Influence of polymer architecture and molecular weight of poly(2-(dimethylamino)ethyl methacrylate) polycations on transfection efficiency and cell viability in gene delivery

Nonviral gene delivery with the help of polycations has raised considerable interest in the scientific community over the past decades. Herein, we present a systematic study on the influence of the molecular weight and architecture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) on the transfection efficiency and the cytotoxicity in CHO-K1 cells. A library of well-defined homopolymers with a linear and star-shaped topology (3- and 5-arm stars) was synthesized via atom transfer radical polymerization (ATRP). The molecular weights of the polycations ranged from 16 to 158 kDa. We found that the cytotoxicity at a given molecular weight decreased with increasing number of arms. For a successful transfection a minimum molecular weight was necessary, since the polymers with a number-average molecular weight, M(n), below 20 kDa showed negligible transfection efficiency at any of the tested polyelectrolyte complex compositions. From the combined analysis of cytotoxicity and transfection data, we propose that polymers with a branched architecture and an intermediate molecular weight are the most promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable transfection results.     

Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro.

This paper describes the effects of whole seminal plasma and of dialysed seminal plasma on the fertilizing ability of fowl spermatozoa stored for 24 h at 4 degrees C. The fertilizing ability of fowl semen diluted 1:1 with Beltsville Poultry Semen Extender and stored for 24 h at 4 degrees C was enhanced after replacement of the homologous seminal plasma by the diluent (89 versus 77% fertilization rate). Better results were obtained with seminal plasma dialysed against water before sperm storage to discard the less than 1 kDa or the less than 50 kDa fractions. It was concluded that low molecular weight seminal plasma fractions could damage the fertilizing ability of spermatozoa during storage at 4 degrees C, whereas high molecular weight fractions appeared to enhance fertilizing ability.     

Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants

Two proteases (P1 and P2) and a chitinase (C1) were purified from the culture supernatant of Serratia ureilytica TKU013 with squid pen as the sole carbon/nitrogen source. The molecular masses of P1, P2 and C1 determined by SDS-PAGE were approximately 50 kDa, 50 kDa and 60 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of P1, P2 and C1 were (pH 10, 40 degrees C, pH 7-11, and <50 degrees C), (pH 10, 40 degrees C, pH 8-11, and <40 degrees C) and (pH 6, 50 degrees C, pH 5-8, and <50 degrees C), respectively. P1 and P2 were inhibited by Mg(2+), EDTA and C1 was inhibited completely by Cu(2+). The antioxidant activity of TKU013 culture supernatant was 72% per mL (DPPH scavenging ability). With this method, we have shown that squid pen wastes can be utilized and have revealed its hidden potential in the production of functional foods.     

Haemophilus paragallinarum secretes metalloproteases

Haemophilus paragallinarum secretes metalloproteases into different culture media lacking serum. Secreted proteins, concentrated by precipitation with 70% ammonium sulphate ((NH(4))(2)SO(4)) or methanol, displayed proteolytic activity at >100 kDa molecular mass in 10% polyacrylamide gels co-polymerized with porcine gelatin (0.1%). They were active in a broad pH range (4-9); pH 7.5 being the optimum. Protease activity was inhibited by 20 mmol EDTA/L and reactivated by calcium. The proteolytic activity was heat-stable at 40, 50, and 60 degrees C, but its activity diminished at 70 degrees C or higher. Secreted proteins partially degraded chicken immunoglobulin G (IgG) and cross-reacted with a polyclonal serum against a high molecular mass protease secreted by Actinobacillus pleuropneumoniae. Extracellular proteases could play a role in infectious coryza caused by H. paragallinarum.     

The cellular regulation of vesicle exocytosis by Entamoeba histolytica

We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed 125iodine (125I) labeled tyrosine conjugated dextran; 125I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of 125I-dextran released after 120 min at 37 degrees C, 31 degrees C, 25 degrees C, and 4 degrees C, respectively (P less than 0.01 for each). Exocytosis at 37 degrees C was inhibited by cytochalasin D (10 micrograms/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 microM) (P less than 0.01 for each at greater than or equal to 60 min). Calcium ionophore A23187 (1 microM) enhanced exocytosis at 5 and 15 min (P less than 0.01). Elevation of vesicle pH with NH4Cl (10 mM) had no effect on release of 125I-dextran; phorbol myristate acetate (10(-6) M) increased exocytosis by 46% at 30 min (P less than 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased 125I-dextran release into the cell supernatant after 30 and 60 min at 37 degrees C (by 40% and 42%, respectively, P less than 0.01); release of 125I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters.     

Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen.

