Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine.

As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.

Pigment production by a newly isolated strain Pycnoporus sanguineus SYBC-L7 in solid-state fermentation.

Natural pigments are playing important roles in our daily lives. They not only make products colorful but also provide various health benefits for humans. In addition, Pycnoporus genus, listed as food- and cosmetic-grade microorganism, is one of the promising organisms for developing natural pigments. In this study, a new fungal strain with high efficiency in producing intense orange pigments was isolated and identified as Pycnoporus sanguineus SYBC-L7. Different agro-industrial wastes were applied to evaluate the growth and pigment production of strain SYBC-L7. SYBC-L7 can grow rapidly and effectively produce pigments using wood chips as substrate in solid-state fermentation (SSF). Culture conditions were also optimized for value-added pigments production and the optimum production conditions were glucose as carbon source, ammonium tartrate as nitrogen source, initial pH 6.0, and relative humidity of 65%. Pigment components, cinnabarinic acid, tramesanguin, and 2-amino-9-formylphenoxazone-1-carbonic acid were confirmed by liquid chromatography-mass spectrometry. Meanwhile, an agar plate diffusion assay was performed to evaluate the antimicrobial activity of the pigment. These pigments showed more significant inhibition of Gram-positive than Gram-negative bacteria. The results showed that Pycnoporus sanguineus SYBC-L7 was able to cost-effectively produce intense natural orange pigments with antibacterial activity in SSF, which is the basis of their large-scale production and application.

Improving the decolorization activity of Bacillus pumilus W3 CotA-laccase to Congo Red by rational modification.

Congo Red (CR) is a typical azo dye with highly toxic and carcinogenic properties. This study aimed to improve the decolorization activity of Bacillus pumilus W3 CotA-laccase for azo dye CR. This work analyzed the interaction between CotA-laccase and CR based on homology modeling and molecular docking. The three amino acids (Gly323, Thr377, Thr418) in the substrate-binding pocket were rationally modified through saturation mutation. Finally, the obtained multi-site mutants T377I/T418G and G323S/T377I/T418G decolorized 76.59% and 59.37% of CR within 24 h at pH 8.0 without a mediator, which were 3.15- and 2.44-fold higher than the wild-type CotA. The catalytic efficiency of the multi-site mutants T377I/T418G and G323S/T377I/T418G to CR were increased by 2.21- and 2.01-fold compared with the wild-type CotA, respectively. The mechanism of activity enhancement of mutants was proposed by structural analysis. This evidence suggests that the mutants T377I/T418G and G323S/T377I/T418G could be used as novel bioremediation tools.

Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis.

Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5. KEY POINTS: • Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis. • Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively. • The mechanisms of awarding enhanced activity to the mutants were supposed.

Recombinant Horseradish Peroxidase C1A Immobilized on Hydrogel Matrix for Dye Decolorization and Its Mechanism on Acid Blue 129 Decolorization.

In this study, horseradish peroxidase C1A (HRP C1A) from Armoracia rusticana was expressed in Escherichia coli as an inclusion body. Subsequently, an active recombinant HRP C1A was obtained by refolding gradually using dilution-ultrafiltration. The recombinant HRP C1A was immobilized on agarose-chitosan hydrogel at 86.9 ± 2.5% of immobilization efficiency. After immobilization of the recombinant HRP C1A, the pH and temperature stability were improved and the reusability of the recombinant HPR C1A was achieved. The immobilized HRP C1A activity was retained above 80% after 6 cycles. The immobilized recombinant HRP C1A was used for the decolorization of four various dyes, including acid blue 129 (AB129), methyl blue (MB), methyl red (MR), and trypan blue (TB). The decolorization rates are all more than 70%, among which the decolorization effect of AB129 was the most significant (the decolorization rate was 76.3 ± 1.6%). Furthermore, a plausible decolorization pathway for AB129 was proposed based on the identified intermediates by ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS). This is the first report of the putative mechanism on the decolorization of AB129 by HRP.

Extracellular expression of mutant CotA-laccase SF in Escherichia coli and its degradation of malachite green.

In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.

Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis.

