适于非水相催化用细菌脂肪酶基本性质的研究

对假单胞菌产脂肪酶的基本酶学性质进行了研究．该酶水解油脂时的最适作用ｐＨ为９．０,最适作用温度为４５℃;在ｐＨ７．０～１０．０范围内稳定,在６０℃以下热稳定性良好．Ｋ＋、Ｃａ２＋、Ｍｇ２＋等金属离子对酶有明显的激活作用,而Ｈｇ２＋、Ｃｕ２＋、Ｓｎ２＋等重金属离子却对酶有较强的抑制作用;几种表面活性剂和胆汁盐均使酶发生不同程度的失活．该酶在水解油脂时表现出１,３－位置专一性,且对不同种类和来源的油脂水解作用速率不同     

彩绒革盖菌多酚氧化酶活性研究

本文测定了彩绒革盖菌在ＰＤＹ液体培养基中的多酚氧化酶活性。在３０℃,１１０ｒ／ｍｉｎ,恒温振荡培养条件下,多酚氧化酶第１４天达产酶高峰,最高酶活５４９．０ｕ;酶作用的最适酸碱度为ｐＨ５．０;最适作用温度３０℃;Ｍｎ２＋、Ｂａ２＋、Ｍｇ２＋等离子对多酚氧化酶有激活作用,Ａｇ＋、Ｆｅ３＋等离子对酶活则有明显的抑制作用     

彩绒革盖菌漆酶及多酚氧化酶活性研究

测定了彩绒革盖菌在ＰＤＹ液体培养基中的漆酶和多酚氧化酶活性。在３０℃,１１０ｒ／ｍｉｎ,恒温振荡培养条件下,漆酶第９ｄ达产酶高峰,最高酶活３９５．６ｕ;多酚氧化酶第１４ｄ达产酶高峰,峰值５４９．０ｕ;酶作用的最适酸碱度为：漆酶ｐＨ４．６,多酚氧化酶ｐＨ５．０;最适作用温度漆酶为２５℃,多酚氧化酶为３０℃;Ｋ＾＋、Ｚｎ＾２＋等离子对漆酶有激活作用,Ｍｎ＾２＋、Ｂａ＾２＋、Ｍｇ＾２＋等离子对多酚氧化酶     

树状单杆菌变株U—616葡萄糖异构酶的研究

以树状黄杆菌ＮＲＲＬ１１０２２为出发菌株,用紫外线对其进行诱变,经筛选得到一株葡萄糖异构酶的高产菌株Ｕ－６１６,其酶活力提高３１％,经保存三年和多次传代复测,其产酶能力保持稳定,其生长和产酶需较高的溶氧水平,最适产酶温度为３０℃,最适产酶ｐＨ为７．０－７．５,铁离子对其生长和产酶无明显的影响。所产葡萄糖异构酶的最适温度为６０￣８０℃,最适ｐＨ为７．５－８．５,Ｃｏ＾２＋和Ｍｇ＾２＋对酶有激活作用,     

中温碱性脂肪酶的研究：Ⅲ.扩展青霉PF868变株碱性脂肪酶的纯化及其…

扩展青霉ＰＦ８６８变株发酵液经硫酸铵盐析和Ｓｅｐｈａｄｅｘ－Ｇ－２００及Ｓｅｐｈａｒｏｓｅ４Ｂ柱层析纯化,获得纯化倍数为３２．４的酶粉。该酶分子量为２３４４２Ｄａｌ,酶学特性表明：该酶的最适作用温度为３２℃,５０℃保温３０ｍｉｎ仍保留５０％酶活性,最适ｐＨ为９．０,作用ｐＨ稳定范围在７．０－１０．０之间,Ｃａ＾２＋和Ｍｇ＾２＋对酶有激活作用,Ｆｅ＾２＋,Ｃｕ＾２＋和Ｍｎ＾２＋酶活力有抑制作用。     

