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Background:Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed.Objective:In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively).Methods:Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package.Results:According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and β-sheet, β-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme.Conclusion:This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

A novel β-glucosidase gene was isolated from Pseudoalteromonas sp. GXQ-1 and heterologously expressed in Escherichia coli. The activity of the encoded enzyme, PABGL, toward p-nitrophenyl-β-D-glucopyranoside was increased 8.74-fold by the presence of 3 M NaCl relative to the absence of added NaCl. PABGL hydrolyzed a variety of soy isoflavone substrates. For the conversion of daidzin to daidzein, the production rate was 1.44 mM/h. The addition of NaCl enhanced the hydrolytic activity of PABGL toward daidzin and genistein; the maximum activation by NaCl was 3.48- and 6.79-fold, respectively. This is the first report of a halophilic β-glucosidase from Pseudoalteromonas spp., and represents the β-glucosidase with the highest multiple of activation by NaCl. PABGL exhibits strong potential for applications in food processing and industrial production.

Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of &alpha, 2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction,the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.

3D graphene (or graphene-like porous carbon) is prepared from precursor lignin separated and purified from alkaline pulp black liquor. The lignin is pretreated using rapid freeze-dry technique for the porous, soft and dispersive microstructure. A special temperature control program is designed to keep the porous structure of lignin particles in the carbonization and graphitization process, avoiding agglomeration. The 3D graphene from lignin consist of curve graphene sheets in dimensions around dozen micrometers, which merge each other with clear dihedral angle and form wood-ear like architecture, different from fullerene like carbon crystals. The porous carbon is characterized and analyzed with FTIR, Raman, XRD, SEM, TEM, XPS, and SSA. A possible chemical mechanism in the synthesis of 3D graphene from lignin precursor is proposed. The aromatic groups and alkane fragments in lignin are decomposed during pyrolysis under high temperature, then the benzene rings compose the graphene sheets, and the sp3 free carbon atoms join the graphene sheets with chemical bonds along the framework of lignin polymer, forming the self-supporting and self-constructing 3D graphene, or porous carbon.

The aim of this study was to rationally engineer the acidic adaptation of B. subtilis Ca-independent alpha-amylase (Amy7C) by decreasing the pKa values of catalytic residues through mutations at active site. Within 4.5 A of three catalytic residues of Amy7C, three mutations R172 K, A270 K and N271H were identified by computational homology modeling and pKa prediction analyses. Five single and double mutants consisting of these three mutations were constructed and characterized. Compared to the wild-type, all mutants shifted the pH optima and pH-activity profiles toward lower pH values without comprising the thermostablity. Double mutants showed simultaneous accumulation of advantageous mutations. The best mutant, A270 K/N271H showed 2 units decrease in optimum pH and about 3.94-fold increase of catalytic efficiency. Structural analysis suggested that the improved acidic adaptation could be attributed to the decreased pKa values of catalytic nucleophile and proton donor residues. Protein engineering of α-amylase for acidic adaptation here provides a successful example of the extent to which mutations near active site and computational models can be used for industrial enzyme improvements.

A novel process of enzyme pretreatment and semi-simultaneous saccharification and fermentation (SSSF) was developed in this work to improve succinic acid (SA) productivity from duckweed (Landoltia punctata) and achieve low viscosity. Viscosity (83.86%) was reduced by the pretreatment with combined enzymes at 50 °C for 2 h to a greater extent than that by single enzyme (26.19–71.75%). SSSF was an optimal combination with 65.31 g/L of SA content, which was remarkably higher than those obtained through conventional separate hydrolysis and fermentation (62.12 g/L) and simultaneous saccharification and fermentation (52.41 g/L). The combined approach was effective for SA production. Approximately 75.46 g/L of SA content with a yield of 82.87% and a productivity of 1.35 g/L/h was obtained after 56 h in a 2 L bioreactor. Further studies will focus on increasing the working scale of the proposed method.

