Thermostability enhancement of chitosanase CsnA by fusion a family 5 carbohydrate-binding module

Objective

To determine the effects of carbohydrate-binding modules (CBMs) on the thermostability and catalytic efficiency of chitosanase CsnA.

Results

Three CBMs (BgCBM5, PfCBM32-2 and AoCBM35) were engineered at the C-terminus of chitosanase CsnA to create hybrid enzymes CsnA-CBM5, CsnA-CBM32 and CsnA-CBM35. K _m values of all the hybrid enzymes were lower than that of the wild type (WT) enzyme; however, only CsnA-CBM5 had an elevated specific activity and catalytic efficiency. The fusion of BgCBM5 enhanced the thermostability of the enzyme, which exhibited a 8.9 °C higher T₅₀ and a 2.9 °C higher T_m than the WT. Secondary structural analysis indicated that appending BgCBM5 at the C-terminus considerably changed the secondary structure content.

Conclusions

The fusion of BgCBM5 improved the thermal stability of CsnA, and the obtained hybrid enzyme (CsnA-CBM5) is a useful candidate for industrial application.

     

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

Background

Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.

Results

In this study, we have compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-d-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-d-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration.

Conclusion

We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-d-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> protein ybgI,a toroidal structure with a dinuclear metal site

Background

The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results

The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Conclusion

The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

     

Lipopeptide produced from <Emphasis Type="Italic">Bacillus</Emphasis> sp. W112 improves the hydrolysis of lignocellulose by specifically reducing non-productive binding of cellulases with and without CBMs

Background

Surfactants have attracted increasing interest for their capability to improve the enzymatic hydrolysis of lignocellulosic biomass. Compared to chemical surfactants, biosurfactants have a broader prospect for industrial applications because they are more environmentally friendly and more effective in some researches. Commercial cellulase preparations are mainly composed of endoglucanases (EGs) and cellobiohydrolases (CBHs) that possess carbohydrate-binding modules (CBMs). However, the effects of lipopeptide-type biosurfactants on enzymatic saccharification of lignocellulose and adsorption behaviors of cellulases with CBMs remain unclear.

Results

In this study, we found that Bacillus sp. W112 could produce a lipopeptide-type biosurfactant from untreated biomass, such as wheat bran and Jerusalem artichoke tuber. The lipopeptide could enhance the enzymatic hydrolysis of dilute acid pretreated Giant Juncao grass (DA-GJG) by fungal and bacterial enzymes. The enhancement increased over a range of temperatures from 30 to 50 °C. Lipopeptide was shown to be more effective in promoting DA-GJG saccharification than chemical surfactants at low dosages, with a best stimulatory degree of 20.8% at 2% loading of the substrates (w/w). Lipopeptide increased the thermostability of EG and CBH in commercial cellulase cocktails. Moreover, the dual effects of lipopeptide on the adsorption behaviors of cellulases were found. It specifically lowered the non-productive binding of cellulases to lignin and increased the binding of cellulases to cellulose. In addition, we investigated the influence of lipopeptide on the adsorption behaviors of CBHs with CBMs for the first time. Our results showed that lipopeptide reduced the adsorption of CBM-deleted CBH to DA-GJG to a greater extent than that of intact CBH while the non-productive binding of intact CBH to lignin was reduced more, indicating that lipopeptide decreased the binding of CBMs onto lignin but not their combination with cellulose.

Conclusions

In this study, we found that lipopeptide from Bacillus sp. W112 promoted the enzymatic hydrolysis of DA-GJG at relative low loadings. The stimulatory effect could be attributed to increasing the cellulase thermostability, reducing non-productive adsorption of cellulases with CBMs caused by lignin and enhancing the binding of cellulases to cellulose.

     

Application of residual polysaccharide-degrading enzymes in dried shiitake mushrooms as an enzyme preparation in food processing

Objective

To examine the activities of residual enzymes in dried shiitake mushrooms, which are a traditional foodstuff in Japanese cuisine, for possible applications in food processing.

Results

Polysaccharide-degrading enzymes remained intact in dried shiitake mushrooms and the activities of amylase, β-glucosidase and pectinase were high. A potato digestion was tested using dried shiitake powder. The enzymes reacted with potato tuber specimens to solubilize sugars even under a heterogeneous solid-state condition and that their reaction modes were different at 38 and 50 °C.

Conclusion

Dried shiitake mushrooms have a potential use in food processing as an enzyme preparation.

     

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification

Background

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.

Results

In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion^a (AFEX?-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.

Conclusions

Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

     

Immobilization of lactoperoxidase on graphene oxide nanosheets with improved activity and stability

Objectives

The purpose of this study was to develop a facile and efficient method to enhance the stability and activity of lactoperoxidase (LPO) by using its immobilization on graphene oxide nanosheets (GO-NS).

