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Polysialylation is a process of polysialic acid (polySia) addition to neural cell adhesion molecule (NCAM), which is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. Polysialylation can be catalyzed by two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST). It has been proposed that two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs, are possible binding sites for the intermolecular interactions of polyST–NCAM and polyST–polySia, respectively, as well as the intramolecular interaction of PSTD–PBR. In this study, Chou’s wenxiang diagrams of the PSTD and PBR are used to determine the key amino acids of these intermolecular and intramolecular interactions, and thus it may be helpful for the identification of the crucial amino acids in the polyST and for the understanding of the molecular mechanism of NCAM polysialylation by incorporating the wenxiang diagram and molecular modeling into NMR spectroscopy.

The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. It has been proposed that NCAM polysialylation could be inhibited by two types of heparin inhibitors, low molecular heparin (LMWH) and heparin tetrasaccharide (DP4). This review summarizes how the interactions between Polysialyltransferase Domain (PSTD) in ST8SiaIV and CMP-Sia, and between the PSTD and polySia take place, and how these interactions are inhibited by LMWH and DP4. Our NMR studies indicate that LMWH is a more effective inhibitor than DP4 for inhibition of NCAM polysialylation. The NMR identification of heparin-binding sites in the PSTD may provide insight into the design of specific inhibitors of polysialylation.

BACKGROUND Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis. RESULTS Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s-1 and 568.93 L mmol-1 s-1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s-1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol-1 s-1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference. CONCLUSIONS The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry.

This study reported simultaneously improved thermostability and hydrolytic pattern of α-amylase from Bacillus subtilis CN7 by rationally engineering the mostly conserved central beta strands in TIM barrel fold. Nine single point mutations and a double mutation were introduced at the 2nd site of the β7 strand and 3rd site of the β5 strand to rationalize the weak interactions in the beta strands of the TIM barrel of α-amylase. All the five active mutants changed the compositions and percentages of maltooligosaccharides in final hydrolytic products compared to the product spectrum of the wild-type. A mutant Y204V produced only maltose, maltotriose, and maltopentaose without any glucose and maltotetraose, indicating a conversion from typical endo-amylase to novel maltooligosaccharide-producing amylase. A mutant V260I enhanced the thermal stability by 7.1 °C. To our best knowledge, this is the first report on the simultaneous improvement of thermostability and hydrolytic pattern of α-amylase by engineering central beta strands of TIM barrel and the novel "beta strands" strategy proposed here may be useful for the protein engineering of other TIM barrel proteins.

A heterologous pathway for sucrose transport and metabolism was introduced into Clostridium beijerinckii to improve sucrose use for n -butanol production. The combined expression of StSUT1 , encoding a sucrose transporter from potato ( Solanum tuberosum ), and SUC2 , encoding a sucrose invertase from Saccharomyces cerevisiae , remarkably enhanced n -butanol production. With sucrose, sugarcane molasses and sugarcane juice as substrates, the C. beijerinckii strain harbouring StSUT1 and SUC2 increased acetone–butanol–ethanol production by 38.7%, 22.3% and 52.8%, respectively, compared with the wild-type strain. This is the first report to demonstrate enhanced sucrose fermentation due to the heterologous expression of a sucrose transporter and invertase in Clostridium . The metabolic engineering strategy used in this study can be widely applied in other microorganisms to enhance the production of high-value compounds from sucrose-based biomass.

Acetoin is an important four-carbon compound that has many applications in foods, chemical synthesis, cosmetics, cigarettes, soaps, and detergents. Its stereoisomer (S)-acetoin, a high-value chiral compound, can also be used to synthesize optically active drugs, which could enhance targeting properties and reduce side effects. Recently, considerable progress has been made in the development of biotechnological routes for (S)-acetoin production. In this review, various strategies for biological (S)- acetoin production are summarized, and their constraints and possible solutions are described. Furthermore, future prospects of biological production of (S)-acetoin are discussed.

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.

Background:The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies.Objective:To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes?Methods:Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4.Results:The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4.Conclusion:The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

Background: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotechnology. Objective: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. Methods: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. Results: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α- helical contents increasing, and the transition from α-helices to β-sheet structures, which was obviously different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

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.

