脯氨酰内肽酶研究进展

脯氨酰内肽酶(prolyl endopeptidase,PEP)［EC3.4.21.26］是一种能特异性水解多肽链中脯氨酸残基羧基端肽键的丝氨酸蛋白酶,能降解许多多肽类神经递质和激素,其活性的异常会引起精神、认知过程的障碍.一些PEP的专一性抑制剂(如JTP-4819)显示在莨菪胺引起的小鼠记忆障碍模型中有改善记忆的药理作用.猪肌肉PEP晶体结构的解析推动了PEP的研究.     

重组点状产气单胞菌脯氨酰内肽酶的纯化和鉴定

报道重组点状产气单胞菌脯氨酰内肽酶(简称apPEP)的基因工程下游工艺研究。工程菌株E.coli BL21/pKKH\|PEP表达产物apPEP为可溶性蛋白,在NBS BioFlo 3000型5L自控发酵罐中经14h培养每升发酵液可达到22.5g干重菌体,含apPEP 3.0g左右。发酵菌体经超声破碎、硫酸铵沉淀后,依次经Sephadex G-25、High performance Q sepharose FF(HP\|Q)、Phenyl separose 6 FF柱层析分离纯化,每升发酵产物最终可得0.86g纯度达96%的重组apPEP,比活力达到65.5u/mg,整个纯化工艺的蛋白回收率为8.2%,活力回收率为24.4%。纯化的apPEP经电喷雾质谱测定分子量为76464±30D,N端氨基酸序列与基因序列推导的一致。等电点为pI6.0左右。与Aeromonas hydrophila来源的PEP(pI=5.5)相近。     

重组点状产气单胞菌脯氨酰内肽酶的性质

以特异性底物和多态的对大肠杆菌表达的重组点状产气单胞菌脯氨酰内肽酶（简称ａｐＰＥＰ）催化性质的研究表明,ａｐＰＥＰ在４～３２℃比较稳定,最适催化温度为３４℃;在ｐＨ６～１０比较稳定,最适ｐＨ为８．４;ａｐＰＥＰ的比活力为６７．６ｕ／ｍｇ。对Ｚ－Ｇｌｙ－Ｐｒｏ－βＮＡ底物的酶解常数Ｋｍ为０．０３ｍｍｏｌ／Ｌ。一些蛋白酶抑制剂和金属离子的抑制作用结果表明,ａｐＰＥＰ不受ＰＭＳＦ、ＴＬＣＫ、ＴＰＣＫ、胰     

点状产气单胞菌脯氨酰内肽酶基因克隆与序列测定

点状产气单胞菌点状亚种（Ａｅｒｏｍｏｎａｓ ｐｕｎｃａｔａ ｓｕｂｓｐ．ｐｕｎｃｔａｔａ）具有脯氨酰内肽酶（ｐｒｏｌｙｌ ｅｎｄｏｐｅｐｔｉｄａｓｅ ＰＥＰ）活性。将其染色体ＤＮＡ有Ｅｃｏ ＲⅠ部分酶切后回收８－１６ｋｂ的ＤＮＡ片段,与ＥｃｏＲⅠ消化载体ｐＵＣ１８连接后转化Ｅ．ｃｏｉｌ ＤＨ５α,用该酶的专一性底物Ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌ－Ｇｌｙ－β－ｎａｐｈｔｈｙｌａｍｉｄｅ从质粒库中     

点状产气单胞菌脯氨酰内肽酶在大肠杆菌中的高效表达

采用ＰＣＲ分段克隆法将点状产气单胞菌点状亚种（Ａｅｒｏｍｏｎａｓ ｐｕｃｔａｔａ ｓｕｂｓｐ．ｊｐｕｃｔａｔａ）脯氨酰内肽酶（ｐｒｏｌｙｌ ｅｎｄｏｐｅｐｔｉｄａｓｅ,ａｐＰＥＰ）的编码区基因分成３段扩增并拼接成编码６９０个氨基酸的完整基因ａｐＰＥＰ,将其克隆在表达载体ｐＢＬ和ｐＫＫＨ上,构建成温度和ＩＰＴＧ诱导型高效表达ａｐＰＥＰ的重组大肠杆菌ＢＬ２１／ｐＢＬ－ＰＥＰ和ＢＬ２１／ｐＫＫＨ－ＰＥＰ     