Methods

Following the LPO purification from bovine whey, it was immobilized onto functionalized GO-NS using glutaraldehyde as cross-linker. Kinetic properties and stability of free and immobilized LPO were investigated.

Results

LPO was purified 59.13 fold with a specific activity of 5.78 U/mg protein. The successful immobilization of LPO on functionalized GO-NS was confirmed by using dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The overall results showed that the stability of the immobilized LPO was considerably improved compared to free LPO. Apparent K_m and V_max of LPO also indicated that the immobilized enzyme had greater affinity to the substrate than the native enzyme.

Conclusions

Graphene oxide nanosheets are effective means for immobilization of LPO.

     

Regular enzyme recovery enhances cellulase production by <Emphasis Type="Italic">Trichoderma reesei</Emphasis> in fed-batch culture

Objective

To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals.

Results

Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24–62%) and β-glucosidase (22–76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and β-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5–4.5 g l^?1.

Conclusion

Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

     

Plant growth-promoting bacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> FR1 secrets a novel type of extracellular polyhydroxybutyrate polymerase involved in abiotic stress response in plants

Objectives

Identification of novel microbial factors contributing to plant protection against abiotic stress.

Results

The genome of plant growth-promoting bacterium Pseudomonas fluorescens FR1 contains a short mobile element encoding a novel type of extracellular polyhydroxybutyrate (PHB) polymerase (PhbC) associated with a type I secretion system. Genetic analysis using a phbC mutant strain and plants showed that this novel extracellular enzyme is related to the PHB production in planta and suggests that PHB could be a beneficial microbial compound synthesized during plant adaptation to cold stress.

Conclusion

Extracellular PhbC can be used as a new tool for improve crop production under abiotic stress.

     

MiRNA-145 suppresses lung adenocarcinoma cell invasion and migration by targeting N-cadherin

Objective

To investigate the roles of miR-145 in lung adenocarcinoma (LAC) and to clarify the regulation of N-cadherin by miR-145.

Results

In 57 paired clinical LAC tissues, diminished miR-145 was significantly correlated with the lymph node metastasis and was negatively correlated with N-cadherin mRNA level expression. Wound healing and transwell assays revealed a reduced capability of tumor metastasis induced by miR-145 in LAC. miR-145 negatively regulated the invasion of cell lines through targeting N-cadherin by directly binding to its 3′-untranslated region. Silencing of N-cadherin inhibited invasion and migration of LAC cell lines similar to miR-145 overexpression.

Conclusions

MiR-145 could inhibit invasion and migration of lung adenocarcinoma cell lines by directly targeting N-cadherin.

     

Weekly oral alendronate in mevalonate kinase deficiency

Background

Mevalonate kinase deficiency (MKD) is caused by mutations in the MVK gene, encoding the second enzyme of mevalonate pathway, which results in subsequent shortage of downstream compounds, and starts in childhood with febrile attacks, skin, joint, and gastrointestinal symptoms, sometimes induced by vaccinations.

Methods

For a history of early-onset corticosteroid-induced reduction of bone mineral density in a 14-year-old boy with MKD, who also had presented three bone fractures, we administered weekly oral alendronate, a drug widely used in the management of osteoporosis and other high bone turnover diseases, which blocks mevalonate and halts the prenylation process.

Results

All of the patient’s MKD clinical and laboratory abnormalities were resolved after starting alendronate treatment.

Conclusions

This observation appears enigmatic, since alendronate should reinforce the metabolic block characterizing MKD, but is crucial because of the ultimate improvement shown by this patient. The anti-inflammatory properties of bisphosphonates are a new question for debate among physicians across various specialties, and requires further biochemical and clinical investigation.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics

Introduction

Concerning NMR-based metabolomics, 1D spectra processing often requires an expert eye for disentangling the intertwined peaks.

Objectives

The objective of NMRProcFlow is to assist the expert in this task in the best way without requirement of programming skills.

Methods

NMRProcFlow was developed to be a graphical and interactive 1D NMR (¹H & ¹³C) spectra processing tool.

Results

NMRProcFlow (http://nmrprocflow.org), dedicated to metabolic fingerprinting and targeted metabolomics, covers all spectra processing steps including baseline correction, chemical shift calibration and alignment.

Conclusion

Biologists and NMR spectroscopists can easily interact and develop synergies by visualizing the NMR spectra along with their corresponding experimental-factor levels, thus setting a bridge between experimental design and subsequent statistical analyses.

     

<Superscript>123</Superscript>I-ADAM SPET imaging of serotonin transporter in patients with epilepsy and comorbid depression

Background

Purpose of the study was to investigate alterations in midbrain serotonin transporter (SERT) binding in patients with epilepsy and symptoms of depression compared to patients with epilepsy with no symptoms of depression.

