Probing the Internal pH and Permeability of a Carboxysome Shell.

The carboxysome is a protein-based nanoscale organelle in cyanobacteria and many proteobacteria, which encapsulates the key CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a polyhedral protein shell. The intrinsic self-assembly and architectural features of carboxysomes and the semipermeability of the protein shell provide the foundation for the accumulation of CO2 within carboxysomes and enhanced carboxylation. Here, we develop an approach to determine the interior pH conditions and inorganic carbon accumulation within an α-carboxysome shell derived from a chemoautotrophic proteobacterium Halothiobacillus neapolitanus and evaluate the shell permeability. By incorporating a pH reporter, pHluorin2, within empty α-carboxysome shells produced in Escherichia coli, we probe the interior pH of the protein shells with and without CA. Our in vivo and in vitro results demonstrate a lower interior pH of α-carboxysome shells than the cytoplasmic pH and buffer pH, as well as the modulation of the interior pH in response to changes in external environments, indicating the shell permeability to bicarbonate ions and protons. We further determine the saturated HCO3- concentration of 15 mM within α-carboxysome shells and show the CA-mediated increase in the interior CO2 level. Uncovering the interior physiochemical microenvironment of carboxysomes is crucial for understanding the mechanisms underlying carboxysomal shell permeability and enhancement of Rubisco carboxylation within carboxysomes. Such fundamental knowledge may inform reprogramming carboxysomes to improve metabolism and recruit foreign enzymes for enhanced catalytical performance.

Flavobacterium phocarum sp. nov., isolated from soils of a seal habitat in Antarctica.

A Gram-stain-negative, yellow-pigmented, non-flagellated, gliding, rod-shaped, oxidase-negative and catalase-positive bacterium, designated SE14T, was isolated from soil on King George Island, South Shetland Islands, Antarctica. Strain SE14T grew at 4-25 °C (optimum, 20 °C), at pH 6.0-9.0 (optimum, pH 7.0-7.5) and with 0-3.0 % NaCl (optimum, 1.0-1.5 %), and could not produce flexirubin-type pigments. 16S rRNA gene sequence analysis showed the the isolate belonged to the genus Flavobacterium. Strain SE14T had the highest 16S rRNA gene sequence similarity to Flavobacterium antarcticum, F. tegetincola and F. degerlachei with 95.8, 95.5 and 95.2 %, respectively. The strain SE14T consisted of a clade with Flavobacteriumnoncentrifugens (16S rRNA gene sequence similarity 94.9 %) and F. qiangtangense (16S rRNA gene sequence similarity 94.2 %) and simultaneously formed a distinct phyletic lineage in the neighbour-joining phylogenetic tree. Polar lipids of the strain included phosphatidylethanolamine and four unidentified aminolipids. Strain SE14T contained anteiso-C15 : 0, iso-C15 : 0 and a mixture of iso-C15 : 0 2-OH and/or C16 : 1ω7c as the main fatty acids, and the only respiratory quinone was menaquinone-6. The genomic DNA G+C content was 42.3 mol%. The polyphasic taxonomic study revealed that strain SE14T belongs to a novel species within the genus Flavobacterium , and the name Flavobacterium phocarum sp. nov. is proposed. The type strain is SE14T (=CCTCC AB 2017225T=KCTC 52612T).

Flavobacterium ardleyense sp. nov., isolated from Antarctic soil.

A Gram-stain-negative, aerobic, yellow-pigmented, non-flagellated, non-gliding, rod-like, oxidase- and catalase-positive bacterium, designated A2-1T, was isolated from soil on Ardley Island, South Shetland Islands, Antarctica. Strain A2-1T grew at 4-22 °C (optimum, 10 °C), at pH 6.0-8.0 (optimum, pH 6.5) and with 0-1.5 % NaCl (optimum, 0.5 %), but could not produce flexirubin-type pigments. 16S rRNA gene sequence analysis showed that the isolates belonged to the genus Flavobacterium. Strain A2-1T had the highest 16S rRNA gene sequence similarity to Flavobacterium cucumis, F. ahnfeltiae and F. cheniae with 95.7, 95.6 and 95.4 %, respectively. The strain A2-1T consisted of a clade with F. cucumis and F. cheniae and simultaneously formed a distinct phyletic lineage in the neighbour-joining phylogenetic tree. Polar lipids of the strain included phosphatidylethanolamine (PE), four unidentified aminolipids and one unidentified lipid. The strain A2-1T contained anteiso-C15 : 0 (20.2 %), iso-C15 : 0 (16.2 %) and C15 : 1 G (11.0 %) as the main fatty acids and the only respiratory quinone was menaquinone MK-6. The genomic DNA G+C content was 34.0 mol%. The polyphasic taxonomic study revealed that the strain A2-1T belongs to a novel species within the genus Flavobacterium and the name Flavobacterium ardleyense sp. nov. is proposed. The type strain is A2-1T (=CCTCC AB 2017157T=KCTC 52644T).

