Efficient production of GlcNAc in an aqueous-organic system with a Chitinolyticbacter meiyuanensis SYBC-H1 mutant.

OBJECTIVES: Shellfish waste is a primary source for making N-acetyl-D-glucosamine. Thus, establishing a high-efficiency and low-cost bioconversion method to produce N-acetyl-D-glucosamine directly from shellfish waste was promising. RESULTS: A mutant C81 was obtained from Chitinolyticbacter meiyuanensis SYBC-H1 via 60Co-γ irradiation. This mutant C81 showed the highest chitinase activity of 9.8 U/mL that was 85% higher than the parent strain. The mutant C81 exhibted improved antioxidant activities, including total antioxidant capacity, superoxide radical ability, and hydroxyl radical scavenging ability, compared to that of the parent strain. Four out of nine organic solvents increased the chitinase activity by 1.9%, 6.8%, 11.7%, and 15.8%, corresponding to methylbenzene, n-heptane, petroleum ether, and n-hexane, respectively. The biphase system composed of aqueous and hexane presented a five-fold reduction of cell viability compared to the control. Using a continuous fermentation bioconversion process, 4.2 g/L GlcNAc was produced from crayfish shell powder with a yield of 80% of the chitin content. CONCLUSIONS: This study demonstrated that the mutant C81 is suitable for converting crayfish shell powder into GlcNAc in an aqueous-organic system.

Two new 13-hydroxylated milbemycin metabolites from the genetically engineered strain Streptomyces avermitilis AVE-H39.

Two new milbemycin metabolites, 13α-hydroxymilbemycin ß13 (1) and 26-methyl-13α-hydroxymilbemycin ß13 (2), were isolated from the fermentation broth of a genetically engineered strain Streptomyces avermitilis AVE-H39. Their structures were determined by the comprehensive spectroscopic data, including 1 D, 2 D NMR, MS spectral analysis and the comparison with data from the literature. Compounds 1 and 2 not only exhibited potent acaricidal activities against Tetranychus cinnabarinus, but also had nematocidal activity against Bursaphelenchus xylophilus.

Two new milbemycin derivatives from a genetically engineered strain Streptomyces bingchenggensis.

Two new milbemycin derivatives, milbemycin M (1) and milbemycin N (2), were isolated from the culture of a genetically engineered strain Streptomyces bingchenggensis BCJ60. Their structures were elucidated through the interpretation of NMR and HR-ESI-MS spectroscopic data, as well as comparison with previous reports. The acaricidal and nematicidal activities of them against Tetranychus cinnabarinus and Bursaphelenchus xylophilus were tested. The results showed that compounds 1-2 possessed potent acaricidal and nematocidal activities.

Evaluation of the Strain Bacillus amyloliquefaciens YP6 in Phoxim Degradation via Transcriptomic Data and Product Analysis.

Phoxim, a type of organophosphorus pesticide (OP), is widely used in both agriculture and fisheries. The persistence of phoxim has caused serious environmental pollution problems. In this study, Bacillus amyloliquefaciens YP6 (YP6), which is capable of promoting plant growth and degrading broad-spectrum OPs, was used to study phoxim degradation. Different culture media were applied to evaluate the growth and phoxim degradation of YP6. YP6 can grow rapidly and degrade phoxim efficiently in Luria-Bertani broth (LB broth) medium. Furthermore, it can also utilize phoxim as the sole phosphorus source in a mineral salt medium. Response surface methodology was performed to optimize the degradation conditions of phoxim by YP6 in LB broth medium. The optimum biodegradation conditions were 40 °C, pH 7.20, and an inoculum size of 4.17% (v/v). The phoxim metabolites, O,O-diethylthiophosphoric ester, phoxom, and α-cyanobenzylideneaminooxy phosphonic acid, were confirmed by liquid chromatography-mass spectrometry. Meanwhile, transcriptome analysis and qRT-PCR were performed to give insight into the phoxim-stress response at the transcriptome level. The hydrolase-, oxidase-, and NADPH-cytochrome P450 reductase-encoding genes were significantly upregulated for phoxim hydrolysis, sulfoxidation, and o-dealkylation. Furthermore, the phoxim biodegradation pathways by YP6 were proposed, for the first time, based on transcriptomic data and product analysis.