Methods

We studied 12 patients with epilepsy (7 patients had focal and 5 had generalized epilepsy syndromes). The presence of self-reported symptoms of depression was assessed using Beck Depression Inventory (BDI) and the Emotional State Questionnaire (EST-Q). The binding potential of the SERT was assessed by performing brain single photon emission tomography (SPET) using the SERT radioligand 2-((2-((dimethylamino)methyl)phenyl)thio)-5-(123)iodophenylamine (¹²³I-ADAM).

Results

Seven patients had BDI and EST-Q subscale scores greater than 11 points, which was interpreted as the presence of symptoms of depression. We found that ¹²³I-ADAM binding was not significantly different between patients with epilepsy with and without symptoms of depression. In addition, ¹²³I-ADAM binding did not show a significant correlation to either BDI or EST-Q depression subscale scores and did not differ between patients with focal vs. generalized epilepsy.

Conclusion

The results of our study failed to demonstrate alterations of SERT binding properties in patients with epilepsy with or without symptoms of depression.

     

Inhibition of integrin β3, a binding partner of kallistatin,leads to reduced viability,invasion and proliferation in NCI-H446 cells

Background

Kallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear.

Methods

Pull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin β3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used.

Results

Interestingly, kallistatin was capable of binding integrin β3 on the cell surface of NCI-H446 cells. Meanwhile, integrin β3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin β3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation.

Conclusions

These findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin β3, leading to blockade of the related signaling pathway.

     

RNA binding protein Lin28A promotes osteocarcinoma cells progression by associating with the long noncoding RNA MALAT1

Objectives

To explore the effects of Lin28A on progression of osteocarcinoma (OS) cells.

Results

Lin28A mRNA and protein expressions were significantly increased in OS tissues compared with that in normal adjacent tissues. Expressions of Lin28A and long noncoding RNA MALAT1 were positively correlated. Patients with higher Lin28A expression had shorter overall survival. Moreover, Lin28A knockdown inhibited OS cells proliferation, migration, invasion and promoted cell apoptosis; Lin28A was found to harbor binding sites on MALAT1 sequences and associated with MALAT1, and increased MALAT1 stability and expression. Notably, the inhibition of Lin28A knockdown was attenuated or even reversed by MALAT1 overexpression.

Conclusions

RNA binding protein Lin28A could facilitate OS cells progression by associating with the long noncoding RNA MALAT1.

     

Phosphodiesterase 4 inhibitors and drugs of abuse: current knowledge and therapeutic opportunities

Background

Long-term exposure to drugs of abuse causes an upregulation of the cAMP-signaling pathway in the nucleus accumbens and other forebrain regions, this common neuroadaptation is thought to underlie aspects of drug tolerance and dependence. Phosphodiesterase 4 (PDE4) is an enzyme that the selective hydrolyzes intracellular cAMP. It is expressed in several brain regions that regulate the reinforcing effects of drugs of abuse.

Objective

Here, we review the current knowledge about central nervous system (CNS) distribution of PDE4 isoforms and the effects of systemic and brain-region specific inhibition of PDE4 on behavioral models of drug addiction.

Methods

A systematic literature search was performed using the Pubmed.

Results

Using behavioral sensitization, conditioned place preference and drug self-administration as behavioral models, a large number of studies have shown that local or systemic administration of PDE4 inhibitors reduce drug intake and/or drug seeking for psychostimulants, alcohol, and opioids in rats or mice.

Conclusions

Preclinical studies suggest that PDE4 could be a therapeutic target for several classes of substance use disorder. We conclude by identifying opportunities for the development of subtype-selective PDE4 inhibitors that may reduce addiction liability and minimize the side effects that limit the clinical potential of non-selective PDE4 inhibitors. Several PDE4 inhibitors have been clinically approved for other diseases. There is a promising possibility to repurpose these PDE4 inhibitors for the treatment of drug addiction as they are safe and well-tolerated in patients.

     

Pseudomyxoma extraperitonei occurring 35 years after appendicectomy: a case report and review of literature

Background

Pseudomyxoma peritonei is a rare condition consisting of mucinous ascites, most commonly arising from mucinous tumors of the appendix and occasionally from the ovary. Very rarely mucinous implants arise in the retroperitoneum without any intra-peritoneal involvement. This has been termed as pseudomyxoma extraperitonei.

Case presentation

We report a case of a 57 year old man who developed pseudomyxoma extraperitonei, 35 years after undergoing an appendicectomy for a perforated appendix.

Conclusions

Pseudomyxoma extraperitonei has been previously reported, however we report the longest incubation period of 35 years for this condition.

     

Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants

Objectives

To develop and validate a microdilution method for measuring the minimum inhibitory concentration (MIC) of biosurfactants.

Results

A standardized microdilution method including resazurin dye has been developed for measuring the MIC of biosurfactants and its validity was established through the replication of tetracycline and gentamicin MIC determination with standard bacterial strains.

Conclusion

This new method allows the generation of accurate MIC measurements, whilst overcoming critical issues related to colour and solubility which may interfere with growth measurements for many types of biosurfactant extracts.