Structures of the NDP-pyranose mutase belonging to glycosyltransferase family 75 reveal residues important for Mn2+ coordination and substrate binding.

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDP-pyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDP-pyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDP-L-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis.

A Prevalent Group of Actinobacterial Radical SAM/SPASM Maturases Involved in Triceptide Biosynthesis.

Triceptides are ribosomally synthesized and post-translationally modified peptides characterized by three-residue cyclophanes. The cyclophanes are installed by radical SAM/SPASM maturases referred to as 3-residue cyclophane forming enzymes (3-CyFEs) which catalyze C(sp2)-Cß(sp3) bond formation on three residue motifs at the C-terminus of precursor peptides. Here, we bioinformatically map uncharacterized rSAM/SPASM enzymes, referred to as Actinobacterial multiple cyclophane maturases. The enzyme FwwB from Actinospira robinae was selected for in vivo functional studies in Escherichia coli, and was found to catalyze formation of multiple Phe- and Trp-derived 3-residue cyclophanes. FwwB was shown to accept a series of engineered substrates but showed specificity for the native 3-residue motif.

Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.

Discovery and characterization of a novel Dyp-type peroxidase from a marine actinobacterium isolated from Trondheim fjord, Norway.

A new dye-decolorizing peroxidase (DyP) was discovered through a data mining workflow based on HMMER software and profile Hidden Markov Model (HMM) using a dataset of 1200 genomes originated from a Actinobacteria strain collection isolated from Trondheim fjord. Instead of the conserved GXXDG motif known for Dyp-type peroxidases, the enzyme contains a new conserved motif EXXDG which has been not reported before. The enzyme can oxidize an anthraquinone dye Remazol Brilliant Blue R (Reactive Blue 19) and other phenolic compounds such as ferulic acid, sinapic acid, caffeic acid, 3-methylcatechol, dopamine hydrochloride, and tannic acid. The acidic pH optimum (3 to 4) and the low temperature optimum (25 °C) were confirmed using both biochemical and electrochemical assays. Kinetic and thermodynamic parameters associated with the catalytic redox center were attained by electrochemistry.

Structural insights reveal the second base catalyst of isomaltose glucohydrolase.

Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-kcat and pH-kcat /Km profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a ß-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.

Rid7C, a Member of the YjgF/YER057c/UK114 (Rid) Protein Family, Is a Novel Endoribonuclease That Regulates the Expression of a Specialist RNA Polymerase Involved in Differentiation in Nonomuraea gerenzanensis.

The YjgF/YER057c/UK114 (Rid) is a protein family breadth conserved in all domains of life and includes the widely distributed archetypal RidA (YjgF) subfamily and seven other subfamilies (Rid1 to Rid7). Among these subfamilies, RidA is the only family to have been biochemically well characterized and is involved in the deamination of the reactive enamine/imine intermediates. In this study, we have characterized a protein of the Rid7 subfamily, named Rid7C, in Nonomuraea gerenzanensis, an actinomycete that is characterized by the presence of two types of RNA polymerases. This is due to the coexistence in its genome of two RNA polymerase (RNAP) ß chain-encoding genes, rpoB(S) (the wild-type rpoB gene) and rpoB(R) (a specialist, mutant-type rpoB gene) that controls A40926 antibiotic production and a wide range of metabolic adaptive behaviors. Here, we found that expression of rpoB(R) is regulated posttranscriptionally by RNA processing in the 5' untranslated region (UTR) of rpoB(R) mRNA and that the endoribonuclease activity of Rid7C is responsible for mRNA processing, thereby overseeing several tracts of morphological and biochemical differentiation. We also provide evidence that Rid7C may be associated with RNase P M1 RNA, although M1 RNA is not required for rpoB(R) mRNA processing in vitro, and that Rid7C endoribonuclease activity is inhibited by A40926, suggesting the existence of a negative feedback loop in A40926 production and a role of the endogenous synthesis of A40926 in the modulation of biochemical differentiation in this microorganism. IMPORTANCE The YjgF/YER057c/UK114 family includes many proteins with diverse functions involved in detoxification, RNA maturation, and control of mRNA translation. We found that Rid7C is an endoribonuclease that is involved in processing of rpoB(R) mRNA, coding for a specialized RNA polymerase beta subunit that oversees morphological differentiation and A40926 antibiotic production in Nonomuraea gerenzanensis. Rid7C-mediated processing promotes rpoB(R) mRNA translation and antibiotic production, while Rid7C endoribonuclease activity is inhibited by A40926, suggesting a role of the endogenous synthesis of A40926 in modulation of biochemical differentiation in this microorganism. Finally, we show that recombinant Rid7C copurified with M1 RNA (the RNA subunit of RNase P) from Escherichia coli extract, suggesting a functional interaction between Rid7C and M1 RNA activities.

