Expression and Characterization of a Cold-Adapted Alginate Lyase with Exo/Endo-Type Activity from a Novel Marine Bacterium Alteromonas portus HB161718T.

The alginate lyases have unique advantages in the preparation of alginate oligosaccharides and processing of brown algae. Herein, a gene alg2951 encoding a PL7 family alginate lyase with exo/endo-type activity was cloned from a novel marine bacterium Alteromonas portus HB161718T and then expressed in Escherichia coli. The recombinant Alg2951 in the culture supernatant reached the activity of 63.6 U/mL, with a molecular weight of approximate 60 kDa. Alg2951 exhibited the maximum activity at 25 °C and pH 8.0, was relatively stable at temperatures lower than 30 °C, and showed a special preference to poly-guluronic acid (polyG) as well. Both NaCl and KCl had the most promotion effect on the enzyme activity of Alg2951 at 0.2 M, increasing by 21.6 and 19.1 times, respectively. The TCL (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) analyses suggested that Alg2951 could catalyze the hydrolysis of sodium alginate to produce monosaccharides and trisaccharides. Furthermore, the enzymatic hydrolysates displayed good antioxidant activity by assays of the scavenging abilities towards radicals (hydroxyl and ABTS+) and the reducing power. Due to its cold-adapted and dual exo/endo-type properties, Alg2951 can be a potential enzymatic tool for industrial production.

Overexpression and biochemical characterization of a truncated endo-α (1 → 3)-fucoidanase from alteromonas sp. SN-1009.

Endo-fucoidanases are important in structural analysis of fucoidans and preparation of fuco-oligosaccharides. However their enzymological properties and analysis of degradation products are scarcely investigated. Truncated endo-α (1 â†’ 3)-fucoidanase Fda1 (tFda1B from Alteromonas sp. was overexpressed and characterized, showing highest activity at pH 7.0, 35 °C, and 1.0 M NaCl. Its Km and kcat were 3.88 ± 0.81 mg/mL and 0.82 ± 0.17 min-1. Fe3+ and Mn2+ enhanced activity by 100% and 19.5% respectively. Co2+ and Cu2+ completely inactivated tFda1B, whereas Ni2+, Mg2+, Zn2+, Pb2+, Ca2+, Ba2+ and Li+ decreased activity by 58.8%, 56.0%, 50.6%, 47.7%, 28.9%, 15.6% and 37.5%, respectively. Catalytic residues were identified through structure and sequence alignment, and confirmed by mutagenesis. Degradation products of Kjellmaniella crassifolia fucoidan by tFda1B were characterized by LC-ESI-MS/MS, confirming tFda1B belongs to endo-(1 â†’ 3)-fucoidanases, and backbone of K. crassifolia fucoidan is 1 â†’ 3 fucoside linkage. This endo-α (1 â†’ 3)-fucoidanase would be useful for elucidating fucoidan structures, and be used as a food enzyme.

Alteromonas portus sp. nov., an alginate lyase-excreting marine bacterium.

An alginate lyase-excreting bacterium, designated strain HB161718T, was isolated from coastal sand collected from Tanmen Port in Hainan, PR China. Cells were Gram-stain-negative rods and motile with a single polar flagellum. Its major isoprenoid quinone was ubiquinone 8 (Q-8), and its cellular fatty acid profile mainly consisted of C16 : 1 ω7c and/or C16 : 1 ω6c, C18 : 1 ω6c and/or C18 : 1 ω7c, C16 : 0, C17 : 0 10-methyl and C16 : 0 N alcohol. The G+C content of the genomic DNA was 44.1 mol%. 16S rRNA gene sequence analysis suggested that strain HB161718T belonged to the genus Alteromonas, sharing 99.5, 99.4, 99.2, 98.9 and 98.5 % sequence similarities to its closest relatives, Alteromonas macleodii JCM 20772T, Alteromonas gracilis 9a2T, Alteromonas australica H17T, Alteromonas marina SW-47T and Alteromonas mediterranea DET, respectively. The low values of DNA-DNA hybridization and average nucleotide identity showed that it formed a distinct genomic species. The combined phenotypic and molecular features supported the conclusion that strain HB161718T represents a novel species of the genus Alteromonas, for which the name Alteromonas portus sp. nov. is proposed. The type strain is HB161718T (=CGMCC 1.13585T=JCM 32687T).

Characterization of Novel Salt-Tolerant Esterase Isolated from the Marine Bacterium Alteromonas sp. 39-G1.

