γ-Glutamyltranspeptidase (γ-GT) from human esophageal cancer, normal esophageal mucosa and normal kindney were partially purified and their biochemical and immunological properties were studied. (1) The γ-GT activi...γ-Glutamyltranspeptidase (γ-GT) from human esophageal cancer, normal esophageal mucosa and normal kindney were partially purified and their biochemical and immunological properties were studied. (1) The γ-GT activity of esophageal cancer was higher than that of normal mucosa, yet still much lower than that of normal kidney. (2) The Michaelis constant and optimum pH of esophageal cancer γ-GT were the same as those of normal kidney tissue. (3) After staining of γ-GT activity, two molecular clusters, i.e. 100kD and 380kD γ-GT were shown in esophageal cancer by 4-20% linear gradient PAGE and the lOOkD γ-GT showed higher activity. (4) The γ-GT of esophageal cancer was immunologically identical with normal enzyme in double immunodif-fusion and immunoelectrophoresis. (5) In double lectin-diffusion, affinity column and crossed immuno-affino-electrophoresis, it was demonstrated that the carbohydrate components of γ-GT from esophageal cancer were heterogeneous and rich in D-Mannose, D-Glucose and N-Acetyl-Glucosamine. The relationship between the appearance of 100kD γ-GT cluster and the heterogeneous carbohydrate components will be further studied.展开更多
<span style="font-family:Verdana;">L-Theanine (</span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glut...<span style="font-family:Verdana;">L-Theanine (</span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glutamylethylamide) is a naturally occurring amino acid derivative known to have several beneficial physiological effects as a diet supplement, and to give an umami taste when used as a food additive. The compound is industrially produced by </span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glutamyltranspeptidase from </span><i><span style="font-family:Verdana;">Pseudomonas nitroreducens</span></i><span style="font-family:Verdana;"> (PnGGT). Using recombinant PnGGT, we have shown previously that Trp385, Phe417, and Trp525 are key amino acid residues for recognition of acceptor substrates at the PnGGT active site. Here, we demonstrate that a recombinant W525D mutant of PnGGT produces L-theanine from ethylamine and L-glutamine more efficiently than wild-type PnGGT, attributable to an increased ratio of transfer activity to hydrolysis activity. An efficient production of L-theanine was achieved by immobilizing </span><i><span style="font-family:Verdana;">Escherichia coli</span></i><span style="font-family:Verdana;"> cells expressing the W525D PnGGT mutant (</span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D) using 2% alginate as the supporting material. The highest L-theanine production using immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D, representing a conversion rate of 90%, w</span><span style="font-family:Verdana;">as</span><span style="font-family:Verdana;"> achieved in optimal reaction conditions of pH 10, 40<span style="white-space:nowrap;">°</span></span><span style="font-family:;" "=""><span><span style="font-family:Verdana;">C, and a substrate molar ratio of L-glutamine to ethylamine of 1:10. The immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D retains 85% and 78% relative activity after storage for a month at 4<span style="white-space:nowrap;">°</span></span></span><span><span style="font-family:Verdana;">C and room temperature, respectively. Immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D thus has strong potential for use in the future commercial production of L-theanine on a large scale.</span></span></span>展开更多
针对γ-谷氨酰转肽酶（GGT）p H耐受性差的缺陷,首先以硅烷化改性的介孔氧化钛晶须（s MTw）为载体对重组枯草芽胞杆菌GGT进行固定化,再以p H 8.0-10.5的载体两性电解质Pharmalyte（CA,p H 8.0-10.5）对其进行后修饰,得到固定化酶s MTw-...针对γ-谷氨酰转肽酶（GGT）p H耐受性差的缺陷,首先以硅烷化改性的介孔氧化钛晶须（s MTw）为载体对重组枯草芽胞杆菌GGT进行固定化,再以p H 8.0-10.5的载体两性电解质Pharmalyte（CA,p H 8.0-10.5）对其进行后修饰,得到固定化酶s MTw-GGT-CA。结果发现：s MTw-GGT-CA的p H耐受性较游离酶明显提高,可在p H 6.0-11.0范围内保持稳定的催化活性。同时,s MTw-GGT-CA的热稳定性也较游离酶有所提高,其最适作用温度为50℃左右,热失活反应活化能Ed为49.88 k J/mol。s MTw-GGT-CA对γ-谷氨酰对硝基苯胺（Gp NA）的亲和力常数Km为0.579 mmol/L,与游离酶相近。展开更多