Acetoin is an important platform chemical with a variety of applications in foods, cosmetics, chemical synthesis, and especially in the asymmetric synthesis of optically active pharmaceuticals. It is also a useful breath biomarker for early lung cancer diagnosis. In order to enhance production of optical (S)-acetoin and facilitate this building block for a series of chiral pharmaceuticals derivatives, we have developed a systematic approach using in situ-NADH regeneration systems and promising diacetyl reductase. Under optimal conditions, we have obtained 52.9 g L−1 of (S)-acetoin with an enantiomeric purity of 99.5% and a productivity of 6.2 g (L h)−1. The results reported in this study demonstrated that the production of (S)-acetoin could be effectively improved through the engineering of cofactor regeneration with promising diacetyl reductase. The systematic approach developed in this study could also be applied to synthesize other optically active α-hydroxy ketones, which may provide valuable benefits for the study of drug development.

// Qi-Shi Du 1, 4, * , Shu-Qing Wang 2, * , Neng-Zhong Xie 1, * , Qing-Yan Wang 1 , Ri-Bo Huang 1 and Kuo-Chen Chou 3, 4 1 State Key Laboratory of China for Biomass Energy Enzyme Technology, National Engineering Research Center of China for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China 2 School of Pharmacy, Tianjin Medical University, Tianjin 300070, China 3 Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China 4 Gordon Life Science Institute, Boston, MA 02478, USA * These authors have contributed equally to this work Correspondence to: Qi-Shi Du, email: qsdu@gordonlifescience.org Kuo-Chen Chou, email: kcchou@gordonlifescience.org Keywords: drug design, PCA, molecular fragments, physicochemical properties, peptides Received: April 26, 2017 Accepted: June 30, 2017 Published: August 01, 2017 ABSTRACT A two-level principal component predictor (2L-PCA) was proposed based on the principal component analysis (PCA) approach. It can be used to quantitatively analyze various compounds and peptides about their functions or potentials to become useful drugs. One level is for dealing with the physicochemical properties of drug molecules, while the other level is for dealing with their structural fragments. The predictor has the self-learning and feedback features to automatically improve its accuracy. It is anticipated that 2L-PCA will become a very useful tool for timely providing various useful clues during the process of drug development.

An interesting possibility is explored: storing the mixture of oxygen and hydrogen in clathrate hydrate in molar ratio 1:2. The interaction energies between oxygen, hydrogen, and clathrate hydrate are calculated using high level quantum chemical methods. The useful conclusion points from this study are summarized as follows. (1) The interaction energies of oxygen-hydrogen mixed cluster are larger than the energies of pure hydrogen molecular cluster. (2) The affinity of oxygen molecules with water molecules is larger than that of the hydrogen molecules with water molecules. (3) The dimension of O2-2H2 interaction structure is smaller than the dimension of CO2-2H2 interaction structure. (4) The escaping energy of oxygen molecules from the hydrate cell is larger than that of the hydrogen molecules. (5) The high affinity of the oxygen molecules with both the water molecules and the hydrogen molecules may promote the stability of oxygen-hydrogen mixture in the clathrate hydrate. Therefore it is possible to store the mixed (O2+2H2) cluster in clathrate hydrate.

The Saccharomyces cerevisiae strain UV02_HG, which has faster growth and a higher ethanol yield in 40% (w/v) glucose culture medium than the wild type, is a mutant generated from the MF02 wild-type strain by UV irradiation. Transcriptome sequencing was performed to analyze the differential gene expression between the two strains. 1203 genes had significantly different expression levels in response to high sugar stress. Based on KEGG enrichment analysis, the identified genes were involved in pathways such as the ribosome, oxidative phosphorylation, protein processing, protein export, carbon metabolism, and the MAPK pathway. Real-time quantitative PCR (qPCR) was used to validate the reliability of the results obtained by RNA-Seq. The network-based gene expression Quantitative Trait Locus (eQTL) tool was used to interpret the driver mutations that elicited the adaptive phenotype. According to this network, we speculated that mutations in STE12 and TUP1 are the main drivers of the adaptive phenotype. Sixteen poorly understood genes whose expression was downregulated by STE12 were annotated by gene ontology, all of which are highly associated with the cell membrane.

