木瓜蛋白酶催化的不对称杂环酮的醇醛缩合反应研究

采用木瓜蛋白酶多功能性催化不对称杂环酮与芳香醛之间的醇醛缩合(Aldol)反应。通过对溶剂、含水量、投料比、酶载量以及反应温度等条件的筛选,发现木瓜蛋白酶在该反应体系中展示了较好的催化活性和底物适应性。在优化反应条件下,获得了最高37%的产率和78%的ee值。本文报道的木瓜蛋白酶在有机溶剂中具有类似天然醛缩酶的催化功能。     

化学修饰木瓜蛋白酶的酶学性质研究

采用邻苯二甲酸酐(PA)对木瓜蛋白酶进行了化学修饰,通过三硝基苯磺酸法、紫外光谱、荧光光谱及FT-IR光谱对修饰效果进行了初步表征,采用动力学方法考察了pH和温度对修饰酶水解活性和稳定性的影响,并计算了一系列动力学和热力学参数.实验结果表明:PA对木瓜蛋白酶的平均氨基修饰度为43%,未对酶的活性基团-SH发生修饰,修饰酶较原酶的紫外吸收峰和最大荧光发射峰均发生蓝移,紫外吸收强度降低、荧光强度增大;PA修饰未改变木瓜蛋白酶的最适反应温度,但将其最适反应pH由7.0提高到8.5,且酶活力也提高了约20%;PA修饰有效提高了酶的催化水解效率和酶与底物的亲和力,如40℃、最适pH条件下修饰酶的转化数kcat(3.03 s-1)和亲和力kcat/Km(1.70 s.L.g-1)均较原酶的(2.28 s-1、1.15 s.L.g-1)高,修饰酶催化水解反应的活化能Ea(25.4 kJ.mol-1)较原酶的(29.3 kJ.mol-1)低;PA修饰还明显提高了酶的pH稳定性和热稳定性,半衰期t1/2延长,酶分子的热变性活化能Ea,d由77.0 kJ.mol-1提高到94.5 kJ.mol-1.可见PA化学修饰法是一种有效改善木瓜蛋白酶的催化性质和稳定性的方法.     

钾催化的石油焦/水蒸气气化反应活性及产氢特性

在固定床反应器中考察了钾盐对金山石油焦/水蒸气气化反应活性和产氢特性的影响,并与石油焦/水蒸气非催化气化反应特性进行了对比研究。研究结果表明,钾盐不仅能有效降低气化反应温度、改善石油焦的气化反应活性,而且能够促进气化反应过程中发生的各种反应(碳/水反应、水煤气变换和甲烷/水蒸气重整反应)。随着气化温度的升高,非催化气化的产物气中H₂的含量逐渐增加,而催化气化的产物气中H₂的含量则有所降低,这是由于非催化和催化气化条件下水煤气变换反应处于不同的状态。与非催化气化相比,催化气化条件下单位质量石油焦完全气化生成的产物气中H₂不仅产率高,而且含量也高(非催化和催化气化条件下H₂的含量分别为47.2%~54.1%和55.0%~60.4%)。各种钾盐对石油焦/水蒸气气化反应的催化活性顺序为:K₂CO₃>KAc>KNO₃>K₂SO₄>KCl,但不同的钾盐对气体产物分布的影响较小。     

固定化木瓜蛋白酶的制备和性质研究

多孔硅球固定化木瓜蛋白酶具有热增活性 .本文在前文研究的基础上 ,用载体交联法制备了甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶 .考察了固定化pH值、戊二醛浓度和给酶量对固定化木瓜蛋白酶活力的影响 .研究了固定化木瓜蛋白酶的性质 ,特别是热稳定性和耐热性 ,并与溶液酶和多孔硅球固定化木瓜蛋白酶进行了比较 .所制得的甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶的最适反应温度均达到了 80℃ ;90℃温育 1h后固定化酶的活力保持在 95 %以上 ;70℃温育处理 5h和 6h后固定化酶的活力也仍能保持在 90 %以上 .固定化木瓜蛋白酶的热稳定性和耐热性得到了显著提高     

离子液体中酶促Michael加成反应的研究

以离子液体作为反应介质,研究了嗜热酯酶APE1547在不同离子液体中催化咪唑与丙烯酸乙酯的Mi-clasel加成反应,并对反应条件进行了优化.在最适反应条件下,Michael加成反应产率可达98.8%,另外发现嗜热酯酶APE1547在该反应体系中可重复使用8次,其催化性能没有明显下降.     