黑曲霉W3350脯氨酰内肽酶在大肠杆菌中的表达

目的：构建pET-PEP重组原核表达载体,并对表达产物进行鉴定。方法：采用RT-PCR技术从黑曲霉（Aspergillus niger）W3350中扩增出脯氨酰内肽酶（PEP）的基因,插入表达载体pET-22b（＋）,经EcoRⅠ和HindⅢ双酶切和DNA序列测定验证,将正确的重组质粒转入大肠杆菌BL21（DE3）中,IPTG（终浓度1mmol/L）诱导获得表达。结果：表达产物进行超声破碎,经SDS-PAGE电泳检测,PEP分子质量大约为57kDa,与理论值相符,通过酶活方法测定脯氨酰内肽酶酶活为0.656U/ml,约是出发菌株的5倍。结论：首次成功构建表达载体pET-PEP并在原核细胞中表达,为进一步研究PEP的生物学功能奠定了基础。     

点状产气单胞菌脯氨酰内肽酶基因的克隆与表达

用活性筛选法从产气单胞菌点状亚种ST7833 (Aeromonas puctata subsp.puctata ST7833)的基因组中克隆了脯氨酰内肽酶 (Prolyl Endopeptidase,简称apPEP)的基因,测定了含有PEP基因的33kb DNA片段的序列,第202092bp编码了690个氨基酸组成的脯氨酰内肽酶,经检索是一种新的PEP基因。并构建了一株组成性高效表达PEP的基因工程菌BL21/pGEMPEP。BL21/pGEMPEP在 YH培养基中apPEP的表达量占菌体总蛋白的30%左右,活力是野生菌的112倍,表达产物主要为可溶性的胞内蛋白,约5%分泌到胞外。非还原SDSPAGE显示为单体,分子量为76kD,与基因序列预测的分子量一致。试管培养后纯化得到了纯度大于90%的重组脯氨酰内肽酶,比活力为67U/mg。     

新型高密度发酵基因工程菌G830

运用PCR方法,从磷酸乙酰转移酶（Pta）－乙酸激酶（Ack)代谢途径缺失菌株E.coliPA1染色体上,扩增出天氨酸激酶－1-高丝氨酸脱氢酶－I（thrA）和高丝氨酸激酶（thrB）基因部分序列,构建了整合型重组质粒pVHb-Kan;应用染色体－质粒同源重组的方法,将透明颤菌血红蛋白（Vitreoscila haemoglobin,VHb)基因整合到大杆菌PA1染色体上的thr操纵子,构建了新型整合工程菌G830。在高密度发酵条件下,G830的细胞呼吸强度、能量代谢、最高菌密度和细胞干重,均明显优于对照菌株PA1和BL21;重组蛋白脯氨酰内肽酶在G830和PA1中获得稳定高表达;重组菌生长状况及发酵指标均与空宿主菌基本一致且表达质粒能维持较好的稳定性。整合型vhb的表达及乙酸代谢途径（Pta-Ack）的缺陷,改善了宿主在贫氧条件下的生长,且促进了重组蛋白的表达。该工程菌具有良好的氧耐受力,且乙酸积累得到大幅度降低,可作为适于高密度发酵的基因工程菌。     

