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木糖异构酶的结构及蛋白质工程肖亚中,伍传金,崔涛（中国科学技术大学生物系合肥２３００２６）关键词木糖异构酶,结构与功能,蛋白质工程木糖异构梅（Ｄ－ＸｙｌｏｓｅＩｓｏｍｅｒａｓｅ,ＥＣ５。３。１。５,下简称ＸＩ）催化五碳Ｄ－木糖转化为Ｄ－木酮糖,在体外亦能催化六碳Ｄ－葡萄糖至Ｄ－果糖的异构化反应［１,２］。该反应是工业上大规模以淀粉制备高果糖浆的关键步骤［３］,故习惯上将木糖异构酶称为葡萄糖异构酶 。 相似文献
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取云南腾冲温泉水样在以木糖为唯一碳源的平板上进行培养,分离纯化得到11 株可以代谢木糖的菌株,分别进行液体摇床培养,并以半胱氨酸-咔唑法测定木糖异构酶活性,筛选得到一株产木糖异构酶活性较高的菌株RD-1,经形态学及16S rRNA 鉴定命名为Anoxybacillus .avithermus WL,该菌在24h 时显示最高酶活6.4 U/mL. 相似文献
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木糖异构酶在酿酒酵母细胞表面的展示 总被引：2，自引：0，他引：2
将来源于嗜热细菌Thermus thermophilus的木糖异构酶基因xylA,与酿酒酵母(Sac-charomyces cerevisiae)a-凝集素表面展示载体pYD1的Aga2p亚基C端序列融合。编码融合蛋白的基因序列前接上半乳糖诱导型启动子。用LiAc完整细胞法转化酿酒酵母EBY100。含重组质粒的菌株EBY100/pYD-xylA经半乳糖诱导表达外源融合蛋白,免疫荧光显微镜结果显示外源蛋白被锚定在细胞壁上,木糖异构酶活性测定结果表明,细胞壁上酶活测定值为1.52U,木糖异构酶在酿酒酵母细胞壁上得到活性表达。 相似文献
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天然球蛋白分子表面暴露的柔性的环是对蛋白质水解作用最敏感的部位,可用蛋白质部分水解来确定木糖异构酶突变体上的这些部位,枯草杆菌蛋白酶对Ｗ１３６Ｅ单体的水解在一级反应图上呈折线,对Ｔ８９Ｓ和Ｖ１３４１单体的部分水解为直线。对镁－酶的水解速度低于对脱辅基酶的水解速度。枯草杆菌蛋白酶对Ｗ１３６Ｅ的第一个水解位点在Ａｌａ２８,Ｔｈｒ２９之间,第二个水解位点在肽链的Ｃ端。 相似文献
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链霉菌M1033染色体DNA经BamH Ⅰ酶解后电泳,Southern转移。根据自测的链霉菌M1033 D－木糖异构酶氨基酸序列设计合成寡聚核苷酸探针x－2、x－3,以x－2、x－3及Am－pullariella sp3876 D-木糖异构酶基因(1．17kb)为探针进行杂交,确定与上述探针杂交最强处在15kb左右。从胶上分离出g-20kb大小的片段,克隆到EMBL 3载体中,经杂交筛选后,得到0．6％的阳性噬斑,其插人大小为13kh。将插入DNA的saIⅠ酶解片段(2．5kb)进一步亚克隆于pucl8,得到重组质粒puB l,经酶解图谱、部分序列分析和互补实验确定puBl含有完整的M1033 D-木糖异构酶基因 相似文献
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The crystal structures of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase (SDXyI) have been analysed and refined at 0.19nm. The crystal space group is I222, with unit cell dimensions of a=9.884 ran, b=9.393nm and c=8.798nm. Based on the coordinates of the Streptomyces rubiginosus xylose isomerase (SRXyI), the initial model of SDXyl was built up by the dose packing analysing and R-factor searching and refined by PROLSQ to a final R-factor of 0.177 with the rms deviations of bond lengths and bond angles of 0.001 9nm and 2.1°, respectively. No significant global conformation change existed between SRXyI and SDXyI except the local conformation in the active site. 相似文献
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Chong Xu Xue‐yong Zhu Mai‐kun Teng Li‐wen Niu Yu‐zhen Wang 《Acta Crystallographica. Section D, Structural Biology》2000,56(2):129-136
The structure of xylose isomerase (XyI) from Streptomyces diastaticus No. 7 strain M1033 (SDXyI) has been refined at 1.85 Å resolution to conventional and free R factors of 0.166 and 0.219, respectively. SDXyI was crystallized in space group P21212, with unit‐cell parameters a = 87.976, b = 98.836, c = 93.927 Å. One dimer of the tetrametric molecule is found in each asymmetric unit. Each monomer consists of two domains: a large N‐terminal domain (residues 1–320), containing a parallel eight‐stranded α/β barrel, and a small C‐terminal loop (residues 321–387), containing five helices linked by random coil. The four monomers are essentially identical in the tetramer, possessing non‐crystallographic 222 symmetry with one twofold axis essentially coincident with the crystallographic twofold axis in the space group P21212, which may explain why the diffraction pattern has strong pseudo‐I222 symmetry even at medium resolution. The crystal structures of XyIs from different bacterial strains, especially from Streptomyces, are similar. The α2 helix of the α/β barrel has a different position in the structures of different XyIs. The conformation of C‐terminal fragment 357–364 in the SDXyI structure has a small number of differences to that of other XyIs. Two Co2+ ions rather than Mg2+ ions exist in the active site of the SDXyI structure; SDXyI seems to prefer to bind Co2+ ions rather than Mg2+ ions. 相似文献
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Graciela N. Roman Norman B. Jansen Humg-Yu Hsiao George T. Tsao 《Enzyme and microbial technology》1985,7(3):129-133
d-Xylose isomerase catalyses the conversion of the common pentose, d-xylose, to its keto-isomer, d-xylose. This reaction is of interest because many microorganisms that are unable to metabolize d-xylose can utilize d-xylulose. The kinetics of a commonly used immobilized whole-cell isomerase, Sweetzyme Q, have been determined from initial rate studies on the forward and reverse reactions. The effect of pH, temperature, and substrate and product concentration on enzyme activity have all been examined. Reaction rates were modelled with the Michaelis-Menten equation. Using constants determined from Lineweaver-Burk plots, the rate equation accurately simulated experimental conversion data. 相似文献
