In a marine green starch-producing microalga Tetraselmis subcordiformis, the role of starch phosphorylase (SP) in the starch biosynthesis was disclosed by characterizing the enzyme properties and activity variations during the starch accumulation process. TsSP4, a SP isoform accounting for the major SP activity in T. subcordiformis, was unique to be active in a monomer form with a molecular weight of approximately 110 kDa. It resembled one of the chloroplast-located SPs (PhoA) in Chlamydomonas reinhardtii with a similarity of 63.3% in sequence, though it possessed the typical L78/80 domain found in the plastidial SPs (Pho1) of higher plants that was absent in PhoA. TsSP4 exhibited moderate sensitivity to ADP-Glc inhibition and had a high activity for longer-chain linear maltooligosacchride (MOS) and amylopectin against highly branched glycogen as the substrates. TsSP4 had 2-fold higher affinity for Glc-1-P in the synthetic direction than for Pi in the phosphorolytic direction, and the catalytic constant kcat for Glc-1-P was 2-fold of that for Pi. Collectively, TsSP4 preferred synthetic rather than phosphorolytic direction. TsSP4 could elongate MOSs even initially with Pi alone in the absence of Glc-1-P, which further supported its synthetic role in the starch biosynthesis. TsSP4 displayed increased activities in the developing and mature stage of starch biosynthesis under nitrogen-starvation conditions, indicating its possible contribution to the amylopectin amplification.

The marine microalga Tetraselmis subcordiformis could photoproduce hydrogen under the regulation of carbonyl cyanide m-chlorophenylhydrazone (CCCP), and a hydrogen production process kinetic analysis was characterized by two peaks, suggesting that two distinct mechanisms might exist in this alga. Therefore, 2D nanoliquid chromatography−tandem mass spectrometry (LC-MS/MS) was introduced to analyze the proteome of samples from different time points. A total of 912 proteins were identified, providing a global view of the cellular responses at the proteomic level. These proteins can be divided into multiple functional groups including stress responses, energy metabolism and redox homeostasis. The quantitative proteomic data provided more details on the electron donors for hydrogen production. During the first stage, photosystem II produced electrons for hydrogen production; during the second stage, metabolites were the major electron donors via nonphotochemical plastoquinone reduction by NADH dehydrogenase.

There was no direct correlation in substrate specificity between the metabolism of Pseudomonas stutzeri DEH138 and its corresponding dehalogenase. Dehalogenase substrates that could be dehalogenated might not be degraded by DEH138 or vice versa. Basing on this, different approaches to enhance L-2-haloacid dehalogenase (L-DEX) production in DEH138 via the combination of non-halogenated compounds with different inducers were applied. The optimum approach to obtain more L-DEX from DEH138 was the combination of DL-lactate and DL-2-chlorobutyrate, with 5.7-fold greater production and 11.7-fold greater productivity of the enzyme after optimization.

A low-temperature-active alkaline esterase, Est12, from a marine sediment metagenomic fosmid library was identified. Est12 prefers short- and middle-chain p-nitrophenol esters as substrate with optimum temperature and pH value of 50 °C and 9.0, respectively, and nearly 50 % of maximum activity retained at 5 °C. The hydrolysis activity of Est12 was stable at 40 °C. Ca2+ especially activated the activity of Est12 to about 151 % of the control. DEPC and PMSF inhibited the activity of Est12 to 34 and 25 %, respectively. In addition, Est12 was more tolerable to methanol compared to other organic solvents tested. The crystal structure of Est12 at 1.39 Å resolution showed that the cap domain which is composed of an α-helix and a flexible region resulted in a relatively wide spectrum of substrate, with p-nitrophenol caproate as the preferred one. Furthermore, the flexible cap domain and the high percentage of Gly, Ser, and Met may play important roles in the adaptation of Est12 to low temperature.

Fatty acid composition is an important physiological parameter of microalgae, which is taken as the third generation alternative resource of biodiesel. To boost microalgal research and applications, a convenient, rapid, and acid-catalyzed transesterification procedure that satisfies the demand for the analysis of the fatty acid composition of lipids with micro-scale samples in the high-throughput screening of microalgal strains is needed, along with the evaluation of the physiological status of microalgae in response to nutrient stress.The reaction conditions of transesterification via a micro-mixer reactor were optimized as follows: 90 °C reaction temperature, 20 min reaction time, 6:1 volume ratio of H2SO4-methanol to lipid-in-hexane, and a Y-type micro-mixer with a 20-m-long extended loop that has a 0.3 mm diameter. The minimum amount of sample was decreased to 30 µg lipids. The new approach was successfully applied to the fatty acid composition analysis of soybean oil and microalgal lipids. Definitely, it could be applied to acyl related oils from different sources.Here, we have developed a simple and rapid method for the analysis of the fatty acid composition of lipids. The new method requires less than 20 min for transesterification and a minimum of only 30 µg lipid sample. Furthermore, a high-throughput process can be easily realized by numbering up the micro-mixer reactors. The micro-mixer reactor has great potential for applications not only in large-scale biodiesel production but also for the micro-scale analysis of microalgae fatty acid compositions.