树状黄杆菌变株U-616葡萄糖异构酶的研究

以树状黄杆菌（Ｆｌａｖｏｂａｃｔｅｒｉｕｍａｒａｂｏｒｅｓｃｅｎｓ）ＮＲＲＬ１１０２２为出发菌株,用紫外线对其进行诱变,经筛选得到一株葡萄糖异构酶的高产菌株Ｕ－６１６,其酶活力提高３１％。经保存三年和多次传代复测,其产酶能力保持稳定。其生长和产酶需较高的溶氧水平,最适产酶温度为３０℃,最适产酶ｐＨ为７．０－７．５,铁离子对其生长和产酶无明显的影响。所产葡萄糖异构酶的最适温度为６０－８０℃,最适ｐＨ为７．５－８．５,Ｃｏ２＋和Ｍｇ２＋对酶有激活作用,对金属离子耐受性较强,对Ｃａ２＋不敏感,热稳定性较好。树状黄杆菌变株Ｕ－６１６是一株产胞内葡萄糖异构酶的优良菌株。     

青霉胞外菊粉酶的纯化和性质研究

青霉菌（ＰｅｎｉｃｉｌｌｉｕｍＳＰ．９１－４）产生的胞外菊粉酶（ｅｘｔｒａｃｅｌｌｕｌａｒｉｎｕｌｉｎａｓｅ）粗酶液,经硫酸铵沉淀,超滤浓缩,Ｓｅｐｈａｄｅｘ－Ｇ－１００凝胶过滤,ＤＥＡＥ－Ｓｅｐｈａｃｅｌ离子交换柱层析等步骤,得到提纯３０倍的酶Ｅ。酶Ｅ反应时最适ｐＨ４．５,最适温度为５０℃,在ｐＨ４．７～７．６范围内,温度５０℃以下酶Ｅ活性稳定;其活性受Ａｇ＾＋,Ｃｕ＾２＋ＰＣＭＢ强烈抑制。采用     

微小毛霉凝乳酶的分离纯化研究

研究微小毛霉(HL-1)凝乳酶的分离纯化条件及方法。研究酶的最适浸提温度、酶的浸提pH值和最适浸提时间,探讨离子浓度、加水量对浸提效率的影响,利用高速冷冻离心法、有机溶剂沉淀法,膜分离法和层析法等对粗酶液进行了分离。利用光谱法对纯化样品进行检测。酶的最适浸提温度为30℃;最适pH为6.0;浸提10 h活力最高;1%的氯化钠有利于酶的分离,加水比例为15时有利于提取,在10 000 r/min下离心10min澄清效果最好,95%的酒精沉淀效果最好,利用0.2μm的微滤膜可除去发酵液中的菌体,8 000的超滤膜可拦截凝乳酶蛋白,S300的填料可有效分离凝乳酶,纯度达95%以上。     

角质蛋白酶固态发酵工艺及酶解条件的研究

曲霉Ａ２８－８是一株优良的角蛋白酶分泌突变菌株。其最佳固体发酵培养基为：２０％羽毛粉,８０％麸皮和微量无机氮（每克培养基中加０．５ｍｇ）;最适产酶条件为：起始ｐＨ７．５～８．０,温度２８℃～３０℃,时间为６０～７０小时,酶活高达２５００ＫＵ／ｇ曲;最适酶解条件为ｐＨ７．０～９．０,温度４５℃～５０℃。     

粘质赛氏菌胞外蛋白酶的提取、纯化及部分性质研究

经过硫酸铵３０％～５０％分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达５３％,并制备了酶的结晶,该酶以ＳｅｐｈａｄｅｘＧ１００柱层析及ＳＤＳ－ＰＡＧＥ测得分子量约为８１０００,该酶的最适ｐＨ为７．０,最适温度为４５℃,Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋、Ｃｕ２＋、Ｃｏ２＋等重金属离子不同程度地抑制酶活性。     

Relationship between enzyme heterozygosity and quaternary structure

The need for proteins to maintain particular quaternary structures constrains variability in amino acid sequence. Monomeric enzymes are then expected to be more variable than dimeric forms, which in turn are expected to be more variable than tetrameric forms. These predictions are confirmed by analysis of available data on enzyme variation. Theories relating enzyme heterozygosity to metabolic function are discussed in the light of these findings.Financial support for part of the work described in this article was derived from NERC Grant GR3/1558 to J. A. Beardmore.     

Detoxification,antioxidant, and digestive enzyme activities and gene expression analysis of Lymantria dispar larvae under carvacrol

Carvacrol is a terpene compound with various biological activities. However, few studies have specifically focused on its insecticidal activity and mechanism of carvacrol. The larvae of Lymantria dispar are seriously harmful herbivorous insect. This study measured the antifeedant, growth-inhibitory, and toxic effects of carvacrol on L. dispar larvae. To further clarify the insecticidal mechanism of carvacrol, the effects of carvacrol on detoxifying enzymes, antioxidative enzymes, digestive enzyme activities, and the mRNA expression of the above-mentioned enzyme genes were investigated. The results of the study showed that the median lethal concentration (LC₅₀) and the sublethal concentration (LC₂₀) of carvacrol were 1.120 mg/mL and 0.297 mg/mL, respectively, at 72 h. After LC₂₀ treatment of L. dispar larvae for 72 h, food intake and weight gain were significantly lower compared with the control. Enzyme activity assays showed that carvacrol significantly inhibited the activities of carboxylesterase (CarE), glutathione S-transferase (GST), and acetylcholinesterase (AchE), and the inhibition rate of AchE activity was highest (66.51%). Carvacrol also activated the activities of superoxide dismutase (SOD) and catalase (CAT), while it inhibited the activities of lipase (LIP) and amylase (AMS), and first inhibited and then activated protease. In addition, qRT-PCR tests showed that carvacrol affected the mRNA expression levels of CarE, GST, AchE, SOD, CAT, LIP, AMS, and protease. This study helps to clarify the insecticidal mechanism of carvacrol on L. dispar larvae.     

Inducible catalase in Pseudomonas fluorescens

The catalase activity of a non-proliferating suspension of Pseudomonas fluorescens doubled after six hours incubation in a 50 mM phosphate buffer medium (pH 7.3). The same effect was observed in a peptone medium. The increased activity was due to induced enzyme synthesis, and not to activation of preexisting catalase. Induced catalase was separated by electrophoresis from deuterium labelled constitutive catalase. The enzyme was also induced under anaerobic conditions in phosphate buffer or in culture when nitrate was supplied as an electron acceptor. Induction was considerably increased by the addition of various nucleotides and amino acids to the incubation medium.     

Biosynthesis of the C-terminal amide in peptide hormones

Recent developments in the study of peptide amidation are reviewed. The main areas covered are assay procedures, purification of amidating enzymes, co-fact0rs and regulation; mechanism and specificity of the amidating reaction, and multiple forms of the amidating enzyme and glycosylation. Discussion is presented on aspects that are poorly understood and new areas open to investigation are indicated.     

Partial purification and kinetic properties of three different d-glucosamine 6-P:N-acetyltransferase forms from human placenta

Three distinct forms of -glucosamine 6-P (Gm 6-P):N-acetyltransferases (EC 2.3.1.4) were partially purified from human placental homogenates by carboxy methyl-Sephadex chromatography. Purification of forms I and II were 13.5-fold, while that of form III was 114-fold. All three forms had a pH optimum value of 9.7 in glycine–NaOH buffer. Enzymes II and III had a K_m value for Gm 6-P of 3.0 mM, which was less than half of that observed for form I (7.1 mM). The corresponding K_m values for acetyl CoA were 0.157 (form I), 0.187 (form II) and 0.280 mM (form III), respectively. Activities of all three forms were inhibited at high concentrations of either substrate. These enzymes were inhibited from 82 to 92% by 2.5 mM p-chloromercuribenzoate. The inhibition was largely reversible by inclusion of 2.5 mM dithiothreitol in the incubation mixtures. There was no requirement for divalent cations, as demonstrated by lack of inhibition of enzyme activity by ethylene diamine tetraacetate. The results are discussed in terms of differences among the enzyme properties of human placental, rodent and porcine liver forms.     

