Abstract:The O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a post-translational modification that transfers N-acetylglucosamine to serine and/or threonine residues of target proteins. O-GlcNAcylation has been the research hotspot since it was first reported by Gerald Hart in the early 1980s. O-GlcNAcylation is so dynamical, inducible and active that it satisfies the prerequisites of the role of protein post-translational modification in signal transductions. In most cases, a competitive inhibition occurs between O-GlcNAcylation and phosphorylation due to their same modification sites in proteins, which is also called "Yin-yang" balance. O-GlcNAcylation has been proved to be associated with multiple cellular signaling pathways, including the regulation of growth, the effect on proliferation, and the response to hormone. Also, O-GlcNAcylation plays a key role in the development of metabolic diseases such as diabetes, neurodegenerative diseases and cancers. Based on the above, it is of great significance to explicate the bio-function of O-GlcNAcylation in physiological and pathological processes.

Abstract:The glmS ribozyme is predominantly present in gram-positive bacteria, and it is a riboswitch that inhibits the synthesis of glucosamine-6-phosphate. In addition, glmS riboswitch is a self-cleft ribozyme located in the 5' untranslated region of the glmS gene. Study on the structure and function of glmS riboswitch will be beneficial to develop new targets for antibiotic action. In this paper, we reviewed the structure and function of glmS riboswitch. In addition, we also introduce recent research progress and application of glmS riboswitch.

Abstract:In nature, the biological degradation of carbohydrates is one of the most important reactions. Carbohydrate-active enzymes are enzymes involved in the synthesis, degradation and modification of carbohydrates. Due to the insolubility of some high-molecular weight polysaccharide substrates, the catalytic efficiency of some carbohydrate-active enzymes is low. Carbohydrate-binding modules (CBM) specifically bind carbohydrate substrates and improve the catalytic efficiency of carbohydrate-active enzymes by elevating the substrate concentrations around the catalytic modules. This review summarizes the knowledge of CBM, including their nomenclature and classification, the relationship between CBM structures and functions, and so on. Furthermore, some applications of CBM in biological and medical engineering are also presented.

Abstract:Organophosphorus compounds are highly toxic chemicals and widely used as insecticides, plasticizers and flame retardants. Due to the difficulty of degradation these toxic chemicals have gradually accumulated in agricultural products, waters and soils, raising serious public concerns regarding health, environment and food safety. The enzymatic degradation of organophosphorus compounds is highly efficient and environmental friendly, which is the current research hotspot. This paper reviewed recent advances in discovery, engineering and applications of organophosphorus hydrolases, and highlighted the current challenges and the future research directions with the aim to provide an insight into the biodegradation of organophosphorus compounds.

Abstract:Halophilic enzymes are derived naturally from halophilic bacteria that survive in high salt environment, as it can maintain the structural stability of the enzyme only in high salt environment. These enzymes can withstand high temperature, pH and organic solvents, so they can be widely used for their catalytic activity in high salt, water/organic and non-aqueous environment. In this review, we address the effect of salt on the activity and stability of halophilic enzymes, and the role of metal ions and organic solvents on the halophilic enzymes. In addition, molecular modification and the application of halophilic enzymes were introduced.

Abstract:β-N-Acetylhexosaminidases (EC.3.2.1.52) are an important class of glycosidases that catalyze hydrolysis of terminal N-acetyl-β-D-hexosamine from various oligosaccharides and polysaccharides. These enzymes are widely distributed in microorganisms, plants, and animals, and play crucial roles in nature. Some β-N-acetylhexosaminidases can catalyze glycosyl transfer to form β-N-acetyl-D-hexosaminides, which have shown great potentiality in enzymatic synthesis of functional glycans. In this review, catalytic mechanisms and biological functions of β-N-acetylhexosaminidases, and potential applications of these enzymes in the synthesis of β-N-acetyl-D-hexosaminide are summarized.

Abstract:Biofilms have been associated with a variety of persistent fungal infections that respond poorly to conventional treatments. Forming biofilm could help fungi escape from host immune system defense and antimicrobial treatment. Fungal biofilms have emerged as a clinical problem associated with persistent infections, causing significant morbidity and mortality. Candida albicans is the most common fungal pathogen in humans, causing mucosal infections as well as life-threatening systemic infections. Biofilm of Candida albicans has been relatively well studied among fungal pathogens. Recent studies show that the extracellular matrix of Candida albicans biofilm consists of proteins, DNA and polysaccharides. Moreover, quorum sensing, environmental nutrition and surface materials affect the formation of Candida albicans biofilms. Other studies reveal that physical barrier function of the extracellular matrix, specific genetic manipulation and other mechanisms might contribute to the drug-resistance of fungal biofilms. This review discusses the recent advances in the understanding of Candida albicans biofilms, including the formation process, structural components, factors of formation, research models, drug-resistance mechanisms and potential treatment strategies.

Abstract:Glycosides widely distributed in nature have many pharmacological activities. A lot of drugs belong to glycoside and the sugar chains are closely related to its pharmacological activities. The modification of sugar chains can change the pharmacological activities of glycosides, which provides tremendous glycoside resource for drug development. Because of high efficiency and low pollution, biotransformation by glycosidase is widely used in the preparation of active glycosides. This review summarized recent advances in the preparation of active glycosides by glycosidase transformation, and provided useful references for preparing active glycosides and related research work.

Abstract:Conversion from lignocellulose to biofuel with enzyme hydrolysis will be beneficial for sustainable development. Most microorganisms used in the industrial production of cellulase are filamentous fungi. However, isolation of cellulase hyper producing strain is limited by the lack of proven technology on genetic modification of filamentous fungi and the mechanism of cellulase synthesis. In this paper, recent progress in the genetic modification of cellulase hyper producing strains are summarized. Specially, we discussed the influences of signal induction, signal transduction and transcriptional regulation on cellulase synthesis in filamentous fungi. Finally, the industrial application of filamentous fungi was also introduced.

Abstract:Amino acid dehydrogenases catalyze the reversible oxidative deamination of amino acids and reductive amination of keto acids, with thermodynamic equilibrium favoring the reductive amination direction. This is one of the most attractive methods for the synthesis of amino acids with high atomic economy and less environmental impact. This review addresses recent advances in studies on reaction mechanism, molecular modification and synthetic applications of amino acid dehydrogenases.

Abstract:[Objective] Instead of standard PCR setting a single annealing temperature (S-T_m), we studied double annealing temperature PCR (D-T_m PCR) setting respective annealing temperature for forward and reverse primers from higher to lower. [Methods] A 4.3 kb pET20b-Xyn (Aspergillus niger xylanase gene) model DNA was amplified with Q5 DNA polymerase by using PxF61 and VPel as forward and reverse primers. The PCR procedure was:pre-denaturation at 98℃ for 3 min, and 30 cycles of denaturation at 98℃ for 30 s, annealing at T_m1 70℃ (PxF61) for 15 s and at T_m2 62℃ (VPel) for 15 s, extension at 72℃ for 130 s. [Results] The 4.3 kb target DNA band of D-T_m PCR was a little brighter, whereas non-specific DNA bands were two less than those of the S-T_m PCR (T_m=61℃). Twenty-five cycles of amplification created the brightest target DNA band in the D-T_m PCR. A 5.3 kb recombinant plasmid DNA was clearly amplified in the D-T_m PCR than the S-T_m PCR. [Conclusion] The D-T_m PCR amplified directly target DNA band without demanding for investigation of an optimal annealing temperature and for setting closer annealing temperatures between forward and reverse primers. Moreover, two respective annealing steps were clearly elucidated from theoretical viewpoint.

Abstract:[Objective] We intended to discover and characterize a novel beta-N-acetylhexosaminidase from Solitalea canadensis. [Methods] Genomic DNA extracted from Solitalea canadensis was used as the template for gene cloning of the beta-N-acetylhexosaminidase using PCR reaction. The PCR product was digested with restriction endonucleases Nde I and Xho I, then ligated to pET-30a vector. After plasmid was transformed into E.coli BL21 (DE3) cells, the recombinant enzyme was expressed by IPTG induction and purified with nickel affinity chromatography. Characterization of recombinant SoCaHexNAc including optimal pH and temperature, metal ions dependency and inhibitor was done using pNP-β-GlcNAc as the substrate. Effect of different chemical compounds and disaccharides on the enzyme activity was also measured. [Results] A beta-N-acetylhexosaminidase gene with an open reading fragment of 2586 bp was successfully obtained, which encodes 856 amino acids with a putative molecular size of 97 kDa. The results of SDS-PAGE revealed that the recombinant SoCaHexNAc (GeneBank accession number:WP_014682183.1) was expressed and purified successfully. Characterization of the enzyme showed that the optimum pH of SoCaHexNAc is 6.0, and the optimum temperature is 42℃ with a half-life being less than 5 minutes. The recombinant SoCaHexNAc was sensitive to SDS and could be partly inhibited by Trition X-100 and urea. Different concentrations of lactose, maltose and cellobiose could also inhibit the activity of SoCaHexNAc to different extends. The IC₅₀ of a specific β-N-acetylhexosaminidase inhibitor, PugNAc, was 2 μmol/L. The substrate specificity result showed that the recombinant SoCaHexNAc was active to pNP-GlcNAc and pNP-GalNAc. When being used for the hydrolysis of GlcNAc from natural glycans, the recombinant SoCaHexNAc exhibited linkage specificity evidenced by the fact that only β 1,6-linked GlcNAc in Core Ⅱ structure, but not the β 1,4-linked GlcNAc in NGA2 structure, was removed, although the terminal GlcNAc was the exceptional terminal sugar in both substrates. [Conclusion] A beta-N-actylhexosamindase with activity specifically towards β 1,6-linked but not β 1,4-linked GlcNAc was discovered and characterized from Solitalea canadensis for the first time. The results of characterization and substrate specificity showed it might be a potential novel tool enzyme which could be used in structure analysis of glycans.

Abstract:[Objective] The aim of this study was to identify a novel L-cysteine sulfinate decarboxylase from alkaline polluted soil metagenome and to use non-rational design method to improve the enzyme. [Methods] L-cysteine sulfinate decarboxylase gene undec1A was cloned into pETBlue-2 vector and expressed in Escherichia coli Tuner (DE3) pLacI. The recombinant Undec1A protein was purified to homogeneity. The original Undec1A protein was characterized and a mutagenesis library was constructed with sequential error-prone PCR method, then an interesting variant was identified. [Results] Multiple sequence alignment analysis showed that Undec1A protein shared the similar pyridoxal 5'-phosphate binding sites and the substrate recognition motif with the other known L-cysteine sulfinate decarboxylases. Molecular docking results indicated that amino acid residues Val237, Asp239, Asp266, Ile267, Ala268 and Lys298 contributed to the decarboxylation of L-cysteine sulfinate acid. Recombinant Undec1A protein had an apparent K_m of (1.557±0.015) mmol/L, a V_max of (49.07±3.19) μmol/(L·min), and a k_cat of (45.80 ±1.32)/min at the optimal reaction conditions of 35℃ and pH 7.0 when using L-cysteine sulfinate acid as the substrate. Furthermore, the protein engineering approach of random mutagenesis via sequential error-prone PCR was applied on the original Undec1A protein. Compared with the original Undec1A protein, the best variant of Undec1A-1180 in the random mutagenesis library, exhibited 5.62-folds at the optimal reaction conditions of 35℃ and pH 7.0. [Conclusion] These results are the first step towards a better understanding of the properties of Undec1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.

Abstract:[Objective] To obtain mutants of Aspergillus niger with high glucoamylase activity, we developed a screening method. [Methods] We mutagenized the starting strain A. niger X1 using diethyl sulfate, then cultured the mutant library on agar plate containing 2-deoxyglucose. By increasing the concentration of 2-deoxyglucose, we obtained mutants with high resistance to 2-deoxyglucose and then studied glucoamylase activities. [Results] In shake flask fermentation, glucoamylase activity of the mutant strain A. niger DG36 increased by 22.2% to 33.8%. In a 50 m³ fermenter, glucoamylase activity of the strain DG36 reached up to 49094 U/mL at 128 h, 32.8% higher than that of the starting strain A. niger X1. As a result, the fermentation period of the strain DG36 was reduced by 16.9% compared with A. niger X1. [Conclusion] Mutant strain DG36 exhibited higher glucoamylase activity, shorter fermentation period and more suitable for the purification treatment than the starting strain A. niger X1.

Abstract:[Objective] To obtain efficient raw rice starch-digesting enzymes. [Methods] α-Amylase AmyP can hydrolyze raw rice starch efficiently. We constructed a chimeric α-amylase (AmyP-Clo) by fusion of the catalytic domain of AmyP and a starch binding domain of α-amylase from Clostridium butyricum T-7. [Results] AmyP-Clo retained the advantages of AmyP, and increased catalytic efficiency towards raw rice starch. The specific activity was (373.9±8.4) U/mg. The final hydrolysis degree was (42.7±1.1)% for the hydrolysis of 5% raw rice starch suspension after 4 h. The maximal fraction of bound protein was (71.1±1.6)%. [Conclusion] AmyP-Clo could efficiently hydrolyze raw rice starch.