烟曲霉脯氨酰内肽酶在巴斯德毕赤酵母中的分泌表达与重组酶性质

【目的】研究烟曲霉脯氨酰内肽酶cDNA基因的异源表达及重组酶性质。【方法】以烟曲霉CICIM F0044总RNA为模板,反转录合成cDNA;再以cDNA为模板,通过PCR扩增去除自身信号肽的脯氨酰内肽酶基因,构建表达载体pPIC9K-PEP;电转化酵母宿主菌Pichia pastoris GS115,获得重组菌PEP-09;纯化并分析重组酶性质。【结果】重组菌摇瓶发酵酶活力最高可达647.3 U/L。表达产物纯化后的分子量为63 kD左右。重组酶最适反应温度为65°C,有较好的温度稳定性,在55°C保温8 h能保留90%以上的酶活力。该酶最适pH为5.5,在pH 3.0 9.0范围内有很好稳定性,在pH 6.0 8.0的缓冲液中37°C保温10 d酶活没有明显变化。【结论】烟曲霉脯氨酰内肽酶cDNA基因在巴斯德毕赤酵母中实现了分泌表达,重组酶活性稳定,有一定的应用潜力。     

黄杆菌肝素酶Ⅱ重组菌培养条件的优化及高密度发酵

黄杆菌肝素酶Ⅱ(HepⅡ)是一类可特异性切割肝素、硫酸乙酰肝素类分子内连接键的酶。文中对黄杆菌肝素酶Ⅱ重组菌的诱导时机、诱导剂添加量、诱导温度、诱导时间等诱导产酶条件进行优化。经过优化最佳摇瓶发酵产酶条件为:37℃培养重组菌至对数生长前期,添加诱导剂IPTG至终浓度为0.3 g/L,20℃下诱导10 h,酶活达到最高,为570 U/L。在此基础上通过发酵罐高密度培养手段将菌体浓度OD600进一步提高到98,酶活大幅度提高到9 436 U/L,该研究结果为HepⅡ的工业化生产与应用奠定了良好的基础。     

Subcellular Distribution of Prolyl Endopeptidase and Cation-Sensitive Neutral Endopeptidase in Rabbit Brain

Abstract: The subcellular distribution of prolyl endopeptidase, and of cationsensitive neutral endopeptidase, two enzymes actively metabolizing many neuropeptides, was determined in homogenates of rabbit brain. The subcellular distribution of both enzymes was more similar to lactate dehydrogenase, a cytoplasmic enzyme marker, than to choline acetyltransferase, a synaptosomal marker. Only 35% of the activity of these two neutral endopeptidases was found in the crude mitochondrial fraction (P₂), the bulk of the remaining activity being associated with the high-speed supernatant. Prolyl endopeptidase and cation-sensitive neutral endopeptidase thus can be regarded as mainly cytoplasmic enzymes in the rabbit brain.     

Inhibition of Rabbit Brain Prolyl Endopeptidase by N-Benzyloxycarbonyl-Prolyl-Prolinal, a Transition State Aldehyde Inhibitor

Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin-releasing hormone, and luteinizing hormone-releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl-prolyl-proline, with a Ki of 50 microM, prompted the synthesis of benzyloxycarbonyl-prolyl-prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a Ki of 14 nM. This Ki is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S1 subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the Ki for prolyl endopeptidase. The data indicate that benzyloxycarbonyl-prolyl-prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

优化益生菌Lactobacillus casei Zhang高密度培养条件

为实现L. casei Zhang的高密度培养,在之前优化增殖培养基的基础上进一步寻求适宜该菌的培养条件。研究了不同中和剂、缓冲盐浓度、葡萄糖浓度、pH值控制、通气条件和补料分批培养对菌体在恒pH条件下发酵的影响,根据不同条件下菌体的比生长速率、菌体密度和活菌数情况,确定L. casei Zhang较适宜的高密度培养条件为：培养基葡萄糖浓度为80 g/L~100 g/L,以氨水为中和剂使pH保持5.9,采用间歇通氮气的方法保持环境厌氧,分批培养方式下37°C保温发酵10 h~12 h后,L. casei Zhang细胞干重达到7 g/L,活菌数3.5×1010 CFU/mL,比优化前提高7倍以上,能够满足益生菌制品生产要求的高菌体密度。     

Effect of a Novel Prolyl Endopeptidase Inhibitor, JTP-4819, on Prolyl Endopeptidase Activity and Substance P- and Arginine-Vasopressin-Like Immunoreactivity in the Brains of Aged Rats