Genome information of the cellulolytic soil actinobacterium Isoptericola dokdonensis DS-3 and comparative genomic analysis of the genus Isoptericola.

The actinobacterial group is regarded as a reservoir of biologically active natural products and hydrolytic enzymes with the potential for biomedical and industrial applications. Here, we present the complete genome sequence of Isoptericola dokdonensis DS-3 isolated from soil in Dokdo, small islets in the East Sea of Korea. This actinomycete harbors a large number of genes encoding carbohydrate-degrading enzymes, and its activity to degrade carboxymethyl cellulose into glucose was experimentally evaluated. Since the genus Isoptericola was proposed after reclassification based on phylogenetic analysis, strains of Isoptericola have been continuously isolated from diverse environments and the importance of this genus in the ecosystem has been suggested by recent culturomic or metagenomic studies. The phylogenic relationships of the genus tended to be closer among strains that had been isolated from similar habitats. By analyzing the properties of published genome sequences of seven defined species in the genus, a large number of genes for carbohydrate hydrolysis and utilization, as well as several biosynthetic gene clusters for secondary metabolites, were identified. Genomic information of I. dokdonensis DS-3 together with comparative analysis of the genomes of Isoptericola provides insights into understanding this actinobacterial group with a potential for industrial applications.

Alkalic dextranase produced by marine bacterium Cellulosimicrobium sp. PX02 and its application.

The enzyme dextranase is widely used in the sugar and food industries, as well as in the medical field. Most land-derived dextranases are produced by fungi and have the disadvantages of long production cycles, low tolerance to environmental conditions, and low safety. The use of marine bacteria to produce dextranases may overcome these problems. In this study, a dextranase-producing bacterium was isolated from the Rizhao seacoast of Shandong, China. The bacterium, denoted as PX02, was identified as Cellulosimicrobium sp. and its growing conditions and the production and properties of its dextranase were investigated. The dextranase had a molecular weight of approximately 40 kDa, maximum activity at 40°C and pH 7.5, with a stability range of up to 45°C and pH 7.0-9.0. High-performance liquid chromatography showed that the dextranase hydrolyzed dextranT20 to isomaltotriose, maltopentaose, and isomaltooligosaccharides. Hydrolysis by dextranase produced excellent antioxidant effects, suggesting its potential use in the health food industry. Investigation of the action of the dextranase on Streptococcus mutans biofilm and scanning electron microscopy showed that it to be effective both for removing and inhibiting the formation of biofilms, suggesting its potential application in the dental industry.

Discovery, Biocatalytic Exploration and Structural Analysis of a 4-Ethylphenol Oxidase from Gulosibacter chungangensis.

The vanillyl-alcohol oxidase (VAO) family is a rich source of biocatalysts for the oxidative bioconversion of phenolic compounds. Through genome mining and sequence comparisons, we found that several family members lack a generally conserved catalytic aspartate. This finding led us to study a VAO-homolog featuring a glutamate residue in place of the common aspartate. This 4-ethylphenol oxidase from Gulosibacter chungangensis (Gc4EO) shares 42 % sequence identity with VAO from Penicillium simplicissimum, contains the same 8α-N3 -histidyl-bound FAD and uses oxygen as electron acceptor. However, Gc4EO features a distinct substrate scope and product specificity as it is primarily effective in the dehydrogenation of para-substituted phenols with little generation of hydroxylated products. The three-dimensional structure shows that the characteristic glutamate side chain creates a closely packed environment that may limit water accessibility and thereby protect from hydroxylation. With its high thermal stability, well defined structural properties and high expression yields, Gc4EO may become a catalyst of choice for the specific dehydrogenation of phenolic compounds bearing small substituents.

