共查询到19条相似文献,搜索用时 156 毫秒
1.
随着生物产业的发展,生物酶催化发挥着越来越重要的作用。然而,部分酶在应用过程中仍然存在诸多问题,影响了生物催化的进一步发展。本文以酶的底物特异性为切入点,回顾了酶的专一性、高效性和环保性;介绍了酶在药物合成和天然产物改性领域的应用以及所遇到的问题;综述了酶的底物特异性改造过程中各种方法的应用,包括化学修饰、非理性和理性设计。化学修饰作为一种直观的修饰方法,通过化学反应对酶分子进行改造;非理性设计是利用易错PCR和DNA Shuffling等手段获得底物特异性提高的突变体;理性设计是基于序列和结构信息对酶分子进行改造。本文从重塑活性口袋提高酶的底物特异性和重塑活性口袋改变酶促反应类型两个方面出发,详述了理性设计改变酶的底物特异性的方法,为酶的特异性改造提供借鉴。 相似文献
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准确地讲,酶的专一性是指酶催化某一种或者某一类底物发生特定的反应生成特定的产物这个性质,而不能将之简单地表述为酶作用于某一种或者某一类底物的性质。因为,同样的底物完全可能经不同的酶催化而生成不同的产物。 相似文献
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分子识别 (MolecularRecognition)是超分子科学研究中的一个基本问题 .在生命科学中酶的特殊催化功能不仅表现为其催化能力的高效性 ,更重要的是在于它能特征性的对某些化合物分子发生专一性的反应 .这种能力的产生是建立在酶对于底物分子识别基础上的 ,因此设计和合成有光谱响 相似文献
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详细综述了非水相酶催化合成蔗糖酯的研究进展。主要介绍了酶的种类、反应介质、底物性质、外加辅助手段等因素对蔗糖酯产率以及对酶区域选择性的影响。酶催化区域选择性合成蔗糖酯的难点在于蔗糖具有多个可酰化羟基及其与酰基供体的不相溶性,提出通过介质工程、底物修饰改性、外加物理场如微波和超声波辅助等改善底物相溶性,通过筛选酶、蔗糖修饰等可以得到高区域选择性的蔗糖酯。最后指出酶催化合成蔗糖酯存在的问题、发展前景等。 相似文献
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高效液相色谱-固定化酶反应器 总被引:1,自引:0,他引:1
酶是一种具有特殊的三度空间构象的蛋白质,它能催化构成生命活动的许多化学反应。有些酶需要具备一种或多种称为辅助因子的非蛋白组分方能显示其活性,这种辅助因子,可以是称作辅酶的有机物分子或金属离子,这类具有催化活性的酶-辅助因子复合物叫做全酶。酶能催化某一底物进行特异性反应,生成特有的反应产物。1860年Emil Fisher提出酶和底物的反应是“一把钥匙开一个锁”的机理,即一种酶对应于一个反应,因此,酶的催化反应具有高度的专一性。例如葡糖氧化酶只催化β-D-葡葡糖,使其氧化生成葡糖酸和过氧化氢。这就是最常用的葡萄糖分析法。酶试剂通常是较贵的,作为催化剂虽然理论上酶自身并不消耗,但在溶液中,酶很容易变性而失活。如果将酶固定到某一载体上,就不需要在分析前配置各种浓度的酶溶液,还能提高其热稳定性,增加可储存的时间,使得一次性应用的酶试剂变为可重复使用而不会降低其功效。1916年,Nelson和Griffin首先报导 相似文献
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酶催化剂具有高效性,多样性,底物专一性,区域选择性、化学选择性、对映选择性以及反应条件温和的特点。而酶的固定化后除了保持原有的特点外,易与反应物和产物分离,可回收重复使用,降低生产成本。本文对酶催化剂的固定化方法以及在有机催化反应中的应用作了部分简述。并对固定化方法进行了比较和评价。 相似文献
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Edge M; Forder C; Hennam J; Lee I; Tonge D; Hardern I; Fitton J; Eckersley K; East S; Shufflebotham A; Blakey D; Slater A 《Protein engineering, design & selection : PEDS》1998,11(12):1229-1234
Variants of human pancreatic carboxypeptidase B (HCPB), with specificity
for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared
by site-directed mutagenesis of the human gene and expressed in the
periplasm of Escherichia coli. By changing residues in the lining of the
S1' pocket of the enzyme, it was possible to reverse the substrate
specificity to give variants able to hydrolyse prior to C- terminal acidic
amino acid residues instead of the normal C-terminal basic residues. This
was achieved by mutating Asp253 at the base of the S1' specificity pocket,
which normally interacts with the basic side- chain of the substrate, to
either Lys or Arg. The resulting enzymes had the desired reversed polarity
and enzyme activity was improved significantly with further mutations at
residue 251. The [G251T,D253K]HCPB double mutant was 100 times more active
against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the
single mutant, [D253K]HCPB. Triple mutants, containing additional changes
at Ala248, had improved activity against hipp-Glu substrate when position
251 was Asn. These reversed-polarity mutants of a human enzyme have the
potential to be used in antibody-directed enzyme prodrug therapy of cancer.
