Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

نویسندگان

  • G Dong
  • C Vieille
  • J G Zeikus
چکیده

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extrac...

متن کامل

Production of Recombinant Proline Dehydrogenase Enzyme from Pseudomonas fluorescens pf-5 in E. coli System

Proline dehydrogenase (ProDH; 1.5.99.8) belongs to superfamily of amino acid dehydrogenase, which plays a significant role in the metabolic pathway from proline to glutamate. The goal of this research was gene cloning and characterization of ProDH enzyme from Pseudomonas fluorescens pf-5 strain. The gene encoding ProDH was isolated by means of PCR amplification and cloned in an IPTG inducible T...

متن کامل

Bacterial Expression and Functional Characterization of A Naturally Occurring Exon6-less Preprochymosin cDNA

Chymosin (Rennin EC 3.4.23.4), an aspartyl proteinase, is the major proteolytic enzyme in the fourthstomach of the unweaned calf, and it is formed by proteolytic activation of its zymogene, prochymosin.Following the cloning of synthesized cDNAs on mRNA pools extracted from the mucosa of the calf fourthstomach, we have identified an alternatively spliced form of preprochymosin ...

متن کامل

Cloning of EprA1 gene of Aeromonas hydrophila in Lactococcus lactis

Bacterial-based systems as live vectors for the delivery of heterologous antigens offer a number of advantages as vaccination strategies. Developments in genetic engineering have given Gram-positive lacticacid bacteria (LAB) the advantage of being used as a host expression system for antigen delivery to inducethe immune response. A fragment containing the full length of the “eprA1” ...

متن کامل

The tRNA(guanine-26,N2-N2) methyltransferase (Trm1) from the hyperthermophilic archaeon Pyrococcus furiosus: cloning, sequencing of the gene and its expression in Escherichia coli.

The structural gene pfTRM1 (GenBank accession no. AF051912), encoding tRNA(guanine-26, N 2- N 2) methyltransferase (EC 2.1.1.32) of the strictly anaerobic hyperthermophilic archaeon Pyrococcus furiosus, has been identified by sequence similarity to the TRM1 gene of Saccharomyces cerevisiae (YDR120c). The pfTRM1 gene in a 3.0 kb restriction DNA fragment of P.furiosus genomic DNA has been cloned ...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • Applied and environmental microbiology

دوره 63 9  شماره 

صفحات  -

تاریخ انتشار 1997