摘要：【中文】本发明涉及一种重组R‑ω‑转氨酶及突变体，以及其在不对称合成西他列汀中的应用。本发明筛选新型R‑ω‑转氨酶，并与现有的高效转氨酶重组，获得的重组体的氨基酸序列如SEQ ID:15所示，其突变体是将SEQ ID:15所示的氨基酸序列的第71、135、或292中的一个或者多个进行单位点突变或多位点突变获得的，最佳的R‑ω‑转氨酶突变株的氨基酸序列如SEQ ID:17所示，对应的核苷酸序列如SEQ ID:18所示。本发明提供了具有更高活性(582.4U/g)和立体选择性(e.e.值99.9％)的R‑ω‑TA突变体，可催化500mM底物转化为西他列汀，转化率高达99％，对于提升西他列汀生物催化制备技术具有重要意义。 【EN】The invention relates to a recombinant R-omega-transaminase and a mutant thereof, and application of the recombinant R-omega-transaminase and the mutant in asymmetric synthesis of sitagliptin. The invention screens novel R-omega-transaminase and recombines with the prior high-efficiency transaminase, the obtained recombinant has an amino acid sequence shown in SEQ ID:15, the mutant is obtained by carrying out single-site mutation or multi-site mutation on one or more of 71 th, 135 th or 292 th amino acid sequences shown in the SEQ ID:15, the optimal amino acid sequence of the R-omega-transaminase mutant is shown in the SEQ ID:17, and the corresponding nucleotide sequence is shown in the SEQ ID: 18. The invention provides the R-omega-TA mutant with higher activity (582.4U/g) and stereoselectivity (e.e. value 99.9%), which can catalyze 500mM substrate to be converted into sitagliptin, the conversion rate is up to 99%, and the R-omega-TA mutant has important significance for improving the sitagliptin biocatalytic preparation technology.

摘要：【中文】本发明涉及一种D‑阿洛酮糖3‑差向异构酶突变体，及其在微生物催化D‑果糖异构化制备D‑阿洛酮糖中的应用。所述D‑阿洛酮糖3‑差向异构酶突变体，由所示氨基酸经定点突变而得，所述点突变位点为下列中的一个或多个：(1)第242位、(2)第105位、(3)第210位、(4)第147位、(5)第184位。本发明有益效果主要体现在：本发明提供了一种全新的D‑阿洛酮糖3‑差向异构酶及其突变体，该突变体具有高最适反应温度85℃，解决了现有酶无法在高温生产D‑psicose的技术难题。使用本突变体生产D‑psicose，产物得率最高可达40.1％，优于原始酶和其他突变酶的转化效果，具有较好工业应用前景。 【EN】The invention relates to a D-psicose3-epimerase mutant and application thereof in preparing D-psicose by catalyzing D-fructose isomerization by microorganisms. The D-psicose3-epimerase mutant is obtained by site-directed mutagenesis of the amino acids shown, wherein the site of: (1) 242 th bit, (2) 105 th bit, (3) 210 th bit, (4) 147 th bit, and (5) 184 th bit. The invention has the following beneficial effects: the invention provides a brand-new D-psicose3-epimerase and a mutant thereof, wherein the mutant has a high optimal reaction temperature of 85 ℃, and solves the technical problem that the existing enzyme cannot produce D-psicose at high temperature. The mutant is used for producing D-psicose, the yield of the product can reach 40.1 percent at most, the conversion effect of the D-psicose is better than that of the original enzyme and other mutant enzymes, and the D-psicose has better industrial application prospect.