木瓜蛋白酶的化学修饰及对其酶活力的影响

用不同酸酐对木瓜蛋白酶进行化学修饰, 以三硝基苯磺酸法(TNBS)测定修饰酶的平均氨基修饰度, 对修饰前后的木瓜蛋白酶分别纯化并通过UV-vis和IR对其结构进行了表征. 考察了温度、pH值和表面活性剂SDS对化学修饰的木瓜蛋白酶活力的影响, 并与天然木瓜蛋白酶进行了比较, 对天然酶和修饰酶进行了动力学研究. 结果表明, 化学修饰木瓜蛋白酶的最适反应温度为80 ℃; 最适pH值为9.0; 在SDS浓度为5 mg•mL－1时修饰酶酶活仍能保持在50%左右; 在所有酶中, 均苯四甲酸酐修饰木瓜蛋白酶的催化效率最高, 为2.442×102. 与天然木瓜蛋白酶相比, 化学修饰木瓜蛋白酶的热稳定性、耐碱性和耐洗涤性得到了显著提高.     

常压两步法催化丙三醇脱水-加氢制备 1,2-丙二醇

 在常压 H2 气氛下催化丙三醇脱水-加氢制备了 1,2-丙二醇. 首先在 220 oC 和常压 H2 条件下, 以 Cu/Al2O3 为催化剂催化丙三醇脱水生成中间体丙酮醇, 其选择性高达 86%. 考察了 Cu 负载量、反应温度和反应气氛对催化剂性能的影响. 在随后的丙酮醇加氢反应中, Raney Ni 催化剂显示出优异的催化性能, 在 120 oC 和常压 H2 条件下, 1,2-丙二醇选择性可达 99% 以上, 催化剂连续使用 5 h 未出现失活现象. 考察了反应温度、空速及反应时间对催化剂性能的影响.     

TS-1/H_2O_2体系催化香茅醛肟化反应

将TS-1/H2O2催化体系用于催化香茅醛氨肟化制备香茅醛肟反应中,结果表明,在优化的反应条件下,香茅醛转化率和香茅醛肟选择性分别达99.9%和99.2%以上.反应过程中主要存在两对竞争反应:TS-1催化的醛肟化反应和碱催化的羟醛缩合反应;TS-1催化的醛肟化反应和双键环氧反应.通过调节体系中氨含量可有效抑制副反应.     

基于木瓜蛋白酶过氧化物模拟酶活性的尿酸检测

木瓜蛋白酶具有过氧化物模拟酶活性,可以催化过氧化氢氧化3,3',5,5'-四甲基联苯胺(TMB)产生蓝色的显色反应。结合尿酸酶和木瓜蛋白酶构建了一个比色检测尿酸(UA)的新方法。其检测过程受pH、温度、TMB浓度和反应时间的影响,最优的检测条件分别为pH 3.0、温度为30℃、TMB浓度0.80 mmol/L及反应时间2 h,线性范围为20~200μmol/L,检出限2.4μmol/L。实验证实了该方法具有较好的选择性并且已经成功地应用于人血清样品的尿酸测定。     

木瓜蛋白酶交联聚体的制备及性质

采用交联酶聚体(CLEAs)技术制得了木瓜蛋白酶CLEAs, 优化了制备条件. 以纯乙醇为蛋白沉淀剂, 质量分数40%戊二醛为交联剂, 于4 ℃下对酶沉淀聚体交联16 h; 所得木瓜蛋白酶CLEAs的最适pH为6.0(游离酶最适pH=7.0), 最适温度范围由游离酶的80 ℃拓宽为50～80 ℃, 热稳定性和溶液稳定性亦明显提高; 微观形貌分析证明木瓜蛋白酶CLEAs优良的催化效能及稳定性来自于CLEAs单元所具有的高比表面积及单元内部多点共价固定的结合方式.     