CotA-laccases are potential enzymes that are widely used in decolorization of dyes and degradation of toxic substances. In this study, a novel CotA-laccase gene from Bacillus pumilus W3 was applied for rational design. After a series of site-directed genetic mutations, the mutant S208G/F227A showed a 5.1-fold higher catalytic efficiency (kcat/Km) than the wild-type CotA-laccase did. The optimal pH of S208G/F227A was 3.5 with ABTS as substrate. The residual activity of mutant S208G/F227A was more than 80% after incubated for 10 h at pH 7-11. Mutant S208G/F227A showed optimal temperature at 80°C with ABTS as substrate. The thermal stability of mutant laccase S208G/F227A was lower than that of wild-type CotA-laccase. This study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis.

Bacterial laccases: promising biological green tools for industrial applications.

Multicopper oxidases (MCOs) are a pervasive family of enzymes that oxidize a wide range of phenolic and nonphenolic aromatic substrates, concomitantly with the reduction of dioxygen to water. MCOs are usually divided into two functional classes: metalloxidases and laccases. Given their broad substrate specificity and eco-friendliness (molecular oxygen from air as is used as the final electron acceptor and they only release water as byproduct), laccases are regarded as promising biological green tools for an array of applications. Among these laccases, those of bacterial origin have attracted research attention because of their notable advantages, including broad substrate spectrum, wide pH range, high thermostability, and tolerance to alkaline environments. This review aims to summarize the significant research efforts on the properties, mechanisms and structures, laccase-mediator systems, genetic engineering, immobilization, and biotechnological applications of the bacteria-source laccases and laccase-like enzymes, which principally include Bacillus laccases, actinomycetic laccases and some other species of bacterial laccases. In addition, these enzymes may offer tremendous potential for environmental and industrial applications.

Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis.

Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications.

Establishment of a markerless multiple-gene deletion method based on Cre/loxP mutant system for Bacillus pumilus.

In this study, we established a Cre/loxP mutant recombination system (Cre/lox71-66 system) for markerless gene deletion to facilitate our follow-up rational genetic engineering to the strain Bacillus pumilus W3. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. Two selected genes, cotA and sigF, were continuously knocked out and verified at different levels using this method. This method is simple and efficient and can be easily implemented for multiple gene deletions in B. pumilus.

Production of spore laccase from Bacillus pumilus W3 and its application in dye decolorization after immobilization.

Given that spore laccase from the Bacillus genus is heat- and alkali-resistant, it is more suitable for industrial applications than fungal laccase. To determine the optimal culture conditions for spore laccase production, the effects of Cu2+ concentration, oxygen content, and culture time on spore laccase production from Bacillus pumilus W3 were investigated. The optimal production parameters were 0.2 mM of Cu2+, 200 rpm shaking speed, 100 mL liquid loading, and 5 days of cultivation. Spore laccase was efficiently immobilized on amino-functionalized celite. When used in dye decolorization, the immobilized spore laccase removed 84.15% of methyl green and 69.70% of acid red 1 after 48 h of treatment. Moreover, the immobilized spore laccase retained 87.04% of its initial decolorization activity after six cycles in the decolorization of acid red 1. These insights into the culture conditions and immobilization of spore laccases should be useful in the development of spore laccase as a biocatalyst in the treatment of textile wastewater.

Improving the catalytic efficiency of Bacillus pumilus CotA-laccase by site-directed mutagenesis.

Bacterial laccases are potential enzymes for biotechnological applications because of their remarkable advantages, such as broad substrate spectrum, various reactions, high thermostability, wide pH range, and resistance to strongly alkaline environments. However, the use of bacterial laccases for industrialized applications is limited because of their low expression level and catalytic efficiency. In this study, CotA, a bacterial laccase from Bacillus pumilus, was engineered through presumptive reasoning and rational design approaches to overcome low catalytic efficiency and thermostability. L386W/G417L, a CotA double-mutant, was constructed through site-directed mutagenesis. The catalytic efficiency of L386W/G417L was 4.3 fold higher than that of wild-type CotA-laccase, but the thermostability of the former was decreased than that of the latter and other mutants. The half-life (t 1/2) of wild-type and G417L were 1.14 and 1.47 h, but the half-life of L386W/G417L was only 0.37 h when incubating the enzyme at 80 °C. Considering the high catalytic efficiency of L386W/G417L, we constructed L386W/G417L/G57F, another mutant, to improve thermostability. Results showed that the half-life of L386W/G417L/G57F was 0.54 h when incubating the enzyme at 90 °C for 2 h with about 34% residual activity, but the residual activity of L386W/G417L was less than 40% when incubating the enzyme at 90 °C for 5 min. L386W/G417L was more efficient in decolorizing various industrial dyes at pH 10 than other mutants. L386W/G417L/G57F also exhibited an efficient decolorization ability. L386W/G417L/G57F is appropriate for biotechnological applications because of its high activity and thermostability in decolorizing industrial dyes. CotA-laccase may be further subjected to molecular modification and be used as an enhancer to improve decolorization efficiency for the physical and chemical treatment of dye wastewater.

