蓝圆鲹骨骼肌内源性脯氨酸内肽酶抑制剂的纯化及其抑制机理

对蓝圆鲹（Decapterus maruadsi）骨骼肌采用热处理、硫酸铵分级沉淀、DEAE-Sepharose弱阴离子交换柱层析、Sephacryl S-200 HR凝胶过滤层析和HiTrap Q HP强阴离子交换柱层析相结合方法,分离、纯化出一种内源性脯氨酸内肽酶抑制剂（prolyl endopeptidase inhibitor,PEPI）。研究了PEPI对脯氨酸内肽酶（prolyl endopeptidase,PEP）活性的影响及抑制机理。Tricine-十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明纯化的PEPI分子质量约为10 kDa,具有较强的热稳定性和酸碱耐受性,其为丝氨酸蛋白酶类PEP的专一性、可逆竞争型抑制剂,抑制常数Ki为0.34 μmol/L。PEPI与PEP形成复合物后,α-螺旋、无规卷曲比例增加,β-折叠比例减少,PEP活性中心构象的改变是酶活力被抑制的主要原因。     

霍氏肠杆菌β-胡萝卜素9,10'双加氧酶的制备及其应用

以霍氏肠杆菌（Enterobacter hormaechei）基因组DNA为模板，通过聚合酶链式反应（PCR）法扩增β-胡萝卜素9,10'双加氧酶基因，构建重组质粒，在大肠杆菌（Escherichia coli）BL21（DE3）中表达，并采用高效液相色谱（HPLC）法检测9,10'双加氧酶活性。结果表明，通过镍柱亲和层析和分子筛Sephacryl TM S-200，得到纯化重组β-胡萝卜素9,10'双加氧酶，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果表明，该酶分子质量约57 kDa，最适反应温度为40 ℃，最适反应pH值为8.5；当底物β-胡萝卜素质量浓度为500 mg/L，β-胡萝卜素9,10'双加氧酶酶活为0.4 U/mL时，β-紫罗兰酮产量为142.3 mg/L，产率达到79.4%。     

超声波、超高压对白鲢鱼肌肉脂肪氧合酶构象及酶活力的影响

本研究采用超声波、超高压对白鲢鱼肌肉中的脂肪氧合酶（lipoxygenase,LOX）进行处理,通过测定 LOX活力、圆二色谱、荧光光谱变化,考察超声波、超高压处理对白鲢鱼肌肉LOX构象及活力的影响。实验结 果表明：随着超声波功率的增加、超声时间的延长,LOX分子内α-螺旋和β-折叠含量降低,荧光强度不断增强, 说明其二级结构和三级结构明显发生变化,使LOX活力不断降低,最适超声条件为300 W、3 h,此时酶活力降低 66.07%;随着超高压压力的增加、超高压时间的延长,LOX分子内α-螺旋和β-折叠含量降低,荧光强度不断减弱, 说明LOX分子二级结构和三级结构发生显著变化,LOX活力迅速降低,最适超高压条件为300 MPa、20 min,此时 酶活力降低93.10%。     

巴氏葡萄球菌TS-82类胡萝卜素裂解酶的分离纯化及其酶学特性

巴氏葡萄球菌TS-82类胡萝卜素裂解酶经强阴离子柱、高效制备液相色谱和多肽分子筛纯化得到液相级纯酶(95.6%)。该酶比活力为125 U/g,纯化倍数为446,回收率为2.39%。纯化后的类胡萝卜素裂解酶经液相色谱-质谱联用测定,得其分子质量为655.093 D。关于酶学特性,研究发现该酶对C_(40)类胡萝卜素底物的最适温度为60℃,而作用于β-阿朴-8’-胡萝卜醛的最适温度是50℃,该酶的稳定温度为50℃以下;该酶对所测定底物的最适p H值为3.0;该酶与5种底物亲和力排列为:玉米黄质虾青素β-胡萝卜素角黄质β-阿朴-8’-胡萝卜醛;Al~(3+)和Fe~(3+)是该酶的强效催化剂,Fe~(2+)是该酶的强效抑制剂;H_2O_2在低浓度范围内(0~16 mmol/L)可促进酶活性;低体积分数乙醇(4%~16%)的添加对酶活性无明显抑制作用。结果表明该酶具有很好的耐酸性和热稳定性,能够适应果酒环境,为其工业化应用提供依据。     