Biochemical identification of a hyperthermostable l-ribulose 3-epimerase from Labedella endophytica and its application for d-allulose bioconversion.

Currently, low sugar and low energy have become an important trend in the food industries. Therefore, the bioconversion of the functional low-calorie rare sugars attracts more and more attention. l-Ribulose 3-epimerase (LREase) belongs to the ketose 3-epimerase (KEase) family, which could not only efficiently catalyze the reversible C-3 epimerization between l-ribulose and l-xylulose but also between d-fructose and d-allulose. In this paper, a hyperthermostable LREase from Labedella endophytica was identified and characterized. It exhibited maximum catalytic activity at pH 6.0 and 80 °C with 1 mM Ni2+. In the presence of Co2+, the t1/2 values at 60, 65, and 70 °C were 37.7, 9.0, and 4.6 h, respectively, and Tm value was 80.9 °C. From 500 g/L d-fructose, it could produce 154.2 g/L d-allulose with a conversion rate of 30.8% in 10 h. In view of its strong thermostability and high catalytic efficiency, L. endophytica LREase might be a good potential alternative for d-allulose industrial production.

Development of molecular driven screening for desulfurizing microorganisms targeting the dszB desulfinase gene.

COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) were developed for the detection of the dszB desulfinase gene (2'-hydroxybiphenyl-2-sulfinate desulfinase; EC 3.13.1.3) by polymerase chain reaction (PCR), which allow to reveal larger diversity than traditional primers. The new developed primers were used as molecular monitoring tool to drive a procedure for the isolation of desulfurizing microorganisms. The primers revealed a large dszB gene diversity in environmental samples, particularly in diesel-contaminated soil that served as inoculum for enrichment cultures. The isolation procedure using the dibenzothiophene sulfone (DBTO2) as sole sulfur source reduced drastically the dszB gene diversity. A dszB gene closely related to that carried by Gordonia species was selected. The desulfurization activity was confirmed by the production of desulfurized 2-hydroxybiphenyl (2-HBP). Metagenomic 16S rRNA gene sequencing showed that the Gordonia genus was represented at low abundance in the initial bacterial community. Such observation highlighted that the culture medium and conditions represent the bottleneck for isolating novel desulfurizing microorganisms. The new developed primers constitute useful tool for the development of appropriate cultural-dependent procedures, including medium and culture conditions, to access novel desulfurizing microorganisms useful for the petroleum industry.

Characterization of the Glutathione S-Transferases Involved in Styrene Degradation in Gordonia rubripertincta CWB2.

