Molecular engineering of transketolase from Escherichia coli and tartaric semialdehyde biosynthesis / 生物工程学报

Transketolase (EC 2.2.1.1, TK) is a thiamine diphosphate-dependent enzyme that catalyzes the transfer of a two-carbon hydroxyacetyl unit with reversible C-C bond cleavage and formation. It is widely used in the production of chemicals, drug precursors, and asymmetric synthesis by cascade enzyme catalysis. In this paper, the activity of transketolase TKTA from Escherichia coli K12 on non-phosphorylated substrates was enhanced through site-directed saturation mutation and combined mutation. On this basis, the synthesis of tartaric semialdehyde was explored. The results showed that the optimal reaction temperature and pH of TKTA_M (R358I/H461S/R520Q) were 32 ℃ and 7.0, respectively. The specific activity on d-glyceraldehyde was (6.57±0.14) U/mg, which was 9.25 times higher than that of the wild type ((0.71±0.02) U/mg). Based on the characterization of TKTA_M, tartaric acid semialdehyde was synthesized with 50 mmol/L 5-keto-d-gluconate and 50 mmol/L non-phosphorylated ethanolaldehyde. The final yield of tartaric acid semialdehyde was 3.71 g with a molar conversion rate of 55.34%. Hence, the results may facilitate the preparation of l-(+)-tartaric acid from biomass, and provide an example for transketolase-catalyzed non-phosphorylated substrates.

Cloning,expression and immunity analysis of transketolase of Echinococcus granulosus / 中国血吸虫病防治杂志

Objective To obtain the prokaryotic expression of transketolase genes and analyze its value as a diagnostic anti-gen for echinococcosis.Methods TK gene was amplified by PCR and cloned into prokaryotic vector pMD19-EgTK,and then subcloned into the expression vector pET-28a.The target gene TK prokaryotic expression plasmid pET-28a was constructed and transferred into BL21. The purified protein was identified by SDS-PAGE and Western blotting. The blood samples of patients with cystic echinococcosis(CE group),alveolar echinococcosis(AE group)and healthy people(healthy group)were collected and detected by ELISA with the recombinant EgTK protein as a diagnostic antigen.Results The recombinant plasmid pET-28a (+)-EgTK was constructed successfully,and there was a band around 70 kDa by using Western blotting.ELISA showed that the difference among the 3 groups of sera reaction A450was significantly different(F=44.47,P<0.01),and the A450values of the CE group(1.46±0.41)and AE group(1.28±0.29)were higher than that of the healthy group(0.66 ± 0.23),but there was no significant difference between the former two.Conclusion The recombinant EgTK protein is better to distinguish the echinococ-cosis group and healthy group,but it can't do a differential diagnosis between CE and AE cases.

Implantação de um método para determinação da atividade da transcetolase eritrocitária para avaliação indireta da tiamina / Implementation of a method for determining the erythrocyte transketolase activity for indirect evaluation of thiamin

O teste de ativação da transcetolase eritrocitária (TK-E) pelo pirofosfato de tiamina (TPP) exógeno é um método indireto para mensurar a tiamina (vitamina B1). A diminuição da atividade da transcetolase eritrocitária e o aumento da estimulação in vitro com o TPP maior do que 17% indicam deficiência de tiamina. Este é um método plausível, pois são nos eritrócitos que estão concentradas a maior parte desta vitamina. Em virtude de surtos de beribéri que tem ocorrido no Brasil desde 2006, o Instituto Adolfo Lutz (IAL), como Laboratório Central de Saúde Pública, propôs a implantação desse método para auxiliar na investigação de novos surtos ou de casos isolados. Foram avaliados o teste de precisão, a linearidade, a estabilidade do hemolisado e da amostra, e estimados os limites de detecção e de quantificação. A atividade da TK-E sem ativação pelo TPP foi de 0,732 UI/gHb e com ativação foi de 0,827 UI/gHb. Todos os resultados dos parâmetros avaliados neste estudo apresentaram-se dentro dos critérios de aceitabilidade garantindo-se a confiabilidade do método. Fica, assim, disponível mais um ensaio bioquímico para a Rede Pública de Saúde, mas ainda necessário definir os valores de referência para estabelecer os limites clínicos da deficiência de tiamina.

Erythrocyte transketolase activation test (TK-E) by exogenous thiamine pyrophosphate (TPP)is an indirect method to measure thiamine (vitamin B1). The decrease in the erythrocyte transketolase activity and the increase of in vitro stimulation with TPP greater than 17 % indicate thiamine deficiency. It is a reasonable method as the major portion of this vitamin are concentrated in erithrocytes. Due to the beriberi outbreaks that have occurred in Brazil since 2006, the Adolfo Lutz Institute (IAL), as a Central Public Health Laboratory, proposed the implementation of this method to give support to the investigation on the new outbreaks or isolated cases. The evaluated parameters were precision, linearity, hemolysate and sample stability, and the limits of detection and quantification were estimated. The TK-E activity without activation by TPP was 0.732 UI/gHb, and with activation was 0.827 UI/gHb. All of the results obtained from the evaluated parameters showed to be within the eligibility criteria, ensuring the reliability of the proposed methods.Thus, this method showed to be adequate as biochemical assay for the Public Health Network. However, there is a need to define the reference values to establish the clinical limits of thiamine deficiency.

Implantação de um método para determinação da atividade da transcetolase eritrocitária para avaliação indireta da tiamina / Implementation of a method for determining the erythrocyte transketolase activity for indirect evaluation of thiamin

O teste de ativação da transcetolase eritrocitária (TK-E) pelo pirofosfato de tiamina (TPP) exógeno é um método indireto para mensurar a tiamina (vitamina B1). A diminuição da atividade da transcetolase eritrocitária e o aumento da estimulação in vitro com o TPP maior do que 17 % indicam deficiência de tiamina. Este é um método plausível, pois são nos eritrócitos que estão concentradas a maior parte desta vitamina. Em virtude de surtos de beribéri que tem ocorrido no Brasil desde 2006, o Instituto Adolfo Lutz (IAL), como Laboratório Central de Saúde Pública, propôs a implantação desse método para auxiliar na investigação de novos surtos ou de casos isolados. Foram avaliados o teste de precisão, a linearidade, a estabilidade do hemolisado e da amostra, e estimados os limites de detecção e de quantificação. A atividade da TK-E sem ativação pelo TPP foi de 0,732 UI/gHb e com ativação foi de 0,827 UI/gHb. Todos os resultados dos parâmetros avaliados neste estudo apresentaram-se dentro dos critérios de aceitabilidade garantindo-se a confiabilidade do método. Fica, assim, disponível mais um ensaio bioquímico para a Rede Pública de Saúde, mas ainda necessário definir os valores de referência para estabelecer os limites clínicos da deficiência de tiamina.

Erythrocyte transketolase activation test (TK-E) by exogenous thiamine pyrophosphate (TPP) is an indirect method to measure thiamine (vitamin B1). The decrease in the erythrocyte transketolase activity and the increase of in vitro stimulation with TPP greater than 17 % indicate thiamine deficiency. It is a reasonable method as the major portion of this vitamin are concentrated in erithrocytes. Due to the beriberi outbreaks that have occurred in Brazil since 2006, the Adolfo Lutz Institute (IAL), as a Central Public Health Laboratory, proposed the implementation of this method to give support to the investigation on the new outbreaks or isolated cases. The evaluated parameters were precision, linearity, hemolysate and sample stability, and the limits of detection and quantification were estimated. The TK-E activity without activation by TPP was 0.732 UI/gHb, and with activation was 0.827 UI/gHb. All of the results obtained from the evaluated parameters showed to be within the eligibility criteria, ensuring the reliability of the proposed methods. Thus, this method showed to be adequate as biochemical assay for the Public Health Network. However, there is a need to define the reference values to establish the clinical limits of thiamine deficiency.

Research progress of TKTL1 in malignant neoplasms / 肿瘤研究与临床

Up to now,one transketolase (TKT) and two transketolase-like genes (TKTL1 and TKTL2) have been identified in the human genome.People have conducted a series of basic and clinical researches of TKTL1 in malignant neoplasms since Coy found TKTL1 has a high expression of protein and gene levels in the malignant tumors in 2005.Although the biochemical and functional mechanism have not been clarified clearly,a positive role of TKTL1 in promoting tumorigenesis and tumor progression has been proved.