ST8Sia II (STX) is a highly homologous mammalian polysialyltransferase (polyST), which is a validated tumor-target in the treatment of cancer metastasis reliant on tumor cell polysialylation. PolyST catalyzes the synthesis of α2,8-polysialic acid (polySia) glycans by carrying out the activated CMP-Neu5Ac (Sia) to N- and O-linked oligosaccharide chains on acceptor glycoproteins. In this review article, we summarized the recent studies about intrinsic correlation of two polybasic domains, Polysialyltransferase domain (PSTD) and Polybasic region (PBR) within ST8Sia II molecule, and suggested that the critical amino acid residues within the PSTD and PBR motifs of ST8Sia II for polysialylation of Neural cell adhesion molecules (NCAM) are related to ST8Sia II activity. In addition, the conformational changes of the PSTD domain due to point mutations in the PBR or PSTD domain verified an intramolecular interaction between the PBR and the PSTD. These findings have been incorporated into Zhou’s NCAM polysialylation/cell migration model, which will provide new perspectives on drug research and development related to the tumor-target ST8Sia II.

Enzyme specificity and particularity is needed not only in enzymatic separation methods, but also in enzymatic determination methods for plant compound extraction. Stevioside, rubusoside, and rebaudioside A are natural sweet compounds from plants. These compounds have the same skeleton and only contain different side-chain glucosyl groups, making them difficult to separate. However, enzymes that target diterpenoid compounds and show specific activity for side-chain glucosyl groups are rare. Herein, we report the identification and characterization of an enzyme that can target both diterpenoid compounds and sophorose, namely, β-glucosidase SPBGL1 from Sphingomonas elodea ATCC 31461. SPBGL1 displayed high specificity toward sophorose, and activity toward stevioside, but not rebaudioside A. The stevioside conversion rate was 98%. SPBGL1 also operated at high substrate concentrations, such as in 50% crude steviol glycoside extract. Glucose liberated from stevioside was easy to quantify using the glucose oxidase method, allowing the stevioside content to be determined.

In order to produce rubusoside, enzymes with preferential specificity for the saccharide sophorose were tested for ability to produce rubusoside from stevioside. We identified BGL1, a β-glucosidase from Streptomyces sp. GXT6, as an enzyme for rubusoside production. Out of several saccharide substrates, BGL1 showed the most affinity to sophorose. This enzyme only hydrolyzes the glucose moiety of the sophoroside at C-13 in stevioside. Production of rubusoside was determined by (1)H and (13)C nuclear magnetic resonance (NMR). Thus, rubusoside was produced from stevioside and the stevioside conversion rate was 98.2 %. The production yield of rubusoside was 78.8 % in 6 h.

As a subset of glycosyltransferases, the family of sialyltransferases catalyze transfer of sialic acid (Sia) residues to terminal non-reducing positions on oligosaccharide chains of glycoproteins and glycolipids, utilizing CMP-Neu5Ac as the activated sugar nucleotide donor. In the four known sialyltransferase families (ST3Gal, ST6Gal, ST6GalNAc and ST8Sia), the ST8Sia family catalyzes synthesis of α2, 8-linked sialic/polysialic acid (polySia) chains according to their acceptor specificity. We have determined the 3D structural models of the ST8Sia family members, designated ST8Sia I (1), II(2), IV(4), V(5), and VI(6) using the Phyre2 server. Accuracy of these predicted models are based on the ST8Sia III crystal structure as the calculated template. The common structural features of these models are: (1) Their parallel templates and disulfide bonds are buried within the enzymes and are predominately surrounded by helices; (2) The anti-parallel β-sheets are located at the N-terminal region of the enzymes; (3) The mono-sialytransferases (mono-STs), ST8Sia I and ST8Sia VI, contain only a single pair of disulfide bonds, and there are no anti-parallel β-sheets in ST8Sia VI; (4) The Nterminal region of all of the mono-STs are located some distant away from their core structure; (5) These conformational features show that the 3D structures of the mono-STs are less compact than the two polySTs, ST8Sia II and ST8Sia IV, and the oligo-ST, ST8Sia III. These structural features relate to the catalytic specificity of the monoSTs; (6) In contrast, the more compact structural features of ST8Sia II, ST8Sia IV and ST8Sia III relate to their ability to catalyze the processive synthesis of oligo- (ST8Sia III) and polySia chains (ST8Sia IIamp; ST8Sia IV); (7) Although ST8Sia II, III and IV have similar conformations in their corresponding polysialyltransferase domain (PSTD) and polybasic region (PBR) motifs, the structure of ST8Sia III is less compact than ST8Sia II and ST8Sia IV, and the amino acid components of the several three-residue-loops in the two motifs of ST8Sia III are different from that in ST8Sia II and ST8Sia IV. This is likely the structural basis for why ST8Sia III is an oligoST and not able to polysialylate and; (8) In contrast, essentially all amino acids within the threeresidue- loops in the PSTD of ST8Sia II and ST8Sia IV are highly conserved, and many amino acids in the loops and the helices of these two motifs are critical for NCAM polysialylation, as determined by mutational analysis and confirmed by our recent NMR results. In summary, these new findings provide further insights into the molecular mechanisms underlying polyST-NCAM recognition, polySTpolySia/ oligoSia interactions, and polysialylation of NCAM.

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α2β2γ2) structure with a native molecular mass of 210 kDa. It requires coenzyme B12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K m values for coenzyme B12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B12-dependent diol dehydratase and not a glycerol dehydratase.

A β-glucosidase gene unbgl1A was isolated by the function-based screening of a metagenomic library and the enzyme protein was expressed in Escherichia coli, purified, and biochemically characterized. The enzyme Unbgl1A had a Km value of 2.09 ± 0.31 mM, and a Vmax value of 183.90 ± 9.61 μmol min−1 mg−1 under the optimal reaction conditions, which were pH 6.0 at 50°C. Unbgl1A can be activated by a variety of monosaccharides, disaccharides, and NaCl, and exhibits a high level of stability at high concentration of NaCl. Two prominent features for this enzyme are: (i) high glucose tolerance. It can be tolerant to glucose as high as 2000 mM, with Ki = 1500 mM; (ii) high NaCl tolerance. Its activity is not affected by 600 mM NaCl. The enzyme showed transglucosylation activities resulting in the formation of cellotriose from cellobiose. These properties of Unbgl1A should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass. Keywords β-glucosidase; glucose tolerance; metagenome

A new trehalose synthase (TreS) gene from Thermomonospora curvata DSM 43183 was cloned and expressed in Escherichia coli XL10-Gold. The purified recombinant enzyme (TreS-T.C) could catalyze the reversible interconversion of maltose and trehalose of sucrose into trehalulose without other disaccharides including isomaltulose at an optimum temperature of 35 °C and a pH of 6.5. The Km of TreS-T.C for maltose (96 mM) was lower than those for trehalose (198 mM) and sucrose (164 mM), suggesting that maltose is the optimum substrate. The maximum trehalose and trehalulose yields were 70% and >80%, respectively. Active TreS-T.C is a trimer comprising three identical 60 kDa subunits. Homology modeling analysis revealed that TreS-T.C had a GH13-typical (β/α)8 barrel catalytic domain. Two sites, one determining substrate specificity (L116) and the other affecting product formation (E330), were found near the active center by homology modeling combined with site-directed mutagenesis. TreS-T.C may be used effectively as a potential biocatalyst for the production of trehalose and trehalulose from maltose and sucrose in a one-step reaction, respectively. This study also provides a feasible and effective method for studying functional amino acid residues around TreS without performing crystal structure analysis and high-throughput screening.

Sucrose hydrolytic enzymes, widely used in a variety of food industries, employ sucrose as a substrate. In addition to their hydrolysis activities, they have other recently discovered characteristics that should make them useful for medical applications. Here, the two enzymes sucrose phosphorylase and invertase are discussed. Sucrose phosphorylase glycosylates non-carbohydrate small molecules and invertase can be used in a portable and personal biosensor to quantify a variety of analytical targets.

Endo-polygalacturonases play an important role on depectinization in fruit juices industry. A putative endo-polygalacturonase gene PoxaEnPG28A was cloned from Penicillium oxalicum CZ1028. PoxaEnPG28A consisted of a putative signal peptide and a catalytic domain belonging to glycoside hydrolase family 28, and it shared 72% identity with that of a functionally characterized endo-polygalacturonase from Trichoderma harzianum. Gene PoxaEnPG28A was successfully expressed in Pichia pastoris with a high yield of 1828.7 U/mL. The purified recombinant enzyme PoxaEnPG28A hydrolyzed polygalacturonic acid in endo-manner releasing oligo-galacturonates. PoxaEnPG28A showed maximal activity at pH 5.5 and 55°C, and was stable between pH 3.0 to 10.0 and below 45°C. The kinetic constants Km and Vmax of PoxaEnPG28A were calculated as 1.57 g/L and 14,641.29 U/mg, respectively. PoxaEnPG28A significantly improved the yields of fruit juices from banana, plantain, papaya, pitaya and mango. The high production level of the recombinant enzyme PoxaEnPG28A by P. pastoris and remarkable catalytic activity of PoxaEnPG28A toward five kinds of fruit juices made the enzyme a potential application in agriculture and food industries.

Acidic endo-polygalacturonases are the major part of pectinase preparations and extensively applied in the clarification of fruits juice, vegetables extracts, and wines. However, most of the reported fungal endo-polygalacturonases are active and stable under narrow pH range and low temperatures. In this study, an acidic endo-polygalacturonase (EPG4) was purified and characterized from a mutant strain of Penicillium oxalicum. The N-terminal amino acid sequence of EPG4 (ATTCTFSGSNGAASASKSQT) was different from those of reported endopolygalacturonases. EPG4 displayed optimal pH and temperature at 5.0 and 60-70°C towards polygalacturonic acid (PGA), respectively, and was notably stable at pH 2.2-7.0. When tested against pectins, EPG4 showed enzyme activity over a broad acidic pH range (>15.0% activity at pH 2.2-6.0 towards citrus pectin; and >26.6% activity at pH 2.2-7.0 towards apple pectin). The Km and Vmax values were determined as 1.27 mg/ml and 5,504.6 U/mg, respectively. The enzyme hydrolyzed PGA in endo-manner, releasing oligo-galacturonates from PGA, as determined by TLC. Addition of EPG4 (3.6 U/ml) significantly reduced the viscosity (by 42.4%) and increased the light transmittance (by 29.5%) of the papaya pulp, and increased the recovery (by 24.4%) of the papaya extraction. All of these properties make the enzyme a potential application in the beverage industry.

Sucrose phosphorylase, an important enzyme mainly involved in the generic starch and sucrose pathways, has now caught the attention of researchers due to its transglycosylation activity. A novel sucrose phosphorylase, unspase, has been isolated, and its transglycosylation properties were characterized. Compared with Bisp, the sucrose phosphorylase from Bifidobacterium adolescentis, unspase had two deleted regions in its C: -terminal. These deleted regions were probably equivalent to the important five-stranded anti-parallel β-sheet domain in sucrose phosphorylase. Unspase has a k(m) of 21.12 mM, a V(max) of 69.24 μmol min(-1) mg(-1) and a k(cat) of 31.19 s(-1) with sucrose as substrate. In 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, unspase transferred the glycosyl moiety to L-arabinose, D-fructose and L-sorbose. Much to our surprise, unspase can catalyze the transglycosylation in which a glycosyl moiety was transferred to L-arabinose in the presence of phosphate, which is an interesting exception to the generally accepted fact that transglycosylation can only occur under the condition of phosphate absence. The final yield of the transglycosylation product (37.9 %) in phosphate buffer was even higher than that (5.8 %) in MOPS buffer. This is a novel phenomenon that a sucrose phosphorylase can catalyze a transglycosylation reaction in the presence of phosphate.

1, 3-propanediol (1, 3-PD) is biologically synthesized by glycerol dehydratase (GDHt) and 1, 3-propanediol dehydrogenase (PDOR). In present study, the gldABC gene, encoding GDHt from Klebsiella pneumoniae and the yqhD gene, encoding PDOR isoenzyme from E.coli BL21 were cloned and co-expressed in E.coli JM109 using plasmid pSE380. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration to study the properties. Optimal temperature and pH of recombinant GDHt with specific activity of 85.8 U/mg were 45 °C and 9.0; and optimal temperature and pH of recombinant YqhD with specific activity of 80.0 U/mg were 37 °C, 7.0. The microbial conversion of 1,3-PD from glycerol by this recombinant E. coli strain was studied and the production of 1,3-PD was about 28.0 g/l.

Synthesis of 1,3-propanediol (1,3-PD) from glycerol through the biotransformation process requires two steps, catalyzed by glycerol dehydratase (GDHt) and 1,3-PD oxidoreductase. GDHt is the rate-limiting enzyme in this process. All recombinant microorganisms for production of 1,3-PD so far utilized the natural genes that may not have been optimized. Two positions, which are 19.3A and 29.6A away from the active site in GDHt from Klebsiella pneumoniae, were subjected to saturation-mutagenesis and 38 mutants were characterized. The catalytic activity of a mutant in beta-subunit (beta-Q42F, 29.6A from the active site) was 8.3-fold higher than the wild type, and the enzyme efficiency of other two mutants beta-Q42L and beta-Q42S for substrate glycerol was 336-fold and 80-fold higher than that for 1,2-propanediol. This investigation supplied further evidence that distant mutations could be a good source of diversity and therefore, made a contribution to the toolbox of industrial enzyme improvement.

A gene encoding Uninv, an invertase active in the neutral pH range, was isolated from a metagenomic library and the enzyme was biochemically characterized. The enzyme had an optimum pH of 6.5 and an optimum temperature of 50°C. It showed maximal ~37% sequence identity to all invertases identified so far. Uninv displayed typical Michaelis–Menten kinetics with substrate inhibition of sucrose, but it retained 50% activity at sucrose concentrations up to 1,000 mM. Uninv had no transfructosylating activity even at high sucrose concentrations. These properties will make this enzyme useful in the food industry.

It has tremendous values for both drug discovery and basic research to develop a solid bioinformatical tool for guiding peptide reagent design. Based on the physical and chemical properties of amino acids, a new strategy for peptide reagent design, the so‐called AABPD (amino acid based‐peptide design), is proposed. The peptide samples in a training dataset are described by a series of HMLP (heuristic molecular lipophilicity potential) parameters and other physicochemical properties of amino acid residues that form a three‐dimensional data matrix where each component is defined by three indexes: the first index refers to the peptide samples, the second to the amino acid positions, and the third to the amino acid parameters. The binding free energy between a peptide ligand and its protein receptor is calculated by a linear free energy equation through the physicochemical parameters, resulting in a set of simultaneous linear equations between the bioactivity of the peptides and the physicochemical properties of amino acids. An iterative double least square technique is developed for the solution of the three‐dimensional simultaneous linear equation set to determine the amino acid position coefficients of peptide sequence and the physicochemical parameter coefficients of amino acid residues alternately. The two sets of coefficients thus obtained are used for predicting the bioactivity of other query peptide reagents. Two calculation examples, the peptide substrate specificity of the SARS coronavirus 3C‐like proteinase and the affinity prediction for epitope‐peptides with Class I MHC molecules are studied by using the peptide reagent design strategy. © 2007 Wiley Periodicals, Inc. J Comput Chem, 2007, Wiley Periodicals, Inc.This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

1,3-propanediol (1,3-PD) is an important material for chemical industry, and there has been always much interest in the production of 1,3-PD using all possible routes. The genes encoding glycerol dehydratase (GDHt) from Citrobacter freundii, Klebsiella pneumoniae and metagenome were cloned and expressed in E. coli. All glycerol dehydratases but the one from metagenome could be detected to show enzyme activities. In order to improve the enzymatic properties of GDHts, the genes encoding α and β-γ subunits were cloned, and the enzyme characteristics were evolved by rational design based on their 3D structures which were constructed by homology modeling. Six heteroenzymes were obtained by swapping the α subunit genes of these three different-source-derived GDHts. The pH, thermal stability and V max of some heteroenzymes were dramatically improved by 2–5 times compared with the wild one (GDHtKP). The GDHt cloned from metagenome, originally proved to be with no enzyme activity, was converted into active enzyme by swapping its subunits with other different GDHts. In addition, the effect of subtle 3D structural changes on the properties of the enzyme was also observed.

A new open reading frame in Thermobifida fusca sequenced genome was identified to encode a new trehalose synthase, annotated as "glycosidase" in the GenBank database, by bioinformatics searching and experimental validation. The gene had a length of 1830 bp with about 65% GC content and encoded for a new trehalose synthase with 610 amino acids and deduced molecular weight of 66 kD. The high GC content seemed not to affect its good expression in E. coli BL21 in which the target protein could account for as high as 15% of the total cell proteins. The recombinant enzyme showed its optimal activities at 25 degrees and pH 6.5 when it converted substrate maltose into trehalose. However it would divert a high proportion of its substrate into glucose when the temperature was increased to 37 degrees, or when the enzyme concentration was high Its activity was not inhibited by 5 mM heavy metals such as Cu2+, Mn2+, and Zn2+ but affected by high concentration of glucose. Blasting against the database indicated that amino acid sequence of this protein had maximal 69% homology with the known trehalose synthases, and two highly conserved segments of the protein sequence were identified and their possible linkage with functions was discussed.

A method, so called “active hydrogen bond network” (AHBN), is proposed for site-directed mutations of hydrolytic enzymes. In an enzyme the AHBN consists of the active residues, functional residues, and conservative water molecules, which are connected by hydrogen bonds, forming a three dimensional network. In the catalysis hydrolytic reactions of hydrolytic enzymes AHBN is responsible for the transportation of protons and water molecules, and maintaining the active and dynamic structures of enzymes. The AHBN of pullulanase BNPulA324 from Bacillus naganoensis was constructed based on a homologous model structure using Swiss Model Protein-modeling Server according to the template structure of pullulanase BAPulA (2WAN). The pullulanase BNPulA324 are mutated at the mutation sites selected by means of the AHBN method. Both thermal stability and pH-sensitivity of pullulanase BNPulA324 were successfully improved. The mutations at the residues located at the out edge of AHBN may yield positive effects. On the other hand the mutations at the residues inside the AHBN may deprive the bioactivity of enzymes. The AHBN method, proposed in this study, may provide an assistant and alternate tool for protein rational design and protein engineering.

Synthesis of α2,8-polysialic acid (polySia) glycans are catalyzed by two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), which are two members of the ST8Sia gene family of sialytransferases. During polysialylation, both STX and PST catalyze the transfer of multiple Sia residues from the activated sugar nucleotide precursor, CMP-Neu5Ac (Sia), to terminal Sia residues on N- and Olinked oligosaccharide chains on acceptor glycoproteins, including the neural cell adhesion molecule (NCAM), which is the major carrier protein of polySia. Based on our new findings and previously published studies, this review summarizes the present concepts regarding the molecular mechanism underlying regulation of protein-specific polysialylation of NCAM that includes the following: (1) Determination of the catalytic domains and specific regions within ST8Sia IV for recognizing and catalyzing the efficient polysialylation of NCAM; (2) Identification of key amino acid residues within the PSTD motif of ST8Sia IV that are essential for polysialylation; (3) Verification of key amino acids in the PBR domain of ST8Sia IV required for NCAM-specific polysialylation; and (4) a 3D conformational study of ST8Sia IV based on the Phyre2 server to discover the relationship between the structure and its functional domains of the polyST. Based on these results, our 3D model of ST8Sia IV was used to identify and characterize the catalytic domains and amino acid residues critical for catalyzing polysialylation, and have provided new structural information for supporting a detailed mechanism of polyST-NCAM interaction required for polysialylation of NCAM, findings that have not been previously reported.

Due to the low toxicity, easy synthesis, rapid elimination, and less side effect, more and more peptide inhibitors are emerging as the effective drugs that are clinically used in therapies of a number of diseases. At the same time the computer-aided drug design (CADD) methods have remarkably developed. In this mini review the newly developed peptide inhibitors and drugs are introduced, including peptide vaccines for cancers, peptide inhibitors for HIV, Alzheimer's disease and related diseases, and the peptides as the leading compounds of drugs. The recent progress in the theory and methodology of peptide inhibitor design is reviewed. (1) The flexible protein-peptide docking model is introduced, in which the peptide structures are treated as segment-flexible chains using genetic algorithm and special force field parameters. (2) The "Wenxiang diagram" is illustrated for protein-peptide interaction analysis that has been successfully used in the coiled-coil interaction analysis. (3) The "Distorted key" theory is reviewed, which is an effective method to convert the peptide inhibitors to the small chemical drugs. (4) The amino acid property-based peptide prediction method (AABPP) is described that is a twolevel QSAR prediction network for the bioactivity prediction of peptide inhibitors. (5) Finally, several types of molecular interactions between protein and peptide ligands are summarized, including cation-π interactions; polar hydrogen-π interactions; and π-π stocking interactions.

Background Among the 20 natural amino acids histidine is the most active and versatile member that plays the multiple roles in protein interactions, often the key residue in enzyme catalytic reactions. A theoretical and comprehensive study on the structural features and interaction properties of histidine is certainly helpful. Results Four interaction types of histidine are quantitatively calculated, including: (1) Cation-π interactions, in which the histidine acts as the aromatic π-motif in neutral form (His), or plays the cation role in protonated form (His+); (2) π-π stacking interactions between histidine and other aromatic amino acids; (3) Hydrogen-π interactions between histidine and other aromatic amino acids; (4) Coordinate interactions between histidine and metallic cations. The energies of π-π stacking interactions and hydrogen-π interactions are calculated using CCSD/6-31+G(d,p). The energies of cation-π interactions and coordinate interactions are calculated using B3LYP/6-31+G(d,p) method and adjusted by empirical method for dispersion energy. Conclusions The coordinate interactions between histidine and metallic cations are the strongest one acting in broad range, followed by the cation-π, hydrogen-π, and π-π stacking interactions. When the histidine is in neutral form, the cation-π interactions are attractive; when it is protonated (His+), the interactions turn to repulsive. The two protonation forms (and pKa values) of histidine are reversibly switched by the attractive and repulsive cation-π interactions. In proteins the π-π stacking interaction between neutral histidine and aromatic amino acids (Phe, Tyr, Trp) are in the range from -3.0 to -4.0 kcal/mol, significantly larger than the van der Waals energies.

In drug design and enzyme engineering, the information of interactions between receptors and ligands is crucially important. In many cases, the protein structures and drug-target complex structures are determined by a delicate balance of several weak molecular interaction types. Among these interaction forces several unconventional interactions play important roles, however, less familiar for researchers. The cation-π interaction is a unique noncovalent interaction only acting between aromatic amino acids and organic cations (protonated amino acids) and inorganic cations (proton and metallic). This article reports new study results in the interaction strength, the behaviors and the structural characters of cation-π interactions between aromatic amino acids (Phe, Tyr, and Trp) and organic and inorganic cations (Lys + , Arg + , H + , H3O + , Li + , Na + , K + , Ca 2+ , and Zn 2+ ) in gas phase and in solutions (water, acetonitrile, and cyclohexane). Systematical research revealed that the cation-π interactions are point-to-plane (aromatic group) interactions, distance and orientationdependent, and the interaction energies change in a broad range. In gas phase the cation-π interaction energies between aromatic amino acids (Phe, Tyr, and Trp) and metallic cations (Li + , Na + , K + , Ca 2+ , and Zn 2+ ) are in the range -12 to -160 kcal/mol, and the interaction energies of protonated amino acids (Arg + and Lys + ) are in the range from -9 to -18 kcal/mol. In solutions the cation-π energies decrease with the dielectric constant e of solvents. However, in aqueous solution the cation-π energies of H 3 O + and protonated amino acids are less affected by solvation effects. The applications of unconventional interaction forces in drug design and in protein engineering are introduced.

1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.

The inhibitions of enzymes (proteins) are determined by the binding interactions between ligands and targeting proteins. However, traditional QSAR (quantitative structure-activity relationship) is a one-side technique, only considering the structures and physicochemical properties of inhibitors. In this study, the structure-based and multiple potential three-dimensional quantitative structure-activity relationship (SB-MP-3D-QSAR) is presented, in which the structural information of host protein is involved in the QSAR calculations. The SB-MP-3D-QSAR actually is a combinational method of docking approach and QSAR technique. Multiple docking calculations are performed first between the host protein and ligand molecules in a training set. In the targeting protein, the functional residues are selected, which make the major contribution to the binding free energy. The binding free energy between ligand and targeting protein is the summation of multiple potential energies, including van der Waals energy, electrostatic energy, hydrophobic energy, and hydrogen-bond energy, and may include nonthermodynamic factors. In the foundational QSAR equation, two sets of weighting coefficients {aj} and {bp} are assigned to the potential energy terms and to the functional residues, respectively. The two coefficient sets are solved by using iterative double least-squares (IDLS) technique in the training set. Then, the two sets of weighting coefficients are used to predict the bioactivities of inquired ligands. In an application example, the new developed method obtained much better results than that of docking calculations.

BACKGROUND: The proteins in a family, which perform the similar biological functions, may have very different amino acid composition, but they must share the similar 3D structures, and keep a stable central region. In the conservative structure region similar biological functions are performed by two or three catalytic residues with the collaboration of several functional residues at key positions. Communication signals are conducted in a position network, adjusting the biological functions in the protein family. METHODOLOGY: A computational approach, namely structural position correlation analysis (SPCA), is developed to analyze the correlation relationship between structural segments (or positions). The basic hypothesis of SPCA is that in a protein family the structural conservation is more important than the sequence conservation, and the local structural changes may contain information of biology functional evolution. A standard protein P(0) is defined in a protein family, which consists of the most-frequent amino acids and takes the average structure of the protein family. The foundational variables of SPCA is the structural position displacements between the standard protein P(0) and individual proteins P(i) of the family. The structural positions are organized as segments, which are the stable units in structural displacements of the protein family. The biological function differences of protein members are determined by the position structural displacements of individual protein P(i) to the standard protein P(0). Correlation analysis is used to analyze the communication network among segments. CONCLUSIONS: The structural position correlation analysis (SPCA) is able to find the correlation relationship among the structural segments (or positions) in a protein family, which cannot be detected by the amino acid sequence and frequency-based methods. The functional communication network among the structural segments (or positions) in protein family, revealed by SPCA approach, well illustrate the distantly allosteric interactions, and contains valuable information for protein engineering study.

Predicting the pH-activities of residues in proteins is an important problem in enzyme engineering and protein design. A novel predictor called 'Pred-pK(a)' was developed based on the physicochemical properties of amino acids and protein 3D structure. The Pred-pK(a) approach considers the influence of all other residues of the protein to predict the pK(a) value of an ionizable residue. An empirical equation was formulated, in which the pK(a) value was a distance-dependent function of physicochemical parameters of 20 amino acid types, describing their electrostatic and van der Waals interaction, as well as the effects of hydrogen bonds and solvation. Two sets of coefficients, {a(alpha)} and {b(l)}, were used in the predictor: {a(alpha)} is the weight factors of 20 amino acid types and {b(l)} is the weight factors of physicochemical properties of amino acids. An iterative double least square procedure was proposed to solve the two sets of weight factors alternately and iteratively in a training set. The two coefficient sets {a(alpha)} and {b(l)} thus obtained were used to predict the pK(a) values of residues in a protein. The average predictive error is +/-0.6 pH in less than a minute in common personal computer.

A new peptide design strategy, the amino acid-based peptide prediction (AABPP) approach, is applied for predicting the affinity of epitope-peptides with class I MHC molecule HLA-A*0201. The AABPP approach consists of two sets of predictive coefficients. The former is the coefficients for the physicochemical properties of amino acids and the latter is the weight factors for the residue positions in a peptide sequence. An iterative double least square technique is introduced to determine the two sets of coefficients alternately through a benchmark dataset. The coefficients converged through such an iterative process are further used to predict the bioactivities of query peptides. In the AABPP algorithm, the following eight physicochemical properties are used as the descriptors of amino acids: (i) lipophilic indices, (ii) hydrophilic indices, (iii) lipophilic surface area, (iv) hydrophilic surface area, (v) alpha-potency indices, (vi) beta-potency indices, (vii) coil-potency indices and (viii) volume of amino acid side chains. In comparison with the existing methods in this area, a remakable advantage of the current approach is that there is no need to know the exact conformation of a query peptide and its alignment with a template. The two steps are indispensable but cannot always be successfully realized otherwise. It is anticipated that the AABPP approach will become a powerful tool for peptide drug design, or at least play a complemetary role to the existing methods.

Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (T(m), the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.

Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.