Marine-Derived Fungi as a Valuable Resource for Amylases Activity Screening.

Marine microbial enzymes including amylases are important in different industrial production due to their properties and applications. This study was focused on the screening of marine-derived fungi for amylase activities. First, we isolated a number of fungi from the sediments of the South China Sea. By the method of dish screening (in vitro), we subsequently obtained a series of amylase-producing fungal strains. The cell-lysate activities of amylases produced by marine fungi toward starch hydrolysis were achieved with the dinitrosalyicylic acid (DNS) method. In addition, the effect of pH and temperature on amylase activities, including thermal and pH stability were discussed. Results showed that out of the 57 isolates with amylase-producing activities, fungi Aspergillus flavus 9261 was found to produce amylase with the best activity of 10.7482 U/mg (wet mycelia). The amylase of Aspergillus flavus 9261 exhibited remarkable thermostability and pH stability with no activity loss after incubation at 50 °C and pH 5.0 for 1 h, respectively. The results provide advances in discovering enzymes from marine-derived fungi and their biotechnology relevance.

Bioinformatics-based study reveals that AP2M1 is regulated by the circRNA-miRNA-mRNA interaction network and affects Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurological disease that worsens with time. The hallmark illnesses include extracellular senile plaques caused by ß-amyloid protein deposition, neurofibrillary tangles caused by tau protein hyperphosphorylation, and neuronal loss accompanying glial cell hyperplasia. Noncoding RNAs are substantially implicated in related pathophysiology, according to mounting data. However, the function of these ncRNAs is mainly unclear. Circular RNAs (circRNAs) include many miRNA-binding sites (miRNA response elements, MREs), which operate as miRNA sponges or competing endogenous RNAs (ceRNAs). The purpose of this study was to look at the role of circular RNAs (circRNAs) and microRNAs (miRNAs) in Alzheimer's disease (AD) as possible biomarkers. The Gene Expression Omnibus (GEO) database was used to obtain an expression profile of Alzheimer's disease patients (GSE5281, GSE122603, GSE97760, GSE150693, GSE1297, and GSE161435). Through preliminary data deletion, 163 genes with significant differences, 156 miRNAs with significant differences, and 153 circRNAs with significant differences were identified. Then, 10 key genes, led by MAPT and AP2M1, were identified by the mediation center algorithm, 34 miRNAs with obvious prognosis were identified by the cox regression model, and 16 key circRNAs were selected by the database. To develop competitive endogenous RNA (ceRNA) networks, hub circRNAs and mRNAs were used. Finally, GO analysis and clinical data verification of key genes were carried out. We discovered that a down-regulated circRNA (has_circ_002048) caused the increased expression of numerous miRNAs, which further inhibited the expression of a critical mRNA (AP2M1), leading to Alzheimer's disease pathology. The findings of this work contribute to a better understanding of the circRNA-miRNA-mRNA regulating processes in Alzheimer's disease. Furthermore, the ncRNAs found here might become novel biomarkers and potential targets for the development of Alzheimer's drugs.

Photobiocatalytic Decarboxylation for the Synthesis of Fatty Epoxides from Renewable Fatty Acids.

Fatty epoxides are unique building blocks in organic transformations and materials production; however, their synthetic methodologies are currently not accessible from renewable fatty acids. Herein, a photoenzymatic decarboxylation of epoxy fatty acids into fatty epoxides was demonstrated using fatty acid photodecarboxylase (FAP) from Chlorella variabilis NC64A (CvFAP). Various fatty epoxides were synthesized in excellent selectivity by wild-type CvFAP. The decarboxylation reaction was also achieved with four new FAP homologues, potentially suggesting a broad availability of the biocatalysts for this challenging decarboxylation reaction. By combining CvFAP with lipase and peroxygenase, a multienzymatic cascade to transform oleic acid and its triglyceride into the corresponding fatty epoxides was established. The obtained fatty epoxides were further converted into rather unusual fatty compounds including diol, alcohol, ether, and chain-shortened carboxylic acids. The present photobiocatalytic synthesis of fatty epoxides from natural starting materials excels by its intrinsic selectivity, mild conditions, and independence of nicotinamide cofactors.

Recent Advances in the Synthesis of Marine-Derived Alkaloids via Enzymatic Reactions.

Alkaloids are a large and structurally diverse group of marine-derived natural products. Most marine-derived alkaloids are biologically active and show promising applications in modern (agro)chemical, pharmaceutical, and fine chemical industries. Different approaches have been established to access these marine-derived alkaloids. Among these employed methods, biotechnological approaches, namely, (chemo)enzymatic synthesis, have significant potential for playing a central role in alkaloid production on an industrial scale. In this review, we discuss research progress on marine-derived alkaloid synthesis via enzymatic reactions and note the advantages and disadvantages of their applications for industrial production, as well as green chemistry for marine natural product research.