摘要：【中文】本发明涉及一种L‑阿拉伯糖差向异构酶突变体，及其在微生物催化D‑半乳糖异构化制备D‑塔格糖中的应用。所述L‑阿拉伯糖差向异构酶突变体，由所示氨基酸经定点突变而得，所述点突变位点为下列中的一个或多个：(1)第97位、(2)第189位、(3)第301位、(4)第409位和(5)第462位。本发明有益效果主要体现在：本发明提供了一种全新的差向异构酶及其突变体，以及其在制备塔格糖(D‑tagatose)中的应用。该突变体具有高最适反应温度80℃，解决了现有酶无法在高温生产D‑tagatose的技术难题。使用本突变体生产D‑tagatose，底物浓度为600g/L时，产物得率最高可达79.7％，优于原始酶和其他突变酶的转化效果，具有较好工业应用前景。 【EN】The invention relates to an L-arabinose epimerase mutant and application thereof in preparing D-tagatose by isomerizing D-galactose under the catalysis of microorganisms. The L-arabinose epimerase mutant is obtained by site-directed mutagenesis of the shown amino acid, and the site of: (1) 97 th bit, (2) 189 th bit, (3) 301 th bit, (4) 409 th bit, and (5) 462 th bit. The invention has the following beneficial effects: the invention provides a brand-new epimerase, a mutant thereof and application thereof in preparing tagatose (D-tagatose). The mutant has a high optimal reaction temperature of 80 ℃, and solves the technical problem that the existing enzyme can not produce D-tagatose at high temperature. When the mutant is used for producing D-tagatose, the highest product yield can reach 79.7 percent when the substrate concentration is 600g/L, and the mutant is superior to the conversion effect of original enzyme and other mutant enzymes, and has better industrial application prospect.

摘要：【中文】本发明涉及一种新型耐高温D‑阿洛酮糖3‑差向异构酶及突变体，及其在微生物催化D‑果糖异构化制备D‑阿洛酮糖(D‑psicose)中的应用。所属异构酶氨基酸序列如SEQ ID NO.3所示；所述异构酶突变体由SEQ ID NO.3所示氨基酸经定点突变而得，所述点突变位点为下列中的一个或多个：(1)第106位、(2)第183位、(3)第211位、(4)第276位。本发明有益效果主要体现在：本发明提供了一种全新的耐高温D‑阿洛酮糖3‑差向异构酶突变体，该突变体具有超高酶活和底物转化率，最适反应温度可达90℃。当底物浓度为700g/L，反应温度为85℃时，产物得率最高达67.14％。将重组酶进行固定化并连续催化300批次，转化率均大于67％。本发明解决了现有酶催化制备D‑psicose效率低下的难题，获得的优于文献报道的连续转化效果对提升D‑psicose工业化水平具有重要意义。 【EN】The invention relates to a novel high-temperature-resistant D-psicose3-epimerase, a mutant and application thereof in preparation of D-psicose (D-psicose) by catalyzing D-fructose isomerization by microorganisms. The amino acid sequence of the isomerase is shown as SEQ ID NO. 3; the isomerase mutant is obtained by site-directed mutagenesis of amino acid shown in SEQ ID NO.3, and the site of the site mutation is one or more of the following: (1) 106 th bit, (2) 183 th bit, (3) 211 th bit, and (4) 276 th bit. The invention has the following beneficial effects: the invention provides a brand-new high-temperature-resistant D-psicose3-epimerase mutant which has ultrahigh enzyme activity and substrate conversion rate and the optimal reaction temperature can reach 90 ℃. When the substrate concentration is 700g/L and the reaction temperature is 85 ℃, the product yield reaches 67.14 percent. The recombinant enzyme is immobilized and continuously catalyzed for 300 batches, and the conversion rate is higher than 67 percent. The method solves the problem of low efficiency of the existing enzyme catalysis preparation of D-psicose, and the obtained continuous conversion effect superior to that reported in the literature has important significance for improving the industrialization level of D-psicose.

摘要：【中文】本发明涉及一种具备D‑阿洛酮糖3‑差向异构酶功能的耐高温TIM barrel蛋白突变体，及其在催化D‑果糖异构化制备D‑阿洛酮糖中的应用。所述TIM Barrel蛋白突变体，由序列如SEQ ID NO.1所示氨基酸经定点突变而得，所述点突变位点为下列中的一个或多个：(1)第241位、(2)第182位、(3)第201位、(4)第105位、(5)第30位。本发明有益效果主要体现在：本发明提供了一种全新的TIM Barrel蛋白酶突变体，该突变体具有超高酶活、较高最适反应温度和底物转化率，解决了现有酶催化制备D‑psicose效率低下的技术难题。使用本突变体生产D‑psicose，产物得率最高可达53.68％，优于原始酶和其他突变酶的转化效果，具有较好工业应用前景。 【EN】The invention relates to a high-temperature-resistant TIM barrel protein mutant with a D-psicose3-epimerase function and application thereof in preparation of D-psicose by catalyzing isomerization of D-fructose. The TIM Barrel protein mutant is obtained by site-directed mutagenesis of amino acid with a sequence shown as SEQ ID NO.1, wherein the site of the site mutation is one or more of the following: (1) 241 th bit, (2) 182 th bit, (3) 201 th bit, (4) 105 th bit, and (5) 30 th bit. The invention has the following beneficial effects: the invention provides a brand-new TIM Barrel protease mutant which has ultrahigh enzyme activity, higher optimal reaction temperature and substrate conversion rate and solves the technical problem of low efficiency of D-psicose prepared by the existing enzyme catalysis. The mutant is used for producing D-psicose, the product yield can reach 53.68 percent at most, the conversion effect of the mutant is better than that of the original enzyme and other mutant enzymes, and the mutant has better industrial application prospect.