•The first transcriptome for Tetraselmis subcordiformis' special hydrogen inducing behavior.•Overall changing pattern of transcription for microalgal H2 evaluation process.•Quantitative analysis unveiled the functions of different hydrogenase subunits.•Postulated mechanism for anaerobic dark induction of T. subcordiformis.•Global gene function analysis showed T. subcordiformis a good candidate for biofuel.It's the first comparative transcriptome study of Tetraselmis subcordiformis' H2 production with carbonyl cyanide m-chlorophenylhydrazone (CCCP) by de novo RNA-seq. The global analysis of the annotated genes and pathways unveiled it a good candidate for biofuels. Focusing on H2 production, the genes coding active center subunit (HydA) and assembly subunits (HydEF and HydG) of hydrogenase proportionally expressed during the induction but stopped after illuminated; on contrast, the gene of the active centre stabilization subunit (Hyd3) highly expressed during the H2 evolution, indicating its key role in the prolonged H2 production. The results also showed genes of HCP4, ppGpp phosphohydrolase and RpoD relating to the hydrogenase expression regulation. On the energy aspect, genes of CF-ATPases and ferredoxin-NADP reductase in chloroplast down-regulated while Mito-ATPase in mitochondria and pyruvate metabolism pathway up-regulated during the H2 production, consisting with our previous reports on physiological level.

A marine bacterial strain, designated OB44-3T, was isolated from a crude oil-contaminated seawater sample collected near Dalian Bay, China. Cells of strain OB44-3T were Gramnegative, aerobic, rod-shaped, and oxidase- and catalasepositive. The major fatty acids were branched-chain saturated iso-C15:0 (27.9%) and unsaturated iso-C17:1ω9c (14.8%). The DNA G+C content was 64.6 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain OB44-3T was a member of the genus Luteimonas (95–96% 16S rRNA gene sequence similarity); its closest neighbors were the type strains of Luteimonas terricola (96% sequence similarity), Luteimonas mephitis (96%), and Luteimonas lutimaris (96%). On the basis of phenotypic, chemotaxonomic, and phylogenetic distinctiveness, strain OB44-3T was considered to represent a novel species of the genus Luteimonas. The name Luteimonas dalianensis sp. nov. is proposed, with strain OB44-3T (=CGMCC 1.12191T =JCM 18136T) as the type strain.

Under stressful conditions, the non-model marine microalga Tetraselmis subcordiformis can accumulate a substantial amount of starch, making it a potential feedstock for the production of fuel ethanol. Investigating the interactions of the enzymes and the regulatory factors involved in starch metabolism will provide potential genetic manipulation targets for optimising the starch productivity of T. subcordiformis. For this reason, the proteome of T. subcordiformis was utilised to predict the first protein–protein interaction (PPI) network for this marine alga based on orthologous interactions, mainly from the general PPI repositories. Different methods were introduced to evaluate the credibility of the predicted interactome, including the confidence value of each PPI pair and Pfam-based and subcellular location-based enrichment analysis. Functional subnetworks analysis suggested that the two enzymes involved in starch metabolism, starch phosphorylase and trehalose-phosphate synthase may be the potential ideal genetic engineering targets.

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu2+, Zn2+, and Co2+. The Km and Vmax for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.

•The environment-induced conformational and functional changes of l-2-haloacid dehalogenase were firstly studied by CD spectroscopy.•Decreased α-helix and increased β-sheet contents were observed along with activity loss caused by pH, temperature and inhibitors.•More than 65.0% of the enzyme activity could be remained if its α-helix content was over 12.0%.•The maintenance of α-helical structure is indispensable to the enzymatic activity, while β-sheet increase restricts the activity.2-Haloacid dehalogenases have been highly studied due to their potential applications in chemical industries and bioremediation. Although biochemical and structural characterizations of the enzyme have been detailed, no information was available regarding environmental effects on the structure–function relationship. Here, circular dichroism spectroscopy (CD) was used to investigate the correlation between changes on the conformation and the function of l-2-haloacid dehalogenase (HadL AJ1) from the Pseudomonas putida induced by the environmental factors. Decreased α-helix and increased β-sheet contents were observed in the structure of HadL AJ1 along with activity losses caused by pH, temperature and inhibitors. Regardless of which factor above-mentioned existed, more than 65.0% of HadL AJ1 activity could be remained if its α-helix content was over 12.0%. The maintenance of α-helical structure in HadL AJ1 was indispensable to its catalysis, while β-sheet increase restricts its activity. This study revealed the variation of enzymatic activity due to environmental conditions resulting in structural changes monitored by CD, which contributed to rational modification and was instructive for predicting changes of the enzymatic activity during application.