芽枝霉菌Lcxs9产两种耐热β-葡萄糖苷酶的快速纯化及酶学特性研究

以高产β-葡萄糖苷酶的芽枝霉菌（Cladosporium cladosporioides）Lcxs9为研究对象,对其进行固态发酵,采用硫酸铵沉淀及强阴离子交换柱对其所产β-葡萄糖苷酶进行分离纯化,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测其分子质量,并研究其酶学性质。结果表明,快速纯化获得两种β-葡萄糖苷酶,分别命名为BG CC1和BG CC2,分子质量分别为41 kDa、53 kDa,比酶活力分别为8.6 U/mg、12.2 U/mg。两种酶都具有水解活性,可以水解纤维二糖得到葡萄糖;同时具有转苷活性,可以利用低分子质量的单糖合成纤维三糖和纤维四糖。两种酶的最适作用pH及温度均分别为6.0、60 ℃,均在pH 4～10和20～80 ℃条件下较稳定,Ag2+、Co2+、Cu2+、Hg2+对两种酶均有抑制作用,而Mn2+、Ca2+和Mg2+均有明显的激活作用,Zn2+和Ni+无明显影响。利用4-硝基苯基-β-D-吡喃葡萄糖苷（pNPG）为底物,两种酶的动力学常数Km和最大反应速率（Vmax）均分别为2.76 mg/mL、20.6 U/mg。     

重组Taxus chinensis苯丙氨酸变位酶性质表征及R-β-芳香丙氨酸合成

为实现酶法合成高附加值的β-苯丙氨酸,首先化学合成来源于紫衫（Taxus chinensis）的苯丙氨酸变位酶（phenylalanine aminomutase,PAM）基因（Tcpam）,构建大肠杆菌重组质粒Pet-sumo-Tcpam,转入大肠杆菌中进行异源诱导表达,采用镍柱亲和层析制备电泳纯的重组酶TcPAM,用于催化合成R-β-苯丙氨酸。结果表明：重组质粒Pet-sumo-Tcpam成功在大肠杆菌中实现高效表达,获得可溶性的重组TcPAM,经过亲和层析制备出电泳纯的重组TcPAM。质谱和圆二色谱检测分析结果表明,TcPAM能够催化α-苯丙氨酸异构化为R构型的β-苯丙氨酸。TcPAM在最适温度30 ℃、pH 9的条件下,酶活力达到4.11 U/mg,金属离子K＋、Fe2＋和Ca2＋对TcPAM的活性影响较小,而Cu2＋和Zn2＋有强烈抑制性,表面活性剂十六烷基三甲基溴化铵、十二烷基硫酸钠、Triton X-100和Tween 80对重组酶活力影响较小,相对酶活力保持在90%以上。进一步利用TcPAM催化α-芳香丙氨酸异构合成β-芳香丙氨酸,结果表明：苯环上携带不同基团的α-芳香丙氨酸为底物时,苯环上携带供电子基团比吸电子基团的底物转化率更高,底物的α-氨基容易转移至β位,其中4-MeO-β-苯丙氨酸的产率最高,达到45%,为建立酶法合成R-β-芳香丙氨酸提供了参考。     

辐照对带鱼鱼糜内源性转谷氨酰胺酶及凝胶特性的影响

内源性转谷氨酰胺酶(transglutaminase,TGase)与鱼糜凝胶性能密切相关,辐照处理会引起鱼糜TGase结构与活性的变化。为考察电子束辐照对带鱼鱼糜品质的影响,采用不同辐照剂量电子束处理带鱼鱼糜,测定鱼糜内源性TGase的活力及其最适反应温度、最适反应pH值,通过傅里叶变换红外光谱和圆二色光谱分析TGase构象单元,结合鱼糜凝胶强度和持水性的变化,探究电子束辐照对鱼糜内源性TGase及凝胶性能的影响机理。结果显示,随着电子束辐照剂量的增加,鱼糜内源性TGase活力呈先上升后下降趋势,5 kGy辐照能明显提高鱼糜TGase活力,鱼糜凝胶强度及其保水性达到最大值;各组TGase的最适反应pH值均为8.0,除9 kGy组外,其余剂量辐照组和对照组TGase的最适反应温度均为40℃;辐照引起鱼糜TGase分子中的二级结构单元转变,5 kGy组TGase的α-螺旋和β-转角相对含量达最小值,而β-折叠及无规卷曲相对含量达最大值。结论:电子束辐照影响带鱼鱼糜内源性TGase二级结构及其活力,适宜剂量辐照促进TGase分子中α-螺旋和β-转角结构转化为β-折叠及无规卷曲,有利于提高鱼糜TGase活性,促进带鱼鱼糜凝胶的形成。     

