 2 GB11345-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-2002 industrial pipe welding engineering code for construction and acceptance of field equipment 4 HGJ222-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-2004 petrochemical JB/ T4708-2000 of welding procedure qualification for steel pressure vessels 7 JB/4709-2000 8 JB4730-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 2.3.6.1 分离胶制备 ?配分离胶成分:12%分离胶 ?各成分加入小烧杯,加入每一种应立即用塑料吸管抽吸慢慢混匀,不要有过多气泡 ?TEMED最后加入,马上灌胶 ?用塑料吸管从一边加入玻璃板之间,防止气泡,不要全部挤入 (2/3高度,梳子下端距分离胶上边缘 1.5cm ?加水覆盖,加满水,出现界面,再次出现界面聚合完成, ?倒出水,用滤纸条吸出面的水 ?聚合30~60 min 2.3.6.2 浓缩胶制备 ?用滤纸吸干水层 ?配制5%浓缩胶:5% ? 各成分加入小烧杯,加入每一种应立即用塑料吸管抽吸慢慢混匀,不要有过多气泡 ? TEMED最后加入,马上灌胶 ?用塑料吸管从一边加入玻璃板之间,混匀加入在分离胶上层 ?插入梳子,不要插反 ?聚合 ?取出梳子(注意用力平衡,不要左右摇摆) ?用滤纸条除去样品槽内的水分。 ?撕去胶条,玻璃板方向:凹槽向内,不要用力压 2.3.6.3 加样品:加入样品和marker,进行电泳。电泳结束后,染色4h,然后用脱色液脱色三次。以maker中标准蛋白质相对分子量的对数(lgMW)为纵坐标,相对迁移距离为横坐标作图,得到标准曲线。然后根据样品蛋白质分子的相对迁移距离,从标准曲线上查出其相对分子质量。 3.结果与分析 3.1 蔗糖酶的提取及初提纯~蔗糖酶的纯化——Q Sepharose- 柱层析法 3.1.1 实验结果 V=18.8ml V=18.5ml Vc=5.8ml V=5.8ml ABD V= V =18.8ml A总A V=V[V/(V-3)]= 18.5[18.8/(18.8-3)]=22.01ml 总BB?AA? V=Vc[V/(V-3)][V/(V-3)]=V[V/(V-3)] = 总c?AA ?BBC?B总B5.8[22.01/(18.5-3)]=8.34ml ? V=VV总/V 总DD?C样=5.8*8.8/0.5=96.75ml 3.1.2 结果分析 -92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-struction and acceptance of field equipment 4 HGJ2222002 industrial pipe welding engineering code for con-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-Ray lighting and quality rating of 2 GB11345-2005 steel fusion welded butt joints, welding engineering-3323andis. Eight, should perform in the construction standards and specifications, serial number a 1 GBattempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutrovisional protective measures have been taken, the responsible unit finedand p . Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the openingusing an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan2000 construction installation engineering construction standard 7, HG ... Pressure gauges, -98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-ion and acceptance specification for 5 GB502752009 mechanical equipment installation engineering construct-1997 steel welded atmospheric pressure vessel 4 GB50231-T47352005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ -GB50128 98 2-ing 1 GB1502000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineer-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 8 JB4730-re vessels 7 JB/47092000 of welding procedure qualification for steel pressu-2004 petrochemical JB/ T47088 浙江工业大学2013届硕士学位论文 A. VB较大是因为在离心前平衡时加入了较多的蒸馏水是V增大。 B B、在同一温度下,溶液中各物质的量不同,所以溶解度不同,对蔗糖酶进行纯化。 C、Q sepharose 为在PH 7左右为强阴离子交换剂且带正电,可与蔗糖酶很好结合,使之洗脱下来。采用Q sepharose-柱层析法提高了实验效率,是蔗糖酶与杂志蛋白得到了很好的分离,提高了纯度,体现了离子交换蛋白纯化的优越性。 3.2 蔗糖酶活力测定 项目 A B C D 葡萄糖/mg 0.178 0.202 0.357 0.345 V总/ml 18.80 22.00 8.34 96.74 V测/ml 0.2 0.3 0.1 0.3 酶总活力单位数 10039.2 8197.2 7256.9 5066.3 酶回收率/, 100 81.65 72.28 49.86 A、本实验采用3,5-二硝基水杨酸与还原糖共热被还原成棕红色的氨基化合物,在一定条件范围内还原糖与反应液的颜色深度成正比,因此利用分光光度计进行测定,计算样品中含糖量,操作简便、迅速,杂志感染小。 