A convenient and effective sucrose transport assay for Clostridium strains is needed. Traditional methods, such as 14C-sucrose isotope labelling, use radioactive materials and are not convenient for many laboratories. Here, a sucrose transporter from potato was introduced into Clostridium, and a fluorescence assay based on esculin was used for the analysis of sucrose transport in Clostridium strains. This showed that the heterologously expressed potato sucrose transporter is functional in Clostridium. Recombinant engineering of high-level sucrose transport would aid sucrose fermentation in Clostridium strains. The assay described herein provides an important technological platform for studying sucrose transporter function following heterologous expression in Clostridium.

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.

Polysialic acid (polySia) is a novel glycan that posttranslationally modifies neural cell adhesion molecules (NCAMs) in mammalian cells. Up-regulation of polySia-NCAM expression or NCAM polysialylation is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. It has been known that two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), can catalyze polysialylation of NCAM, and two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs play key roles in affecting polyST activity or NCAM polysialylation. However, the molecular mechanisms of NCAM polysialylation and cell migration are still not entirely clear. In this minireview, the recent research results about the intermolecular interactions between the PBR and NCAM, the PSTD and cytidine monophosphate-sialic acid (CMP-Sia), the PSTD and polySia, and as well as the intramolecular interaction between the PBR and the PSTD within the polyST, are summarized. Based on these cooperative interactions, we have built a novel model of NCAM polysialylation and cell migration mechanisms, which may be helpful to design and develop new polysialyltransferase inhibitors.

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.

Background: The relationship between protein structure and its bioactivity is one of the fundamental problems for protein engineering and pharmaceutical design. Method: A new method, called SPTD (Simulated Protein Thermal Detection), was proposed for studying and improving the thermal stability of enzymes. The method was based on the evidence observed by conducting the MD (Molecular Dynamics) simulation for all the atoms of an enzyme vibrating from the velocity at a room temperature (e.g., 25°C) to the desired working temperature (e.g., 65°C). According to the recorded MD trajectories and the coordinate deviations of the constituent residues under the two different temperatures, some new strategies have been found that are useful for both drug delivery and starch industry. Conclusion: The SPTD technique presented in this paper may become a very useful tool for pharmaceutical design and protein engineering.

In this study, a sugarcane bagasse cellulose bundle crystal is converted to a cellulose few-layer two-dimensional (2D) crystal by a specially designed technique scheme, which consists of (1 → 4)-β-D-glucan chains bound to each other by hydrogen bonds in a plane. A graphene few-layer 2D crystal is fabricated from the cellulose 2D crystal by pyrolysis reaction. The fabricated graphene 2D crystal is further analysed and characterised using scanning electron microscopy, high-resolution transmission electron microscopy (HRTEM), atomic force microscopy, and Raman, X-ray diffraction and X-ray photoelectric spectroscopies. A very clear 2D crystal pattern of graphene is observed in the HRTEM image of the carbon product prepared from the cellulose 2D crystal. As a control, in a parallel experiment, multi-layer graphene (or graphite) is obtained from the cellulose bundle crystal. The following possible mechanism is proposed. During carbonization of the cellulose 2D crystal, the glucose units (C6H10O5)n lose oxygen and hydrogen atoms in the form of water, and the six carbon units (C6)n reorganise, forming the graphene 2D crystal at the structural basis of the 2D crystal of cellulose. The findings from this study provide an economical and environmentally friendly approach to the fabrication of graphene and extend the applications of sustainable biomass cellulose.

Acetoin is an important platform chemical with a variety of applications in foods, cosmetics, chemical synthesis, and especially in the asymmetric synthesis of optically active pharmaceuticals. It is also a useful breath biomarker for early lung cancer diagnosis. In order to enhance production of optical (