An esterase gene, estA1, was cloned from Alteromonas sp. 39-G1 isolated from the Beaufort Sea. The gene is composed of 1,140 nucleotides and codes for a 41,190 Da protein containing 379 amino acids. As a result of a BLAST search, the protein sequence of esterase EstA1 was found to be identical to Alteromonas sp. esterase (GenBank: PHS53692). As far as we know, no research on this enzyme has yet been conducted. Phylogenetic analysis showed that esterase EstA1 was a member of the bacterial lipolytic enzyme family IV (hormone sensitive lipases). Two deletion mutants (Δ20 and Δ54) of the esterase EstA1 were produced in Escherichia coli BL21 (DE3) cells with part of the N-terminal of the protein removed and His-tag attached to the C-terminal. These enzymes exhibited the highest activity toward p-nitrophenyl (pNP) acetate (C2) and had little or no activity towards pNP-esters with acyl chains longer than C6. Their optimum temperature and pH of the catalytic activity were 45°C and pH 8.0, respectively. As the NaCl concentration increased, their enzyme activities continued to increase and the highest enzyme activities were measured in 5 M NaCl. These enzymes were found to be stable for up to 8 h in the concentration of 3-5 M NaCl. Moreover, they have been found to be stable for various metal ions, detergents and organic solvents. These salt-tolerant and chemical-resistant properties suggest that the enzyme esterase EstA1 is both academically and industrially useful.

Biochemical characterization and structural analysis of ulvan lyase from marine Alteromonas sp. reveals the basis for its salt tolerance.

Marine macroalgae have gained considerable attention as renewable biomass sources. Ulvan is a water-soluble anionic polysaccharide, and its depolymerization into fermentable monosaccharides has great potential for the production of bioethanol or high-value food additives. Ulvan lyase from Alteromonas sp. (AsPL) utilizes a ß-elimination mechanism to cleave the glycosidic bond between rhamnose 3-sulfate and glucuronic acid, forming an unsaturated uronic acid at the non-reducing end. AsPL was active in the temperature range of 30-50 °C and pH values ranging from 7.5 to 9.5. Furthermore, AsPL was found to be halophilic, showing high activity and stability in the presence of up to 2.5 M NaCl. The apparent Km and kcat values of AsPL are 3.19 ±â€¯0.37 mg mL-1 and 4.19 ±â€¯0.21 s-1, respectively. Crystal structure analysis revealed that AsPL adopts a ß-propeller fold with four anti-parallel ß-strands in each of the seven propeller blades. The acid residues at the protein surface and two Ca2+ coordination sites contribute to its salt tolerance. The research on ulvan lyase has potential commercial value in the utilization of algal resources for biofuel production to relieve the environmental burden of petrochemicals.

Characterization of membrane vesicles secreted by seaweed associated bacterium Alteromonas macleodii KS62.

Recent studies have reported abundant presence of bacterial extracellular membrane vesicles in the marine environment. However, the ecological significance of these bacterial vesicles in the marine environment is only beginning to be explored. In present study, for the first time we report and characterize membrane vesicles secreted by a seaweed associated bacterium, Alteromonas macleodii KS62. Proteomics studies revealed that the vesicle proteome was rich in hydrolytic enzymes (30%) like glycoside hydrolases, proteases, sulphatases, lipases, nucleases and phosphatases. Zymography experiments and enzyme assays established that the vesicles carry active κ-carrageenan degrading enzymes. κ-carrageenan is a major polysaccharide of cell walls of certain red seaweeds like Kappaphycus. Purified membrane vesicles were successfully able to degrade Kappaphycus biomass. Based on these results, we discuss how the hydrolase-rich vesicles may play a role in red seaweed cell wall degradation so that the bacteria can invade and colonise the seaweed biomass establishing a saprophytic lifestyle. We also discuss the role of these vesicles in nutrient acquisition and their ecological significance in the marine environment.

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 ß-glucosidase from the marine bacterium Alteromonas sp. L82.

A novel gene (bgl) encoding a cold-adapted ß-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni2+ affinity chromatography. The purified recombinant ß-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant ß-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant ß-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a Km of 0.55 mM, a Vmax of 83.6 U/mg, a kcat of 74.3 s-1 and kcat/Km of 135.1 at 40°C, pH 7 with 4-nitrophenyl-ß-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.

DNA binding with a minimal scaffold: structure-function analysis of Lig E DNA ligases.

DNA ligases join breaks in the phosphodiester backbone of DNA by catalysing the formation of bonds between opposing 5'P and 3'OH ends in an adenylation-dependent manner. Catalysis is accompanied by reorientation of two core domains to provide access to the active site for cofactor utilization and enable substrate binding and product release. The general paradigm is that DNA ligases engage their DNA substrate through complete encirclement of the duplex, completed by inter-domain kissing contacts via loops or additional domains. The recent structure of a minimal Lig E-type DNA ligase, however, implies it must use a different mechanism, as it lacks any domains or loops appending the catalytic core which could complete encirclement. In the present study, we have used a structure-guided mutagenesis approach to investigate the role of conserved regions in the Lig E proteins with respect to DNA binding. We report the structure of a Lig-E type DNA ligase bound to the nicked DNA-adenylate reaction intermediate, confirming that complete encirclement is unnecessary for substrate engagement. Biochemical and biophysical measurements of point mutants to residues implicated in binding highlight the importance of basic residues in the OB domain, and inter-domain contacts to the linker.

Efficient production of trans-4-hydroxy-l-proline from glucose using a new trans-proline 4-hydroxylase in Escherichia coli.

trans-4-Hydroxy-l-proline (trans-4Hyp) is widely used as a valuable building block for the organic synthesis of many pharmaceuticals such as carbapenem antibiotics. The major limitation for industrial bioproduction of trans-4Hyp is the low titer and productivity by using the existing trans-proline 4-hydroxylases (trans-P4Hs). Herein, three new trans-P4Hs from Alteromonas mediterranea (AlP4H), Micromonospora sp. CNB394 (MiP4H) and Sorangium cellulosum (ScP4H) were discovered through genome mining and enzymatic determination. These trans-P4Hs were introduced into an l-proline-producing chassis cell, and the recombinant strain overexpressing AlP4H produced the highest concentration of trans-4Hyp (3.57 g/L) from glucose in a shake flask. In a fed-batch fermentation with a 5 L bioreactor, the best strain SEcH (pTc-B74A-alp4h) accumulated 45.83 g/L of trans-4Hyp within 36 h, with the highest productivity (1.27 g/L/h) in trans-4Hyp fermentation from glucose, to the best of our knowledge. This study provides a promising hydroxylase candidate for efficient industrial production of trans-4Hyp.

Characterization of an Alteromonas long-type ulvan lyase involved in the degradation of ulvan extracted from Ulva ohnoi.

Ulvan is a sulfated polysaccharide found in the cell wall of the green algae Ulva. We first isolated several ulvan-utilizing Alteromonas sp. from the feces of small marine animals. The strain with the highest ulvan-degrading activity, KUL17, was analyzed further. We identified a 55-kDa ulvan-degrading protein secreted by this strain and cloned the gene encoding for it. The deduced amino acid sequence indicated that the enzyme belongs to polysaccharide lyase family 24 and thus the protein was named ulvan lyase. The predicted molecular mass of this enzyme is 110 kDa, which is different from that of the identified protein. By deletion analysis, the catalytic domain was proven to be located on the N-terminal half of the protein. KUL17 contains two ulvan lyases, one long and one short, but the secreted and cleaved long ulvan lyase was demonstrated to be the major enzyme for ulvan degradation.

Purification and characterization of polysaccharides degradases produced by Alteromonas sp. A321.

Two different degradases from Alteromonas sp. A321 for polysaccharides from Enteromorpha prolifera (DPE-L and DPE-P) were purified to homogeneity. The molecular weights of purified DPE-L and DPE-P were 75.2 and 102.5 kDa, respectively, and their internal sequences were analysed by LC-MS-MS. The enzymes exhibited an optimum temperature of 30-40 °C (DPE-L) and 35-45 °C (DPE-P), an optimum pH of 7.0 (DPE-L) and 6.0 (DPE-P). DPE-P was highly stable in the presence of EDTA and 1,10-phenanthroline while DPE-L was inhibited by 1,10-phenanthroline. The Km values of DPE-L and DPE-P were 2.93 mg/ml and 0.31 mg/ml and the Vmax values were 6.11 µmol/min/ml and 2.88 µmol/min/ml, respectively. Results of HPLC and ESI-MS analyses showed that enzymatic products were: Rha1(SO3H)1, Rha1(SO3H)1Gluc1, Rha2(SO3H)2Gluc1, and Rha3(SO3H)3Gluc1Xyl1 by DPE-L, and Glu2, Glu3, plus Glu4 by DPE-P, respectively. Thus DPE-L and DPE-P can be used to produce oligosaccharides which potentially revealed more of structure of polysaccharides from E. prolifera.

Engineering the Organophosphorus Acid Anhydrolase Enzyme for Increased Catalytic Efficiency and Broadened Stereospecificity on Russian VX.

The enzyme organophosphorus acid anhydrolase (OPAA), from Alteromonas sp. JD6.5, has been shown to rapidly catalyze the hydrolysis of a number of toxic organophosphorus compounds, including several G-type chemical nerve agents. The enzyme was cloned into Escherichia coli and can be produced up to approximately 50% of cellular protein. There have been no previous reports of OPAA activity on VR {Russian VX, O-isobutyl S-[2-(diethylamino)ethyl] methylphosphonothioate}, and our studies reported here show that wild-type OPAA has poor catalytic efficacy toward VR. However, via application of a structurally aided protein engineering approach, significant improvements in catalytic efficiency were realized via optimization of the small pocket within the OPAA's substrate-binding site. This optimization involved alterations at only three amino acid sites resulting in a 30-fold increase in catalytic efficiency toward racemic VR, with a strong stereospecificity toward the P(+) enantiomer. X-ray structures of this mutant as well as one of its predecessors provide potential structural rationales for their effect on the OPAA active site. Additionally, a fourth mutation at a site near the small pocket was found to relax the stereospecificity of the OPAA enzyme. Thus, it allows the altered enzyme to effectively process both VR enantiomers and should be a useful genetic background in which to seek further improvements in OPAA VR activity.

Distinct Metal Isoforms Underlie Promiscuous Activity Profiles of Metalloenzymes.

Within a superfamily, functionally diverged metalloenzymes often favor different metals as cofactors for catalysis. One hypothesis is that incorporation of alternative metals expands the catalytic repertoire of metalloenzymes and provides evolutionary springboards toward new catalytic functions. However, there is little experimental evidence that incorporation of alternative metals changes the activity profile of metalloenzymes. Here, we systematically investigate how metals alter the activity profiles of five functionally diverged enzymes of the metallo-ß-lactamase (MBL) superfamily. Each enzyme was reconstituted in vitro with six different metals, Cd(2+), Co(2+), Fe(2+), Mn(2+), Ni(2+), and Zn(2+), and assayed against eight catalytically distinct hydrolytic reactions (representing native functions of MBL enzymes). We reveal that each enzyme metal isoform has a significantly different activity level for native and promiscuous reactions. Moreover, metal preferences for native versus promiscuous activities are not correlated and, in some cases, are mutually exclusive; only particular metal isoforms disclose cryptic promiscuous activities but often at the expense of the native activity. For example, the L1 B3 ß-lactamase displays a 1000-fold catalytic preference for Zn(2+) over Ni(2+) for its native activity but exhibits promiscuous thioester, phosphodiester, phosphotriester, and lactonase activity only with Ni(2+). Furthermore, we find that the five MBL enzymes exist as an ensemble of various metal isoforms in vivo, and this heterogeneity results in an expanded activity profile compared to a single metal isoform. Our study suggests that promiscuous activities of metalloenzymes can stem from an ensemble of metal isoforms in the cell, which could facilitate the functional divergence of metalloenzymes.

Isolation and identification of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from the red-brown pigment-producing bacterium Alteromonas stellipolaris LMG 21856.

We describe the isolation and identification of a gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD (EC 1.13.11.27)) from the red-brown pigment-producing bacterium Alteromonas stellipolaris LMG 21856. HPPD directs the synthesis of 2,5-dihydroxyphenylacetic acid (homogentisic acid (HGA)). The sequence of the deduced peptide showed homology to HPPDs from other strains of Alteromonas; the greatest similarity was to the hypothetical HPPD from Alteromonas sp. SN2. As observed for HPPDs from other sources, expression of the A. stellipolaris HPPD gene in Escherichia coli cells could be detected by the gradual development of a brown pigment in cultures as a result of the spontaneous oxidation and polymerization of HGA.

Designed surface residue substitutions in [NiFe] hydrogenase that improve electron transfer characteristics.

Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii "Deep Ecotype" [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons necessary for hydrogen production. Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons. Thus, we engineered a hydrogenase-ferredoxin fusion that also contained several other modifications. In addition to the C-terminal ferredoxin fusion, we truncated the C-terminus of the hydrogenase small subunit, identified as the available terminus closer to the electron transfer region. We also neutralized an anionic patch surrounding the interface Fe-S cluster to improve transfer kinetics with the negatively charged ferredoxin. Initial screening showed the enzyme tolerated both truncation and charge neutralization on the small subunit ferredoxin-binding face. While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor. Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme.

Modeling three-dimensional structure of two closely related Ni-Fe hydrogenases.

The results of homology modeling of HydSL, a NiFe-hydrogenase from purple sulfur bacterium Thiocapsa roseopersicina BBS, and deep-water bacterium Alteromonas macleodii deep ecotype are presented in this work. It is shown that the models have larger confidence level than earlier published ones; full-size models of these enzymes are presented for the first time. The C-end fragment of small subunit of T. roseopersicina hydrogenase is shown to have random orientation in relation to the main protein globule. The obtained models of this enzyme have a large number of ion pairs, as well as thermostable HydSL hydrogenase from Allochromatium vinosum, in contrast to thermostable HydSL hydrogenase from Alt. macleodii and thermolabile HydAB hydrogenase from Desulfovibrio vulgaris. The possible determinant of oxygen stability of studied hydrogenases could be the lack of several intramolecular tunnels. Hydrophobic and electrostatic surfaces were mapped in order to find out possible pathways of coupling hydrogenase to electron-transferring chains, as well as methods for construction of artificial photobiohydrogen-producing systems.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase.

BACKGROUND: In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii "deep ecotype" as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported "G1" (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. RESULTS: Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the "G1" enzyme. CONCLUSIONS: We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii "deep ecotype", to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme.

Cloning, expression, and biochemical characterization of a novel GH16 ß-agarase AgaG1 from Alteromonas sp. GNUM-1.

Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-ß-D-galactopyranoside but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K m and V max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and (13)C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type ß-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.

Gene cloning and characterization of an α-amylase from Alteromonas macleodii B7 for Enteromorpha polysaccharide degradation.

Enteromorpha polysaccharides (EP) extracted from green algae have displayed a wide variety of biological activities. However, their high molecular weight leads to a high viscosity and low solubility, and therefore, greatly restrains their application. To solve this problem, bacteria from the surface of Enteromorpha were screened, and an Alteromonas macleodii strain B7 was found to be able to decrease the molecular weight of EP in culture media. Proteins harvested from the supernatant of the A. macleodii B7 culture were subjected to native gel electrophoresis, and a band corresponding to the Enteromorpha polysaccharide lyase (EPL) was detected by activity staining. The enzyme identity was subsequently confirmed by MALDI-TOF/TOF mass spectrometry as the putative α-amylase reported in A. macleodii ATCC 27126. The amylase gene (amySTU) from A. macleodii B7 was cloned into Escherichia coli, resulting in highlevel expression of the recombinant enzyme with EP-degrading activity. AmySTU was found to be cold-adapted; however, its optimal enzyme activity was detected at 40°C. The α-amylase was highly stable over a broad pH range (5.5-10) with the optimal pH at 7.5-8.0. The highest enzyme activity was detected when NaCl concentration was 2%, which dropped by 50% when the NaCl concentration was increased to 16%, showing an excellent nature of halotolerance. Furthermore, the amylase activity was not significantly affected by tested surfactants or the presence of some organic solvents. Therefore, the A. macleodii strain B7 and its α-amylase can be useful in lowering EP molecular weight and in starch processing.

Production of enzymes by Alteromonas sp. A321 to degrade polysaccharides from Enteromorpha prolifera.

Polysaccharides from Enteromorpha prolifera (PE) are becoming increasingly popular due to its bioactivity and abundant source. Screening novel microorganisms which could secrete enzymes to degrade PE efficiently for oligosaccharides production is a promising solution to improve its application. In this study, a marine bacterium that can produce enzymes to degrade PE specifically was selected. It was identified as Alteromonas sp. A321, based on the biochemical properties and 16S rDNA gene sequencing. In order to maximize the activity of degradase for polysaccharides from E. prolifera (DPE), the effects of medium composition and culture conditions were investigated. The highest DPE production was obtained in the medium consisting of K2HPO4 0.15%, PE 0.9%, NaNO3 0.4%, NaCl 1.0% and MgSO4 0.05%. The degradase activity was enhanced from original 0.391 U/ml to 0.744 U/ml. DPE show high efficiency and substrate specificity to PE with 63.53% of reducing sugar production in the 7 h hydrolysis.