B、葡萄糖标准曲线制作时加样一定要非常准确,因为分光光度后,测定酶活力时要根据标准曲线来计算葡萄糖的毫克数。本实验结果较好,因为所有点都落在一条直线上且R^2较高接近0.999。 attempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutandis. Eight, should perform in the construction standards and specifications, serial number a 1 GB3323-2005 steel fusion welded butt joints, welding engineering-Ray lighting and quality rating of 2 GB11345-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-2002 industrial pipe welding engineering code for construction and acceptance of field equipment 4 HGJ222-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-2004 petrochemical JB/ T4708-2000 of welding procedure qualification for steel pressure vessels 7 JB/4709-2000 8 JB4730-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined C、由结果发现酶活力逐渐下降,C的酶活力总数较高,可能是由于在加入蔗糖后摇匀时不是很均匀,使一些酶的反应不充分。 3.3 蔗糖酶蛋白质含量的测定及比活力计算 蛋白回总酶活总蛋白比活力 蛋白质 纯化倍 项目 V总/ml 收 /U /mg U/mg 质量/mg 数/倍 率/, A 18.8 10039.2 666.35 15.07 100 0.177 1 B 22 8197.2 407.14 20.13 61.1 0.0925 1.34 C 8.34 7256.9 46.82 154.99 7.03 0.1403 10.29 D 96.74 5066.3 1.62 3124.71 0.243 0.0419 207.35 A、本实验采用Folin-酚法测定蛋白质含量,反应产物的颜色随着蛋白质浓度的增高而加深,本法极为灵敏,课测定范围非常精确,且蛋白质经过纯化后对本法的影响较小。 B、本实验的蛋白质标准曲线制作的比较准确,所有点都落在直线上,且R^2=0.9989,非常接近1,所以对后面的蛋白质含量计算比较准确。 C、从结果可以看出A、B、C、D的总蛋白一次减少,说明在每部的纯化过程中蛋白质损失越来越多,蛋白质回收率越来越小,尤其是D非常小。而比活力却随着纯化次数的增加越来越大,纯化倍数也增加,比活力越大说明酶的纯化程度越高。 3.4 微量凯氏定氮法测总蛋白氮计算与分析 样品含氮量(mg/ml)=17.51mg/ml 蛋白质含量(mg/ml)=17.51*6.25=109.344mg/ml 2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 8 JB4730-re vessels 7 JB/47092000 of welding procedure qualification for steel pressu-2004 petrochemical JB/ T4708-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-struction and acceptance of field equipment 4 HGJ2222002 industrial pipe welding engineering code for con-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-Ray lighting and quality rating of 2 GB11345-2005 steel fusion welded butt joints, welding engineering-3323andis. Eight, should perform in the construction standards and specifications, serial number a 1 GBattempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutrovisional protective measures have been taken, the responsible unit finedand p . Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the openingusing an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan2000 construction installation engineering construction standard 7, HG ... Pressure gauges, -98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-ion and acceptance specification for 5 GB502752009 mechanical equipment installation engineering construct-1997 steel welded atmospheric pressure vessel 4 GB50231-T47352005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ -GB50128 98 2-ing 1 GB1502000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineer-10 浙江工业大学2013届硕士学位论文 样品B蛋白质含量(mg)=109.344*22=2407.625mg A、本实验采用微量凯氏定氮法测定蛋白质含量,方法简单、适用范围广,较准确且成本低。 B ,实验用Folin-酚法测定B的蛋白质含量为407.14mg,而本次实验测得的蛋白质含量为2407.625mg,是前次实验的6倍,因为本实验测得的样品中含氮量默认为样品中所有蛋白质的含量,而且蒸馏水中也含有少量氮,试验中对颜色的判别也存在误差,使两次实验结果相差较大。 