1. The neutral collagenase released into the culture medium by explants of ehrumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G-200, Sephadex QAE A-50 and Sephadex G-100 superfine. 2. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibrils of 312 mug collagen degraded min-1 mg enzyme protein-1, representing more than a 1000-fold increase over that of the active culture medium. 3. Electrophoresis in polyacrylamide disc-gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase. 4. The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33000. 5. Data obtained from sutdies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. 6. It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-D-Arg and non-specific protease activity was absent. 7. The collagenase attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). 8. At 37 degrees C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. 9. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta-articular tissues although rates of attack were less than with reconstituted fibrils. 10. The data suggests that pure rheumatoid synovial collagenase at 37 degrees C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non-specific proteases. 11. The different susceptibilities of various collagenous substrates to collagenase attack are discussed.     

Two endoxylanases active and stable at alkaline pH from the newly isolated thermophilic fungus, Myceliophthora sp. IMI 387099

Two extra-cellular endoxylanases (Xyl Ia and Ib) were purified to homogeneity from the newly isolated thermophilic fungus, Myceliophthora sp. IMI 387099. Xyl Ia and Ib, having a molecular mass of approximately 53 kDa and pI of 5.2 and 4.8, respectively, were optimally active at 75 degrees C and at pH 6.0. They were stable at pH 9.2 at 60 degrees C for 2 h, but less stable at pH 6.0 and above 50 degrees C. Mg+2, Zn+2, Ca+2, Co+2 and DTT increased their activity by 1.5-3.0-folds, while SDS and NBS completely inhibited their activity. Both xylanases were active on pNPX and pNPC, but their activity on pNPC was three times higher than that on pNPX. Xyl Ia was more active than Xyl Ib on pNP-alpha-L-Arap, while the latter preferred pNP-alpha-L-Araf. Both xylanases showed two to four times higher activity on rye and wheat arabinoxylans than on birchwood xylan, but Xyl Ib was more active than Xyl Ia on oat spelt xylan. Wheat insoluble pentosan was a good substrate for Xyl Ia, while Xyl Ib preferred wheat soluble arabinoxylan. Xyl Ia had lower Km and higher kcat/Km ratios than Xyl Ib towards all three xylans tested. Both xylanases degraded X4-X6 in an endo-fashion and catalysed hydrolysis and trans-xylosylation reactions. HPLC and LC/MS analysis showed that Xyl Ia and Ib released the unsubstituted X2-X6 as well as mono and di-methyl glucuronic acid substituted X3 and X2 from arabinoxylans.     

Acetal linked oligoethylenimines for use as pH-sensitive gene carriers

Two types of acid-degradable nonviral gene carriers, OEI-MK and OEI-BAA, were synthesized by polymerizing oligoethylenimine of 800 Da (OEI800) with the pH-sensitive acetone ketal cross-linker 2,2-bis(N-maleimidoethyloxy) propane (MK) or the 4-methoxybenzaldehyde bisacrylate acetal cross-linker 1,1-bis-(2-acryloyloxy ethoxy)-[4-methoxy-phenyl]methane) (BAA). Corresponding acid-insensitive counterparts (OEI-BM and LT-OEI-HD) were synthesized as well, representing control polymers. Kinetics of hydrolysis were measured and confirmed the pH-dependent degradation profile of the acetal functions, with short half-lives of 3 min at pH 5.0, and 5 h (OEI-MK) or 3.5 h (OEI-BAA) at physiological pH 7.4 and 37 degrees C. DNA polyplexes of a luciferase expression plasmid were tested for gene transfer efficiency and biocompatibility in two cell lines (B16F10 and Neuro2A). Polyplexes with acid-labile polymers showed an improved toxicity profile compared to those made with acid-stable polymer analogues. At low cation/plasmid (c/p) w/w ratios the transfection efficiency of pH-sensitive polymers was slightly reduced, but it became similar or superior to the efficiency of acid-stable polymers at higher c/p ratios. An improved in vivo biocompatibility of the acid-degradable polymers over the stable control polymers was confirmed by liver histology after systemic administration of polymers in Balb/c mice.     

Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37 degrees C, 33 degrees C and 25 degrees C respectively, although high activities were recorded over the temperature range 20-45 degrees C. The pH range of high activity was between 6.0 and 9.0, with an optimum for each DNase at 8.0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg2+ and Mn2+); however, other metal ions (Fe2+, Ni2+, Zn2+) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).     

Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris

A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C.     

Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization

Hybrid antibacterial peptide CA-MA (cecropinA(1-8)-magainin2(1-12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA-MA in methylotrophic yeast, Pichia pastoris, the gene of CA-MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZalpha-A. The SalI-linearized plasmid pPICZalpha-CA-MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96h with 1.0% methanol at 28 degrees C, pH 5.0. Recombinant CA-MA was purified by reversed-phase HPLC and 22 mg pure active CA-MA was obtained from 1L fermentation culture. Tricine-SDS-PAGE indicated that recombinant CA-MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA-MA demonstrated a single large signal for the molecular ion [M+2H+](2+) at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA-MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA-MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.     

