抗氧化型枯草杆菌碱性蛋白酶高产工程菌的构建：I.抗氧化型碱性蛋白…

枯草芽孢杆菌（ＢａｃｉｌｌｕｓＳｕｂｔｉｌｉｓ）Ｂ１３５工程菌能产生抗氧化型碱性蛋白酶,粗酶经硫酸铵分级沉淀,ＣＭ－５２层析,ＳｅｐｈａｄｅｘＧ－１００层析,得到凝胶电泳均一样品,比活达到１７００Ｕ／ｍｇ,是粗酶比活的７．６９倍,该酶在６０℃时酶活力是最高,最适ｐＨ为１０．２在５０℃时,温浴１０ｍｉｎ后,酶活降低到原来的５０％,该酶受１ＭＨ２Ｏ３作用２０ｍｉｎ,仍保持９６％的酶活。     

中国新分离碱性耐热芽孢杆菌的木聚糖酶产酶特征

目的：检测由中国新分离碱性耐热芽孢杆菌[1]产生的粗木聚糖酶的酶活。方法：通过DNS法测定粗木聚糖酶的酶活。结果：实验表明,以橡树木聚糖为底物培养的新分离菌株在30-50℃处理2h酶活不丧失。其中,XJU-1菌株在60、70和80℃时粗酶酶活分别丧失是最初酶活的1.54%、19.09%和72.59%;而XJU-80的粗酶酶活分别是3.59%、26.43%和72.59%。两个菌株产生的粗木聚糖酶的最适pH是7.5-8.0。将该粗酶在pH 7.0-9.0（50℃）处理24h后,酶活几乎均降低最初酶活的18%。结论：由XJU-1和XJU-80产生的木聚糖酶是生化领域有用的嗜碱耐热酶。     

重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究

将产热稳定性过氧化氢酶的重组大肠杆菌培养后菌体破碎得到的粗酶液经热处理、硫酸铵分级沉淀、DEAE\|Sephadex A\|50离子交换层析、HiPrep16/10 Phenyl疏水作用层析、Superdex200 HR 10/30凝胶层析提纯后得到电泳纯的酶,比酶活达到15629U/mg。此酶的最适温度为70℃,最适pH70,在60℃保温60min酶活力基本不变,在pH3～8的范围内比较稳定。此酶的Km和Vmax分别为775mmol/L和278mmol\5min\+\{-1\}·mg-1。1mmol/L的Zn2+、Ba2+、Mn2+可使该酶完全失活,KCN、NaN\-3、Na\-2S\-2O\-4、巯基乙醇对酶活力有抑制作用,50mmol/L的EDTA不影响酶活性。     

枯草杆菌(Bacillus subtilis)产果胶酶的研究Ⅰ.菌种选育、鉴定及其酶学特征分析

分离到1株高产果胶酶菌株,经形态、生理以及生化指标鉴定,确认为枯草杆菌(Bacillus subtilis)并命名为枯草杆菌18.发酵液用90%硫酸铵沉淀,透析后的粗酶经CM-52柱层析,收集酶峰,再过Shephadex G-100得部分纯化酶.该酶最适pH9.0,在pH9～11稳定,最适反应温度60℃,50℃加热50 min保存60%酶活,60℃加热50 min酶活则保存10%.酶的等电点(pI)4.0,分子量31 000.该酶对苎麻脱胶有较好的特异性.     

包衣微丸型碱性蛋白酶的制备

开发了一种包衣微丸型碱性蛋白酶的制备工艺,结果表明:30 L发酵中罐发酵50 h比酶活可达4.26×104 U/mL,发酵液经絮凝处理、板框压滤、膜浓缩后可制成酶活达300 000 U/mL的酶浓缩液.经流化后制得含酶颗粒,再包裹薄层后,得包衣微丸型碱性蛋白酶.对制备的包衣微丸型碱性蛋白酶的稳定性、去污效果等指标进行了评估.制备的包衣微丸型碱性蛋白酶产品在严苛条件下的稳定性与国外产品( Savinase 8.0T、PuraFast 2000HS)相当,产品暴露在37℃、75％湿度下8周后仍可保持74％的酶活力,产品的去污效果优于国外产品.制备的包衣微丸型碱性蛋白酶颗粒大小均匀,流动性和分散性好,对外界高温、高湿等不良环境具有很强的抵抗能力,适于工业化生产.     