Graphene microcrystal (GMC) is a type of glassy carbon fabricated from lignin, in which the microcrystals of graphene are chemically bonded by sp3 carbon atoms, forming a glass-like microcrystal structure. The lignin is refined from sugarcane bagasse using an ethanol-based organosolv technique which is used for the fabrication of GMC by two technical schemes: The pyrolysis reaction of lignin in a tubular furnace at atmospheric pressure, and the hydrothermal carbonization (HTC) of lignin at lower temperature, followed by pyrolysis at higher temperature. The existence of graphene nanofragments in GMC is proven by Raman spectra and XRD patterns, the ratio of sp2 carbon atoms to sp3 carbon atoms is demonstrated by XPS spectra, and the microcrystal structure is observed in the high-resolution transmission electron microscope (HRTEM) images. Temperature and pressure have an important impact on the quality of GMC samples. With the elevation of temperature, the fraction of carbon increases, while the fraction of oxygen decreases, and the ratio of sp2 to sp3 carbon atoms increases. In contrast to the pyrolysis techniques, the HTC technique needs lower temperatures because of the high vapor pressure of water. In general, with the help of biorefinery, the biomass material, lignin, is found to be qualified and sustainable material for the manufacture of GMC. Lignin acts as a renewable substitute for the traditional raw materials of glassy carbon, copolymer resins of phenol formaldehyde, and furfuryl alcohol-phenol.

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.

Although not being classified as the most fundamental protein structural elements like α-helices and β-strands, the loop segment may play considerable roles for protein stability, flexibility, and dynamic activity. Meanwhile, the protein loop is also quite elusive; i.e. its interactions with the other parts of protein as well as its own shape-maintaining forces have still remained as a puzzle or at least not quite clear yet. Here, we report a molecular force, the so-called polar hydrogen-π interaction (Hp-π), which may play an important role in supporting the backbones of protein loops. By conducting the potential energy surface scanning calculations on the quasi π-plane of peptide bond unit, we have observed the following intriguing phenomena: (1) when the polar hydrogen atom of a peptide unit is perpendicularly pointing to the π-plane of other peptide bond units, a remarkable Hp-π interaction occurs; (2) the interaction is distance and orientation dependent, acting in a broad space, and belonging to the 'point-to-plane' one. The molecular force reported here may provide useful interaction concepts and insights into better understanding the loop's unique stability and flexibility feature, as well as the driving force of the protein global folding.

A method combining a Plackett-Burman design (PBD), the steepest ascent method (SA), and a Box-Behnken design (BBD) was developed to optimize succinic acid production from cane molasses by Actinobacillus succinogenes GXAS137. The important parameters were (g/L): total sugars of cane molasses (85 g/L), yeast extract (8.84 g/L), and MgCO3 (63.1 g/L). Verification experiments indicated that the maximal succinic acid production reached 57.43±0.86 g/L, which agreed with the predicted value (57.12 g/L). In addition, batch and fed-batch fermentations were carried out in a 1.3 L stirred bioreactor. Compared with a batch fermentation that produced 57.96 g/L of succinic acid at 60 h, a fed-batch fermentation, performed to minimize the inhibition effect of the substrate, produced 64.34 g/L of succinic acid at 60 h. The combined method is powerful for selection of optimized conditions for succinic acid production from cane molasses.

(2R,3R)-2,3-Butanediol has many industrial applications, such as it is used as an antifreeze agent and low freezing point fuel. In addition, it is particularly important to provide chiral groups in drugs. In recent years, this valuable bio-based chemical has attracted increasing attention, and significant progress has been made in the development of microbial cell factories for (2R,3R)-2,3-butanediol production. This article reviews recent advances and challenges in microbial routes to (2R,3R)-2,3- butanediol production, and highlights the metabolic engineering and synthetic biological approaches used to improve titers, yields, productivities, and optical purities. Finally, a systematic and integrative strategy for developing high-performance microbial cell factories is proposed.

Clostridium acetobutylicum is an important organism for biobutanol production. Due to frequent exposure to bacteriophages during fermentation, industrial C. acetobutylicum strains require a strong immune response against foreign genetic invaders. In the present study, a novel CRISPR system was reported in a C. acetobutylicum GXAS18-1 strain by whole genome sequencing, and several specific characteristics of the CRISPR system were revealed as follows: (1) multiple CRISPR loci were confirmed within the whole bacterial genome, while only one cluster of CRISPR-associated genes (Cas) was found in the current strain; (2) similar leader sequences at the 5’ end of the multiple CRISPR loci were identified as promoter elements by promoter prediction, suggesting that these CRISPR loci were under the control of the same transcriptional factor; (3) homology analysis indicated that the present Cas genes shared only low sequence similarity with the published Cas families; and (4) concerning gene similarity and gene cluster order, these Cas genes belonged to the csm family and originated from the euryarchaeota by horizontal gene transfer.