Abstract: The effects of a novel prolyl endopeptidase (PEP) inhibitor, ( S )-2-[[( S )-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]- N -(phenylmethyl)-1-pyrrolidinecarboxamide (JTP-4819), on the PEP activity in the brain and on the contents of substance P (SP)- and arginine-vasopressin (AVP)-like immunoreactivity (LI) in the cerebral cortex and hippocampus of young and aged rats were investigated using enzyme immunoassay. JTP-4819 exhibited a concentration-dependent in vitro inhibitory action on PEP activity in the brains of both young and aged rats, with IC₅₀ values of ∼0.7 and 0.8 n M , respectively. A single dose of JTP-4819 (3 mg/kg, p.o.) increased the SPLI content in the cerebral cortex but not the hippocampus of aged rats (23–24 months old). In addition, repeated administration of JTP-4819 (1 mg/kg, p.o., for 21 days) increased the SPLI content in the cerebral cortex and restored the SPLI content in the hippocampus, which had decreased with aging. In contrast, single (1 mg/kg, p.o.) and repeated (1 mg/kg, p.o., for 21 days) administration of JTP-4819 only tended to increase the AVPLI content of the hippocampus and cerebral cortex in aged rats, respectively. These results indicate that JTP-4819 increases the cerebral and hippocampal SPLI content in aged rats by inhibiting the action of PEP.     

重组大肠杆菌产普鲁兰酶的高密度发酵工艺研究

以表达普鲁兰酶的重组大肠杆菌作为出发菌株,在摇瓶培养的基础上,建立了大肠杆菌工程菌产普鲁兰酶的高密度发酵工艺。通过测定细胞密度、细胞干重、分离菌体可溶性成分与不溶性成分及SDS-PAGE电泳,分析重组大肠杆菌的生长和普鲁兰酶的表达情况。摇瓶试验使用合成培养基和LB培养基,重组大肠杆菌在合成培养基生长较慢,诱导5 h的普鲁兰酶表达量高于LB培养基,包涵体比例低于LB培养基。重组大肠杆菌的高密度发酵使用合成培养基,补料阶段采用指数流加的工艺,在设定细胞的比生长速率为0.12的前提下,限制补料中碳源的供应,以阻止乙酸的产生。当细胞密度OD600达到70.0开始诱导,最终细胞密度为每升53.3 g细胞干重,细胞内可溶性普鲁兰酶为每升1.35 g。     

Delineation of a Particulate Thyrotropin-Releasing Hormone-Degrading Enzyme in Rat Brain by the Use of Specific Inhibitors of Prolyl Endopeptidase and Pyroglutamyl Peptide Hydrolase

The degradation of thyrotropin-releasing hormone in rat brain homogenates was studied in the presence of N-benzyloxycarbonyl-prolyl-prolinal and pyroglutamyl diazomethyl ketone, specific and potent active-site-directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin-releasing hormone-degrading enzyme(s). Reports of a thyrotropin-releasing hormone-degrading enzyme with narrow specificity that cleaves the pGlu-His bond of this tripeptide led us to develop a coupled assay using pGlu-His-Pro-2NA as the substrate to measure this activity. Cleavage of the pGlu-His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl-peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o-phenanthroline. The particulate pyroglutamyl-peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin-releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu-His bond of thyrotropin-releasing hormone but is distinct from pyroglutamyl peptide hydrolase.     

链霉菌FIM-0916产安福霉素的发酵条件优化

本文采用单因素和正交试验设计,对链霉菌 Streptomyces canus sp. FIM-0916产安福霉素的发酵培养基配方及发酵条件进行了优化。优化后的最佳培养基配方为：蔗糖1.5%,黄豆粉1.0%,组氨酸0.1%,KNO3 0.1%, CaCO3 0.1%。最佳发酵条件为：种子菌龄54 h,装液量120 mL/500 mL,接种量2%,发酵温度28℃,摇床转速250 r/min;最佳发酵时间为5 d。在该优化条件下,安福霉素的发酵效价比对照提高248%,为安福霉素的后续开发奠定了基础。