氨化大孔球状聚氯乙烯固定化木瓜蛋白酶的研究

以氨化大孔球状聚氯乙烯为载体,采用戊二醛载体交联的方法,将木瓜蛋白酶进行了固定化。以酪蛋白为底物,测定了固定化酶的活力回收。研究了固定化条件对固定化酶活力回收的影响。同时,对所得固定化酶的性质,如温度-活力关系、pH-活力关系、热稳定性以及重复使用性进行了考察。结果表明,所得固定化木瓜蛋白酶具有较好的稳定性和重复使用性。     

无机/金属纳米颗粒与木瓜蛋白酶组装的研究

运用氧化还原反应在二氧化硅表面沉积组装了银纳米粒子,并在该载体表面共价连接木瓜蛋白酶,试图组合超微载体与化学共价固定化酶的优点,进一步提高组装后的酶活性.初步研究了微球尺寸及银纳米粒子对组装酶活力影响.实验结果表明,在给酶量为33.3mg/g时,木瓜蛋白酶直接共价连接在二氧化硅表面时活力仅为402u/mg,而在组装有银的载体上时活力为536u/mg,催化活性提高30%左右.同时在高给酶量条件下,酶活力回收提高了60%左右,活力回收最大值达到了30.5%,明显优于戊二醛直接共价固定的文献报道值.     

Protease-catalyzed Synthesis of Bz-Arg-Gly-Asp-OMe in Full Aqueous Medium

Synthesis of N-benzoyl-argininylglycylasparagine methyl ester(Bz-Arg-Gly-Asp-OMe),a precursor tripeptide of Arg-Gly-Asp)was catalyzed by papain under kinetic control,at alkaline pH,in a full aqueous medium.The substrates were N-benzoyl-argininylglycine ethyl ester and asparagine dimethyl ester.An aqueous solution of 0.1 mol/L KCl/NaOH containing 8 mmol/L EDTA and 2 mmol/L DTT was selected as the reaction medium.The synthesized hydrophilic tripeptide was soluble in the reaction medium during the reaction process,however,the secondary hydro-lysis of the tripeptide product was not considerable.The effects of different factors,including water content,temperature,reaction time,and molar ratio of the substrates,on the yield of Bz-Arg-Gly-Asp-OMe were examined.The optimal reaction conditions were 0.05 mol/L Bz-Arg-Gly-OEt and 0.15 mol/L Asp(-OMe)2·HCl in 0.1 mol/L KCl/NaOH solution(pH 8.5),at 40 ℃,and a reaction time of 60 min,with a maximum conversion yield of 62.4%.     

Papain incorporated chitin dressings for wound debridement sterilized by gamma radiation

Wound debridement is essential for the removal of necrotic or nonviable tissue from the wound surface to create an environment conducive to healing. Nonsurgical enzymatic debridement is an attractive method due to its effectiveness and ease of use. Papain is a proteolytic enzyme derived from the fruit of Carica papaya and is capable of breaking down a variety of necrotic tissue substrates. The present study was focused on the use of gamma radiation for sterilization of papain dressing with wound debriding activity. Membranes with papain were prepared using 0.5% chitin in lithium chloride/dimethylacetamide solvent and sterilized by gamma radiation. Fluid absorption capacity of chitin–papain membranes without glycerol was 14.30±6.57% in 6 h. Incorporation of glycerol resulted in significant (p<0.001) increase in the absorption capacity. Moisture vapour transmission rate of the membranes was 4285.77±455.61 g/m²/24 h at 24 h. Gamma irradiation at 25 kGy was found suitable for sterilization of the dressings. Infrared (IR) spectral scanning has shown that papain was stable on gamma irradiation at 25–35 kGy. The irradiated chitin–papain membranes were impermeable to different bacterial strains and also exhibited strong bactericidal action against both Gram-positive and Gram-negative bacteria. The fluid handling characteristics and the antimicrobial properties of chitin–papain membranes sterilized by gamma radiation were found suitable for use as wound dressing with debriding activity.     