Degradation of raw feather by a novel high molecular weight extracellular protease from newly isolated Bacillus cereus DCUW

Biotreatment of feather wastes and utilization of the degraded products in feed and foodstuffs has been a challenge. In the present study, we have demonstrated the degradation of feather waste by Bacillus cereus DCUW strain isolated during a functional screening based microbial diversity study on East Calcutta Wetland Area. A high molecular weight keratinolytic protease from feather degrading DCUW strain was purified and characterized. Moreover, utilization of degraded products during feather hydrolysis was developed and demonstrated. The purified keratinolytic protease was found to show pH and temperature optima of 8.5 and 50 degrees C, respectively. PMSF was found to inhibit the enzyme completely. The purified enzyme showed molecular weight of 80 kDa (from SDS-PAGE). The protease was found to have broad range substrate specificities that include keratin, casein, collagen, fibrin, BAPNA and gelatin. The protease was identified as minor extracellular protease (Vpr) by RT-PCR and northern blotting techniques. This is the first report describing the characterization of minor extracellular protease (Vpr) and its involvement in feather degradation in B. cereus group of organisms.     

Purification and functional characterization of a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667

The gene encoding a novel alpha-L-arabinofuranosidase from Bifidobacterium longum B667, abfB, was cloned and sequenced. The deduced protein had a molecular mass of about 61 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with alpha-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. Regions flanking the gene comprised two divergently transcribed open reading frames coding for hypothetical proteins involved in sugar metabolism. A histidine tag was introduced at the C terminus of AbfB, and the recombinant protein was overexpressed in Lactococcus lactis under control of the tightly regulated, nisin-inducible nisA promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 260 kDa, suggesting a homotetrameric structure. AbfB was active at a broad pH range (pH 4.5 to 7.5) and at a broad temperature range (20 to 70 degrees C), and it had an optimum pH of 6.0 and an optimum temperature of 45 degrees C. The enzyme seemed to be less thermostable than most previously described arabinofuranosidases and had a half-life of about 3 h at 55 degrees C. Chelating and reducing agents did not have any effect on its activity, but the presence of Cu(2+), Hg(2+), and Zn(2+) markedly reduced enzymatic activity. The protein exhibited a high level of activity with p-nitrophenyl alpha-L-arabinofuranoside, with apparent K(m) and V(max) values of 0.295 mM and 417 U/mg, respectively. AbfB released L-arabinose from arabinan, arabinoxylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. No endoarabinanase activity was detected. These findings suggest that AbfB is an exo-acting enzyme and may play a role, together with other glycosidases, in the degradation of L-arabinose-containing polysaccharides.     

Enzymatic properties of a neutral endo-1,3(4)-beta-xylanase Xyl II from Bacillus subtilis

A Bacillus sp. CCMI 966, characterised as Bacillus subtilis, has a duplication time of about 24 min. It produces at least two extracellular xylanases, Xyl I and Xyl II. The extracellular xylanase activity seems to be strongly correlated with the biomass growth profile. The Xyl II isoenzyme was purified by ammonium sulphate precipitation and anionic exchange chromatography, with a purification factor of 8.3. The molecular weight of the isoenzyme was estimated by SDS-PAGE revealing that Xyl II is a multimeric enzyme with a catalytic subunit of about 20 kDa. Under non-denaturing conditions, a molecular weight of about 340 kDa was obtained by native PAGE gel and of 20 kDa by gel filtration chromatography. The enzyme showed an optimum pH and temperature of 6.0 at 60 degrees C. Xyl II was stable at 40 degrees C for 180 min at pH 6.0. The specificity of Xyl II for different substrates was evaluated. Xyl II presents a higher affinity towards OSX, with a K(m) of 1.56 g l(-1) and showed the ability to hydrolyse laminarin, with a K(m) of 1.02 g l(-1). Xylotetraose is the main product of xylan degradation. The Xyl II ability for binding to cellulose and/or xylan was also studied.     

高温α-淀粉酶基因突变体在大肠杆菌、毕赤酵母中的表达

对地衣芽孢杆菌(Bacillus licheniformis)高温α-淀粉酶(amyE)基因进行改造获得的基因突变体(amyEM),通过PCR扩增,将此基因分别克隆至大肠杆菌表达载体pBV220和毕赤酵母表达载体pPIC9K上,并分别转化大肠杆菌DH5α和毕赤酵母GS115感受态细胞,获得重组大肠杆菌和重组毕赤酵母。通过表达产物的酶活性检测和SDS-PAGE分析,证明突变α-淀粉酶(AmyEM)在大肠杆菌、毕赤酵母中获得有效表达。对重组大肠杆菌产生的α-淀粉酶的粗酶性质分析表明,此酶分子量约为55kDa。其最适反应温度为80℃~90℃,与野生型基因相比,其最适pH均为6.0,但不同的是突变体在pH 5.0~5.5时表现出较高的酶活力;在毕赤酵母细胞的表达产物可分泌至胞外。由于酵母可对蛋白进行糖基化,酶分子量增加到60kDa,最适pH也改变为5.5。此高温α-淀粉酶突变体所具有的在微酸性环境具有较高酶活力的性质,具有重要的潜在工业应用价值。     

