A Novel Cold-Adapted and High-Alkaline Alginate Lyase with Potential for Alginate Oligosaccharides Preparation.

Alginate oligosaccharides (AOs) prepared through enzymatic reaction by diverse alginate lyases under relatively controllable and moderate conditions possess versatile biological activities. But widely used commercial alginate lyases are still rather rare due to their poor properties (e.g., lower activity, worse thermostability, ion tolerance, etc.). In this work, the alginate lyase Alyw208, derived from Vibrio sp. W2, was expressed in Yarrowia lipolytica of food grade and characterized in order to obtain an enzyme with excellent properties adapted to industrial requirements. Alyw208 classified into the polysaccharide lyase (PL) 7 family showed maximum activity at 35 °C and pH 10.0, indicating its cold-adapted and high-alkaline properties. Furthermore, Alyw208 preserved over 70% of the relative activity within the range of 10-55 °C, with a broader temperature range for the activity compared to other alginate-degrading enzymes with cold adaptation. Recombinant Alyw208 was significantly activated with 1.5 M NaCl to around 2.1 times relative activity. In addition, the endolytic Alyw208 was polyG-preferred, but identified as a bifunctional alginate lyase that could degrade both polyM and polyG effectively, releasing AOs with degrees of polymerization (DPs) of 2-6 and alginate monomers as the final products (that is, DPs 1-6). Alyw208 has been suggested with favorable properties to be a potent candidate for biotechnological and industrial applications.

[A cross-sectional study on the prevalence rate and influencing factors of non-alcoholic fatty liver disease in overweight/obese children]. / è¶ é‡/è‚¥èƒ–å„¿ç«¥éžé ’ç²¾æ€§è„‚è‚ªè‚ç— æ‚£ç— çŽ‡åŠå ¶å½±å“å› ç´ çš„æ¨ªæ–­é¢ç ”ç©¶.

OBJECTIVES: To investigate the prevalence rate of non-alcoholic fatty liver disease (NAFLD) in overweight/obese children who visit a hospital, and to explore the influencing factors of NAFLD, in order to provide a basis for the prevention of NAFLD in overweight/obese children. METHODS: Overweight/obese children who visited Hunan Children's Hospital from June 2019 to September 2021 were recruited. The prevalence rate of NAFLD was examined. Logistic regression analysis was used to explore the factors influencing the development of NAFLD [non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH)]. Receiver operating characteristic curve analysis was used to evaluate the predictive value of the influencing factors for NAFL and NASH. RESULTS: A total of 844 overweight/obese children aged 6-17 years were enrolled. The prevalence rate of NAFLD in overweight/obese children was 38.2% (322/844), among which the prevalence rates of NAFL and NASH were 28.8% (243/844) and 9.4% (79/844), respectively. Multivariate logistic regression analysis showed that the increase of waist-to-hip ratio (WHR) and low high-density lipoprotein cholesterol (HDL-C) were associated with the development of NAFL and NASH (P<0.05). The receiver operating characteristic curve analysis showed that the combined measurement of WHR and HDL-C had a predictive value for NAFL (area under the curve: 0.653, 95%CI: 0.613-0.694), and for NASH (area under the curve: 0.771, 95%CI: 0.723-0.819). CONCLUSIONS: The prevalence rate of NAFLD in overweight/obese children who visit a hospital is high. WHR and HDL-C are associated with the development of NAFLD and the combined measurement of WHR and HDL-C has a certain value for predicating the development of NAFLD.

Expression and Characterization of a Novel Cold-Adapted Chitosanase from Marine Renibacterium sp. Suitable for Chitooligosaccharides Preparation.

(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni-NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0-50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20-30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn2+ and Co2+. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.

Characterization and Secretory Expression of a Thermostable Tannase from Aureobasidium melanogenum T9: Potential Candidate for Food and Agricultural Industries.

Being a key industrial enzyme, tannase is extensively applied in various fields. Despite the characterizations of a large number of tannases, there are hardly a few tannases with exceptional thermostability. In this detailed study, a tannase-encoding gene named tanA was identified from Aureobasidium melanogenum T9 and heterologously expressed in Yarrowia lipolytica host of food grade. The purified tannase TanA with a molecular weight of above 63.0 kDa displayed a specific activity of 941.4 U/mg. Moreover, TanA showed optimum activity at 60°C and pH 6.0. Interestingly, TanA exhibited up to 61.3% activity after incubation for 12 h at 55°C, signifying its thermophilic property and distinguished thermostability. Additionally, TanA was a multifunctional tannase with high specific activities to catalyze the degradation of various gallic acid esters. Therefore, this study presents a novel tannase, TanA, with remarkable properties, posing as a potential candidate for food and agricultural processing.

Characterization of a New Bifunctional and Cold-Adapted Polysaccharide Lyase (PL) Family 7 Alginate Lyase from Flavobacterium sp.