一种测定酱油曲中蛋白酶活力方法研究

以底物Suc-Ala-Ala-Pro-Leu-pNA(琥珀酰-丙氨酰-丙氨酰-脯氨酰-亮氨酰-对硝基苯胺)在蛋白酶催化下生成显色基团对硝基苯胺为基础,结合多功能酶标仪检测显色基团对硝基苯胺(pNA)含量,建立酱油曲中蛋白酶活力的新检测方法。在单因素试验的基础上,选定底物浓度、反应pH值、反应温度3个因素的3个水平进行中心组合试验,通过响应面分析得出酶活测定最优条件。结果表明,底物浓度、反应温度对酶活有显著影响,酶活测定的最佳条件为:底物浓度2.23mmol/mL,反应温度40.6℃,反应pH值为8.7。同时与GB/T23527-2009《蛋白酶活力的测定福林法》的方法进行了比对,新建立方法与国家标准方法检测结果具有显著相关性(R2=0.903)。     

广东客家娘酒发酵过程中产β-葡萄糖苷酶酵母菌的筛选及酶学性质研究

采用七叶苷分离培养基初筛、4-硝基苯-β-D-吡喃葡萄糖苷（p-NPGal）显色法复筛的方法从广东客家娘酒发酵过程中的酒糟中筛选产β-葡萄糖苷酶能力较强的酵母菌,通过26S rDNA D1/D2区基因序列分析对其进行鉴定,并对其酶学性质进行分析。结果表明,获得1株产β-葡萄糖苷酶能力较强的假丝酵母Candida apicola kj_312。该菌株所产的β-葡萄糖苷酶具有较宽的底物特异性,最适底物为对硝基苯基-β-D-吡喃葡萄糖苷;最适反应温度为60 ℃,在25～65 ℃范围内,相对酶活力＞50%;最适pH值为4.5,在pH值为4.0～7.0酸性范围内,相对酶活力＞80%;1 mmol/L的Ca2+、Mn2+、Zn2+和Fe2+及100 mmol/L的Ca2+、Zn2+和Fe2+能显著提高酶活力。     

Leifsonia菌β-葡萄糖苷酶（Lf18920）的酶学性质研究及对藏红花素转化作用的初步研究

克隆表达了一种赖氏菌（leifsonia spp. ZF2019）的一个GH3家族糖苷酶基因（Lf18920），以pET-28a-HMT为载体构建了表达质粒，在大肠杆菌BL21中诱导表达，用Ni-NTA柱纯化表达蛋白。研究了该酶的底物特异性及动力学参数，分析了温度、pH、金属离子等因素对该酶活性的影响，并采用高效液相色谱和液质联用色谱探究了该酶对藏红花素的转化作用。结果表明：该酶与4-硝基苯基-β-D-吡喃葡萄糖苷（4-nitrophenyl beta-D-glucopyranoside,pNPG）的亲和力最强，对底物pNPG的特征常数Vmax和Km分别为0.155U/mg和1.97 mmol/L。该β-糖苷酶的最适温度为40℃，最适pH为5.0；低浓度的Zn2+使酶活提高了37%左右，SDS和吡啶可以使酶完全失活，可耐受1%的尿素（仍能保持77%的相对酶活）。该酶能部分水解藏红花素，但不能将其彻底水解成藏红花酸。研究结果表明，该酶可用于以藏红花素为底物的不同水解度藏红花苷的制备。     

