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Food Chem ; 305: 125447, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499289


A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2 U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9 kDa via SDS-PAGE. RmAmyA was optimally active at 75 °C and pH 6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16 h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry.

Int J Biol Macromol ; 127: 683-692, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30703426


A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.

Proteínas Fúngicas , Maltose/química , Pichia , Amido/química , alfa-Amilases , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Trissacarídeos/química , alfa-Amilases/biossíntese , alfa-Amilases/química , alfa-Amilases/genética
Biochem Biophys Res Commun ; 493(4): 1510-1517, 2017 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-28986258


Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignancy with an estimated 5 year survival rate of approximately 5% of all stages combined. High potential of PDAC metastasis is a leading cause for high mortality and poor prognosis. The majority of patients present with distant metastasis at diagnosis. Fractalkine (FKN) is recognized as a chemokine and a specific ligand of CX3CR1. It has been reported that FKN/CX3CR1 system was upregulated in many types of solid tumors. However, role of FKN/CX3CR1 in PDAC development remains unclear. In the current investigation, we found that FKN and CX3CR1 expression was significantly increased in PDAC tissues, especially in the metastatic samples, and was highly-correlated with severity of PDAC. Ectopic expression of FKN promoted the proliferation and migration of PDAC, while knockdown of CX3CR1 reversed the function of FKN. In addition, PDAC cells with FKN-deficiency showed impaired proliferation and migration activity. The underlying mechanism is that FKN/CX3CR1 activated JAK/STAT signaling, which in turn regulated cell growth. Consistently, in vivo tumorigenesis assay validated the regulatory role of FKN/CX3CR1 in PDAC growth. Our investigation helped understanding the pathogenesis of PDAC occurrence, and demonstrated critical role of FKN/CX3CR1 in PDAC development.

Carcinoma Ductal Pancreático/metabolismo , Quimiocina CX3CL1/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Quimiocina CX3CL1/antagonistas & inibidores , Quimiocina CX3CL1/genética , Técnicas de Silenciamento de Genes , Humanos , Janus Quinases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/genética , Interferência de RNA , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais