α-Amylases from Bacillus licheniformis (BLA) and Bacillus amyloliquefaciens (BAA) are both important industrial enzymes with high similarity in structure but significant differences in thermostability. The mechanisms underlying this discrepancy are still poorly understood. Here, we investigated the role of two amino acids’ insertion on the thermostability of these two group amylases. A newly obtained thermophilic amylase AMY121 was found much closer to BLA in both primary structure and enzymological properties. Two amino acids’ insertion widespread among BAA group α-amylases was identified as one of the key factors leading to the thermostability differences, since thermostability of insertion mutants (AMY121-EG and AMY121-AA) from AMY121 significantly decreased, while that of deletion mutant from BAA increased. Moreover, we proposed that conformational disturbance caused by insertion mutation might weaken the calcium-binding affinity and consequently decrease the enzyme thermostability.

High value utilization of byproduct of slaughterhouses is regarded as beneficial to both economy and environment. A hemoglobin degrading protease PA720 was purified from fermentation broth of a Porites lutea associated strain Streptomyces rutgersensis SCSIO 11720 (SCSIO 11720) and exhibited maximal activity at 70°C, pH 10.5. The isolated strain was identified by genotypic method and 16 s RNA gene sequence was deposited in GenBank under accession No. KC414842. The enzyme exhibited excellent thermostability since it was stable at 70°C with t1/2 value of 1,386.3 min. Kinetic and thermodynamic study showed the activation energy (Ea), ΔG* (free energy), ΔH* (enthalpy) and ΔS* (entropy) for protease deactivation were 62.71 kJ/mol, 74.26 kJ/mol, 59.86 kJ/mol, and −41.97 J/mol/K, respectively. Highest specific activity of protease PA720 was observed when using casein (9,953 U/mg) and hemoglobin (9,854 U/mg) as substrates. Hemoglobin hydrolysate prepared by protease PA720 showed significant antibacterial activity towards Escherichia coli, Staphylococcus aureus and Bacillus subtilis, indicating this protease could be used as an instrumental enzyme for production of hemoglobin-based antibacterial peptides. Based on partial amino acid sequences of the enzyme PA720, the full gene encoding this protease was obtained by degenerate primer PCR and has been deposited in GenBank under accession No. KC414842. The deduced amino acid sequence exhibits homology with other microbial thermophilic proteases in subtilisin family.

A novel endoglucanase gene, mgcel44, was isolated from a mangrove soil metagenomic library by functional-based screening. It encodes a 648-aa peptide with a catalytic domain of glycosyl hydrolase family 44. The deduced amino acid sequence of mgcel44 shares less than 50 % identity with endoglucanases in GenBank database. mgcel44 was cloned and overexpressed in Escherichia coli. The recombinant enzyme, MgCel44, has a molecular mass of 70.8 kDa as determined by SDS-PAGE. Its optimal pH and temperature for activity were 6 and 45 °C, respectively. It was highly active at 25–45 °C and pH 5–8. Its activity was enhanced in 0.5 M NaCl by >1.6-fold and stable up to 1.5 M NaCl. MgCel44 was resistant to several organic solvents and had high activity at 15 % (v/v) solvent after incubating for 24 h at 25 °C.

The diversity and properties of actinobacteria, predominant residents in coral holobionts, have been rarely documented. In this study, we aimed to explore the species diversity, antimicrobial activities and biosynthetic potential of culturable actinomycetes within the tissues of the scleractinian corals Porites lutea, Galaxea fascicularis and Acropora millepora from the South China Sea. A total of 70 strains representing 13 families and 15 genera of actinobacteria were isolated. The antimicrobial activity and biosynthetic potential of fifteen representative filamentous actinomycetes were estimated. Crude fermentation extracts of 6 strains exhibited comparable or greater activities against Vibrio alginolyticus than ciprofloxacin. Seven of the 15 actinomycetes strains possess type I polyketide synthases (PKS-I) and/or nonribosomal peptide synthetases (NRPS) genes. Nine tested strains possess type II polyketide synthases (PKS-II). Phylogenetic analysis based on 16S rRNA gene sequences indicated that these PKS and NRPS gene screening positive strains belong to genera Nocardiopsis, Pseudonocardia, Streptomyces, Micromonospora, Amycolatopsis and Prauserella. One PKS-I and four NRPS fragments showed <70 % similarity to their closest relatives, which suggested the novelty of these genes. This study helps uncover the genetic capacity of stony coral-associated actinomycetes to produce bioactive molecules.

A Gram-positive bacterium, designated SCSIO 15042T, was isolated from a sediment of the South China Sea and was subjected to a polyphasic taxonomic study. The isolate grew at 20–60 °C, pH 6.0–10.0 and it could grow with up to 10 % (w/v) NaCl. The cell-wall diamino acid was found to be meso-diaminopimelic acid. Polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine and an unknown polar lipid. The only menaquinone was determined to be MK-7. The major fatty acids were identified as C16:1ω7c/C16:1ω6c, C16:0, iso-C15:0, anteiso-C15:0, and iso-C16:0. The DNA G+C content of strain SCSIO 15042T was determined to be 43.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain SCSIO 15042T to the genus Bacillus. Levels of 16S rRNA gene sequence similarities between strain SCSIO 15042T and Bacillus herbersteinensis D-1-5aT, Bacillus infantis SMC 4352-1T, Bacillus novalis LMG 21837T and Bacillus drentensis LMG 21831T were 96.2, 96.2, 96.1 and 96.1 %, respectively. Based on the evidence of the present polyphasic study, strain SCSIO 15042T is considered to represent a novel species of the genus Bacillus, for which the name Bacillus abyssalis sp. nov. is proposed. The type strain is SCSIO 15042T (=DSM 25875T = CCTCC AB 2012074T = NBRC 109102T).

The gene BglNH encoding a β-glucosidase was cloned from a marine streptomycete. Sequence analysis revealed that BglNH encoded a 456-aa peptide with a calculated mass of 51 kDa. The deduced amino acid sequence of BglNH showed the highest identities of 61 % with known β-glucosidases and contained a catalytic domain which belonged to the glycoside hydrolase family 1. The gene BglNH was expressed in Escherichia coli and the recombinant enzyme (r-BglNH) was purified. The optimum pH and temperature of r-BglNH were pH 6.0 and 45 °C, respectively. The r-BglNH displayed the typical salt-tolerant and glucose-enhanced characteristics. Its activity was remarkably enhanced in the presence of 0.5 M NaCl (rose more than 1.6-fold) and 0.1 M glucose (rose more than 1.4-fold). Moreover, r-BglNH displayed good pH stability and metal tolerance. It remained stable after incubating with buffers from pH 4.0 to 10.0, and most metal ions had no significant inhibition on its activity. These properties indicate that r-BglNH is an ideal candidate for further research and industrial applications.

Two new indole diketopiperazines, namely luteoalbusins A–B (1–2), along with eight known ones (3–10), were isolated from the fungus Acrostalagmus luteoalbus SCSIO F457 originated from deep-sea sediment. Their structures were determined by 1D/2D NMR, MS, and CD data analyses. Each of these compounds was evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 cell lines, and compounds 1–5 showed significant cytotoxicties against all four cancer cell lines. Moreover, new compounds 1 and 2 had more potent cytotoxicity than the other ones and cisplatin.

A novel actinobacterium, designated strain SCSIO 10480T, was isolated from a gorgonian coral sample of Anthogorgia sp. Phylogenetic and phenotypic properties of the organism supported that it belonged to the genus Janibacter. Phylogenetic analysis indicated that the levels of 16S rRNA gene sequence similarity between strain SCSIO 10480T and other type strains of recognized members of the genus Janibacter were 96.0–97.8 %. Growth in the presence of up to 17 % (w/v) NaCl and optimally at pH 9.0–10.0 was a distinctive characteristic of strain SCSIO 10480T. Other biochemical and physiological properties and the fatty acid profile also differentiated the isolate from other members of Janibacter species. Based on the results obtained in this study, we propose that strain SCSIO 10480T should be classified within a novel species of the genus Janibacter, for which the name Janibacter alkaliphilus sp. nov. is proposed, with SCSIO 10480T (=CCTCC AB 2011027T = DSM 24723T) as the type strain.

A novel aerobic actinomycete strain, designated as SCSIO 02100T, was isolated from a deep sea sediment sample collected from Northern South China Sea at a depth of 578 m. This isolate requires sea water or a sodium-supplemented medium for growth. BLAST searches based on the almost full length of the 16S rRNA gene sequence, showed that strain SCSIO 02100T had the highest similarities with Streptomyces armeniacus (JCM 3070T) (97.1 %). Phylogenetic trees reconstructed on the basis of 16S rRNA gene sequences revealed that strain SCSIO 02100T formed a distinct lineage with S. nanshensis SCSIO 01066T with 96.9 % similarity. Further analysis of the polyphasic taxonomic data, including morphological, phenotypic and chemotaxonomic properties, showed that strain SCSIO 02100T could be readily distinguished from the most closely related members of the genus Streptomyces. Thus, based on the polyphasic taxonomic data, a novel species, Streptomyces oceani sp. nov., is proposed, with the type strain SCSIO 02100T (=DSM 42043T = CGMCC 4.7007T).

Three new γ-pyrones named marinactinones A–C (1–3) were isolated from marine-derived actinomycete Marinactinospora thermotolerans SCSIO 00606. These structures were elucidated by extensive spectroscopic methods. All three new compounds were evaluated for cytotoxic effects on six cancer cell lines and inhibitory activities of DNA topoisomerase II. Compounds 1–3 exhibited moderate cytotoxicities against SW1990, HepG2 and SMCC-7721 cell lines, and compound 2 showed weak DNA topoisomerase II inhibition activity. This is the first report on the chemical constituents and their biological activities from Marinactinospora, a novel genus of marine actinomycetes.

A pair of flavanone glucoside diastereomers, (2R)- and (2S)-eriodictyol-5-O-β-d-glucopyranoside (1a, 1b), was successfully separated by RP-C18 high-performance liquid chromatography from Balanophora involucrata Hook. f. Some other compounds, including a pair of flavanone enantiomers, (2R)- and (2S)-eriodictyol (2a, 2b), and a pair of flavanone glucoside diastereomers, (2R)- and (2S)-eriodictyol-7-O-β-d-glucopyranoside(3a, 3b), were separated by capillary electrophoresis from the same plant. The absolute configurations at C-2 of 1a and 1b were determined based on their circular dichroism spectra. Enzymatic hydrolysis of 1a and 1b by β-d-glucosidase afforded (2R)- and (2S)-eriodictyol, respectively, which were used as the authentic standards for co-elution to determine the migration order of the enantiomers, 2a and 2b. We also report the first example of identifying the migration order of 2a and 2b and resolving the separation of 3a and 3b by capillary electrophoresis. In addition, 1a was unambiguously characterized for the first time by NMR spectra.

Marine sediment is a distinctive habitat of cold enzyme producing bacteria. A protease producing strain Halobacillus sp. SCSIO 20089 was isolated from a marine sediment of South China Sea. Using chromatographic techniques, the extracellular protease was purified to homogeneity from the culture supernatant. The purified protease exhibited maximal activity at 30°C, pH 8.0, and remained more than 20% of its activity at 0°C. Its activation energy was calculated to be 34.4 kJ/mol, suggesting it is a cold-adapted protease. Based on the N-terminal amino acid sequence of the purified enzyme, full gene encoding the enzyme was obtained by combination of degenerate primer PCR and hiTAIL-PCR. The deduced amino acid sequence showed 57% and 52% identity with mesothermal and thermophilic protease in thermolysin family respectively. All these indicate the enzyme is a unique cold-active thermolysin-like protease with potential in both basic research and industrial application areas.

•Lys209 was identified as thermostability conferring residue for the first time.•Systematic mutagenesis on Lys209 revealed its structural role.•Thermostability of AMY121 improved 3.4 folds by semi-rational protein design.Protein engineering on hotspot residues is acknowledged as an effective way to improve the stability and activity of enzymes. Searching for the key spot of a protein is a critically important but time-consuming process. In our previous study, an InDel site neighbouring residue Lys209 of the deep sea bacterial amylase AMY121 was proposed as one putative determinant for thermostability by homology sequence comparison and biochemical analysis [1]. Here, the structural stabilizing role of residue Lys209 was verified, and semi-rational protein design was applied to further improve capacity of higher temperature adaptation. Systematic mutagenesis study on Lys209 of amylase AMY121 revealed that all of the nineteen substitution mutants on the 209th site led to stability loss compared with wild type enzyme, indicating the crucial role of residue Lys209 in protein structural stabilizing. Four Lys209 neighboring sites were then selected and site directed saturation mutagenesis libraries were constructed for high through put thermostability screening. Two thermostability enhanced mutants Y187E and K205L were obtained from 1600 mutant colonies. Temperature properties comparison of wild type AMY121 and its variants showed Y187E and K205L possessed better kinetic thermal stability. The optimum temperature Y187E and K205L both increased by 5 °C. T5015 values of Y187E and K205L also increased to 0.63 and 8.52 °C, respectively. Structural basis for the difference in thermostability of wild type enzyme and its variants was further analyzed by computer modeling, and increasing in number of salt bridges and hydrophobic interactions around Lys209 was proposed as the main driving force for better compact protein structure of the enzyme.Download full-size image