STUDY ON THE IMMOBILIZATION OF PAPAIN WITH A MACROPOROUS BEAD CARRIER OF COPOLYMER CONTAINING MONOMER UNITS OF NAMINOETHYL ACRYLAMIDE AND VINYL ALCOHOL*

 A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde as the couplingagent. The enzymatic activity of the immobilized papain was compared with free papain using casein as a substrate, and theeffects of glutaraldehyde concentration, pH, temperature, time and papain amount added on the activity recovery were alsoinvestigated. The results show that the MR-AA carrier contains reactive primary amine groups,hydrophilic amido links and hydroxyl groups,as well as macroporous structures based on its matrix (MR-AV matrix),furthermore,the activity recovery of papain in the immobilization could reach 48%～58%.In comparison with free papain,the resulting immobilized papain exhibits a remarkable thermostability and better reusability.     

Papain Catalyzed Synthesis of Aspartyl Dipeptides

Procedures have been developed for the preparation of aspartyl containing dipeptides P-L-Asp-AA₂ (P=Boc, Cbz and Moz; AA₂=Phe-R or Ala-R; R=OH, OBzl and OTMB) using papain as a catalyst. The pH dependent of esterase activity by papain catalyzed hydrolysis have been studied. The papain catalyzing reaction can be reacted in aqueous solution, buffer solution or cosolvent system in a thermodynamically controlled approach. A procedure for the conversion of the artificial sweetner precursor P-Asp-Phe-OH to the aspartame is studied also.     

Immobilization of Modified Papain with Anhydride Groups on Activated Cotton Fabric

Papain (EC 3.4.22.2) has been chemically modified using two novel reagents including different anhydrides of 1,2,4-benzenetricarboxylic and pyromellitic acids. Then, the modified papain was immobilized on the activated cotton fabric by a two-step method. The number of free amino groups in the modified protein was investigated through the 2,4,6-trinitrobenzenesulfonic acid method. Energy dispersive spectrometer was used to characterize papain immobilization. Some parameters of both modified and native papain immobilized on cotton fabric, such as optimum temperature, optimum pH, and the stabilities for reservation in various detergents were studied and compared. The resultant papain had its optimum pH shifted from 6.0 to 9.0. Compared with immobilized native papain, the thermal stability and the resistance to alkali and washing detergent of immobilized modified enzyme were improved considerably. When the concentration of detergent was 20 mg/ml, the activity of the immobilized pyromellitic papain retained about 40% of its original activity, whereas the native papain was almost inhibited. This work demonstrated that the cotton fabric immobilized modified papain has potential applications in the functional textiles field.     

吸附-纤维素覆膜联合固定化酶

以食品工业中常用的木瓜蛋白酶为模式酶, 建立了吸附-纤维素覆膜联合固定化酶方法. 通过对吸附载体类别、 纤维素种类及溶剂、 保护剂种类及其浓度、 干燥方式及时间等的优化, 得到最佳的吸附-纤维素覆膜联合固定化酶工艺. 以硅藻土或HPD-417(大孔树脂)作为吸附载体, 甲基纤维素(分子量40000~50000)丙酮溶液作为覆膜溶液, 加入6%(质量分数)的聚乙二醇或麦芽糖作为覆膜保护剂, 于4 ℃干燥9 h, 制得固定化木瓜蛋白酶, 硅藻土吸附-纤维素覆膜固定化酶酶活回收率达到96.50%, HPD-417吸附-纤维素覆膜固定化酶酶活回收率达到93.92%. 对吸附-纤维素覆膜固定化酶的性质进行了研究, 发现纤维素覆膜后固定化酶具有良好的热稳定性, 于80 ℃下保存12 h后, 固定化酶活残余率仍然能保持90%左右; 在pH=4.5~9.5的范围内, 固定化酶的稳定性较好; 连续使用9次后, 固定化酶活残余率仍能保持95%左右.