Euzebyella marina sp. nov., isolated from seawater.

A Gram-stain-negative, aerobic, yellow-pigmented, non-flagellated, non-gliding, oxidase- and catalase-positive bacterium, designated CY01T, was isolated from seawater of the Yellow Sea. CY01T grew at 15-37 °C (optimum, 30 °C), pH 5-8 (optimum, 6.5-7.5) and with 0.5-12 % (w/v) NaCl (optimum, 0.5-3.5 %). It could not produce flexirubin-type pigment or reduce nitrate to nitrite. CY01T showed the highest 16S rRNA gene sequence similarity to the type strain of Euzebyella saccharophila (97.0 %) and clustered tightly with the species of the genus Euzebyella in the phylogenetic trees based on the 16S rRNA gene sequences. The major cellular fatty acids of CY01T were iso-C15 : 0, iso-C15 : 1G and iso-C17 : 0 3-OH and the major respiratory quinone was menaquinone MK-6. Polar lipids included phosphatidylethanolamine (PE), four unidentified lipids and one unidentified aminolipid. The genomic DNA G+C content was 38.2 mol%. Based on the results of the polyphasic characterization of CY01T, it represents a novel species of the genus Euzebyella, for which the name Euzebyella marina sp. nov. is proposed. The type strain is CY01T (=CCTCC AB 2014348T=KCTC 42440T).

Structure and ecological roles of a novel exopolysaccharide from the arctic sea ice bacterium Pseudoalteromonas sp. Strain SM20310.

The structure and ecological roles of the exopolysaccharides (EPSs) from sea ice microorganisms are poorly studied. Here we show that strain SM20310, with an EPS production of 567 mg liter(-1), was screened from 110 Arctic sea ice isolates and identified as a Pseudoalteromonas strain. The EPS secreted by SM20310 was purified, and its structural characteristics were studied. The predominant repeating unit of this EPS is a highly complicated α-mannan with a molecular mass greater than 2 × 10(6) Da. The backbone of the EPS consists of 2-α-, 6-α-mannosyl residues, in which a considerable part of the 6-α-mannosyl residues are branched at the 2 position with either single t-mannosyl residues or two mannosyl residues. The structure of the described EPS is different from the structures of EPSs secreted by other marine bacteria. Analysis of the ecological roles of the identified EPS showed that the EPS could significantly enhance the high-salinity tolerance of SM20310 and improve the survival of SM20310 after freeze-thaw cycles. These results suggest that the EPS secreted by strain SM20310 enables the strain to adapt to the sea ice environment, which is characterized by low temperature, high salinity, and repeated freeze-thaw cycles. In addition to its functions in strain SM20310, this EPS also significantly improved the tolerance of Escherichia coli to freeze-thaw cycles, suggesting that it may have a universal impact on microorganism cryoprotection.

Genome sequences of type strains of seven species of the marine bacterium Pseudoalteromonas.

There are over 30 species in the marine bacterial genus Pseudoalteromonas. However, our knowledge about this genus is still limited. We sequenced the genomes of type strains of seven species in the genus, facilitating the study of the physiology, adaptation, and evolution of this genus.

Characterization of bacterial polysaccharide capsules and detection in the presence of deliquescent water by atomic force microscopy.

We detected polysaccharide capsules from Zunongwangia profunda SM-A87 with atomic force microscopy (AFM). The molecular organization of the capsules at the single-polysaccharide-chain level was reported. Furthermore, we found that with ScanAsyst mode the polysaccharide capsules could be detected even in the presence of deliquescent water covering the capsule.