重组鲍鱼肌肉脯氨酰内肽酶的性质研究

为研究皱纹盘鲍（Haliotis discus hannai）中脯氨酰内肽酶（Prolyl endopeptidase,Hdh-PEP)的酶学特性与结构特性,利用基因工程技术重组并在大肠杆菌中高效表达了皱纹盘鲍PEP。原核表达的Hdh-PEP分子量为85 kDa,在pH2～6、温度20～60 ℃条件下,Hdh-PEP的表面疏水性明显升高。氨基酸序列同源性分析结果表明,Hdh-PEP催化结构域中有三个高度保守的氨基酸序列:Seq 1:K-D-G-T-K/R-I-P、Seq 2:Y-G-Y-G-G-F和Seq 3:I-R-G-G-E-Y/F。酶动力学研究表明,Hdh-PEP的米氏常数K_m为5.32 μmol/L,催化常数k_cat值为15.7 s?1。PEP的特异性抑制剂SUAM-14746和ZPP对Hdh-PEP酶活力具有强抑制作用,丝氨酸蛋白酶抑制（PMSF）对Hdh-PEP酶活力也有较大程度的抑制作用。本实验制备了高特异性抗Hdh-PEP多克隆抗体,可检测鲍鱼肌肉中天然PEP的存在情况。Hdh-PEP的体外高效表达和特异性多克隆抗体制备为后续深入研究Hdh-PEP的性质提供了重要参考。     

蓝圆鲹骨骼肌脯氨酸内肽酶的分离纯化及其对胶原肽的作用

以蓝圆鲹为研究对象,探讨脯氨酸内肽酶(Prolyl endopeptidase,PEP)对鱼类肌肉胶原蛋白的作用及其机理。通过硫酸铵分级盐析,DEAE-Sephacel阴离子交换,Phenyl-Sepharose疏水层析和Q-Sepharose阴离子交换,从蓝圆鲹骨骼肌中分离纯化得到一种脯氨酸内肽酶。SDS-PAGE结果显示,PEP的分子量为82 ku,肽质量指纹图谱分析得到16个肽片段,共169个氨基酸残基。片段序列与墨西哥鲷鱼(Neolamprologus brichardi)PEP的同源性达98.8%,证明纯化得到的酶是PEP。PEP的最适温度为35℃,但热稳定性较差;最适p H为6.0,在p H 5.0~7.5之间有较好的稳定性。将PEP与合成的鱼胶原蛋白小肽反应,利用反相高效液相色谱对产物进行分离,电喷雾质谱分析结果显示PEP的水解位点在脯氨酸残基的羧基端。以上结果表明,鱼类肌肉中的PEP能够协同金属蛋白酶,通过切割脯氨酸残基进一步降解胶原小肽从而参与到鱼肌肉胶原蛋白的新陈代谢中,是鱼死后参与胶原蛋白降解的重要酶类。     

脯氨酰内肽酶水解啤酒蛋白的研究

将脯氨酰内肽酶(PEP)添加在啤酒后酵液中,通过冷混浊实验分析脯氨酰内肽酶对啤酒非生物稳定性的影响。结果表明,添加5mg/L的脯氨酰内肽酶就能有效地提高啤酒的非生物稳定性;采用75%硫酸铵沉淀啤酒蛋白,进行SDS-PAGE电泳分析,结果显示,啤酒蛋白电泳图谱的分布主要表现为8～14.4 ku的窄带和35～45 ku的宽带,而经脯氨酰内肽酶处理的啤酒蛋白电泳图谱中8～14.4 ku的条带消失,说明此蛋白被脯氨酰内肽酶水解。     

Optimization of process conditions for the production of a prolylendopeptidase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 11414 in solid state fermentation

The effect of 8 factors [(with/without) daily mixing and moisture control, incubation time (t), temperature, ratio between dry substrate mass and bed’s cross section area (MA), inoculum size (spores/g), wheat germ content (WG), initial pH, and moisture content (M)] in the production of a prolyl endopeptidase (PEP) by Aspergillus niger ATCC 11414 in solid state fermentation (SSF) was tested. Contribution of all the factors was significant (p<0.05); main effects were those of MA, t, and M. The 4 interactions that presented high interaction severity indexes involved the WG. Under optimized conditions PEP and protease activity were 9.76±0.06 and 3.6×10⁶±1.5×10⁵ U/kg, respectively. The enzyme was partially purified (ammonium sulfate precipitation, dialysis, DEAE-Sepharose ionexchange); it has a molecular weight of 66 kDa (SDS-PAGE), and maximum activity was exhibited at pH 4 and 50°C. The enzyme is stable in a wide pH range (2.2–10) and at temperatures lower than 70°C.     