产低温碱性蛋白酶海洋适冷菌SY的筛选

从连云港海域、港口、远洋捕捞船及鱼市等地采集的海水和各类海鱼、贝类等样品中分离到217株产蛋白酶的细菌,并从中得到1株产低温碱性蛋白酶的海洋适冷细菌—SY。研究表明,该菌株最适生长温度和最适产酶温度均在15℃左右,0℃下仍可生长;具有一定的耐盐性和嗜盐性,无盐条件不能生长,在3%的NaCl盐浓度时,生长达到最高峰;最适生长和最适产酶pH均为8.0;SY菌所产的蛋白酶可能为一种丝氨酸蛋白酶,酶最适作用温度为50℃,最适作用pH为9.0;酶的热稳定性差,50℃保温20min,酶活下降40%。     

宇佐美曲霉酸性蛋白酶的研究II．537酸性蛋白酶提纯及性质

由字佐美曲霉(Aspergillus usamii)537产生的酸性蛋白酶粗酶制剂,通过硫酸铵饱和度为45--65％的分部沉淀、离子交换树脂脱色、Sephadex G 2 5脱盐、DEAE‘$ep“ad。‘A一25层析可得到比活为2037u,mg的纯酶,为原粗酶比活的12倍,在Davis凝胶电泳中得到一条带。     

短小芽孢杆菌碱性蛋白酶BP的纯化和性质

短小芽孢杆菌产生的碱性蛋白酶BP经CM—Sephadex-C-50和Sephadex-G75两个柱层析,得到了聚丙烯酰胺凝胶电泳纯的酶组分,比活力从1307μ/mg提高到5538μ/mg.活力回收为21%,酶水解酪蛋白的最适反应温度为50℃,最适pH为9.5,Mn^2+、Ca^2+对酶有激活作用,Hg2+、Ag^+对酶有抑制作用.酶的热稳定性不高,但在Ca^2+保护下,热稳定性明显提高.酶的最适作用底物为酪蛋白,对血红蛋白、蛇毒蛋白、牛血清蛋白、卵蛋白、核糖核酸酶也有水解作用.对酪蛋白的Km为0.62%,V_max为50μg/min.DFP可完全抑制酶活性,PMSF和NBS也严重抑制酶活力,PCMB、_o-PTH和EDTA几乎不抑制酶活力.纯酶的分子量为25000Dal.该酶蛋白含有17种氨基酸,其中甘氨酸(Gly)和丙氨酸(Ala)为主要氨基酸.     

M-26碱性木聚糖酶的分离纯化及酶学性质研究

从Bacillus pumilus M-26发酵液中分离纯化碱性木聚糖酶,进行酶学性质研究,同时制备工业用碱性木聚糖酶制剂。首先将M-26发酵液进行硫酸铵盐析,制备工业用碱性木聚糖酶干品;然后进行sephadexG-25层析脱盐和cellulose DE-52层析得以纯化。硫酸铵的饱和度50%,酶制剂的酶活可达9 000 IU/g,收率为85%;分离纯化使酶的比活为126.32 IU/mg蛋白,纯化倍数为19.89,酶的回收率12.83%;分子量约为20 ku;M-26碱性木聚糖酶的最适温度和pH分别是55℃和pH 8.0,具有一定的耐碱性;该酶无纤维素酶活性,Fe2＋对其有激活作用;Mn2＋、Zn2＋、Fe3＋、Cu2＋对其具有抑制作用。短小芽胞杆菌M-26碱性木聚糖酶具有纸浆生物漂白应用前景。     

雅致放射毛霉AS3.2778碱性蛋白酶的纯化及性质研究

利用盐析,离子交换,疏水层析及凝胶过滤的方法从雅致放射毛霉AS3.2778的发酵麸曲中分离纯化出一碱性蛋白酶,其纯化提高了22.7倍,酶活回收率16.1%,最终比酶活可达到6094u/mg。电泳分析发现,该蛋白酶是一单体蛋白,其分子量大约在32KDa。性质分析表明：该蛋白酶在60℃、pH8.5～10.5具有最大催化活性;在40℃以下,pH6.0～9.0的范围有很好的稳定性;1mM的PMSF可以完全抑制其活性,显示该蛋白酶属于丝氨酸蛋白酶家族。底物专一性的研究发现,该蛋白酶有相当广泛的肽键选择性,对绝大多数由疏水性氨基酸（尤其是亮氨酸）构成的肽键有很强的水解能力。     

枯草蛋白酶E的突变体

Ser236位于横贯枯草蛋白酶E的α螺旋末端,远离催化活性中心,Ser236的突变不会对酶的活性产生大的影响。用定点突变的方法对枯草蛋白酶E的基因进行改造引入Ser236Cys,可能会形成分子间二硫键,有利于提高酶的稳定性。Ser236Cys变体酶(BP1)活性是野生型蛋白酶E的15倍,热稳定性提高3倍;进一步在其他位点引入突变的变体酶BU1(A1a15Asp/Gly20His/Ser236Cys)和BW1(Ser24His/Lys27Asp/Ser236Cys)活性都比野生型蛋白酶E低,但BW1的稳定性稍高于野生型蛋白酶E。     

Subtilisin modification of monodeamidated ribonuclease-A

Limited proteolysis of RNAase-Aa₁ (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa₁S. RNAase-Aa₁S was chromatographically distinct from RNAase-S, but exhibited very nearly the same enzymic activity, antigenic conformation and susceptibility to trypsin as did RNAase-S. Fractionation of RNAase-Aa₁S by trichloroacetic acid yielded RNAase-Aa₁S-protein and RNAase-Aa₁S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa₁S′ when mixed together. RNAase-Aa₁S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa₁S-protein with S-peptide exhibited slight but noticeably weaker binding of the peptide to the deamidated S-protein as compared with that of native protein. Unlike the subtilisin digestion of RNAase-A, which gives nearly 100% conversion into RNAase-S, the digestion of RNAase-Aa₁ gives only a 50% conversion. The resistance of RNAase-Aa₁ to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa₁ with its subtilisin-modified product. RNAase-S was also found to undergo activity and structural changes in acidic solutions, similar to those of RNAase-A. The initial reaction product (RNAase-Sa₁) isolated by chromatography was not homogeneous. Unlike the acid treatment of RNAase-A, which affected only the S-protein part, the acid treatment of RNAase-S affected both the S-protein and the S-peptide region of the molecule.     