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为了使酿酒酵母较好地利用木糖产生乙醇,将来自Thermus thermophilus的木糖异构酶基因XYLA和酿酒酵母自身的木酮糖激酶基因XKS1,构建到酵母表达载体pESC-LEU中,导入酿酒酵母YPH499中,同时成功表达了两种酶基因。该菌以木糖为唯一碳源进行限氧发酵,木糖的利用率为9.64%,为宿主菌的4.17倍,产生2.22 mmol.L-1的乙醇。同时初步探讨了两种酶基因的表达量对酿酒酵母发酵木糖生成乙醇的影响。木糖异构酶对木糖的利用起关键性的作用,木酮糖激酶的过量表达不利于乙醇生成。 相似文献
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Gabriel de Souza Colombo Isis Viana Mendes Betúlia de Morais Souto Cristine Chaves Barreto Luana Assis Serra Eliane Ferreira Noronha Nádia Skorupa Parachin João Ricardo Moreira de Almeida Betania Ferraz Quirino 《Letters in applied microbiology》2022,74(6):941-948
The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions. 相似文献
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L. F. Delboni S. C. Mande F. Rentier-Delrue V. Mainfroid S. Turley F. M. Vellieux J. A. Martial W. G. Hol 《Protein science : a publication of the Protein Society》1995,4(12):2594-2604
The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the \"closed\" conformation in either subunit. The crystallographic R-factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 A for 248 C alpha atoms and have average temperature factors of 18.9 and 15.9 A2, respectively. In both subunits, the active site Lys 10 adopts an unusual phi, psi combination. A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearothermophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearothermophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures. 相似文献
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Jea‐Won Cho Byeong‐Gu Han Sang Youn Park Seung Jun Kim Myoung‐Dong Kim Byung Il Lee 《Acta Crystallographica. Section F, Structural Biology Communications》2013,69(10):1127-1130
Bacteroides thetaiotaomicron BT0793, a putative xylose isomerase, was overexpressed in Escherichia coli, purified and crystallized using polyethylene glycol monomethyl ether 550 as the precipitant. X‐ray diffraction data were collected to 2.10 Å resolution at 100 K using synchrotron X‐rays. The crystal was found to belong to space group P1, with unit‐cell parameters a = 96.3, b = 101.7, c = 108.3 Å, α = 82.8, β = 68.2, γ = 83.0°. The asymmetric unit contained eight subunits of xylose isomerase with a crystal volume per protein weight (VM) of 2.38 Å3 Da−1 and a solvent content of 48.3%. 相似文献
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Summary Plant genetic transformation technologies rely upon the selection and recovery of transformed cells. Selectable marker genes
used so far have been either antibiotic resistance genes or herbicide tolerance genes. There is a need to apply alternative
principles of selection, as more transgenic traits have to be incorporated into a transgenic crop and because of concern that
the use of conventional marker genes may pose a threat to humans and the environment. New classes of marker genes are now
available, conferring metabolic advantage of the transgenic cells over the non-transformed cells. The new selection systems,
as described in this review, are being used with success and superior performance over the traditional marker systems. 相似文献
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L Lama B Nicolaus V Calandrelli I Romano R Basile A Gambacorta 《Journal of industrial microbiology & biotechnology》2001,27(4):234-240
Xylose isomerase produced by Bacillus thermoantarcticus was purified 73-fold to homogeneity and its biochemical properties were determined. It was a homotetramer with a native molecular
mass of 200 kDa and a subunit molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K
m of 33 mM for xylose and also accepted D-glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a temperature of 85°C.
Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme required divalent cations for
its activity and thermal stability. Mn2+, Co2+ or Mg2+ were of comparable efficiency for xylose isomerase reaction, while Mg2+ was necessary for glucose isomerase reaction. Journal of Industrial Microbiology & Biotechnology (2001) 27, 234–240.
Received 18 March 2001/ Accepted in revised form 03 July 2001 相似文献