摘要：【中文】本发明涉及一种新型L‑阿拉伯糖异构酶(LAI)及其突变体，及其在连续催化D‑半乳糖异构化制备D塔格糖(D‑tagatose)的应用。所述异构酶突变体，由SEQ ID NO.1所示所示氨基酸经定点突变而得，所述点突变位点为下列中的一个或多个：(1)第107位、(2)第199位、(3)第59位。本发明有益效果主要体现在：本发明提供了一种全新的LAI及其突变体，85℃，700g/L D‑半乳糖反应体系下，产物得率最高达89.2％。将重组酶进行固定化并连续催化200批次，转化率均仍大于89％。本发明解决了现有酶催化制备D‑tagatose效率低下的难题，获得的优于文献报道的连续转化效果对提升D‑tagatose工业化水平具有重要意义。 【EN】The invention relates to a novel L-arabinose isomerase (LAI) and a mutant thereof, and application thereof in preparing D tagatose (D-tagatose) by continuously catalyzing D-galactose isomerization. The isomerase mutant is obtained by site-directed mutagenesis of amino acid shown in SEQ ID NO.1, and the site of the site-directed mutagenesis is one or more of the following: (1) 107 th bit, (2) 199 th bit, and (3) 59 th bit. The invention has the following beneficial effects: the invention provides a brand-new LAI and a mutant thereof, and the highest product yield can reach 89.2 percent under the reaction system of 85 ℃ and 700g/L D-galactose. The recombinase is immobilized and continuously catalyzed for 200 batches, and the conversion rate is still more than 89%. The method solves the problem of low efficiency of the existing enzyme-catalyzed preparation of D-tagatose, and the obtained continuous conversion effect superior to that reported in the literature has important significance for improving the industrialization level of D-tagatose.

摘要：【中文】本发明涉及一种L‑阿拉伯糖差向异构酶突变体，及其在微生物催化D‑半乳糖异构化制备D‑塔格糖中的应用。所述L‑阿拉伯糖差向异构酶突变体，由所示氨基酸经定点突变而得，所述点突变位点为下列中的一个或多个：(1)第304位、(2)第75位、(3)第274位、(4)第167位。本发明有益效果主要体现在：本发明提供了一种全新的L‑阿拉伯糖差向异构酶及其突变体，该突变体具有高最适反应温度75℃，其酶活与原始酶相比提高280％。使用本突变体生产D‑tagatose，产物得率最高可达73.3％。将重组酶进行固定化并连续催化30批次，转化率均大于73％。该突变体的转化水平展示了优良的工业应用前景。 【EN】The invention relates to an L-arabinose epimerase mutant and application thereof in preparing D-tagatose by isomerizing D-galactose under the catalysis of microorganisms. The L-arabinose epimerase mutant is obtained by site-directed mutagenesis of the shown amino acid, and the site of: (1) 304 th bit, (2) 75 th bit, (3) 274 th bit, and (4)167 th bit. The invention has the following beneficial effects: the invention provides a brand-new L-arabinose epimerase and a mutant thereof, wherein the mutant has the high optimal reaction temperature of 75 ℃, and the enzyme activity of the mutant is improved by 280 percent compared with the original enzyme. The mutant is used for producing D-tagatose, and the highest product yield can reach 73.3 percent. The recombinant enzyme is immobilized and continuously catalyzed for 30 batches, and the conversion rate is more than 73 percent. The transformation level of the mutant shows excellent industrial application prospect.