Cloning, Expression and 3D Structure Prediction of Chitinase from Chitinolyticbacter meiyuanensis SYBC-H1.

Two CHI genes from Chitinolyticbacter meiyuanensis SYBC-H1 encoding chitinases were identified and their protein 3D structures were predicted. According to the amino acid sequence alignment, CHI1 gene encoding 166 aa had a structural domain similar to the GH18 type II chitinase, and CHI2 gene encoding 383 aa had the same catalytic domain as the glycoside hydrolase family 19 chitinase. In this study, CHI2 chitinase were expressed in Escherichia coli BL21 cells, and this protein was purified by ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G-100 chromatography. Optimal activity of CHI2 chitinase occurred at a temperature of 40 °C and a pH of 6.5. The presence of metal ions Fe(3+), Fe(2+), and Zn(2+) inhibited CHI2 chitinase activity, while Na⁺ and K⁺ promoted its activity. Furthermore, the presence of EGTA, EDTA, and ß-mercaptoethanol significantly increased the stability of CHI2 chitinase. The CHI2 chitinase was active with p-NP-GlcNAc, with the Km and Vm values of 23.0 µmol/L and 9.1 mM/min at a temperature of 37 °C, respectively. Additionally, the CHI2 chitinase was characterized as an N-acetyl glucosaminidase based on the hydrolysate from chitin. Overall, our results demonstrated CHI2 chitinase with remarkable biochemical properties is suitable for bioconversion of chitin waste.

A novel spiro-heterocycle milbemycin metabolite from a genetically engineered strain of Streptomyces bingchenggensis.

Milbemycin R, a novel spiro-heterocycle milbemycin, was obtained from the metabolites produced by the mutant strain S. bingchenggensis BCJ60B11. Its structure was determined by spectroscopic and spectrometric analyses including 1 D, 2 D NMR, IR, HR-ESI-MS data. The acaricidal and nematicidal activities of milbemycin R against Tetranychus cinnabarinus and Bursaphelenchus xylophilus were tested. Milbemycin R possessed better acaricidal activity than milbemycins A3/A4.

Development of an HPLC method to analyze and prepare elsinochrome C and hypocrellin A in the submerged fermentation broth of Shiria sp. SUPER-H168.

A rapid and sensitive analytical method based on reverse-phase high-performance liquid chromatography was first developed to simultaneously determine elsinochrome C (EC) and hypocrellin A (HA) in the submerged fermentation. The mobile phase consisted of acetonitrile-water 60:40 (v/v) with a flow-rate of 1 mL/min. The calibration curves were as follows: y = 37,625x + 249,775 for EC, y = 30,813x + 556,409 for HA and linear at the investigated concentration. The correlation coefficients (R(2) ) were 0.9989 and 0.9998 respectively for EC and HA. The limits of detection and quantification were 175 and 585 µg/L for EC and 205 and 610 µg/L for HA. The precisions of concentration and retention times were less than 2.5 and 0.3%. The recovery of the method was greater than 95.0%. The methodology was applied to analyze simultaneously EC and HA concentrations in a submerged fermentation, and was adequate for analysis of biosynthesis of perylenequinones. The method was also amplified to separate and purify EC and HA using a semi-preparative C(18) column. In addition, elsinochrome C was first identified in the submerged fermentation broth of Shiraia sp. SUPER-H168.

Simultaneous laccase production and color removal by culturing fungus Pycnoporus sp. SYBC-L3 in a textile wastewater effluent supplemented with a lignocellulosic waste Phragmites australis.

We conducted experiments to culture Pycnoporus sp. SYBC-L3 in a medium comprising an industrial waste (dye-containing textile effluent) and a lignocellulosic waste (Phragmites australis) that achieved laccase production while having the color removed from the wastewater. Our experimental results showed that the fungus grew well in liquid submerged cultivation with the diluted textile effluent as the sole culture medium, but relatively low extracellular laccase activity (1.8 U/mL) was produced. Addition of the lignocellulosic biomass enhanced laccase production and color removal. The highest laccase activity was found to be 6.5 U/mL in the presence of Phragmites australis stem. Under this condition, 70 % color removal occurred in the culture medium. This study provided an alternative novel scheme to remove color in textile wastewater while having an economic value added by producing laccase.