Mechanistic insight into the function of the C-terminal PKD domain of the collagenolytic serine protease deseasin MCP-01 from deep sea Pseudoalteromonas sp. SM9913: binding of the PKD domain to collagen results in collagen swelling but does not unwind the collagen triple helix.

Deseasin MCP-01 is a bacterial collagenolytic serine protease. Its catalytic domain alone can degrade collagen, and its C-terminal PKD domain is a collagen-binding domain (CBD) that can improve the collagenolytic efficiency of the catalytic domain by an unknown mechanism. Here, scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential, and circular dichroism spectroscopy were used to clarify the functional mechanism of the PKD domain in MCP-01 collagenolysis. The PKD domain observably swelled insoluble collagen. Its collagen-swelling ability and its improvement to the collagenolysis of the catalytic domain are both temperature-dependent. SEM observation showed the PKD domain swelled collagen fascicles with an increase of their diameter from 5.3 mum to 8.8 mum after 1 h of treatment, and the fibrils forming the fascicles were dispersed. AFM observation directly showed that the PKD domain bound collagen, swelled the microfibrils, and exposed the monomers. The PKD mutant W36A neither bound collagen nor disturbed its structure. Zeta potential results demonstrated that PKD treatment increased the net positive charges of the collagen surface. PKD treatment caused no change in the content or the thermostability of the collagen triple helix. Furthermore, the PKD-treated collagen could not be degraded by gelatinase. Therefore, though the triple helix monomers were exposed, the PKD domain could not unwind the collagen triple helix. Our study reveals the functional mechanism of the PKD domain of the collagenolytic serine protease MCP-01 in collagen degradation, which is distinct from that of the CBDs of mammalian matrix metalloproteases.

Phylogenetic Distribution of Polysaccharide-Degrading Enzymes in Marine Bacteria.

Deconstruction is an essential step of conversion of polysaccharides, and polysaccharide-degrading enzymes play a key role in this process. Although there is recent progress in the identification of these enzymes, the diversity and phylogenetic distribution of these enzymes in marine microorganisms remain largely unknown, hindering our understanding of the ecological roles of marine microorganisms in the ocean carbon cycle. Here, we studied the phylogenetic distribution of nine types of polysaccharide-degrading enzymes in marine bacterial genomes. First, we manually compiled a reference sequence database containing 961 experimentally verified enzymes. With this reference database, we annotated 9,335 enzyme sequences from 2,182 high-quality marine bacterial genomes, revealing extended distribution for six enzymes at the phylum level and for all nine enzymes at lower taxonomic levels. Next, phylogenetic analyses revealed intra-clade diversity in the encoding potentials and phylogenetic conservation of a few enzymes at the genus level. Lastly, our analyses revealed correlations between enzymes, with alginate lyases demonstrating the most extensive correlations with others. Intriguingly, chitinases showed negative correlations with cellulases, alginate lyases, and agarases in a few genera. This result suggested that intra-genus lifestyle differentiation occurred many times in marine bacteria and that the utilization of polysaccharides may act as an important driver in the recent ecological differentiation of a few lineages. This study expanded our knowledge of the phylogenetic distribution of polysaccharide enzymes and provided insights into the ecological differentiation of marine bacteria.

Oyster (Crassostrea gigas) hydrolysates produced on a plant scale have antitumor activity and immunostimulating effects in BALB/c mice.

Oyster extracts have been reported to have many bioactive peptides. But the function of oyster peptides produced by proteolysis is still unknown. In this study, the oligopeptide-enriched hydrolysates from oyster (Crassostrea gigas) were produced using the protease from Bacillus sp. SM98011 at laboratory level, and scaled up to pilot (100 L) and plant (1,000 L) levels with the same conditions. And the antitumor activity and immunostimulating effects of the oyster hydrolysates in BALB/c mice were investigated. The growth of transplantable sarcoma-S180 was obviously inhibited in a dose-dependent manner in BALB/c mice given the oyster hydrolysates. Mice receiving 0.25, 0.5 and 1 mg/g of body weight by oral gavage had 6.8%, 30.6% and 48% less tumor growth, respectively. Concurrently, the weight coefficients of the thymus and the spleen, the activity of natural killer (NK) cells, the spleen proliferation of lymphocytes and the phagocytic rate of macrophages in S180-bearing mice significantly increased after administration of the oyster hydrolysates. These results demonstrated that oyster hydrolysates produced strong immunostimulating effects in mice, which might result in its antitumor activity. The antitumor and immunostimulating effects of oyster hydrolysates prepared in this study reveal its potential for tumor therapy and as a dietary supplement with immunostimulatory activity.

Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production.

Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biological elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diameter) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the production of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen production of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.

Ecological function of myroilysin, a novel bacterial M12 metalloprotease with elastinolytic activity and a synergistic role in collagen hydrolysis, in biodegradation of deep-sea high-molecular-weight organic nitrogen.

Nearly all high-molecular-weight (HMW) dissolved organic nitrogen and part of the particulate organic nitrogen in the deep sea are present in hydrolysis-resistant amides, and so far the mechanisms of biodegradation of these types of nitrogen have not been resolved. The M12 family is the second largest family in subclan MA(M) of Zn-containing metalloproteases and includes most enzymes from animals and only one enzyme (flavastacin) from a human-pathogenic bacterium (Flavobacterium meningosepticum). Here, we characterized the novel M12 protease myroilysin with elastinolytic activity and collagen-swelling ability from the newly described deep-sea bacterium Myroides profundi D25. Myroilysin is a monomer enzyme with 205 amino acid residues and a molecular mass of 22,936 Da. It has the same conserved residues at the four zinc ligands as astacin and very low levels of identity (

Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China sea.

Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

Purification and functional characterization of endo-beta-mannanase MAN5 and its application in oligosaccharide production from konjac flour.

MAN5, the main extracellular saccharide hydrolase from Bacillus sp. MSJ-5, is an endo-beta-mannanase with a demand of at least five sugar moieties for effective cleavage. It has a pH optimum of 5.5 and a temperature optimum of 50 degrees C and is stable at pH 5-9 or below 65 degrees C. MAN5 has a very high ability to hydrolyze konjac flour, 10 U/mg of which could completely liquefy konjac flour gum in 10 min at 50 degrees C. HPLC analysis showed that most glucomannan in the konjac flour was hydrolyzed into a large amount of oligosaccharides with DP of 2-6 and a very small amount of monosaccharide. With the culture supernatant as enzyme source, the optimum condition to prepare oligosaccharides from konjac flour was obtained as 10 mg/ml konjac flour incubated with 10 U/mg enzyme at 50 degrees C for 24 h. With this condition, more than 90% polysaccharides in the konjac flour solution were hydrolyzed into oligosaccharides and a little monosaccharide (2.98% of the oligosaccharides). Konjac flour is an underutilized agricultural material with low commercial value in China. With MAN5, konjac flour can be utilized to generate high value-added oligosaccharides. The high effectiveness and cheapness of this technique indicates its potential in industry.

Predicting protein interaction interfaces from protein sequences: case studies of subtilisin and phycocyanin.

Identification of protein interaction interfaces is very important for understanding the molecular mechanisms underlying biological phenomena. Here, we present a novel method for predicting protein interaction interfaces from sequences by using PAM matrix (PIFPAM). Sequence alignments for interacting proteins were constructed and parsed into segments using sliding windows. By calculating distance matrix for each segment, the correlation coefficients between segments were estimated. The interaction interfaces were predicted by extracting highly correlated segment pairs from the correlation map. The predictions achieved an accuracy 0.41-0.71 for eight intraprotein interaction examples, and 0.07-0.60 for four interprotein interaction examples. Compared with three previously published methods, PIFPAM predicted more contacting site pairs for 11 out of the 12 example proteins, and predicted at least 34% more contacting site pairs for eight proteins of them. The factors affecting the predictions were also analyzed. Since PIFPAM uses only the alignments of the two interacting proteins as input, it is especially useful when no three-dimensional protein structure data are available.

Pilot and plant scaled production of ACE inhibitory hydrolysates from Acetes chinensis and its in vivo antihypertensive effect.

