Engineering non-conservative substrate recognition sites of extradiol dioxygenase: Computation guided design to diversify and accelerate degradation of aromatic compounds.

Extradiol dioxygenases (EDOs) catalyzing meta-cleavage of catecholic compounds promise an effective way to detoxify aromatic pollutants. This work reported a novel scenario to engineer our recently identified Type I EDO from Tcu3516 for a broader substrate scope and enhanced activity, which was based on 2,3-dihydroxybiphenyl (2,3-DHB)-liganded molecular docking of Tcu3516 and multiple sequence alignment with other 22 Type I EDOs. 11 non-conservative residues of Tcu3516 within 6 Å distance to the 2,3-DHB ligand center were selected as potential hotspots and subjected to semi-rational design using 6 catecholic analogues as substrates; the mutants V186L and V212N returned with progressive evolution in substrate scope and catalytic activity. Both mutants were combined with D285A for construction of double mutants and final triple mutant V186L/V212N/D285A. Except for 2,3-DHB (the mutant V186L/D285A gave the best catalytic performance), the triple mutant prevailed all other 5 catecholic compounds for their degradation; affording the catalytic efficiency kcat/Km value increase by 10-30 folds, protein Tm (structural rigidity) increase by 15 °C and the half-life time enhancement by 10 times compared to the wild type Tcu3516. The molecular dynamic simulation suggested that a stabler core and a more flexible entrance are likely accounting for enhanced catalytic activity and stability of enzymes.

Efficient One-Pot Synthesis of Uridine Diphosphate Galactose Employing a Trienzyme System.

The limited availability of high-cost nucleotide sugars is a significant constraint on the application of their downstream products (glycosides and prebiotics) in the food or pharmaceutical industry. To better solve the problem, this study presented a one-pot approach for the biosynthesis of UDP-Gal using a thermophilic multienzyme system consisting of GalK, UGPase, and PPase. Under optimal conditions, a 2 h reaction resulted in a UTP conversion rate of 87.4%. In a fed-batch reaction with Gal/ATP = 20 mM:10 mM, UDP-Gal accumulated to 33.76 mM with a space-time yield (STY) of 6.36 g/L·h-1 after the second feeding. In repetitive batch synthesis, the average yield of UDP-Gal over 8 cycles reached 10.80 g/L with a very low biocatalyst loading of 0.002 genzymes/gproduct. Interestingly, Galk (Tth0595) could synthesize Gal-1P using ADP as a donor of phosphate groups, which had never been reported before. This approach possessed the benefits of high synthesis efficiency, low cost, and superior reaction system stability, and it provided new insights into the rapid one-pot synthesis of UDP-Gal and high-value glycosidic compounds.

Selective enhanced cytotoxicity of amino acid deprivation for cancer therapy using thermozyme functionalized nanocatalyst.

BACKGROUND: Enzyme therapy based on differential metabolism of cancer cells has demonstrated promising potential as a treatment strategy. Nevertheless, the therapeutic benefit of reported enzyme drugs is compromised by their uncontrollable activity and weak stability. Additionally, thermozymes with high thermal-stability suffer from low catalytic activity at body temperature, preventing them from functioning independently. RESULTS: Herein, we have developed a novel thermo-enzymatic regulation strategy for near-infrared (NIR)-triggered precise-catalyzed photothermal treatment of breast cancer. Our strategy enables efficient loading and delivery of thermozymes (newly screened therapeutic enzymes from thermophilic bacteria) via hyaluronic acid (HA)-coupled gold nanorods (GNRs). These nanocatalysts exhibit enhanced cellular endocytosis and rapid enzyme activity enhancement, while also providing biosafety with minimized toxic effects on untargeted sites due to temperature-isolated thermozyme activity. Locally-focused NIR lasers ensure effective activation of thermozymes to promote on-demand amino acid deprivation and photothermal therapy (PTT) of superficial tumors, triggering apoptosis, G1 phase cell cycle arrest, inhibiting migration and invasion, and potentiating photothermal sensitivity of malignancies. CONCLUSIONS: This work establishes a precise, remotely controlled, non-invasive, efficient, and biosafe nanoplatform for accurate enzyme therapy, providing a rationale for promising personalized therapeutic strategies and offering new prospects for high-precision development of enzyme drugs.

Increased Expression Levels of Thermophilic Serine Protease TTHA0724 through Signal Peptide Screening in Bacillus subtilis and Applications of the Enzyme.

The thermostable protease TTHA0724 derived from Thermus thermophilus HB8 is an ideal industrial washing enzyme due to its thermophilic characteristics; although it can be expressed in Escherichia coli via pET-22b, high yields are difficult to achieve, leading to frequent autolysis of the host. This paper details the development of a signal peptide library in the expression system of B. subtilis and the optimization of signal peptides for enhanced extracellular expression of TTHA0724. When B. subtilis was used as the host and the optimized signal peptide was used, the expression level of TTHA0724 was 16.7 times higher compared with E. coli. B. subtilis as an expression host does not change the characteristics of TTHA0724. The potential application fields of TTHA0724 are studied. TTHA0724 can be used as a detergent additive at 60 °C, which can sterilize and eliminate mites while thoroughly cleaning protein stains. Soybean meal enzymatic hydrolysis with TTHA0724 at a high temperature produced a higher content of antioxidant peptides. These results indicate that TTHA0724 has great potential for industrial applications.

Selective and Sustainable Production of Sub-terminal Hydroxy Fatty Acids by a Self-Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens.

Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs.

Thermophilic Inorganic Pyrophosphatase Ton1914 from Thermococcus onnurineus NA1 Removes the Inhibitory Effect of Pyrophosphate.

Pyrophosphate (PPi) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PPi can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from Thermococcus onnurineus NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg2+ at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32-41%, while that of short-chain DNA was only improved by 9.5-15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.

Characterization of a Novel Fe2+ Activated Non-Blue Laccase from Methylobacterium extorquens.

Herein, a novel laccase gene, Melac13220, was amplified from Methylobacterium extorquens and successfully expressed in Escherichia coli with a molecular weight of approximately 50 kDa. The purified Melac13220 had no absorption peak at 610 nm and remained silent within electron paramagnetic resonance spectra, suggesting that Melac13220 belongs to the non-blue laccase group. Both inductively coupled plasma spectroscopy/optical emission spectrometry (ICP-OES) and isothermal titration calorimetry (ITC) indicated that one molecule of Melac13220 can interact with two iron ions. Furthermore, the optimal temperature of Melac13220 was 65 °C. It also showed a high thermolability, and its half-life at 65 °C was 80 min. Melac13220 showed a very good acid environment tolerance; its optimal pH was 1.5. Cu2+ and Co2+ can slightly increase enzyme activity, whereas Fe2+ could increase Melac13220's activity five-fold. Differential scanning calorimetry (DSC) indicated that Fe2+ could also stabilize Melac13220. Unlike most laccases, Melac13220 can efficiently decolorize Congo Red and Indigo Carmine dyes even in the absence of a redox mediator. Thus, the non-blue laccase from Methylobacterium extorquens shows potential application value and may be valuable for environmental protection, especially in the degradation of dyes at low pH.

Superanionic Solvent-Free Liquid Enzymes Exhibit Enhanced Structures and Activities.

The surface of a carboxylate-enriched octuple mutant of Bacillus subtilis lipase A (8M) is chemically anionized to produce core (8M)-shell (cationic polymer surfactants) bionanoconjugates in protein liquid form, which are termed anion-type biofluids. The resultant lipase biofluids exhibit a 2.5-fold increase in hydrolytic activity when compared with analogous lipase biofluids based on anionic polymer surfactants. In addition, the applicability of the anion-type biofluid using Myoglobin (Mb) that is well studied in anion-type solvent-free liquid proteins is evaluated. Although anionization resulted in the complete unfolding of Mb, the active α-helix level is partially recovered in the anion-type biofluids, and the effect is accentuated in the cation-type Mb biofluids. These highly active anion-type solvent-free liquid enzymes exhibit increased thermal stability and provide a new direction in solvent-free liquid protein research.

Current Strategies for Real-Time Enzyme Activation.

Enzyme activation is a powerful means of achieving biotransformation function, aiming to intensify the reaction processes with a higher yield of product in a short time, and can be exploited for diverse applications. However, conventional activation strategies such as genetic engineering and chemical modification are generally irreversible for enzyme activity, and they also have many limitations, including complex processes and unpredictable results. Recently, near-infrared (NIR), alternating magnetic field (AMF), microwave and ultrasound irradiation, as real-time and precise activation strategies for enzyme analysis, can address many limitations due to their deep penetrability, sustainability, low invasiveness, and sustainability and have been applied in many fields, such as biomedical and industrial applications and chemical synthesis. These spatiotemporal and controllable activation strategies can transfer light, electromagnetic, or ultrasound energy to enzymes, leading to favorable conformational changes and improving the thermal stability, stereoselectivity, and kinetics of enzymes. Furthermore, the different mechanisms of activation strategies have determined the type of applicable enzymes and manipulated protocol designs that either immobilize enzymes on nanomaterials responsive to light or magnetic fields or directly influence enzymatic properties. To employ these effects to finely and efficiently activate enzyme activity, the physicochemical features of nanomaterials and parameters, including the frequency and intensity of activation methods, must be optimized. Therefore, this review offers a comprehensive overview related to emerging technologies for achieving real-time enzyme activation and summarizes their characteristics and advanced applications.

Molecular dynamics simulation guided distal mutation of Thermotoga naphthophila ß-glucosidase for significantly enhanced synthesis of galactooligosaccharides and expanded product scope.

For efficient enzymatic production of health-beneficial galactooligosaccharides (GOSs), a glycone (-1)/aglycone (+2) subsite mutation strategy to engineer a thermophilic GH1 ß-glucosidase (Tn0602) from Thermotoga naphthophila RKU-10 was introduced. Six single mutation variants (F226G, N246G, N246E, N222F, N222Y, G224T) and two double mutants (F226GF414S, F226GF414Y) were designed. The +2-subsite variant F226G produced 136 mM galactooligosaccharide 1.2-fold more than the wild type (115 mM). More significantly, a superimposed mutation of the -1/+2 subsites F226G/F414S gave a total GOS production of 314 mM (82.16% lactose conversion), 2.7-fold higher than the total GOS production of the wild type. Furthermore, the variant F226GF414S was profiled 241 mM of trisaccharide (galß (1 â†’ 3)/(1 â†’ 4) lactose) and 73 mM tetrasaccharide (galß (1 â†’ 3)/(1 â†’ 4) galß (1 â†’ 3)/(1 â†’ 4) lactose). According to a 300-ns molecular dynamic simulation, the superimposed mutation increased GOS productivity and expanded the scope of products by changing the structural flexibility and reducing the steric hindrance of the substrate tunnel. Overall, our study successfully demonstrated that a - 1/+2 subsite mutagenesis method could be used in ß-glucosidases Tn0602 to improve enzyme productivity and expand product scope, which could be a potential route to evolve retaining glycosidases towards the desired direction.

A new thermophilic extradiol dioxygenase promises biodegradation of catecholic pollutants.

Extradiol dioxygenases (EDOs) catalyze the meta cleavage of catechol into 2-hydroxymuconaldehyde, a critical step in the degradation of aromatic compounds in the environment. In the present work, a novel thermophilic extradiol dioxygenase from Thermomonospora curvata DSM43183 was cloned, expressed, and characterized by phylogenetic and biochemical analyses. This enzyme exhibited excellent thermo-tolerance, displaying optimal activity at 50 °C, remaining >40% activity at 70 °C. Structural modeling and molecular docking demonstrated that both active center and pocket-construction loops locate at the C-terminal domain. Site-specific mutants D285A, H205V, F301V based on a rational design were obtained to widen the entrance of substrates; resulting in significantly improved catalytic performance for all the 3 mutants. Compared to the wild-type, the mutant D285A showed remarkably improved activities with respect to the 3,4-dihydroxyphenylacetic acid, catechol, and 3-chlorocatechol, by 17.7, 6.9, and 3.7-fold, respectively. The results thus verified the effectiveness of modeling guided design; and confirmed that the C-terminal loop structure indeed plays a decisive role in determining catalytic ring-opening efficiency and substrate specificity of the enzyme. This study provided a novel thermostable dioxygenase with a broad substrate promiscuity for detoxifying environmental pollutants and provided a new thinking for further enzyme engineering of EDOs.

Mild Hypothermia Therapy Lowers the Inflammatory Level and Apoptosis Rate of Myocardial Cells of Rats with Myocardial Ischemia-Reperfusion Injury via the NLRP3 Inflammasome Pathway.

OBJECTIVE: To explore the protective effects and mechanism of mild hypothermia treatment in the treatment of myocardial ischemia-reperfusion injury. Material and Methods. A total of 20 Sprague-Dawley (SD) rats were assigned to 4 groups: the blank control group, sham operation group, ischemia reperfusion group, and mild hypothermia therapy group (each n = 5). Some indexes were detected. In addition, myocardial cell models of oxygen-glucose deprivation/reoxygenation injury (OGD) were established. The expression of mRNA IL-6 and TNF-α and the key enzyme levels of apoptosis (cleaved-Caspase-3) and the NLRP3 inflammasome/p53 signaling pathway in the models were determined. RESULTS: The expression of serum IL-6 and TNF-α in the mild hypothermia therapy group was significantly lower than that in the ischemia reperfusion group. The mild hypothermia therapy group also showed a significantly lower TUNEL cell count and NLRP3 and p53 phosphorylation levels than the ischemia reperfusion group (all p < 0.05). The in vitro mild hypothermia + OGD group also showed significantly lower mRNA expression of IL-6 and TNF-α and levels of cleaved Caspase-3, NLRP3, and phosphorylated p53 protein than the OGD group (all p < 0.05). CONCLUSION: In conclusion, mild hypothermia therapy can inhibit the apoptosis and myocardial inflammation of cells induced by MI/R injury in rats and inhibiting the activity of the NLRP3 inflammasome pathway and p53 signaling pathway may be the mechanism.

Galactosyldiacylglycerols: From a Photosynthesis-Associated Apparatus to Structure-Defined In Vitro Assembling.

Being ubiquitously present in plants, microalgae, and cyanobacteria and as the major constituents of thylakoid membranes, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) make up approximately 52 and 26%, respectively, of chloroplast lipids. Thylakoid membranes harbor the photosynthetic complexes and numerous essential biochemical pathways where MGDG and DGDG play a central role in facilitating photosynthesis light reaction, maintaining chloroplast morphology, and responding to abiotic stresses. Furthermore, these galactolipids are also bioactive compounds with antitumor, antimicrobial, antiviral, immunosuppressive, and anti-inflammatory activities and important nutritional value. These characteristics are strictly dependent upon their fatty acyl chain length, olefinic nature, and stereoconfiguration. However, their application potentials are practically untapped, largely as a result of the fact that their availability in large quantity and high purity (structured galactolipids) is challenging. In addition to laborious extraction from natural sources, in vitro assembling of these molecules could be a promising alternative. Thus, this review updates the latest advances in elucidating biosynthesis paths of MGDG and DGDG and related enzyme systems, which present invaluable inspiration to design approaches for a retrosynthesis of galactolipids. More critically, this work summarizes recent developments in the biological and enzymatic syntheses of galactolipids, especially the strategic scenarios for the construction of in vitro enzymatic and/or chemoenzymatic synthesis routes. Protein engineering of enzymes involved in the synthesis of MGDG and DGDG to improve their properties is highlighted, and the applications of galactolipids in foods and medicine are also discussed.

Read-across: Principle, case study and its potential regulatory application in China.

Read-across, has generated much attention and has been used in many regulatory schemes as an alternative approach to testing globally. The regulatory application of read-across in the chemical management in China is progressing but still limited. A workshop on the "Read-across: Principle, case study and its potential regulatory application in China", organized by the Chemical Risk Assessment Specialty Group under the Committee of Industrial Toxicology of Chinese Society of Toxicology, was held on May 28, 2019 to discuss the potential broader application and acceptance of read-across to support chemical risk assessment in China. The Workshop included global experts from regulatory agencies, academia and industry. Scientific presentations and constructive discussions raised awareness on the use of read-across in different regions, identified barriers to regulatory acceptance, and participants also brainstormed on practical strategies to help facilitate the further regulatory application of read-across approaches in China.

Using near-infrared enhanced thermozyme and scFv dual-conjugated Au nanorods for detection and targeted photothermal treatment of Alzheimer's disease.

Investigation of targeting inhibitors of Aß aggregation, heme-Aß peroxidase-like activity and efficient detectors of Aß aggregation, are of therapeutic value and diagnostics significance for the treatment of Alzheimer's disease (AD). Due to the complex pathogenesis of AD, theranostics treatment with multiple functions are necessary. Herein we constructed the NIR absorption property of gold nanorods (GNRs) loaded with single chain variable fragment (scFv) 12B4 and thermophilic acylpeptide hydrolase (APH) ST0779 as a smart theranostic complex (GNRs-APH-scFv, GAS), which possesses both rapid detection of Aß aggregates and NIR photothermal treatment that effectively disassembles Aß aggregates and inhibits Aß-mediated toxicity. Methods: We screened targeting anti-Aß scFv 12B4 and thermophilic acylpeptide hydrolase as amyloid-degrading enzyme, synthesized GAS gold nanorods complex. The GAS was evalued by Aß inhibition and disaggregation assays, Aß detection assays, Aß mediated toxicity assays in vitro. In vivo, delaying Aß-induced paralysis in AD model of Caenorhabditis elegans was also tested by GAS. Results: In vitro, GAS has a synergistic effect to inhibit and disassociate Aß aggregates, in addition to decrease heme-Aß peroxidase-like activity. In cultured cells, treatment with GAS reduces Aß-induced cytotoxicity, while also delaying Aß-mediated paralysis in CL4176 C.elegans model of AD. Furthermore, the photothermal effect of the GAS upon NIR laser irradiation not only helps disassociate the Aß aggregates but also boosts APH activity to clear Aß. The GAS, as a targeting detector and inhibitor, allows real-time detection of Aß aggregates. Conclusion: These results firstly highlight the combination of scFv, APH and nanoparticles to be theranostic AD drugs. Taken together, our strategy provides a new thought into the design of smart compounds for use as efficiently therapeutic and preventive agents against AD. Moreover, our design provides broad prospects of biomedical strategy for further theranostics application in those diseases caused by abnormal protein.

Insight into the Structure and Activity of Surface-Engineered Lipase Biofluids.

Despite a successful application of solvent-free liquid protein (biofluids) concept to a number of commercial enzymes, the technical advantages of enzyme biofluids as hyperthermal stable biocatalysts cannot be fully utilized as up to 90-99% of native activities are lost when enzymes were made into biofluids. With a two-step strategy (site-directed mutagenesis and synthesis of variant biofluids) on Bacillus subtilis lipase A (BsLA), we elucidated a strong dependency of structure and activity on the number and distribution of polymer surfactant binding sites on BsLA surface. Here, it is demonstrated that improved BsLA variants can be engineered via site-mutagenesis by a rational design, either with enhanced activity in aqueous solution in native form, or with improved physical property and increased activity in solvent-free system in the form of a protein liquid. This work answered some fundamental questions about the surface characteristics for construction of biofluids, useful for identifying new strategies for developing advantageous biocatalysts.

ß-Glucosidase from Thermotoga naphthophila RKU-10 for exclusive synthesis of galactotrisaccharides: Kinetics and thermodynamics insight into reaction mechanism.

This work reports a novel thermophilic ß-glucosidase (TN0602) from Thermotoga naphthophila RKU-10, demonstrating exceptionally high catalytic selectivity (100%) for the exclusive synthesis of prebiotic galactotrisaccharides (GOS3) in a high volumetric production yield of 23.28gL-1h-1 (higher than the highest value ever reported) at pH 6.5 and 75°C, with milk processing waste lactose as both the galactosyl donor and acceptor. A comparative study with commercial ß-galactosidase from Aspergillus oryzae (AO) with respect to reaction kinetics, enzyme-substrate thermodynamic binding (substrate induced fluorescence quenching) and molecular docking simulation studies showed that ß-glucosidase TN0602 has a deep catalytic "pocket" with a narrow entrance that prevents simultaneous access of lactose and GOS3 to the catalytic site, explaining its distinct catalytic specificity and reaction kinetics. The findings revealed in this work offer an improved understanding of how enzyme protein structure determines catalytic specificity, which serves as new knowledge to engineer ß-glucosidase for the biosynthesis of designer GOS.

Valorizing Dairy Waste: Thermophilic Biosynthesis of a Novel Ascorbic Acid Derivative.

l-Ascorbic acid (l-AA) is an essential nutrient that is extremely unstable and cannot be synthesized by the human body. Therefore, attempts have been performed to develop biologically active l-AA derivatives with improved stability. This work presents a facile, scalable, and efficient enzymatic transgalactosylation of lactose to l-AA using ß-glucosidase (TN0602) from Thermotoga naphthophila RKU-10. ß-Glucosidase TN0602 displays high transgalactosylation activity at pH 5.0, 75 °C, and l-AA/lactose ratio of 2:1 to form a novel l-AA derivative [2-O-ß-d-galactopyranosyl-l-ascorbic acid (l-AA-Gal)] with a maximal productivity of 138.88 mmol L-1 in 12 h, which is higher than most reports of enzymatic synthesis of l-AA-α-glucoside. Synthetic l-AA-Gal retains most l-AA antioxidant capability and presents dramatically higher stability than l-AA in an oxidative environment (Cu2+). In conclusion, this work reports a new way to valorize dairy waste lactose into a novel molecule l-AA-Gal, which could be a promising l-AA derivative to be used in a wide range of applications.

Synergistic effect of thermostable ß-glucosidase TN0602 and cellulase on cellulose hydrolysis.

Thermophilic enzymes have many potential benefits in industrial production with increased flexibility related to process configurations. A thermostable ß-glucosidase from Thermotoga naphthophila RUK-10 was found to possess catalytic activity for cellobiose hydrolysis with a high potential for application in biomass conversion. The aggregation of cellobiose often has an inhibitory effect on cellobiohydrolases and endoglucanases during cellulose hydrolysis. The presence of ß-glucosidases has a significant effect on reducing inhibition from hydrolytic products by hydrolysing the intermedia cellobiose. In this study, ß-glucosidase TN0602 exhibited a high tolerance to glucose and high thermostability even after a long incubation (>72 h). Additionally, supplementing ß-glucosidase TN0602 with microcrystalline cellulose, untreated corn straw and steam-exploded corn straw hydrolysis reactions containing a commercial cellulase led to an increased conversion rate in released glucose compared to hydrolysis without the addition of ß-glucosidase (15.82, 30.62 and 35.21%, respectively); the increase of conversion rates were 61.86, 93.50 and 94.55%. It was thus shown that an obvious synergistic effect exists between TN0602 and cellulases for cellulose hydrolysis, suggesting its potential as a component of enzymatic cocktails for the conversion of lignocellulosic biomass to other chemicals.

Identification of an archaeal maltooligosyltrehalose trehalohydrolase encoded by an interrupted gene.

Complete genome analysis of the thermoacidophilic Archaeon Sulfolobus tokodaii strain 7 revealed two open reading frames (ORF), namely, ST0926 and ST0927. These ORFs are interrupted by two putative insertions and encode for the N- and C-terminal fragments, respectively, of a putative Sulfolobus sp. maltooligosyltrehalose trehalohydrolase (StMTHase). Two specific deletion mutations, designed on the basis of sequence alignments of the adjacent ORFs and the published Sulfolobus sp. MTHases, allowed soluble expression in Escherichia coli of an active acidic and thermophilic enzyme. The purified enzyme exhibited a maximum amylolytic activity at 70 °C and pH 5.0, which resembled the optimal conditions of the Sulfolobus homologs. Furthermore, we report that these ORFs are actively co-transcribed in vivo, and we confirm the presence of insertions between them at the cDNA level. However, immunization and western blot experiments demonstrated no expression of ST0926 or the putative full-length StMTHase in vivo, indicating that they might exist as nonfunctional pseudogenes.