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Plastics and plasticizers accumulate in the ecological niches affecting biodiversity, and human and environmental health. Bacteria degrading polyethylene terephthalate (PET) were screened and PETases involved in PET degradation were characterized. Here, we identified a carboxylesterase Dkca1 of 48.44 kDa molecular mass from Dietzia kunjamensis IITR165 shown to degrade amorphous PET film into bis(2-hydroxyethyl) terephthalate (BHET) and terephthalic acid (TPA) formed 64.35 μM and 35.26 μM, respectively within 96 h at 37 °C as revealed by LC-MS analysis showed significant PET hydrolase activity similar to reported PETases. SEM analysis confirms the surface erosion as cavities and holes. Dkca1 also hydrolysed BHET and dibutyl phthalate (DBP) at a concentration of 1 mM within 3 h indicating its versatility. Fluorescence quenching shows Dkca1 protein has a maximum affinity (K d ) towards BHET (86.55 μM) than DBP (134.2 μM). The protein demonstrated high stability under temperatures above 40 °C and at the pH range of 6.0-9.0. Moreover, Amino acid composition showed that the Dkca1 enzyme belongs to family VII carboxylesterase containing conserved catalytic triad of Ser183-Glu289-His378 with pentapeptide motif GXSAG and an oxyanion hole H103GGG106, sharing 37.47 % and 32.44 % similarity with a PET hydrolase TfCa from Thermobifida fusca and PAE hydrolase CarEW from Bacillus sp. K91, respectively. A docking study revealed that ligand PET, BHET, and DBP showed favourable binding in the catalytic pocket of the Dkca1 protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gut microbial catechol dehydroxylases are a largely uncharacterized family of metalloenzymes that potentially impact human health by metabolizing dietary polyphenols. Here, we use metatranscriptomics (MTX) to identify highly transcribed catechol-dehydroxylase-encoding genes in human gut microbiomes. We discover a prevalent, previously uncharacterized catechol dehydroxylase (Gp Hcdh) from Gordonibacter pamelaeae that dehydroxylates hydrocaffeic acid (HCA), an anti-inflammatory gut microbial metabolite derived from plant-based foods. Further analyses suggest that the activity of Gp Hcdh may reduce anti-inflammatory benefits of polyphenol-rich foods. Together, these results show the utility of combining MTX analysis and biochemical characterization for gut microbial enzyme discovery and reveal a potential link between host inflammation and a specific polyphenol-metabolizing gut microbial enzyme., Competing Interests: Declaration of interests A.T.C. received research support from Zoe Ltd. for a diet-microbiome study unrelated to this manuscript. C.H. is a scientific advisor for Zoe Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrazidase from Microbacterium hydrocarbonoxydans was revealed to catalyze synthetic hydrazide compounds, enabling the bacteria to grow with them as a sole carbon source, but natural substrates have remained unknown. In this study, kinetic analyses of hydrazidase with parabens showed that the compounds can be substrates. Then, methylparaben induced gene expressions of the operon containing hydrazidase and ABC transporter, and the compound as a sole carbon source was able to grow the bacteria. Furthermore, homology search was carried out revealing that several actinomycetes possess hydrazidase homologs in the operon. Among those bacteria, an amidase from Pseudonocardia acaciae was subjected to a kinetic analysis and a structure determination revealing similar but not identical to those of hydrazidase. Since parabens are reported to exist in plants and soil, and several actinomycetes code the homologous operon, the enzymes with those operons may play a physiologically important role for bacterial survival with use of parabens., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans . Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed K m 2.16 mg/mL and V max 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria .

Background: Rhododendron delavayi is a natural shrub that is distributed at different elevations in the karst region of Bijie, China, and that has an important role in preventing land degradation in this region. In this study, we determined the soil mineral element contents and soil enzyme activities. The composition of the soil bacterial community of R. delavayi at three elevations (1448 m, 1643 m, and 1821 m) was analyzed by high-throughput sequencing, and the interrelationships among the soil bacterial communities, mineral elements, and enzyme activities were determined., Results: The Shannon index of the soil bacterial community increased and then decreased with increasing elevation and was highest at 1643 m. Elevations increased the number of total nodes and edges of the soil bacterial community network, and more positive correlations at 1821 m suggested stronger intraspecific cooperation. Acidobacteria, Actinobacteria and Proteobacteria were the dominant phyla at all three elevations. The Mantel test and correlation analysis showed that Fe and soil urease significantly affected bacterial communities at 1448 m; interestingly, Chloroflexi was positively related to soil urease at 1448 m, and Actinobacteria was positively correlated with Ni and Zn at 1821 m. Fe and soil urease significantly influenced the bacterial communities at lower elevations, and high elevation (1821 m) enhanced the positive interactions of the soil bacteria, which might be a strategy for R. delavayi to adapt to high elevation environments., Conclusion: Elevation significantly influenced the composition of soil bacterial communities by affecting the content of soil mineral elements and soil enzyme activity., (© 2024. The Author(s).)

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides., Competing Interests: Declaration of competing interest Authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Thermophilic actinomycetes are commonly found in extreme environments and can thrive and adapt to extreme conditions. These organisms exhibit substantial variation and garnered significant interest due to their remarkable enzymatic activities. This study evaluated the potential of Streptomyces griseorubens NBR14 and Nocardiopsis synnemataformans NBRM9 strains to produce thermo-stable amylase via submerged fermentation using wheat and bean straw. The Box-Behnken design was utilized to determine the optimum parameters for amylase biosynthesis. Subsequently, amylase underwent partial purification and characterization. Furthermore, the obtained hydrolysate was applied for ethanol fermentation using Saccharomyces cerevisiae. The optimal parameters for obtaining the highest amylase activity by NBR14 (7.72 U/mL) and NBRM9 (26.54 U/mL) strains were found to be 40 and 30 °C, pH values of 7, incubation time of 7 days, and substrate concentration (3 and 2 g/100 mL), respectively. The NBR14 and NBRM9 amylase were partially purified, resulting in specific activities of 251.15 and 144.84 U/mg, as well as purification factors of 3.91 and 2.69-fold, respectively. After partial purification, the amylase extracted from NBR14 and NBRM9 showed the highest activity level at pH values of 9 and 7 and temperatures of 50 and 60 °C, respectively. The findings also indicated that the maximum velocity (V max ) for NBR14 and NBRM9 amylase were 57.80 and 59.88 U/mL, respectively, with K m constants of 1.39 and 1.479 mM. After 48 h, bioethanol was produced at concentrations of 5.95 mg/mL and 9.29 mg/mL from hydrolyzed wheat and bean straw, respectively, through fermentation with S. cerevisiae. Thermophilic actinomycetes and their α-amylase yield demonstrated promising potential for sustainable bio-ethanol production from agro-byproducts., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Many actinobacterial species contain structural genes for iron-dependent enzymes that consume ergothioneine by way of O 2 -dependent dioxygenation. The resulting product ergothioneine sulfinic acid is stable under physiological conditions unless cleavage to sulfur dioxide and trimethyl histidine is catalyzed by a dedicated desulfinase. This report documents that two types of ergothioneine sulfinic desulfinases have evolved by convergent evolution. One type is related to metal-dependent decarboxylases while the other belongs to the superfamily of rhodanese-like enzymes. Pairs of ergothioneine dioxygenases (ETDO) and ergothioneine sulfinic acid desulfinase (ETSD) occur in thousands of sequenced actinobacteria, suggesting that oxidative ergothioneine degradation is a common activity in this phylum., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The isonitrile group is a compact, electron-rich moiety coveted for its commonplace as a building block and bioorthogonal functionality in synthetic chemistry and chemical biology. Hundreds of natural products containing an isonitrile group with intriguing bioactive properties have been isolated from diverse organisms. Our recent discovery of a conserved biosynthetic gene cluster in some Actinobacteria species highlighted a novel enzymatic pathway to isonitrile formation involving a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase. Here, we focus this chapter on recent advances in understanding and probing the biosynthetic machinery for isonitrile synthesis by non-heme iron(II) and α-ketoglutarate-dependent dioxygenases. We will begin by describing how to harness isonitrile enzymatic machinery through heterologous expression, purification, synthetic strategies, and in vitro biochemical/kinetic characterization. We will then describe a generalizable strategy to probe the mechanism for isonitrile formation by combining various spectroscopic methods. The chapter will also cover strategies to study other enzyme homologs by implementing coupled assays using biosynthetic pathway enzymes. We will conclude this chapter by addressing current challenges and future directions in understanding and engineering isonitrile synthesis., (Copyright © 2024. Published by Elsevier Inc.)

Here, we describe hitherto unknown shape-function of S/O-HexNActransferase SvGT (ORF AQF52_3101) instrumental in glycosylation of bacteriocin SvC (ORF AQF52_3099) in Streptomyces venezuelae ATCC 15439. Data from gel filtration, mass spectrometry, analytical ultracentrifugation, and Small Angle X-ray Scattering (SAXS), experiments confirmed elongated dimeric shape in solution for SvGT protein. Enzyme assays confirmed the dependence of SvGT on the availability of Mg 2+ ions to be functionally activated. SAXS data analysis provided that apo and Mg 2+ -activated protein adopt a shape characterized by a radius of gyration and maximum linear dimension of 5.2 and 17.0 nm, and 5.3 and 17.8 nm, respectively. Alphafold2 server was used to model the monomeric chain of this protein which was docked on self to obtain different poses of the dimeric entity. Experimental SAXS data was used to select and refine the structure of SvGT dimer. Results showed that Mg 2+ ions induce reorientation of the GT domain of one chain leading to a dimer with C2 symmetry, and the C-terminal portion entangles with each other in all states. Mutation-rendered alteration in activity profiles confirmed the role of conserved residues around catalytic motif. Global structure analysis puts forth the need to understand the role of constitutionally diverse C-terminal portion in regulating substrate selectivity.Communicated by Ramaswamy H. Sarma.

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 Mn 2+ and GDP, GDP-D-glucopyranose, GDP-L-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn 2+ 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., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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( sp 2 )-Cβ( sp 3 ) 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.

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) β 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.

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-k cat and pH-k cat /K m 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 β-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., (© 2021 Federation of European Biochemical Societies.)

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 ( K D ) 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.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Endo-type chitinase is the principal enzyme involved in the breakdown of N -acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida , Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5-11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N -acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N ' -diacetyl-β-d-chitobiose together with a small amount of N -acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N ' -diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

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-N 3 -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., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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., (© 2021. The Microbiological Society of Korea.)

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., (© 2021 Wiley-VCH GmbH.)

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 Ni 2+ . In the presence of Co 2+ , the t 1/2 values at 60, 65, and 70 °C were 37.7, 9.0, and 4.6 h, respectively, and T m 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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 K m values of 0.0682 ± 0.0074 and 2.0281 ± 0.1301 mM and V max 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.

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 (DBTO 2 ) 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., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2021. Published by Elsevier Ltd.)

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 β-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro . β-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable β-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. IMPORTANCE Caldicellulosiruptor 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.

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.

Aims: Purine-degrading enzymes are favourable as medications and diagnostic tools for hyperuricemia. This study aimed to characterize enzymes isolated from micro-organisms, which may be useful for developing a new prophylaxis for hyperuricemia., Methods and Results: Cellulosimicrobium funkei A153 was found to be a good catalyst for hypoxanthine degradation and could oxidize hypoxanthine to xanthine and further to uric acid. The enzyme catalysing this oxidation was purified, and its partial amino acid sequences were examined. Based on this information and genome sequencing results, this xanthine dehydrogenase family protein was cloned and expressed in Rhodococcus erythropolis L88. The recombinant enzyme with a His-tag was characterized. The enzyme was a xanthine oxidase as it could utilize molecular oxygen as an electron acceptor. It was stable under 50°C and exhibited maximum activity at pH 7·0. The k cat , K m and k cat /K m values for xanthine were 1·4 s -1 , 0·22 mmol l -1 and 6·4 s -1  mmol -1  l, respectively., Conclusions: Xanthine oxidase is favourable for hyperuricemia medication because it oxidizes hypoxanthine, an easily adsorbed purine, to xanthine and further to uric acid, which are hardly adsorbed purines., Significance and Impact of the Study: The enzyme is useful for decreasing serum uric acid levels via conversion of easily absorbed purines to hardly absorbed purines in the intestine. Enzymes from micro-organisms may be used as a novel prophylaxis for hyperuricemia., (© 2020 The Society for Applied Microbiology.)

Covering from 1992 to the end of 2020-11-20.Genetically-encoded polyenic macrolactams, which are constructed by Nature using hybrid polyketide synthase/nonribosomal peptide synthase (PKSs/NRPSs) assembly lines, are part of the large collection of natural products isolated from bacteria. Activation of cryptic (i.e., silent) gene clusters in these microorganisms has more recently allowed to generate and eventually isolate additional members of the family. Having two unsaturated fragments separated by short saturated chains, the primary macrolactam is posited to undergo transannular reactions and further rearrangements thus leading to the generation of a structurally diverse collection of polycyclic (natural) products and oxidized derivatives. The review will cover the challenges that scientists face on the isolation of these unstable compounds from the cultures of the producing microorganisms, their structural characterization, biological activities, optimized biogenetic routes, as well as the skeletal rearrangements of the primary structures of the natural macrolactams derived from pericyclic reactions of the polyenic fragments. The efforts of the synthetic chemists to emulate Nature on the successful generation and structural confirmation of these natural products will also be reported.

Hyaluronate lyases have received extensive attention due to their applications in medical science, drug and biochemical engineering. However, few thermotolerant and pH-stable hyaluronate lyases have been found. In this study, hyaluronate lyase TcHly8B from Thermasporomyces composti DSM22891 was expressed in Escherichia coli BL21(DE3), purified, and characterized. Phylogenetic analysis revealed that TcHly8B belonged to a new subfamily in PL8. The molecular mass of recombinant TcHly8B determined by SDS-PAGE was approximately 86 kDa. The optimal temperature of TcHly8B was 70 °C, which was higher than that of previously reported hyaluronate lyases. TcHly8B was very stable at temperatures from 0 to 60 °C. The optimal pH of TcHly8B was 6.6. It could retain more than 80% of its original enzyme activity after incubation for 12 h in the pH range of 3.0-10.6. TcHly8B degraded hyaluronic acid into unsaturated disaccharides as the end products. The amino acid sequence and structure analysis of TcHly8B demonstrated that the amino acid composition and salt bridges might contribute to the thermostability of TcHly8B. Overall, this study provides an excellent example for the discovery of thermotolerant hyaluronate lyases and can be applied to the industrialized production and basic research of hyaluronate oligosaccharides., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Benzofluorene-containing atypical angucyclines are an important family of natural products with a broad spectrum of antibacterial and cytotoxic properties. Interestingly, symmetric and asymmetric dimers showed better activity than the monomer in this class of compounds. Herein, we reported the isolation of a new asymmetric dimer nenestatin B (2) from the deep sea actinomycete Micromonospora echinospora SCSIO 04089 and a monomer nenestatin C (3) from an NmrA family regulatory protein coding gene nes18 inactivated mutant. The structural elucidation of 3 indicated the essential role of Nes18 in the biosynthetic pathway of 2, specifically in dimerization via C-C bond formation.

The cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca 2+ -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca 2+ to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca 2+ to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70°C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS., (© 2020 Wiley Periodicals LLC.)

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., (Copyright © 2021 Zhang et al.)

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products ("normal" and "abnormal") when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (k cat /K M ) 8.9-fold to 484 s -1  mM -1 for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMO C308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L -1  d -1 . This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils., (© 2020 Wiley-VCH GmbH.)

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d-ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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.

Lysoplasmalogen-specific phospholipase D (LyPls-PLD) catalyzes reactions in a manner similar to those catalyzed by glycerophosphodiester phosphodiesterase (GDPD) and other well-known PLDs. Although these enzymes hydrolyze the glycerophosphodiester bond, their substrate specificities are completely different. Previously, we reported that LyPls-PLD from Thermocrispum sp. RD004668 shows only 53% activity with 1-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF) relative to the 100% activity it shows with choline lysoplasmalogen (LyPlsCho). Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) activity can be used to evaluate for cardiovascular disease. Hence, development of a point-of-care testing kit requires a LysoPAF-specific PLD (LysoPAF-PLD) to measure Lp-PLA 2 activity. Rational site-directed mutagenesis and kinetic analysis were applied to generate LysoPAF-PLD from LyPls-PLD and to clarify the mechanisms underlying the substrate-recognition ability of LyPls-PLD. Our results suggest that LyPls-PLD variants A47, M71, N173, F211, and W282 are possibly involved in substrate recognition and that F211L may substantially alter substrate preference. Moreover, the specific activity ratio LysoPAF/LyPlsCho corresponding to F211L was up to 25-fold higher than that corresponding to the wild-type enzyme. Thus, we succeeded in switching from LyPlsCho- to LysoPAF-PLD. These results suggest that the F211L variant may be utilized to measure Lp-PLA 2 activity. Kinetic analyses demonstrated that product release was the rate-limiting step of the reaction, with flexibility of the sn-1 ether-linked vinyl/alkyl chain of the substrate being essential for substrate binding and product release. Our findings may lead to a better understanding of the differences between homologous enzymes (such as PLD, sphingomyelinase D, and GDPD of the phosphatidylinositol-phosphodiesterase superfamily) in relation to substrate recognition. ENZYME: EC 3.1.4.2 (currently assigned)., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca2+ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190*S176A, in complex with metal ions, Ca2+ and Zn2+, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190*, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca2+ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Better understanding through direct observation of the mechanisms involved in chemical and enzymatic hydrolysis of biomass is of great importance, to implement a substitute for the common cellulose standards. We report the hydrolysis of biomass, using exclusively the parenchyma, to isolate cellulose nanoplatelets using a less harsh pretreatment. Then, we show direct evidence of the effect of endoglucanase on the structure of cellulose nanoplatelets, finding that amorphous cellulose is exclusively digested, loosening the cellulose nanofibrils in the process. The analysis of micrographs demonstrates that when cellulose nanoplatelets are deposited on a silicon wafer, its thickness can be qualitatively measured by the interference color detected using an optical microscope. This finding facilitates further studies of mechanisms involved in lignin removal and cellulose nanofibrils production by specific enzymatic digestion., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Arthrobacter simplex exhibits excellent Δ 1 -dehydrogenation ability, but the acquisition of the desirable strain is limited due to lacking of comprehensive genetic manipulation. Herein, a promoter collection for fine-tuning gene expression was achieved. Next, the expression level was enhanced and directed evolution of the global transcriptional factor (IrrE) was applied to enhance cell viability in systems containing more substrate and ethanol, thus contributing to higher production. IrrE promotes a stronger antioxidant defense system, more energy generation, and changed signal transduction. Using a stronger promoter, the enzyme activities were boosted but their positive effects on biotransformation performance were inferior to cell stress tolerance when exposed to challenging systems. Finally, an optimal strain was created by collectively reinforcing cell stress tolerance and catalytic enzyme activity, achieving a yield 261.8% higher relative to the initial situation. Our study provided effective methods for steroid-transforming strains with high efficiency.

Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator; however, the effects of Pb contamination on P. crinitum rhizosphere soil enzymatic activities and microbial composition remain largely unexplored. Thus, an indoor experiment was conducted by cultivating P. crinitum seedlings and exposing them to four Pb concentrations (0, 1,000, 2000 and 3000 mg/kg Pb). Protease, urease, acid phosphatase and invertase activities were determined using standard methods while soil bacterial composition was determined by 16 S rDNA sequencing. The results showed that rhizosphere soil acid phosphatase activity significantly increased with increasing Pb concentration, while urease activity was significantly greater in rhizosphere soil contaminated with 1000 and 2000 mg/kg than in the control. There was a clear shift in bacterial composition during phytoremediation by P. crinitum. Compared to the control, Bacteroidetes was more abundant in all Pb-contaminated soils, Actinobacteria was more abundant in 1000 mg/kg Pb-treated soil, and Firmicutes was more abundant in 3000 mg/kg Pb-treated soil. Positive correlations were observed between dominant bacterial phyla and soil enzyme activities. Metabolic pathways, such as ABC transporter, quinine reductase, and ATP-binding protein were significantly increased in rhizosphere soil bacteria with Pb contamination. In conclusion, Pb contamination differentially influenced the activities of rhizosphere soil enzymes, specifically increasing acid phosphatase and urease activities, and alters the dominance of soil bacteria through up-regulation of genes related to some metabolic pathways. The strong correlations between dominant bacterial phyla and enzymatic activities suggest synergetic effects on the growth of P. crinitum during Pb contamination., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hyaluronic acid (HA) is an anionic linear polysaccharide abundantly distributed in the extracellular matrix of mammalian connective, growing, and tumor tissues. Hyaluronidase is used as an important drug diffusion promoter and a tool enzyme to produce HA oligosaccharides. However, there is no thermostable hyaluronidase suitable for application to date. In this study, a thermophilic hyaluronate lyase, TcHly8C, from Thermasporomyces composti DSM22891 was expressed in Escherichia coli. The recombinant TcHly8C was most active at 70 °C, and it retained about 30% of initial activity after incubation at 60 °C for 28 days. The half-lives of TcHly8C at 60 °C and 70 °C were 16.1 d and 2.3 h, respectively. The optimum pH of TcHly8C is 5.93, and it was stable at pH 6.15-10.90. The presence of Mg 2+ could enhance its enzymatic activity significantly. K m , k cat , and k cat /K m of TcHly8C towards HA were 3.69 mg∙ml -1 , 17.82 s -1 , and 4.82 ml∙mg -1 ∙s -1 , respectively. TcHly8C degraded HA in an exolytic mode, and the end product was unsaturated HA disaccharide (ΔUA-GlcNAc). Overall, our results show that TcHly8C is the first reported PL8 exo-type hyaluronate lyase with high thermostability, which provides a potential enzyme used in medicine and production of HA oligosaccharides., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.

