Theβ-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A.nidulans A773.The resulting purifiedβ-glucosidase,named AnGH3,is a monomeric enzyme with a molecular weight of approximat...Theβ-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A.nidulans A773.The resulting purifiedβ-glucosidase,named AnGH3,is a monomeric enzyme with a molecular weight of approximately 80 kDa,as confirmed by SDS-PAGE.Circular dichroism further validated its unique canonical barrel fold(β/α),a feature also observed in the 3D homology model of AnGH3.The most striking aspect of this recombinant enzyme is its robustness,as it retained 100%activity after 24 h of incubation at 45 and 50ºC and pH 6.0.Even at 55°C,it maintained 72%of its enzymatic activity after 6 h of incubation at the same pH.The kinetic parameters Vmax,KM,and Kcat/KM forρ-nitrophenyl-β-D-glucopyranoside(ρNPG)and cellobiose were also determined.UsingρNPG,the enzyme demonstrated a Vmax of 212 U mg−1,KM of 0.0607 mmol L−1,and Kcat/KM of 4521 mmol L−1 s−1 when incubated at pH 6.0 and 65°C.The KM,Vmax,and Kcat/KM using cellobiose were 2.7 mmol L−1,57 U mg−1,and 27 mmol-1 s−1,respectively.AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L−1 and 25%,respectively.Even in challenging conditions,at 65°C and pH 6.0,the enzyme maintained its activity,retaining 100%and 70%of its initial activity in the presence of 200 mmol L−1 furfural and 5-hydroxymethylfurfural(HMF),respectively.The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum,where it led to a 48%increase in glucose release after 24 h.These unique characteristics,including high catalytic performance,good thermal stability in hydrolysis temperature,and tolerance to elevated concentrations of ethanol,D-xylose,furfural,and HMF,position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail,thereby opening new avenues in the field of biotechnology and enzymology.展开更多
To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein express...To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.展开更多
基金gratefully acknowledge the São Paulo Research Foundation(FAPESP)(Grant no:2018/07522-6,2021/06679-1,2022/00539-6,and 2023/01547-5FCT(POCI-01-0145-FEDER-032206)-transnational cooperation project EcoTech,Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq)(310340/2021-7)+6 种基金National Institute of Science and Technology of Bioethanol(INCT)(CNPq 465319/2014-9/FAPESP no:2014/50884-5)for financial supportResearch scholarships were granted to RCA by CNPq(Grant no:151187/2023-1)by FAPESP(Grant no:2023/09627-8)to DA by FAPESP(Grant no:2020/15510-8 and 2023/01338-7)to JCSS by CNPq(Grant no:384465/2023-4)FAPESP(Grant no:2019/21989-7)GSA by CAPES(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior,Finance Code 001).
文摘Theβ-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A.nidulans A773.The resulting purifiedβ-glucosidase,named AnGH3,is a monomeric enzyme with a molecular weight of approximately 80 kDa,as confirmed by SDS-PAGE.Circular dichroism further validated its unique canonical barrel fold(β/α),a feature also observed in the 3D homology model of AnGH3.The most striking aspect of this recombinant enzyme is its robustness,as it retained 100%activity after 24 h of incubation at 45 and 50ºC and pH 6.0.Even at 55°C,it maintained 72%of its enzymatic activity after 6 h of incubation at the same pH.The kinetic parameters Vmax,KM,and Kcat/KM forρ-nitrophenyl-β-D-glucopyranoside(ρNPG)and cellobiose were also determined.UsingρNPG,the enzyme demonstrated a Vmax of 212 U mg−1,KM of 0.0607 mmol L−1,and Kcat/KM of 4521 mmol L−1 s−1 when incubated at pH 6.0 and 65°C.The KM,Vmax,and Kcat/KM using cellobiose were 2.7 mmol L−1,57 U mg−1,and 27 mmol-1 s−1,respectively.AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L−1 and 25%,respectively.Even in challenging conditions,at 65°C and pH 6.0,the enzyme maintained its activity,retaining 100%and 70%of its initial activity in the presence of 200 mmol L−1 furfural and 5-hydroxymethylfurfural(HMF),respectively.The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum,where it led to a 48%increase in glucose release after 24 h.These unique characteristics,including high catalytic performance,good thermal stability in hydrolysis temperature,and tolerance to elevated concentrations of ethanol,D-xylose,furfural,and HMF,position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail,thereby opening new avenues in the field of biotechnology and enzymology.
文摘To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.
