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INTRODUCTION: Microbial degradation of cellulose has enormous economic potential for the conversion of plant biomass into fuel and chemicals. Cellulose is a linear polymer composed of D-glucose units linked by 1,4--D-glucosidic bonds. Its enzymatic hydrolysis requires the action of both endoglucanases (1,4--D-glucan glucanohydrolase [EC 3.2.1.4]) and exoglucanases (1,4--D-glucan cellobiohydrolase [EC 3.2.1.91]). A synergic interaction of these enzymes is necessary for the complete hydrolysis of crystalline cellulose. Thermophilic microrganisms have received considerable attention as source of highly active and thermostable cellulolytic enzymes; genes encoding endoglucanases are widely distributed among fungi and Bacteria; recently one was identified in the Archaeon Pyrococcus furiosus suggesting the occurrence of polysaccharides in hydrothermal vent environments (1). Our results report the identification of cellulasic activity in the Archaeon Sulfolobus solfataricus and molecular characterisation of celS, encoding a putative cellulase homologous to thermophilic endoglucanases. MATERIALS AND METHODS: S. solfataricus MT4 strain, kindly provided by Prof. Mario De Rosa, was grown at 82°C in a rotary shaker. A gene bank of S solfataricus MT4 strain was constructed as described previously (2). pGEM1.1 was the recombinant plasmid screened with adh gene as probe and containing celS . It was sequenced using primers constructed ad hoc. Analysis of the putative open reading frames (ORFs), was performed using Blast program. Total RNA was extracted in the late exponentially growth phase and primer extension was performed (3). Detectection of cellulasic activity was determined on CMC-cellulose plates and after SDS-PAGE (4). RESULTS: Cellulase activitiy was detected in the supernatant of S. solfataricus MT4 cultures and specific enzyme staining after SDS-PAGE revealed the presence of two proteins with apparent molecular mass of about 49 kDa and 40 kDa. At the same time we have cloned a DNA fragment from S. solfataricus MT4 containing an ORF (CelS) of 322 aminoacids (molecular weight 36703 Da) with significant homology to the P. furiosus (1), Thermotoga maritima (4) and T. neapolitana (5) endo-1,4--glucanases. The gene was demonstrated to be transcribed in vivo. In order to optimise the expression of celS we tried to grow MT4 on different -glucans, namely in minimal media containing Avicel, lichenan, carboxylmethylcellulose, but they were unable to support growth. Therefore enzymatic and transcriptional analysis were performed on cells cultured in more unspecific minimal and rich media. The results obtained suggested a catabolite repression by glucose and in general a down regulation by complex nutrients. The transcription start site was unambiguously identified by primer extension and revealed coincident with translational initiation. In order to demonstrate the relationship between structure and function of celS, the gene was fused with gst (glutathione-S- tranferase) in the expression vector pGEX-2tk and expressed in E.coli. The purification and the characterisation of the recombinant protein are underway.

Cellulose is one of the most abundant biopolymer on Earth and represents an important source of renewable energy. It is an unbranched polymer composed of D-glucose units linked by 1,4--D-glucosidic bonds. Enzymatic hydrolysis of cellulose requires a consortium of enzymes, including endo--glucanases, exoglucanases, and -glucosidases. Endoglucanases randomly hydrolyse internal glycosidic linkages and are widely distributed among fungi, Bacteria and Archaea. Cellulase activitiy was detected in the supernatant of S. solfataricus MT4 cultures and specific enzyme staining after SDS-PAGE revealed the presence of two proteins with apparent molecular mass of about 49 kDa and 40 kDa. At the same time we have cloned a DNA fragment from Sulfolobus solfataricus MT4 containing an ORF (CelS) of 322 aminoacids (molecular weight 36703 Da) with significant homology to the Pyrococcus furiosus (1), Thermotoga maritima (2) and T. neapolitana (3) endo-1,4--glucanases. The gene was demonstrated to be transcribed in vivo. In order to optimise the expression of celS we tried to grow MT4 on different -glucans, namely in minimal media containing Avicel, lichenan, carboxylmethylcellulose, cellobiose, but they resulted to be unable to support growth. Therefore enzymatic and transcriptional analysis were performed on cells cultured in more unspecific minimal and rich media. The results obtained suggested a catabolite repression by glucose and in general a down regulation by complex nutrients. The transcription start site was unambiguously identified by primer extension and revealed coincident with translational initiation

La cellulosa è uno dei polimeri più abbondanti sulla terra e rappresenta una importante fonte di energia rinnovabile. Il processo di degradazione della cellulosa è complesso e richiede l'intervento di endo--glucanasi, esoglucanasi e glucosidasi. Visto il crescente interesse per gli enzimi termofili abbiamo iniziato la caratterizzazione di una -endoglucanasi extracellulare da Sulfolobus solfataricus. Abbiamo identificato una sequenza di DNA dal ceppo G di S. solfataricus a monte della regione codificante per l'alcool deidrogenasi, studiata nel nostro laboratorio, che contiene una "Open reading Frame" di 322 aminoacidi ,CelSs. Tale ORF, che mostra un peso molecolare, dedotto dalla sequenza aminoacidica, di 36680 Da, presenta una omologia significativa con le 1-4--endoglucanasi di Thermotoga maritima e T. neapolitana. Mediante analisi trascrizionale è stato dimostrato che il gene è trascritto in vivo. L'attività cellulasica è stata identificata nel ceppo G di S. solfataricus; l'analisi su SDS-PAGE-CMC ha mostrato la presenza di due proteine con attività endoglucanasica di cui una del peso molecolare di circa 36 kDa simile a quello dedotto da CelSs. Attualmente è in corso l'espressione e la caratterizzazione della proteina ricombinante prodotta in Escherichia coli.

Cellulose, is an unbranched glucose polymer composed of D-glucose units linked by 1,4--D-glucosidic bonds. It is one of the most abundant biopolymers on Earth and represents an important renewable energy source. Genes encoding endoglucanases are widely distributed among fungi and Bacteria; recently one was identified in the Archaeon Pyrococcus furiosus suggesting the occurrence of polysaccharides in hydrothermal vent environments (1). We have identified a DNA fragment from Sulfolobus solfataricus Gstrain containing an ORF encoding a putative endoglucanase (CelSs) of 322 aminoacids (about 36 kDa) with significant homology to the Thermotoga maritima and T. neapolitana endo-1,4--glucanase. The gene was demonstrated to be transcribed in vivo and the transcription start site was identified by primer extension. The cellulase activity was detected in the supernatant of S. solfataricus G cultures and specific enzyme staining after SDS-PAGE revealed the presence of two proteins with apparent molecular mass of about 43 kDa and 36 kDa. The insertion into suitable vectors for the high level expression of the celSs open reading frame in E.coli and the characterisation of the recombinant protein are underway.

A protein disulfide oxidoreductase from the archaeon Aeropyrum pernix K1 has been overexpressed in Escherichia coli and crystallized at 298 K using the hanging-drop vapour-diffusion method. Crystals belong to the space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 90.59, b = 102.43, c = 128.96 A. A complete data set has been collected at the Elettra synchrotron source in Trieste to 1.93 A resolution using a single frozen crystal.