Integrative analyses of transcriptome and carotenoids profiling revealed molecular insight into variations in fruits color of Citrus Reticulata Blanco induced by transplantation.

Citrus fruits exhibit vivid color and are favored extensively. However, the biochemical and molecular mechanism of Citrus Reticulata Blanco fruits coloring, especially the effect of transplantation on fruits coloring, is unclear. Herein, RNA-Seq and carotenoids profiling were applied to investigate the effect of transplantation on Orah mandarin fruits coloring. Transplantation induces fruit color shallowing, Ca2+ and ACC level declining and IAA level increasing. Transplantation induced variation in fruit skin and pulp carotenoids, mainly ß-citraurin as one of the important pigments of citrus peel. 2253 up-regulated genes, 1103 down-regulated genes in skin and 815 up-regulated genes, 534 down-regulated genes in pulp of transplanted tree fruits were identified by RNA-Seq. The DEGs involved hormone signal, carotenoids biosynthesis and TFs such as MYB and bHLH family TFs. The carotenoid cleavage dioxygenase gene (Ciclev10028113m.g) is positively correlated with ß-citraurin and regulated directly and/or indirectly by MYB1R1, PIF4, ACC and IAA. Integrative analyses revealed potential molecular insights into Orah mandarin peel color variation during transplantation.

Identification of Chitinolytic Enzymes in Chitinolyticbacter meiyuanensis and Mechanism of Efficiently Hydrolyzing Chitin to N-Acetyl Glucosamine.

Chitinolyticbacter meiyuanensis SYBC-H1, a bacterium capable of hydrolyzing chitin and shrimp shell to N-acetyl glucosamine (GlcNAc) as the only product, was isolated previously. Here, the hydrolysis mechanism of this novel strain toward chitin was investigated. Sequencing and analysis of the complete genome of SYBC-H1 showed that it encodes 32 putatively chitinolytic enzymes including 30 chitinases affiliated with the glycoside hydrolase (GH) families 18 (26) and 19 (4), one GH family 20 ß-N-acetylglucosaminidase (NAGase), and one Auxiliary Activities (AA) family 10 lytic polysaccharide monooxygenase (LPMO). However, only eight GH18 chitinases, one AA10 LPMO, and one GH20 NAGase were detected in the culture broth of the strain, according to peptide mass fingerprinting (PMF). Of these, genes encoding chitinolytic enzymes including five GH18 chitinases (Cm711, Cm3636, Cm3638, Cm3639, and Cm3769) and one GH20 NAGase (Cm3245) were successfully expressed in active form in Escherichia coli. The hydrolysis of chitinous substrates showed that Cm711, Cm3636, Cm3638, and Cm3769 were endo-chitinases and Cm3639 was exo-chitinase. Moreover, Cm3639 and Cm3769 can convert the GlcNAc dimer and colloidal chitin (CC) into GlcNAc, which showed that they also possess NAGase activity. In addition, NAGase Cm3245 possesses a very high exo-acting activity of hydrolyzing GlcNAc dimer. These results suggest that chitinases and NAGase from SYBC-H1 both play important roles in conversion of N-acetyl chitooligosaccharides to GlcNAc, resulting in the accumulation of the final product GlcNAc. To our knowledge, this is the first report of the complete genome sequence and chitinolytic enzyme genes discovery of this strain.

The Draft Genome Sequence and Analysis of an Efficiently Chitinolytic Bacterium Chitinibacter sp. Strain GC72.

A novel chitinolytic bacterium Chitinibacter sp. GC72, which produces an enzyme capable of efficiently converting chitin only into N-acetyl-D-glucosamine (GlcNAc), was successfully sequenced and analyzed. The assembled draft genome of strain GC72 is 3,455,373 bp, containing 3346 encoded protein sequences with G + C content of 53.90%. Among these annotated genes, 17 chitinolytic enzymes including 12 glycoside hydrolase family 18 chitinases, three family 19 chitinases, one family 20 ß-hexosaminidase, and one auxiliary activity family 10 lytic polysaccharide monooxygenase, were found to be essential in the production of GlcNAc from chitin. The genomic information of strain GC72 provides a reference genome for Chitinibacter bacteria and abundant novel chitinolytic enzyme resources, and allows researchers to explore potential applications in GlcNAc enzymatic production.

A novel bacterial ß-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities.

BACKGROUND: N-Acetyl glucosamine (GlcNAc) and N-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4-(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a ß-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs. RESULTS: The gene encoding the novel ß-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12-35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The V max, K m, K cat, and K cat/K m of CmNAGase for pNP-GlcNAc were 16,666.67 µmol min-1 mg-1, 0.50 µmol mL-1, 25,555.56 s-1, and 51,111.12 mL µmol-1 s-1, respectively. Analysis of the hydrolysis products of N-Acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-Acetyl COSs ((GlcNAc)3-(GlcNAc)7) from (GlcNAc)2-(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. CONCLUSIONS: The observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.

Degradation and the antioxidant activity of polysaccharide from Enteromorpha linza.

Polysaccharide extracted from Enteromorpha linza possesses excellent antioxidant activities, but its molecular weight was greatly high which influences the activity. In this study, the combination of ascorbic acid and H(2)O(2) was used as degradation reagents in order to obtain the lower molecular weight product. The results suggested that the most effective molar ratio of the two reagents was 1. Three degraded polysaccharides were selected to evaluate their antioxidant activities in vitro and characterized the relationship between antioxidant activity and chemical characteristics. It was found that the degraded sample with lower molecular weight possessed the higher antioxidant activities.