高产高纯度脯氨酰内肽酶黑曲霉工程菌的构建

利用基因工程技术,构建过表达黑曲霉来源带有自身信号肽的脯氨酰内肽酶黑曲霉重组菌株TH2-protAS和过表达以黑曲霉内源高效分泌的葡糖淀粉酶信号肽以及α-淀粉酶信号肽代替自身信号肽的脯氨酰内肽酶黑曲霉重组菌株TH2-SglaA-protA和TH2-SamyA-protA。SDS-PAGE结果显示,脯氨酰内肽酶在重组菌株中分泌表达,但是存在不同程度的糖基化。酶活检测结果表明,重组菌株TH2-protAS、TH2-SglaA-protA和TH2-SamyA-protA的最高酶活分别为1.70、2.19、1.91 U/mL。在重组菌株TH2-SglaA-protA的基础上,利用基因敲除技术,敲除了主要的背景蛋白酸稳定的α-淀粉酶,结合发酵条件优化,获得了高纯度的脯氨酰内肽酶。综合上述结果可得出结论:可在黑曲霉中同源高效分泌表达脯氨酰内肽酶;glaA信号肽和amyA信号肽明显增加黑曲霉中脯氨酰内肽酶的分泌表达量,且glaA信号肽的效果优于amyA信号肽;将基因敲除和发酵调控相结合,可以有效去除分泌的背景蛋白,获得高产高纯度脯氨酰内肽酶的菌株,该重组菌株具有明确的工业应用潜力。     

Purification and characterization of dibenzothiophene sulfone monooxygenase and FMN-dependent NADH oxidoreductase from the thermophilic bacterium Paenibacillus sp. strain A11-2

A dibenzothiophene (DBT) sulfone monooxygenase (TdsA), which catalyses the oxidative CS bond cleavage of DBT sulfone to produce 2-(2-hydroxyphenyl)benzenesulfinate (HPBS) was purified from the thermophilic DBT desulfurizing bacterium Paenibacillus sp. strain A11-2 by multistep chromatography. The molecular mass of the purified enzyme was determined to be 120 kDa by gel filtration and the subunit molecular mass was calculated to be 48 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicating a dimeric structure. The N-terminal amino acid sequence of the purified TdsA was determined to be MRQMHLAGFFAAGNTHH, which revealed no significant similarity to any other known amino acid sequences. The purified TdsA absolutely required an oxidoreductase for its activity. This oxidoreductase (TdsD) was also purified to homogeneity, and its molecular size was calculated to be 50 kDa and 25 kDa by gel filtration and SDS-PAGE, respectively. TdsD was completely FMN-dependent, and FAD could not act as a cofactor. The N-terminal amino acid sequence of the purified TdsD was determined to be TSQTAEQSIAPIVAQYRHPEQPISALFVNR, which showed significant similarity to kinesin-like protein (44% identity). The optimal temperatures for the activity of TdsA and TdsD were 45 degrees C and 55 degrees C, respectively. Both enzymes showed optimal activity at pH 5.5. TdsA was slightly inhibited by sulfate, but not by 2-hydroxybiphenyl (2-HBP), which is another end product of DBT. TdsA showed higher activity toward bulkier substrates than its mesophilic counterpart, DszA. These properties suggest the applicability of biodesulfurization to the processing of actual petroleum fractions.     