相似文献
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It is widely agreed that new enzymes evolve from existing ones through the duplication of genes encoding existing enzymes followed by sequence divergence. While evolution is an inherently random process, studies of divergently related enzymes have shown that the evolution of new enzymes follows one of three general routes in which the substrate specificity, reaction mechanism, or active site architecture of the progenitor enzyme is reused in the new enzyme. Recent developments in structural biology relating to divergently related (beta/alpha)8 enzymes have brought new insight into these processes and have revealed that conserved structural elements play an important role in divergent evolution. These studies have shown that, although evolution occurs as a series of random mutations, stable folds such as the (beta/alpha)8 barrel and structural features of the active sites of enzymes are frequently reused in evolution and adapted for new catalytic purposes. 相似文献
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Sang Jin Park Dr. Jung Min Choi Hyun‐Ho Kyeong Dr. Song‐Gun Kim Prof. Hak‐Sung Kim 《Chembiochem : a European journal of chemical biology》2015,16(5):854-860
Triterpenoids with desired glycosylation patterns have attracted considerable attention as potential therapeutics for inflammatory diseases and various types of cancer. Sugar‐hydrolyzing enzymes with high substrate specificity would be far more efficient than other methods for the synthesis of such specialty triterpenoids, but they are yet to be developed. Here we present a strategy to rationally design a β‐glycosidase with high regiospecificity for triterpenoids. A β‐glycosidase with broad substrate specificity was isolated, and its crystal structure was determined at 2.0 Å resolution. Based on the product profiles and substrate docking simulations, we modeled the substrate binding modes of the enzyme. From the model, the substrate binding cleft of the enzyme was redesigned in a manner that preferentially hydrolyzes glycans at specific glycosylation sites of triterpenoids. The designed mutants were shown to produce a variety of specialty triterpenoids with high purity. 相似文献
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Masayama A Takahashi T Tsukada K Nishikawa S Takahashi R Adachi M Koga K Suzuki A Yamane T Nakano H Iwasaki Y 《Chembiochem : a European journal of chemical biology》2008,9(6):974-981
The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD. 相似文献
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Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae 下载免费PDF全文
Philipp Matzel Lukas Krautschick Prof. Matthias Höhne 《Chembiochem : a European journal of chemical biology》2017,18(20):2022-2027
Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C4–C8) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94–96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source. 相似文献
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A dehydro-thermal treatment has been used to crosslink α-amylase (EC 3.2.1.1)–poly(vinyl alcohol) blends in order to reduce their water solubility and to favour their potential use as bio-artificial polymeric materials based on enzymes. The influence of a thermal treatment on the thermal and biological stability of the enzyme blended with a synthetic polymer was carefully studied by means of calorimetry and biological assays. The values of the kinetic parameters obtained from the biological assays indicate that the enzyme specificity for the substrate in all the blends examined is not affected by the thermal treatment. In addition, the results of the releasing tests showed that the thermally crosslinked α-amylase–poly(vinyl alcohol) blends can be considered suitable for manifacturing delivery systems able to release enzymes. © 1998 Society of Chemical Industry 相似文献
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L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) is characterized by a broad
substrate specificity and utilizes a wide range of 2-oxo acids branched at
the C4 atom. Modifications have been made to the sequence of the
NAD(H)-dependent L-HicDH from Lactobacillus confusus in order to define and
alter the region of substrate specificity towards various 2- oxocarbonic
acids. All variations were based on a 3D-structure model of the enzyme
using the X-ray coordinates of the functionally related L- lactate
dehydrogenase (L-LDH) from dogfish as a template. This protein displays
only 23% sequence identity to L-HicDH. The active site of L- HicDH was
modelled by homology to the L-LDH based on the conservation of
catalytically essential residues. Substitutions of the active site residues
Gly234, Gly235, Phe236, Leu239 and Thr245 were made in order to identify
their unique participation in substrate recognition and orientation. The
kinetic properties of the L239A, L239M, L236V and T245A enzyme variants
confirmed the structural model of the active site of L-HicDH. The
substrates 2-oxocaproate, 2-oxoisocaproate, phenylpyruvate,
phenylglyoxylate, keto-tert-leucine and pyruvate were fitted into the
active site of the subsequently refined model. In order to design
dehydrogenases with an improved substrate specificity towards keto acids
branched at C3 or C4, amino acid substitutions at positions Leu239, Phe236
and Thr245 were introduced and resulted in mutant enzymes with completely
different substrate specificities. The substitution T245A resulted in a
relative shift of substrate specificity for keto-tert-leucine of more than
17000 compared with the 2-oxocaproate (kcat/KM). For the substrates
branched at C4 a relative shift of up to 500 was obtained for several
enzyme variants. A total of nine mutations were introduced and the kinetic
data for the set of six substrates were determined for each of the
resulting mutant enzymes. These were compared with those of the wild-type
enzyme and rationalized by the active site model of L-HicDH. An analysis of
the enzyme variants provided new insight into the residues involved in
substrate binding and residues of importance for the differences between
LDHs and HicDH. After the protein design project was complete the X-ray
structure of the enzyme was solved in our group. A comparison between the
model and the experimental 3D structure proved the quality of the model.
All the variants were designed, expressed and tested before the 3D
structure became available.
相似文献