Optimization of nutrition factors on chitinase production from a newly isolated Chitiolyticbacter meiyuanensis SYBC-H1.

The present study reports statistical medial optimization for chitinase production by a novel bacterial strain isolated from soil recently, which the name Chitinolyticbacter meiyuanensis SYBC-H1 is proposed. A sequential statistical methodology comprising of Plackett-Burman and response surface methodology (RSM) was applied to enhance the fermentative production of chitinase, in which inulin was firstly used as an effective carbon source. As a result, maximum chitinase activity of 5.17 U/mL was obtained in the optimized medium, which was 15.5-fold higher than that in the basal medium. The triplicate verification experiments were performed under the optimized nutrients levels which indicated that it well agreed with the predicted value.

Advances in lytic polysaccharide monooxygenases with the cellulose-degrading auxiliary activity family 9 to facilitate cellulose degradation for biorefinery.

One crucial step in processing the recalcitrant lignocellulosic biomass is the fast hydrolysis of natural cellulose to fermentable sugars that can be subsequently converted to biofuels and bio-based chemicals. Recent studies have shown that lytic polysaccharide monooxygenase (LPMOs) with auxiliary activity family 9 (AA9) are capable of efficiently depolymerizing the crystalline cellulose via regioselective oxidation reaction. Intriguingly, the catalysis by AA9 LPMOs requires reductant to provide electrons, and lignin and its phenolic derivatives can be oxidized, releasing reductant to activate the reaction. The activity of AA9 LPMOs can be enhanced by in-situ generation of H2O2 in the presence of O2. Although scientific understanding of these enzymes remains somewhat unknown or controversial, structure modifications on AA9 LPMOs through protein engineering have emerged in recent years, which are prerequisite for their extensive applications in the development of cellulase-mediated lignocellulosic biorefinery processes. In this review, we critically comment on advances in studies for AA9 LPMOs, i.e., characteristic of AA9 LPMOs catalysis, external electron donors to AA9 LPMOs, especially the role of the oxidization of lignin and its derivatives, and AA9 LPMOs protein engineering as well as their extensive applications in the bioprocessing of lignocellulosic biomass. Perspectives are also highlighted for addressing the challenges.

Two new threonine-containing metabolites from fungus Curvularia inaequalis strain HS-FG-257.

Two new threonine-containing metabolites, N-[4-hydroxy-3-prenyl-benzoyl]-L-threonine (1) and N-[2,2-dimethyl-2H-chromene-6-carbonyl]-L-threonine (2), were isolated from the fermentation broth of the soil fungus Curvularia inaequalis strain HS-FG-257. Their structures were elucidated through the interpretation of HR-ESIMS and extensive NMR spectroscopic data. Both compounds exhibited no cytotoxic activity against the test cell lines A549 and HCT-116.

Chitinolyticbacter meiyuanensis SYBC-H1T, gen. nov., sp. nov., a chitin-degrading bacterium isolated from soil.

A novel aerobic mesophilic bacterial strain SYBC-H1(T) capable of degrading chitin was isolated and classified in this study. The strain exhibited strong chitinolytic activity and was a Gram-negative, curved, rod-shaped, and motile bacterium. Growth of this strain was observed between 10 and 41°C and between pH 3.5 and 9.5. The DNA G + C content of strain SYBC-H1(T) was 53.25 mol%. The cellular fatty acids (>5%) were 12:0 iso 3-OH (5.87%), 16:0 (28.16%), and 18:1ω7c (20.48%). Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain SYBC-H1(T) belonged to the family Neisseriaceae, and was distantly related (95.0% similarity) to the genus Chitiniphilus. Its phenotype was unique and genetic and phylogenetic analysis experiments suggested that strain SYBC-H1(T) represented the type strain (CGMCC 3438(T), ATCC BAA-2140(T)) of a novel genus, for which the name Chitinolyticbacter meiyuanensis SYBC-H1(T) gen. nov., sp. nov. was proposed. The highest enzymatic activity of chitinase (9.6 U/ml) was obtained at 72 h in 250 ml shake flasks. The 16S rRNA gene sequence of SYBC-H1(T) has been deposited in GenBank under the accession number GQ981314.