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multistep process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Here, we solve the three-dimensional structure of TgMIC5 by nuclear magnetic resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost 50-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane-anchored TgSUB1.     

枯草杆菌蛋白酶E的蛋白质工程

用定点突变和随机突变的方法,对枯草杆菌碱性蛋白酶E基因进行改造。突变后的基因插入大肠杆菌-枯草杆菌穿梭质粒pBE-2中,在碱性和中性蛋白酶缺陷型的枯草杆菌DBl04中进行表达,得到突变种的碱性蛋白酶．它们的突变位点分别是(M222A)、(M222A、N118S)、(M222A、N118S、Q103R)、(M222A、N118S、Q103R、D60N)。各突变种酶的性质测定 结果表明．M222A突变使酶抗氧化,N118S突变使酶增加热稳定性,Q103R和D60N突变虽然能增加酶的比活,但使酶的热稳定性大大下降,尤其是D60N突变使酶变得极不稳定。野生型碱性蛋白酶与(M222A)突变种的等电点均为8．92．而M222A,N118S)。(M222A,N118S ,Ql03R)和(M222A,118S．Q103R,D60N)突变酶分别为8.88．9.10和9．17。用Nsuc-AAPF-pNA作为底物时酶反应景适pH值为7.5～9.5,而用酪蛋白底物时最适pH值为10～12。     

枯草杆菌蛋白酶的基因工程改性

枯草杆菌蛋白酶（Ｓｕｂｔｉｌｉｓｉｎ）是一种工业上应用很广的酶,在洗涤剂、制革、丝绸等多种行业上有着广泛的用途。特别是用于生产加酶洗涤剂,帮助去除血渍、奶渍、汗渍及可可等各种蛋白污垢。１９６０年,丹麦人首先利用地衣芽孢杆菌生产了被称为Ｓｕｂｔｉｌｉｓｉｎ Ｃａｒｌｓｂｅｒｇ的碱性蛋白酶,随后该酶被用于生产加酶洗涤剂,目前有资料称国外市场上９０％是加酶洗涤剂。国内１９９０年枯草杆菌蛋白酶产量约为１．３万吨,加酶洗衣粉占洗涤剂总量约１０％。枯草杆菌蛋白酶的生产菌和研究对象主要是地衣芽孢杆菌、解淀粉芽…     

In Vivo Kinetics of Oxidatively Modified HDL

The kinetics of oxidatively modified high-density lipoprotein (HDL) in vivo were investigated. ¹²⁵I-labeled oxidized (Ox) I-IDL and ¹³¹I-labeled native (N) HDL were injected simultaneously into control and WHHL rabbits. The fractional catabolic rates of ¹²⁵I-labeled Ox-HDL were significantly greater than those of ¹³¹I-labeled N-HDL in both control (2.52 ± 0.36/day vs 0.94 ± 0.02/day) and WHHL rabbits (4.07/day vs 1.32/day). Oxidized HDL was catabolized faster than native HDL and was taken up primarily by the liver, spleen, and kidney.     

枯草杆菌蛋白酶基因工程的研究进展

本文介绍了枯草杆菌蛋白酶(Subtilisin)的研究现状,即利用定位诱变和体外重组等技术改变酶的性质,包括催化活性、底物特异性、稳定性、低温适应性以及酶在有机相中的性能等。对枯草杆菌蛋白酶的成功改造不仅有可观的商业价值,而且为蛋白质工程的发展作出了重要的贡献 。     

Limited Proteolysis of Disulfide-reduced Ovalbumin by Subtilisin

Our previous study [Takahashiet al., J. Biochem., 109, 846–851 (1991)] has shown that the disulfide-reduced form of ovalbumin was proteolyzed by subtilisin into three major fragments. It was investigated whether or not these three fragments would be folded into one molecule. Gel permeation and ion-exchange chromatography indicated that the three fragments were eluted in a single peak. The proteolyzed protein had a CD spectrum that was almost indistinguishable from the disulfide-reduced, non-proteolyzed, form of ovalbumin. Differential scanning calorimetry, however, revealed, that the proteolyzed ovalbumin was denatured at a lower temperature than that of the disulfide-reduced, non-proteolyzed. protein. Thus, it is concluded that the three fragments were folded into a native-like conformation with decreased stability. Chemical analyses of the fragments purified by reverse-phase HPLC revealed that there was a cleavage site in the disulfide-reduced form of ovalbumin, at least at the amino-terminal side of Cys⁷³, in addition to the well-known cleavage sites in plakalbumin.