Function of the pentose phosphate pathway and its key enzyme, transketolase, in the regulation of the meiotic cell cycle in oocytes / 대한생식의학회지

OBJECTIVE: Previously, we identified that transketolase (Tkt), an important enzyme in the pentose phosphate pathway, is highly expressed at 2 hours of spontaneous maturation in oocytes. Therefore, this study was performed to determine the function of Tkt in meiotic cell cycle regulation, especially at the point of germinal vesicle breakdown (GVBD). METHODS: We evaluated the loss-of-function of Tkt by microinjecting Tkt double-stranded RNAs (dsRNAs) into germinal vesicle-stage oocytes, and the oocytes were cultured in vitro to evaluate phenotypic changes during oocyte maturation. In addition to maturation rates, meiotic spindle and chromosome rearrangements, and changes in expression of other enzymes in the pentose phosphate pathway were determined after Tkt RNA interference (RNAi). RESULTS: Despite the complete and specific knockdown of Tkt expression, GVBD occurred and meiosis was arrested at the metaphase I (MI) stage. The arrested oocytes exhibited spindle loss, chromosomal aggregation, and declined maturation promoting factor and mitogen-activated protein kinase activities. The modified expression of two enzymes in the pentose phosphate pathway, Prps1 and Rbks, after Tkt RNAi and decreased maturation rates were amended when ribose-5-phosphate was supplemented in the culture medium, suggesting that the Tkt and pentose phosphate pathway are important for the maturation process. CONCLUSION: We concluded that Tkt and its associated pentose phosphate pathway play an important role in the MI-MII transition of the oocytes' meiotic cell cycle, but not in the process of GVBD.

Exploring the Potential of Flunarizine for Cisplatin-Induced Painful Uremic Neuropathy in Rats / 대한배뇨장애요실금학회지

PURPOSE: The present study was designed to explore the potential of flunarizine for cisplatin induced painful uremic neuropathy in rats. METHODS: Cisplatin (2 mg/kg; i.p., for 5 consecutive days) was administered and renal uremic markers i.e., serum creatinine were estimated on days 4 and 25. Behavioral changes were assessed in terms of thermal hyperalgesia (hot plate, plantar, tail immersion, and tail flick tests at different time intervals). Biochemical analysis of total calcium, superoxide anion, DNA, and transketolase, and myeloperoxidase activity in tissue samples was also performed. Furthermore, flunarizine (100, 200, and 300 microM/kg; p.o., for 21 consecutive days) was administered to evaluate its potency on uremic neuropathy, and the results were compared with those for the carbamazepine-treated (30 mg/kg; p.o., for 21 consecutive days) groups. RESULTS: Flunarizine attenuated the cisplatin-induced uremic neuropathy, and the degree of behavioral and biochemical changes in serum and tissue samples in a dose dependent manner. The medium and high doses of flunarizine were shown to produce a significant effect on cisplatin induced painful uremic neuropathy. CONCLUSIONS: Our results indicate the potential of flunarizine for anti-oxidative, anti-inflammatory, and neuroprotective actions. Therefore, it may have use as a novel therapeutic agent for the management of painful uremic neuropathy.

Study on the Breeding of Histidine Producing Mutant and Its Properties / 微生物学通报

A L-histidine producing mutant was derived from Corynebacterium glutamicum HZ4221(TRA R DCP R AMT R histidase - )by means of mutagenesis with N-methy-N′-nitro- N-nitrosoguanidine(NTG).Contrast to original strain,the amount of histidine accumulation reached to a level of 5.31g/L in a medium containing 80g/Lglucose and 30g/L ammonium sulfate after cultured for 72 hours; the transketolase activity reduced to a degree of 15.7%.The utilization of the carbon sources,genetic stability,effect of metal ions were also been investigated in this paper.

Effect of transketolase-like protein 1 on proliferation of human nasopharyngeal carcinoma cells / 中国病理生理杂志

0.05). However,the expression level of TKTL1 gene was significantly downregulated in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector. Cancer cells were arrested in G0/G1 phase,and cancer cell proliferation was significantly inhibited in the CNE cells transfected with siRNA TKTL1 construct. CONCLUSION:TKTL1 plays an important role in the total transketolase activity and cell proliferation of human nasopharyngeal carcinoma. TKTL1 may be considered as a potential target for novel anti-cancer therapy.