The angiotensin-I-converting enzyme (ACE) inhibitory oligopeptide-enriched hydrolysates from Acetes chinensis by treatment with the protease from Bacillus sp. SM98011 were produced at pilot scale (100 L) and plant scale (1000 L). The pilot and plant scaled hydrolysate products almost had the same properties as that at laboratory scale. Spray-drying had little effect on the peptide composition and bioactivity of the hydrolysates. The plant scaled hydrolysates were used to study its blood pressure-depressing effect in vivo. It caused reduce of 18.3-38.6 mmHg of the blood pressure of spontaneously hypertensive rats in dose-dependent manner in the range of 100-1,200 mg/kg/day. Histopathologic study showed that the pathologic changes of heart and brain in SHRs got obvious alleviation after treatment of the hydrolysates.

High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis.

Twelve kinds of marine protein materials, including fish, shrimp, seashell, algae and seafood wastes were selected for the hydrolysis using four different proteases. The IC(50) values for angiotensin-converting enzyme (ACE) inhibitory activity of 48 hydrolysates were rapidly determined by capillary electrophoresis (CE). The values ranged from 0.17 to 501.7mg/ml, and were affected by both the marine protein resources and the selected proteases. Hydrolysates of the lowest IC(50) values were from shrimp (Acetes chinensis), shark meat, mackerel bone, Polysiphonia urceolata and Spirulina platensis, indicating these five kinds of marine food proteins contained beneficial materials for the production of ACE inhibitory peptides by proteolysis. The hydrolysates obtained using proteases Protamex and SM98011 had lower IC(50) values, showing these two proteases were superior to others. The CE method achieved the same sensitivity as the high performance liquid chromatography (HPLC) method. However, the CE method was faster and, as a result, more economical. Therefore, CE had potential for rapid screening of marine protein hydrolysates enriched in ACE inhibitory peptides.

Stabilization of cold-adapted protease MCP-01 promoted by trehalose: prevention of the autolysis.

Protease MCP-01 is similar to other cold-adapted enzymes in that it is a cold-adapted serine protease having high specific activity and low thermostability at low and moderate temperature. Its thermolability and self-autolysis has resulted in difficulties in its purification, preservation and research on its structure and function. The disaccharide trehalose is known to effectively stabilize proteins. Its prevention effect on the autolysis of cold-adapted protease MCP-01 was monitored by capillary electrophoresis. In the absence of trehalose, protease MCP-01 autolyzed rapidly at 35 degrees C. However, when trehalose was added, autolysis was remarkably prevented and the loss of activity reduced. MCP-01 may be a useful model for basic research on the interaction of protein and trehalose.

Review on the angiotensin-I-converting enzyme (ACE) inhibitor peptides from marine proteins.

Hypertension is now a major problem threatening people health in the world. Angiotensin-I-converting enzyme (ACE) plays an important physiological role in regulation of blood pressure via conversion of angiotensin I to angiotensin II. Inhibition of ACE may have an antihypertensive effect as a consequence of a decrease in blood pressure. A number of terrestrial-derived peptides have been reviewed about their in vitro and in vivo ACE-inhibitory activities. Marine organisms are potentially an untapped source of drugs and value-added food production. The aim of this review is to discuss the marine-derived ACE-inhibitory peptides from sources, production, structure aspects, and their future prospects as functional food or novel therapeutic drug candidates.

Tenderization effect of cold-adapted collagenolytic protease MCP-01 on beef meat at low temperature and its mechanism.

The enzymes currently used to increase meat tenderness are all mesophilic or thermophilic proteases. This study provides insight into the tenderization effect and the mechanism of a cold-adapted collagenolytic enzyme MCP-01 on beef meat at low temperatures. MCP-01 (10 U of caseinolytic activity) reduced the meat shear force by 23% and increased the relative myofibrillar fragmentation index of the meat by 91.7% at 4 °C, and it also kept the fresh colour and moisture of the meat. Compared to the commercially used tenderizers papain and bromelain, MCP-01 showed a unique tenderization mechanism. MCP-01 had a strong selectivity for degrading collagen at 4 °C, showed a distinct digestion pattern on the myofibrillar proteins, and had a different disruption pattern on the muscle fibres under scanning electron micrograph. These results suggest that the cold-adapted collagenolytic protease MCP-01 may be promising for use as a meat tenderizer at low and moderate temperatures.