3.5 SDS-GAPE测定蛋白质的相对分子质量 3.5.1 蛋白质迁移距离 迁移距离相对分子量项目 相对迁移率 染料迁移距离/cm /cm 数 0.49 0.1284 97400 0.75 0.2213 66200 标准蛋3.66 白质 1.2 0.3579 4300 1.78 0.4699 3100 0.4 0.1120 99151 A 3.57 1.25 0.3501 45009 B 0.38 0.1053 101391 3.61 0.51 0.1339 92215 C 3.81 0.92 0.2415 65659 attempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutandis. Eight, should perform in the construction standards and specifications, serial number a 1 GB3323-2005 steel fusion welded butt joints, welding engineering-Ray lighting and quality rating of 2 GB11345-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-2002 industrial pipe welding engineering code for construction and acceptance of field equipment 4 HGJ222-92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-2004 petrochemical JB/ T4708-2000 of welding procedure qualification for steel pressure vessels 7 JB/4709-2000 8 JB4730-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 3.5.2结果与分析 标准蛋白质中本来有6种蛋白质,胶体上应该有6条电泳区带,但实验中只有4条,原因可能有如下几点:?另外两种蛋白质分子质量较小,迁移距离较大跑到胶外去了,所以看不到;?可能那两种蛋白质浓度太低,电泳区带太窄以致无法看清;?可能是最后几条重合在一起形成一条电泳区带。 4.结论 通过实验,从提取酵母中的酶到柱层析及测定蛋白质的一些性质这些实验过程中,熟悉了高速离心机、紫外分光光度计,恒温水浴槽,改良式凯氏定氮仪,DYY-60型电泳仪等仪器的使用方法,掌握了酵母自溶法,离心除杂质法、热提取法、乙醇沉淀提取法,Q-Sepharose柱层析法、DNS法、Folin-酚法、SDS聚丙烯酰胺凝胶电泳法等操作方法,并得到了蔗糖酶的一系列数据。通过查阅资料了解了很多课本以外的实验方法,学会了一些简单的数据处理,在实践中有一定的指导意义。 参考文献: [1] 许培雅, 邱乐泉. 离子交换层析纯化蔗糖酶实验方法改进研究[ J] . 实验室研究与探索, 2002. [2] 孙培龙,吴石金.生物化学技术实验指导.化学工业出版社,2008. [3] 陈冰, 林轩. 蔗糖酶水解蔗糖的研究[J] . 湛江师范学院学报, 1997. [4] 孙国志,冯惠勇,徐亲民.蔗糖酶提取方法的研究[J].食品工业科学,2002年04期. -92 technical specification for welding of aluminium and its alloys 5 low temperature steel welding procedure 6 SH3525-struction and acceptance of field equipment 4 HGJ2222002 industrial pipe welding engineering code for con-89 steel welds manual methods of ultrasonic inspection and testing results for grade 3 GB50236-Ray lighting and quality rating of 2 GB11345-2005 steel fusion welded butt joints, welding engineering-3323andis. Eight, should perform in the construction standards and specifications, serial number a 1 GBattempted as the case of responsible for the accident and construction team injuries accident penalty provisions, mutatis mutrovisional protective measures have been taken, the responsible unit finedand p . Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the openingusing an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan2000 construction installation engineering construction standard 7, HG ... Pressure gauges, -98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-ion and acceptance specification for 5 GB502752009 mechanical equipment installation engineering construct-1997 steel welded atmospheric pressure vessel 4 GB50231-T47352005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ -GB50128 98 2-ing 1 GB1502000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineer-2005 pressure vessel welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 8 JB4730-re vessels 7 JB/47092000 of welding procedure qualification for steel pressu-2004 petrochemical JB/ T470812
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