Light-Driven Enzymatic Decarboxylation of Dicarboxylic Acids.

Photodecarboxylase from Chlorella variabillis (CvFAP) is one of the three known light-activated enzymes that catalyzes the decarboxylation of fatty acids into the corresponding C1-shortened alkanes. Although the substrate scope of CvFAP has been altered by protein engineering and decoy molecules, it is still limited to mono-fatty acids. Our studies demonstrate for the first time that long chain dicarboxylic acids can be converted by CvFAP. Notably, the conversion of dicarboxylic acids to alkanes still represents a chemically very challenging reaction. Herein, the light-driven enzymatic decarboxylation of dicarboxylic acids to the corresponding (C2-shortened) alkanes using CvFAP is described. A series of dicarboxylic acids is decarboxylated into alkanes in good yields by means of this approach, even for the preparative scales. Reaction pathway studies show that mono-fatty acids are formed as the intermediate products before the final release of C2-shortened alkanes. In addition, the thermostability, storage stability, and recyclability of CvFAP for decarboxylation of dicarboxylic acids are well evaluated. These results represent an advancement over the current state-of-the-art.

Enzymatic synthesis of enantiopure alcohols: current state and perspectives.

Enantiomerically pure alcohols, as key intermediates, play an essential role in the pharmaceutical, agrochemical and chemical industries. Among the methods used for their production, biotechnological approaches are generally considered a green and effective alternative due to their mild reaction conditions and remarkable enantioselectivity. An increasing number of enzymatic strategies for the synthesis of these compounds has been developed over the years, among which seven primary methodologies can be distinguished as follows: (1) enantioselective water addition to alkenes, (2) enantioselective aldol addition, (3) enantioselective coupling of ketones with hydrogen cyanide, (4) asymmetric reduction of carbonyl compounds, (5) (dynamic) kinetic resolution of racemates, (6) enantioselective hydrolysis of epoxides, and (7) stereoselective hydroxylation of unactivated C-H bonds. Some recent reviews have examined these approaches separately; however, to date, no review has included all the above mentioned strategies. The aim of this mini-review is to provide an overview of all seven enzymatic strategies and draw conclusions on the effect of each approach.

Immobilized and Free Cells of Geotrichum candidum for Asymmetric Reduction of Ketones: Stability and Recyclability.

Marine-derived fungus Geotrichum candidum AS 2.361 was previously reported by our group as an active strain for the enantioselective reduction of ketones. Although some other Geotrichum strains were also found from the terrestrial sources, information on their stability and reusability is scarce. Herein, the stabilities-in terms of pH tolerance, thermostability, and storage stability, and reusability-of G. candidum AS 2.361 were described for the asymmetric reduction of a series of aromatic ketones. Two differently immobilized cells (agar immobilization and calcium alginate immobilization) as well as free cells were prepared. For three substrates (1-(3-bromophenyl) ethan-1-one (1b), 1-(2-chlorophenyl) ethan-1-one (1d), and acetophenone (1g)) immobilized cells on agar showed a great improvement in the bioreduction activities compared to the free cells, increasing yields up to 97% with ee values of 99%. Cells immobilized on agar/calcium alginate could maintain more than 90% of the original activities within the assayed pH ranges of 3.5⁻11, while free cells were highly sensitive to alkaline and acidic conditions. Concerning thermostability, immobilized cells on agar kept 99% of their original activities after incubation at 60 °C for 1 h, while almost no activity was detected for the free cells under the same condition. Immobilized cells were stable at 4 °C for 80 days without any activity loss, while free cells started to decrease the activity after storage at 4 °C for six days. The immobilized cells retained almost 99% activity after four reuse cycles, while free cells lost almost all the activities at on the third cycle.

Rhodococcus strains as source for ene-reductase activity.

Rhodococcus strains are ubiquitous in nature and known to metabolise a wide variety of compounds. At the same time, asymmetric reduction of C=C bonds is important in the production of high-valued chiral building blocks. In order to evaluate if Rhodococci can be used for this task, we have probed several Rhodococcus rhodochrous and R. erythropolis strains for ene-reductase activity. A series of substrates including activated ketones, an aldehyde, an imide and nitro-compound were screened using whole cells of seven Rhodococcus strains. This revealed that whole cells of all Rhodococcus strains showed apparent (S)-selectivity towards ketoisophorone, while most other organisms show (R)-selectivity for this compound. Three putative ene-reductases from R. rhodochrous ATCC 17895 were heterologously expressed in Escherichia coli. One protein was purified and its biocatalytic and biochemical properties were characterised, showing typical (enantioselective) properties for class 3 ene-reductases of the old yellow enzyme family.

A Comparative Study on Asymmetric Reduction of Ketones Using the Growing and Resting Cells of Marine-Derived Fungi.