Identification of Chitinolytic Enzymes in Chitinolyticbacter meiyuanensis and Mechanism of Efficiently Hydrolyzing Chitin to N-Acetyl Glucosamine.

Chitinolyticbacter meiyuanensis SYBC-H1, a bacterium capable of hydrolyzing chitin and shrimp shell to N-acetyl glucosamine (GlcNAc) as the only product, was isolated previously. Here, the hydrolysis mechanism of this novel strain toward chitin was investigated. Sequencing and analysis of the complete genome of SYBC-H1 showed that it encodes 32 putatively chitinolytic enzymes including 30 chitinases affiliated with the glycoside hydrolase (GH) families 18 (26) and 19 (4), one GH family 20 ß-N-acetylglucosaminidase (NAGase), and one Auxiliary Activities (AA) family 10 lytic polysaccharide monooxygenase (LPMO). However, only eight GH18 chitinases, one AA10 LPMO, and one GH20 NAGase were detected in the culture broth of the strain, according to peptide mass fingerprinting (PMF). Of these, genes encoding chitinolytic enzymes including five GH18 chitinases (Cm711, Cm3636, Cm3638, Cm3639, and Cm3769) and one GH20 NAGase (Cm3245) were successfully expressed in active form in Escherichia coli. The hydrolysis of chitinous substrates showed that Cm711, Cm3636, Cm3638, and Cm3769 were endo-chitinases and Cm3639 was exo-chitinase. Moreover, Cm3639 and Cm3769 can convert the GlcNAc dimer and colloidal chitin (CC) into GlcNAc, which showed that they also possess NAGase activity. In addition, NAGase Cm3245 possesses a very high exo-acting activity of hydrolyzing GlcNAc dimer. These results suggest that chitinases and NAGase from SYBC-H1 both play important roles in conversion of N-acetyl chitooligosaccharides to GlcNAc, resulting in the accumulation of the final product GlcNAc. To our knowledge, this is the first report of the complete genome sequence and chitinolytic enzyme genes discovery of this strain.

Mining of alkaline proteases from Bacillus altitudinis W3 for desensitization of milk proteins: Their heterologous expression, purification, and characterization.

In this study, three active alkaline proteases (AprEs) (BaApr1, BaApr2, and BaApr9) from Bacillus altitudinis W3 were obtained through bioinformatics analysis and verification. Multiple sequence alignment showed low identity of 64.60% and suggested that the three AprEs belonged to the S8A subfamily of serine proteases. They showed maximal activity with pH of 9.5 at 55 °C, 8.5 at 50 °C, and 10.5 at 45 °C, respectively. They were stable at alkaline condition and below 50 °C. In the presence of Ca2+, the optimal temperatures and thermostability of them were significantly improved. They were activated by Ca2+ and Mg2+ but inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Surfactants had little effect on them, but most organic solvents had some inhibitory effect except for n-hexane. They were effective in hydrolyzing natural proteins such as casein and NON-fat powdered milk. BaApr1 exhibited the highest catalytic efficiency towards casein and showed an excellent effect on the desensitization of milk proteins. The present study reveals some useful characteristics of the three AprEs, and indicates that AprEs have potential application values in the desensitization process of milk proteins.

Two novel milbemycin derivatives from the genetically engineered strain Streptomyces avermitilis AVE-H39.

Two novel milbemycin derivatives, 5,27-epoxy-13α-hydroxy milbemycin ß11 (1) and 5,27-epoxy-13α-hydroxy-25-ethyl milbemycin ß11 (2), were isolated from the genetically engineered strain Streptomyces avermitilis AVE-H39. Their structures were elucidated through the interpretation of HR-ESIMS and extensive NMR spectroscopic data. Compounds 1 and 2 exhibited moderate acaricidal and nematicidal activities.

The effects of phytoremediation on soil bacterial communities in an abandoned mine site of rare earth elements.