A psychrophilic strain Cryobacterium sp. LW097 was isolated from the subglacial sediments and discovered to show considerable β-galactosidases activity at low temperatures. To provide access to genes predicted to encode cold-adapted glycoside hydrolases with biotechnological relevance, we have sequenced the genome of Cryobacterium sp. LW097. Annotation with CAZy database revealed four β-galactosidase genes, bgal322, bgal435, bgal436, and bgal2567 belonging to the GH-42 family and GH-35 family. All the four β-galactosidases recombinantly expressed retained a high level of relative activity at 5 °C and showed different optimum temperatures ranging from 25 °C to 40 °C. The enzyme kinetics proved that Bgal322, Bgal436, and Bgal2567 had lower K m to both oNPG and lactose at 5 °C, further proving their adaption to low temperature. Substrate specificity analysis showed that these four β-galactosidases owned different preferences. The novel GH-42 β-galactosidases Bgal435 showed β-D-glucosidase activity (33.67 ± 0.28%) in addition to β-D-galactosidase activity. Bgal322 preferred β-D-(1,4)-galactobiose, whereas the other three preferred lactulose. Bgal435 showed the highest k cat value of 68.2 ± 1.7 s -1 at 5 °C toward lactose among these four enzymes. The exquisite substrate specificity of Bgal436 in milk made it a potential candidate for applications in milk lactulose quantification., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes- Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.

While several bioactive natural products that contain tetramate or pyridone heterocycles have been described, information on the enzymology underpinning these functionalities has been limited. Here we biochemically characterize an off-loading Dieckmann cyclase, NcmC, that installs the tetramate headgroup in nocamycin, a hybrid polyketide/nonribosomal peptide natural product. Crystal structures of the enzyme (1.6 Å) and its covalent complex with the epoxide cerulenin (1.6 Å) guide additional structure-based mutagenesis and product-profile analyses. Our results offer mechanistic insights into how the conserved thioesterase-like scaffold has been adapted to perform a new chemical reaction, namely, heterocyclization. Additional bioinformatics combined with docking and modeling identifies likely candidates for heterocycle formation in underexplored gene clusters and uncovers a modular basis of substrate recognition by the two subdomains of these Dieckmann cyclases.

Moutai Jiuqu is a famous aromatic raw material of Maotai flavor liquor in China. It is brewed at high temperature and contains many kinds of bacteria, molds, and yeasts. There are many useful glycoside hydrolases in these microfloras, from which efficient glycoside hydrolases can be screened for biotransformation of natural saponins. In this study, an α-L-arabinofuranosidase gene (CaAraf51, 1524 bp, 507 amino acid, 55.07 kDa, and pI = 4.8) was cloned from Cellulosimicrobium aquatile Lyp51, which was isolated from the Maotai Jiuqu. The CaAraf51 was heterogeneously expressed in E. coli BL21 (DE3) and purified by N-terminal His-tag with the Ni 2+ -affinity column chromatography. The results show that purified CaAraf51 has a 6.8-fold purification factor and specific activity of 15 U/mg. Under optimal conditions (pH 5.0, temperature 40 °C), kinetic parameters K m of CaAraf51 for pNPαAraf and Rc were 1.1 and 0.57 mM, the V max were 25 and 6.25 μmol/min/mg, respectively. 90% of 0.87 mg Rc substrate can be transformed by 9.6 U purified CaAraf51 in 1 mL reaction system under suitable conditions (30 °C, pH 7.5 phosphate buffer, 1 h). In addition, we also tested the effects of metal ions and chemical agents on the activity of CaAraf51. According to systematically studied its function and enzymatic properties, CaAraf51 has excellent value and potential of biotransformation Rc into Rd.

Two bacterial strains isolated from soil, namely Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006, were found to produce a novel oligosaccharide. The oligosaccharide was enzymatically produced from dextran using the culture supernatant of Agreia sp. D1110 or M. trichothecenolyticum D2006. LC-MS and NMR analysis identified the novel oligosaccharide as cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→}, which was named cycloisomaltotetraose, and abbreviated as CI4. CI4 was subsequently crystalized and its X-ray crystallographic structure was determined. CI4 crystals were shown to be pentahydrate, with the CI4 molecules in the crystal structure displaying a unique 3D structure, in which two glucosyl residues in the molecule were facing each other. This unique 3D structure was quite different from the 3D structure of known cyclic tetrasaccharides. This is the first report of CI4 molecules and their unique crystal structure., (Copyright © 2020 Elsevier Ltd. All rights reserved.)