文摘γ-Glutamyltranspeptidase (γ-GT) from human esophageal cancer, normal esophageal mucosa and normal kindney were partially purified and their biochemical and immunological properties were studied. (1) The γ-GT activity of esophageal cancer was higher than that of normal mucosa, yet still much lower than that of normal kidney. (2) The Michaelis constant and optimum pH of esophageal cancer γ-GT were the same as those of normal kidney tissue. (3) After staining of γ-GT activity, two molecular clusters, i.e. 100kD and 380kD γ-GT were shown in esophageal cancer by 4-20% linear gradient PAGE and the lOOkD γ-GT showed higher activity. (4) The γ-GT of esophageal cancer was immunologically identical with normal enzyme in double immunodif-fusion and immunoelectrophoresis. (5) In double lectin-diffusion, affinity column and crossed immuno-affino-electrophoresis, it was demonstrated that the carbohydrate components of γ-GT from esophageal cancer were heterogeneous and rich in D-Mannose, D-Glucose and N-Acetyl-Glucosamine. The relationship between the appearance of 100kD γ-GT cluster and the heterogeneous carbohydrate components will be further studied.
文摘<span style="font-family:Verdana;">L-Theanine (</span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glutamylethylamide) is a naturally occurring amino acid derivative known to have several beneficial physiological effects as a diet supplement, and to give an umami taste when used as a food additive. The compound is industrially produced by </span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glutamyltranspeptidase from </span><i><span style="font-family:Verdana;">Pseudomonas nitroreducens</span></i><span style="font-family:Verdana;"> (PnGGT). Using recombinant PnGGT, we have shown previously that Trp385, Phe417, and Trp525 are key amino acid residues for recognition of acceptor substrates at the PnGGT active site. Here, we demonstrate that a recombinant W525D mutant of PnGGT produces L-theanine from ethylamine and L-glutamine more efficiently than wild-type PnGGT, attributable to an increased ratio of transfer activity to hydrolysis activity. An efficient production of L-theanine was achieved by immobilizing </span><i><span style="font-family:Verdana;">Escherichia coli</span></i><span style="font-family:Verdana;"> cells expressing the W525D PnGGT mutant (</span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D) using 2% alginate as the supporting material. The highest L-theanine production using immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D, representing a conversion rate of 90%, w</span><span style="font-family:Verdana;">as</span><span style="font-family:Verdana;"> achieved in optimal reaction conditions of pH 10, 40<span style="white-space:nowrap;">°</span></span><span style="font-family:;" "=""><span><span style="font-family:Verdana;">C, and a substrate molar ratio of L-glutamine to ethylamine of 1:10. The immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D retains 85% and 78% relative activity after storage for a month at 4<span style="white-space:nowrap;">°</span></span></span><span><span style="font-family:Verdana;">C and room temperature, respectively. Immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D thus has strong potential for use in the future commercial production of L-theanine on a large scale.</span></span></span>
文摘针对γ-谷氨酰转肽酶（GGT）p H耐受性差的缺陷,首先以硅烷化改性的介孔氧化钛晶须（s MTw）为载体对重组枯草芽胞杆菌GGT进行固定化,再以p H 8.0-10.5的载体两性电解质Pharmalyte（CA,p H 8.0-10.5）对其进行后修饰,得到固定化酶s MTw-GGT-CA。结果发现：s MTw-GGT-CA的p H耐受性较游离酶明显提高,可在p H 6.0-11.0范围内保持稳定的催化活性。同时,s MTw-GGT-CA的热稳定性也较游离酶有所提高,其最适作用温度为50℃左右,热失活反应活化能Ed为49.88 k J/mol。s MTw-GGT-CA对γ-谷氨酰对硝基苯胺（Gp NA）的亲和力常数Km为0.579 mmol/L,与游离酶相近。