The glutathione S-transferases carried on the plasmid for the styrene-specific degradation pathway in the Actinobacterium Gordonia rubripertincta CWB2 were heterologously expressed in Escherichia coli. Both enzymes were purified via affinity chromatography and subjected to activity investigations. StyI and StyJ displayed activity toward the commonly used glutathione S-transferase model substrate 1-chloro-2,4-dinitrobenzene (CDNB) with Km values of 0.0682 ± 0.0074 and 2.0281 ± 0.1301 mM and Vmax values of 0.0158 ± 0.0002 and 0.348 ± 0.008 U mg-1 for StyI and StyJ, respectively. The conversion of the natural substrate styrene oxide to the intermediate (1-phenyl-2-hydroxyethyl)glutathione was detected for StyI with 48.3 ± 2.9 U mg-1. This elucidates one more step in the not yet fully resolved styrene-specific degradation pathway of Gordonia rubripertincta CWB2. A characterization of both purified enzymes adds more insight into the scarce research field of actinobacterial glutathione S-transferases. Moreover, a sequence and phylogenetic analysis puts both enzymes into a physiological and evolutionary context. IMPORTANCE Styrene is a toxic compound that is used at a large scale by industry for plastic production. Bacterial degradation of styrene is a possibility for bioremediation and pollution prevention. Intermediates of styrene derivatives degraded in the styrene-specific pathways are precursors for valuable chemical compounds. The pathway in Gordonia rubripertincta CWB2 has proven to accept a broader substrate range than other bacterial styrene degraders. The enzymes characterized in this study, distinguish CWB2s pathway from other known styrene degradation routes and thus might be the main key for its ability to produce ibuprofen from the respective styrene derivative. A biotechnological utilization of this cascade could lead to efficient and sustainable production of drugs, flavors, and fragrances. Moreover, research on glutathione metabolism in Actinobacteria is rare. Here, a characterization of two glutathione S-transferases of actinobacterial origin is presented, and the utilization of glutathione in the metabolism of an Actinobacterium is proven.

Engineered RebH Halogenase Variants Demonstrating a Specificity Switch from Tryptophan towards Novel Indole Compounds.

Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure-guided semi-rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated.

Recombinant expression and characterization of novel P450s from Actinosynnema mirum.

Cytochrome P450 monooxygenases (P450s) are the major contributor in the metabolism of xenobiotics, including therapeutic agents. Thus, P450s find broad application in the pharmaceutical industry to synthesize metabolites of new active pharmaceutical ingredients in order to evaluate toxicity and pharmacokinetics. As an alternative to human hepatic P450s, microbial P450s offer several advantages, such as an easier and more efficient heterologous expression as well as higher stability under process conditions. Recently, the wild-type strain Actinosynnema mirum has been reported to catalyze hydroxylation reactions with high activity on a broad range of substrates. In this study, one of these substrates, ritonavir, was used to analyze the transcriptional response of the wild-type strain. Analysis of the differential gene expression pattern allowed the assignment of genes potentially responsible for ritonavir conversion. Heterologous expression of these candidates and activity testing led to the identification of a novel P450 that efficiently converts ritonavir resembling the activity of the human CYP3A4.

Existence in cellulose shelters: industrial and pharmaceutical leads of symbiotic actinobacteria from ascidian Phallusia nigra, Andaman Islands.

The diversity of actinobacteria associated with marine ascidian Phallusia nigra from Andaman Islands was investigated. A total of 10 actinobacteria were isolated and based on the biochemical and molecular characterization, the isolates were assigned to 7 different actinobacterial genera. Eight putatively novel species belonging to genera Rhodococcus, Kineococcus, Kocuria, Janibacter, Salinispora and Arthrobacter were identified based on 16S rDNA sequence similarity with the NCBI database. The organic extracts of ten isolates displayed considerable bioactivity against test pathogens, which were Gram-positive and Gram-negative in nature. PCR-based screening for type I and type II polyketide synthases (PKS-I, PKS-II) and nonribosomal peptide synthetases (NRPS) revealed that, 10 actinobacterial isolates encoded at least one type of polyketide synthases biosynthesis gene. Majority of the isolates found to produce industrially important enzymes; amylase, protease, gelatinase, lipase, DNase, cellulase, urease, phosphatase and L-asparaginase. The present study emphasized that, ascidians are a prolific resource for novel bioactive actinobacteria with potential for novel drug discovery. This result expands the scope to functionally characterize the novel ascidian associated marine actinobacteria and their metabolites could be a source for the novel molecules of commercial interest.

Coexpression of a ß-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degradation Activity of the Caldicellulosiruptor bescii Exoproteome.

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous ß-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. ß-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and ß-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular ß-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable ß-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCECaldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.

An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG.

The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis.IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

Catalytically Enhanced Cas9 through Directed Protein Evolution.

Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.

Purification and characterization of cycloisomaltotetraose-forming glucanotransferases from Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006.

Glucanotransferases that can synthesize cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.