Novel thermoresponsive nonviral gene vector: P(NIPAAm-co-NDAPM)-b-PEI with adjustable gene transfection efficiency

A thermoresponsive cationic copolymer, poly( N-isopropylacrylamide- co- N-(3-(dimethylamino)propyl)methacrylamide)- b-polyethyleneimine (P(NIPAAm- co-NDAPM)- b-PEI), was designed and synthesized as a potential nonviral gene vector. The lower critical solution temperature (LCST) of P(NIPAAm- co-NDAPM)- b-PEI in water measured by UV-vis spectroscopy was 38 degrees C. P(NIPAAm- co-NDAPM)- b-PEI as the gene vector was evaluated in terms of cytotoxicity, buffer capability determined by acid-base titration, DNA binding capability characterized by agarose gel electrophoresis and particle size analysis, and in vitro gene transfection. P(NIPAAm- co-NDAPM)- b-PEI copolymer exhibited lower cytotoxicity in comparison with 25 kDa PEI. Gel retardation assay study indicated that the copolymer was able to bind DNA completely at N/P ratios higher than 30. At 27 degrees C, the mean particle sizes of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes decreased from 1200 to 570 nm corresponding to the increase in N/P ratios from 10 to 60. When the temperature changed to 37 degrees C, the mean particle sizes of complexes decreased from 850 to 450 nm correspondingly within the same N/P ratio range due to the collapse of thermoresponsive PNIPAAm segments. It was found that the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes was higher than or comparable to that of 25 kDa PEI/DNA complexes at their optimal N/P ratios. Importantly, the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes could be adjusted by altering the transfection and cell culture temperature.     

Cloning, purification and biochemical characterization of metallic-ions independent and thermoactive l-arabinose isomerase from the Bacillus stearothermophilus US100 strain

The araA gene encoding L-arabinose isomerase from Bacillus stearothermophilus US100 strain was cloned, sequenced and over-expressed in E. coli. This gene encodes a 496-amino acid protein with a calculated molecular weight of 56.161 kDa. Its amino acid sequence displays the highest identity with L-AI from Thermus sp. IM6501 (98%) and that of Geobacillus stearothermophilus T6 (97%). According to SDS-PAGE analysis, under reducing and non-reducing conditions, the recombinant enzyme has an apparent molecular weight of nearly 225 kDa, composed of four identical 56-kDa subunits. The L-AI US100 was optimally active at pH 7.5 and 80 degrees C. It was distinguishable by its behavior towards divalent ions. Indeed, the L-AI US100 activity and thermostability were totally independent for metallic ions until 65 degrees C. At temperatures above 65 degrees C, the enzyme was also independent for metallic ions for its activity but its thermostability was obviously improved in presence of only 0.2 mM Co2+ and 1 mM Mn2+. The V(max) values were calculated to be 41.3 U/mg for L-arabinose and 8.9 U/mg for D-galactose. Their catalytic efficiencies (k(cat)/K(m)) for l-arabinose and D-galactose were, respectively, 71.4 and 8.46 mM(-1) min(-1). L-AI US100 converted the d-galactose into D-tagatose with a high conversion rate of 48% after 7 h at 70 degrees C.     

Low molecular weight chitosans: preparation with the aid of papain and characterization

Low molecular weight chitosans (LMWC) of different molecular weight (4.1-5.6 kDa) were obtained by the depolymerization of chitosan using papain (from Carica papaya latex, EC. 3.4.22.2) at optimum conditions of pH 3.5 and 37 degrees C for 1-5 h. Scanning electron microscopy (SEM) showed approximately 15-fold decrease in the particle size after depolymerization. Decrease in the molecular weight was associated with decrease in the degree of acetylation (DA) as evidenced by circular dichroism (CD), FT-IR and solid-state CP-MAS 13C-NMR data. X-ray diffraction pattern revealed slight decrease in the crystallinity index (CrI) whereas the 13C-NMR data showed molecular inhomogeneity. LMWC showed lytic effect towards Bacillus cereus and Escherichia coli more efficiently than native chitosan. The growth inhibitory effect was maximal towards B. cereus, with minimum inhibitory concentration (MIC) of 0.01% (w/v).     

Molecular identification of oligoalginate lyase of Sphingomonas sp. strain A1 as one of the enzymes required for complete depolymerization of alginate

A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37 degrees C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS, algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.