Alginate oligosaccharides produced by enzymatic degradation show versatile physiological functions and biological activities. In this study, a new alginate lyase encoding gene alyS02 from Flavobacterium sp. S02 was recombinantly expressed at a high level in Yarrowia lipolytica, with the highest extracellular activity in the supernatant reaching 36.8 ± 2.1 U/mL. AlyS02 was classified in the polysaccharide lyase (PL) family 7. The optimal reaction temperature and pH of this enzyme were 30 °C and 7.6, respectively, indicating that AlyS02 is a cold-adapted enzyme. Interestingly, AlyS02 contained more than 90% enzyme activity at 25 °C, higher than other cold-adapted enzymes. Moreover, AlyS02 is a bifunctional alginate lyase that degrades both polyG and polyM, producing di- and trisaccharides from alginate. These findings suggest that AlyS02 would be a potent tool for the industrial applications.

High and efficient isomaltulose production using an engineered Yarrowia lipolytica strain.

Isomaltulose is an ideal functional sweetener and has been approved as a safe sucrose substitute. It is produced mainly through sucrose isomerization catalyzed by sucrose isomerase. Here, to produce food-grade isomaltulose and improve its yield, the sucrose isomerase gene from Pantoea dispersa UQ68J was overexpressed in the non-pathogenic yeast Yarrowia lipolytica. When the engineered strain, S47, was fermented on 600 g/L sucrose in a 10-L bioreactor, a maximum isomaltulose concentration of 572.1 g/L was achieved. Sucrose isomerase activity was 7.43 U/mL, and yield reached 0.96 g/g. Moreover, monosaccharide byproducts were simultaneously transformed into intracellular lipids, thus reducing the production of undesirable compounds and resulting in high isomaltulose purity (97.8%) in the final broth. In summary, the bioprocess employed in this study provides an efficient alternative strategy for isomaltulose production.

Hydrocarbons, the advanced biofuels produced by different organisms, the evidence that alkanes in petroleum can be renewable.

It is generally regarded that the petroleum cannot be renewable. However, in recent years, it has been found that many marine cyanobacteria, some eubacteria, engineered Escherichia coli, some endophytic fungi, engineered yeasts, some marine yeasts, plants, and insects can synthesize hydrocarbons with different carbon lengths. If the organisms, especially some native microorganisms and engineered bacteria and yeasts, can synthesize and secret a large amount of hydrocarbons within a short period, alkanes in the petroleum can be renewable. It has been documented that there are eight pathways for hydrocarbon biosynthesis in different organisms. Unfortunately, most of native microorganisms, engineered E. coli and engineered yeasts, only synthesize a small amount of intracellular and extracellular hydrocarbons. Recently, Aureobasidium pullulans var. melanogenum isolated from a mangrove ecosystem has been found to be able to synthesize and secret over 21.5 g/l long-chain hydrocarbons with a yield of 0.275 g/g glucose and a productivity of 0.193 g/l/h within 5 days. The yeast may have highly potential applications in alkane production.

Enhanced production of Ca²âº-polymalate (PMA) with high molecular mass by Aureobasidium pullulans var. pullulans MCW.

BACKGROUND: Polymalic acid (PMA) has many applications in food and medical industries. However, so far it has not been commercially produced by fermentation. Therefore, it is very important how to develop an economical process for a large scale production of PMA by one step fermentation. RESULTS: After over 200 strains of Aureobasidium spp. isolated from the mangrove systems in the South of China were screened for their ability to produce Ca(2+)-polymalate (PMA), it was found that Aureobasidium pullulans var. pullulans MCW strain among them could produce high level of Ca(2+)-PMA. The medium containing only 140.0 g/L glucose, 65.0 g/L CaCO3 and 7.5 g/L corn steep liquor was found to be the most suitable for Ca(2+)-PMA production. Then, 121.3 g/L of Ca(2+)-PMA was produced by A. pullulans var. pullulans MCW strain within 120 h at flask level. During 10-L batch fermentation, 152.52 g/L of Ca(2+)-PMA in the culture and 8.6 g/L of cell dry weight were obtained within 96 h, leaving 4.5 g/L of reducing sugar in the fermented medium. After purification of the Ca(2+)-PMA from the culture and acid hydrolysis of the purified Ca(2+)-PMA, HPLC analysis showed that A. pullulans var. pullulans MCW strain produced only one main component of Ca(2+)-PMA and the hydrolysate of the purified Ca(2+)-PMA was mainly composed of L-malic acid. Mw (the apparent molecular weight) of the purified PMA was 2.054 × 10(5) (g/moL) and the purified PMA was estimated to be composed of 1784 L-malic acids. CONCLUSIONS: It was found that A. pullulans var. pullulans MCW strain obtained in this study could yield 152.52 g/L of Ca(2+)-PMA within the short time, the produced PMA had the highest molecular weight and the medium for production of Ca(2+)- PMA by this yeast was very simple.