Whole-cell biocatalysts offer a highly enantioselective, minimally polluting route to optically active alcohols. Currently, most of the whole-cell catalytic performance involves resting cells rather than growing cell biotransformation, which is one-step process that benefits from the simultaneous growth and biotransformation, eliminating the need for catalysts preparation. In this paper, asymmetric reduction of 14 aromatic ketones to the corresponding enantiomerically pure alcohols was successfully conducted using the growing and resting cells of marine-derived fungi under optimized conditions. Good yields and excellent enantioselectivities were achieved with both methods. Although substrate inhibition might be a limiting factor for growing cell biotransformation, the selected strain can still completely convert 10-mM substrates into the desired products. The resting cell biotransformation showed a capacity to be recycled nine times without a significant decrease in the activity. This is the first study to perform asymmetric reduction of ketones by one-step growing cell biotransformation.

The use of marine-derived fungi for preparation of enantiomerically pure alcohols.

A highly enantioselective and minimally polluting approach to optically pure chiral alcohols is developed using cheap, readily available and sustainable marine-derived fungi as catalysts. An evaluation of the synthetic potential of 13 Chinese marine fungi was performed to screen for enantioselective reduction of 13 aromatic ketones from different compound classes as substrates. Good yields and excellent enantioselectivities were achieved with this method. In details, first the effects of several crucial variables on the bioreduction of aromatic ketones with whole cells of marine fungi were explored systematically. Next, we obtained insight into the substrate scope of the tested fungi under the optimized conditions, and selected reduction processes were performed at a commercial scale of up to 1000 mL to determine scalability, which led to excellent yields and enantioselectivities. Last, ketone reductases from two prioritized fungi exhibited good recyclability, with those of Rhodotorula mucilageinosa giving a > 95% yield with up to 99% ee during 3 cycles and those of Rhodotorula rubra giving a > 95% yield with up to 99% ee during 9 cycles.

Risk assessment of trace metal-polluted coastal sediments on Hainan Island: A full-scale set of 474 geographical locations covering the entire island.

Hainan Island is the second largest island and one of the most famous tourist destinations in China, but sediment contamination by trace metals in coastal areas is a major issue. However, full-scale risk assessments of trace metal-polluted coastal sediments are lacking. In this study, coastal surface sediments from 474 geographical locations covering almost the entire island were collected to identify risk-related variables. Controlling factors and possible sources of trace metals were identified, and the toxicity effects were carefully evaluated. Our results suggest that trace-metal pollution in coastal sediments, which was mainly caused by Pb, Zn and Cu emissions, has primarily resulted from industrial sewage and shipping activities and has threatened the offshore ecosystem of Hainan Island and warrants extensive consideration. This is the first study that has systematically investigated trace metal-polluted coastal sediments throughout the entirety of Hainan Island and provides solid evidence for sustainable marine management in the region.

Stereochemistry of enzymatic water addition to C=C bonds.

Water addition to carbon-carbon double bonds using hydratases is attracting great interest in biochemistry. Most of the known hydratases are involved in primary metabolism and to a lesser extent in secondary metabolism. New hydratases have recently been added to the toolbox, both from natural sources or artificial metalloenzymes. In order to comprehensively understand how the hydratases are able to catalyse the water addition to carbon-carbon double bonds, this review will highlight the mechanistic and stereochemical studies of the enzymatic water addition to carbon-carbon double bonds, focusing on the syn/anti-addition and stereochemistry of the reaction.

Enantioselective Michael addition of water.

The enantioselective Michael addition using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addition of water in water to prepare important ß-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry.

Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

The Michael hydratase - alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,ß-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated.

Draft genome sequence of Rhodococcus rhodochrous strain ATCC 17895.

Rhodococcus rhodochrous ATCC 17895 possesses an array of mono- and dioxygenases, as well as hydratases, which makes it an interesting organism for biocatalysis. R. rhodochrous is a Gram-positive aerobic bacterium with a rod-like morphology. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Based on small subunit rRNA analysis, the strain is more likely to be a strain of Rhodococcus erythropolis rather than Rhodococcus rhodochrous.

Diastereoselective synthesis and bioactivity of long-chain anti-2-amino-3-alkanols.

An improved four-step approach for the stereoselective synthesis of long-chain anti-2-amino-3-alkanols is described. Using this method, the syntheses of antiproliferative (antitumoral) compounds, spisulosine (ES-285, 2), clavaminols A and B (3 and 4), the deacetylated products of clavaminols H and N (7 and 8), as well as (2S,3R)-2-aminododecan-3-ol (9) and xestoaminol C (10), have been achieved in excellent diastereoselectivities. In vitro study showed that these compounds induced cell death and dose-dependently inhibited cell proliferation in human glioblastoma cell line SHG-44, indicating the anti-tumor property of this series of compounds.