Molecular cloning and characterization of gene encoding novel puromycin-inactivating enzyme from blasticidin S-producing Streptomyces morookaensis

Puromycin (PM) is classified into a family of nucleoside antibiotics together with blasticidin S (BS). PM-producing Streptomyces alboniger is known to express a PM-inactivating enzyme as a self-resistance determinant, which catalyzes the acetylation of PM. We have shown that, although BS-producing Streptomyces morookaensis also produces a PM-inactivating enzyme, it catalyzes the hydrolysis of an amide linkage between the aminonucleoside and O-methyl-L-tyrosine moiety of PM. In the present study, we cloned and characterized a gene encoding PM hydrolase (PMH) from BS-producing S. morookaensis JCM4673. The nucleotide sequence analysis suggests that an open reading frame consisting of 1986 bp is a gene for PMH and encodes a protein consisting of 662 amino acids with a calculated molecular mass of 71,260 Da. The molecular mass of the recombinant PMH, which was produced using an Escherichia coli host-vector system, was the same as that of PMH purified from the JCM4673 strain. Our biochemical study of the recombinant PMH confirmed that the enzyme is an aminopeptidase with broad substrate specificity. The putative primary structure of PMH contains a Gly-X-Ser-X-Gly motif, which is commonly observed among serine proteases. In addition, the amino acid sequence of PMH displays a high similarity to that of the Streptomyces acyl-peptide hydrolase (ACPH), which is a member of the prolyl oligopeptidase (POP) family of serine proteases. Furthermore, the catalytic triad (Ser-Asp-His), which is observed in the POP family, is also present in the primary structure of PMH. These results suggest that PMH is an aminopeptidase classified into the POP family.     

Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase

Peptides from hydrolysates of fish proteins and from cheeses were analysed for inhibition of prolyl endopeptidase (PE) isolated from porcine muscle. Muscles of cod, salmon, and trout were homogenised and incubated at pH 4.0 with pepsin and then at pH 7.5 with trypsin to obtain fish protein hydrolysates. Homogenates were incubated without exogenous enzymes at pH 4.0 and 7.5 to obtain fish protein autolysates. Water-soluble extracts from "rakfisk" (a Norwegian fermented/autolysed trout muscle dish) and water-soluble extracts from Cheddar, Norvegia, Jarlsberg, and Blue cheese were also prepared. Peptides in the supernatants obtained after heat-treatment of fish hydrolysates, autolysates and water-soluble extracts of rakfisk and cheeses at 95 degrees C for 15 min were analysed for inhibition of PE. Inhibition was also measured in peptide fractions separated by reversed-phase high-performance chromatography and by gel permeation chromatography. The peptide fractions from fish hydrolysates, fish autolysates, and water-soluble extracts of cheeses inhibited PE in hydrolysing Z-Gly-Pro-amidomethylcoumarin. Inhibition by peptides from rakfisk was negligible. Pepsin + trypsin hydrolysates from the three fish species contained PE inhibitory peptides with a broad range of apparent hydrophobicity and apparent molecular mass. Autolysates from muscles of the 3 fish species contained narrow peptide peaks of different molecular mass and different apparent hydrophobicity with strong PE inhibitory activity. The content of hydrophilic inhibitory peptides was lower in cheeses than in pepsin + trypsin hydrolysates of fish muscle.     

利用坛紫菜藻红蛋白制备脯氨酰内肽酶抑制肽

以坛紫菜（Porphyra haitanensis）为原料,通过硫酸铵盐析和DEAE-Sepharose阴离子交换柱层析等方法分离纯化得到一种高纯度藻红蛋白（R-phycoerythrin,R-PE）（A565 nm/A280 nm=5.4）。利用胃、肠液消化酶对R-PE进行酶解制备脯氨酰内肽酶（prolyl endopeptidase,PEP）抑制肽。酶解条件为：1）胃蛋白酶与R-PE比例0.5%（m/m）,酶解时间30 min,pH 1.2,温度37 ℃;2）胰蛋白酶和胰凝乳蛋白酶质量比为1∶1的混合酶,其与R-PE比例0.25%（m/m）,酶解时间30 min,pH 7.5,温度37 ℃。Tricine-十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析发现,经两步酶解,R-PE被完全降解。采用凝胶过滤色谱法测得R-PE水解液中分子质量在3 kDa以下的小肽含量为86.06%。R-PE水解液对PEP抑制水平IC50为136.35 μg/mL。酶抑制动力学研究发现,R-PE水解液对PEP表现为可逆的非竞争性抑制作用,抑制常数Ki为14 μg/mL。本研究利用坛紫菜R-PE制备活性高的PEP抑制肽,将为坛紫菜深加工及高值化利用提供一定的理论参考。