Overexploitation of rare earth elements (REEs) has caused serious desertification and environmental pollution. The ecological restoration of mining areas has attracted increasing attention in China. Soil microbiota is important for successful ecological remediation of abandoned mine land. In this study, soil samples were collected from a restored REE mine site, and the bacterial community composition and diversity were assessed by Illumina high-throughput sequencing targeting the V3-V4 region of the 16S rRNA gene. Microbiota significantly developed in the remediated land. A total of 663,781 effective 16S rRNA gene sequences were obtained, which were classified into 28 bacterial phyla and 3 archaeal phyla. The dominant phyla across all samples (>5% of total effective sequences) were Proteobacteria, Acidobacteria and Firmicutes. Bacterial diversity indices (OTU number, Shannon index and Chao1 index) of the restored soils were higher than those of the tailings and even surpassed those in the unmined site. Redundancy analysis indicated that soil nutrients (soil organic carbon, available phosphorus and total nitrogen) were the dominant factors, followed by soil pH, affecting bacterial community structure. In general, these results suggested that soil amendment and phytoremediation effectively improved the soil environment of the abandoned mine site, which also increased our understanding of the correlation between microbial variation and soil properties in restored REE mine soils.

Hydrogen Peroxide-Resistant CotA and YjqC of Bacillus altitudinis Spores Are a Promising Biocatalyst for Catalyzing Reduction of Sinapic Acid and Sinapine in Rapeseed Meal.

For the more efficient detoxification of phenolic compounds, a promising avenue would be to develop a multi-enzyme biocatalyst comprising peroxidase, laccase and other oxidases. However, the development of this multi-enzyme biocatalyst is limited by the vulnerability of fungal laccases and peroxidases to hydrogen peroxide (H2O2)-induced inactivation. Therefore, H2O2-resistant peroxidase and laccase should be exploited. In this study, H2O2-stable CotA and YjqC were isolated from the outer coat of Bacillus altitudinis SYBC hb4 spores. In addition to the thermal and alkali stability of catalytic activity, CotA also exhibited a much higher H2O2 tolerance than fungal laccases from Trametes versicolor and Trametes trogii. YjqC is a sporulation-related manganese (Mn) catalase with striking peroxidase activity for sinapic acid (SA) and sinapine (SNP). In contrast to the typical heme-containing peroxidases, the peroxidase activity of YjqC was also highly resistant to inhibition by H2O2 and heat. CotA could also catalyze the oxidation of SA and SNP. CotA had a much higher affinity for SA than B. subtilis CotA. CotA and YjqC rendered from B. altitudinis spores had promising laccase and peroxidase activities for SA and SNP. Specifically, the B. altitudinis spores could be regarded as a multi-enzyme biocatalyst composed of CotA and YjqC. The B. altitudinis spores were efficient for catalyzing the degradation of SA and SNP in rapeseed meal. Moreover, efficiency of the spore-catalyzed degradation of SA and SNP was greatly improved by the presence of 15 mM H2O2. This effect was largely attributed to synergistic biocatalysis of the H2O2-resistant CotA and YjqC toward SA and SNP.

Recent advances in microbial raw starch degrading enzymes.

Raw starch degrading enzymes (RSDE) refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. These promising enzymes can significantly reduce energy and simplify the process in starch industry. RSDE are ubiquitous and produced by plants, animals, and microorganisms. However, microbial sources are the most preferred one for large-scale production. During the past few decades, RSDE have been studied extensively. This paper reviews the recent development in the production, purification, properties, and application of microbial RSDE. This is the first review on microbial RSDE to date.

Optimization of nutrition factors on chitinase production from a newly isolated Chitiolyticbacter meiyuanensis SYBC-H1

The present study reports statistical medial optimization for chitinase production by a novel bacterial strain isolated from soil recently, which the name Chitinolyticbacter meiyuanensis SYBC-H1 is proposed. A sequential statistical methodology comprising of Plackett-Burman and response surface methodology (RSM) was applied to enhance the fermentative production of chitinase, in which inulin was firstly used as an effective carbon source. As a result, maximum chitinase activity of 5.17 U/mL was obtained in the optimized medium, which was 15.5-fold higher than that in the basal medium. The triplicate verification experiments were performed under the optimized nutrients levels which indicated that it well agreed with the predicted value.