Metal-Induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent malic enzyme

Human mitochondrial NAD(P)+-dependent malic enzyme was strongly inhibited by Lu3+. The X-ray crystal structures indicated a structural change between the metal-free and Lu3+-containing enzymes (Yang Z, Batra R, Floyd DL, Hung HC, Chang GG, Tong L. Biochem Biophys Res Commun 2000;274:440-444). We characterized the reversible slow-binding mechanism and the structural interconversion between Mn2+- and Lu3+-containing human mitochondrial malic enzymes. When Lu3+ was added, the activity of the human enzyme showed a downward curve over time, similar to that of the pigeon enzyme. The rate of the transformation (k(obs)) from the initial rate to the steady-state rate increased hyperbolically with the concentration of Lu3+, suggesting the involvement of an isomerization step. Lu3+ had a much higher affinity for the isomerized form (K*(i,Lu (app)) = 4.8 microM) than that of the native form (K(i,Lu (app)) = 148 microM). When an excess of Mn2+ was added to the Lu3+-inhibited enzyme, assays of the kinetic activity showed an upward trend, indicating reactivation. This result also indicated that the reactivation was a slow process. Fluorescence quenching experiments confirmed that the Lu3+-induced isomerization was completely reversible. The dynamic quenching constants for the metal-free, Mn2+-containing, and Lu3+-containing enzyme were 3.08, 3.07, and 3.8 M(-1), respectively. When the Lu3+-containing enzyme was treated with excess Mn2+, the dynamic quenching constant returned to the original value (3.09 M(-1)). These results indicated that binding of Mn2+ did not induce any conformational change in the enzyme. The open form transformed to the closed form only after substrate binding. Lu3+, on the other hand, transformed the open form into a catalytically inactive form. Excess Mn2+ could replace Lu3+ in the metal binding site and convert the inactive form back into the open form. This reversible process was slow in both directions because of the same but opposite structural change involved.     

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α₂β₂ heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5′-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.     

Comparative studies of suidatrestin, a specific inhibitor of trehalases

Suidatrestin, isolated from a Streptomyces strain, was characterized as a new trehalase inhibitor. Its inhibitory potential was 7 to 50-fold higher than that of validamycin when tested against insect, fungal and mammalian trehalases. The kinetic properties of suidatrestin were studied in vitro with trehalases from flight muscle mitochondria of the fly, Protophormia terraenovae, from larval midgut of the moth, Spodoptera littoralis, and from porcine kidney, as well as with maltase from yeast. Suidatrestin was inactive on maltase but inhibited all trehalases with IC₅₀ values of 0.08–0.1 μM; K_i values ranged from 0.02 to 0.05 μM. The very low K_i/K_m ratios (3.9×10⁻⁶–4.9×10⁻⁶) indicated excellent in vitro inhibitory action of suidatrestin. When injected into larvae of S. littoralis, suidatrestin required high and repetitive doses which lead to reversible inhibition of larval growth only. Consecutive omission of the inhibitor even stimulated weight increase above that of controls. Significant mortality was achieved at a rather high dose only. Injection of a growth-inhibiting dose of suidatrestin did not change hemolymph osmolality as a measure of sugar concentration. The discrepancy between in vitro and in vivo potency of suidatrestin may be understood once its chemical structure is fully known.     

酶制剂研制的国内外进展和发展策略

简述了酶工程的研究范围,国际上酶制剂工业发展的研究,特点。概述了近年来我国酶制剂工业发展中存在的问题,包括产品结构,技术含量,研发能力。提出我国酶制剂工业今后发展的一些建议：充分发挥资源优势,加强现代生物技术的应用,调整酶制剂产品的结构。