Stimulating the biosynthesis of antroquinonol by addition of effectors and soybean oil in submerged fermentation of Antrodia camphorata.

Antrodia camphorata is a precious medicinal mushroom that has attracted increasing attentions. Antroquinonol has been considered as being among the most biologically active components of A. camphorata. However, it was hardly biosynthesized via conventional submerged fermentation. Two approaches were applied to stimulate the biosynthesis of antroquinonol in submerged fermentation. On one hand, different kinds of effectors that may involve in the antroquinonol biosynthesis were investigated. Among the tested effectors, camphorwood leach liquor was the most effective for stimulating the antroquinonol production. On the other hand, because of the hydrophobic characteristics of antroquinonol, soybean oil was added to establish an extractive fermentation system for alleviating the product inhibition and resulting in enhanced productivity. The highest antroquinonol concentration could be achieved at 89.06 ± 0.14 mg/L when 10% (v/v) soybean oil was added at the beginning of the fermentation. This study will be of great significance for the study of A. camphorata and the bioprocess regulation of antroquinonol production.

Complete genome sequence of Bacillus pumilus W3: A strain exhibiting high laccase activity.

Here we report the full genome sequence of Bacillus pumilus W3, which was isolated from raw gallnut honey in Nandan County, Guangxi Province of China, showing high CotA-laccase activity. The W3 strain contains 3,745,123bp with GC content of 41.39%, and contains 3695 protein-coding genes, 21 rRNAs and 70 tRNAs.

Efficient secretory production of CotA-laccase and its application in the decolorization and detoxification of industrial textile wastewater.

Fungal laccases are typically unstable at high pH and temperature conditions, which limit their application in the decolorization of textile wastewater. By contrast, the highly stable bacterial laccases can function within a wider pH range and at high temperatures, thus have significant potential in treatment for textile wastewater. In our previous work, a thermo-alkali-stable CotA-laccase gene was cloned from Bacillus pumilus W3 and overexpressed in Escherichia coli. In this study, the robust CotA-laccase achieved efficient secretory expression in Bacillus subtilis WB600 by screening a suitable signal peptide. A maximum CotA-laccase yield of 373.1 U/mL was obtained at optimum culture conditions in a 3-L fermentor. Furthermore, the decolorization and detoxification of textile industry effluent by the purified recombinant CotA-laccase in the presence and absence of redox mediators were investigated. Among the potential mediators that enhanced effluent decolorization, acetosyringone (ACS) was the most effective. The toxicity of the CotA-laccase-ACS-treated effluent was greatly reduced compared with that of the crude effluent. To the best of our knowledge, this study is the first to report on the heterologous expression of CotA-laccase in B. subtilis. The recombinant strain B. subtilis WB600-5 has a great potential in the industrial production of this bacterial enzyme, and the CotA-laccase-ACS system is a promising candidate for the biological treatment of industrial textile effluents.

Molecular cloning, characterization, and dye-decolorizing ability of a temperature- and pH-stable laccase from Bacillus subtilis X1.

Laccases from fungal origin are typically unstable at high temperatures and alkaline conditions. This characteristic limits their practical applications. In this study, a new bacterial strain exhibiting laccase activity was isolated from raw fennel honey samples and identified as Bacillus subtilis X1. The CotA-laccase gene was cloned from strain X1 and efficiently expressed in Escherichia coli in a biologically active form. The purified recombinant laccase demonstrated an extensive pH range for catalyzing substrates and high stability toward alkaline pH and high temperatures. No loss of laccase activity was observed at pH 9.0 after 10 days of incubation, and approximately 21 % of the initial activity was detected after 10 h at 80 °C. Two anthraquinonic dyes (reactive blue 4 and reactive yellow brown) and two azo dyes (reactive red 11 and reactive brilliant orange) could be partially decolorized by purified laccase in the absence of a mediator. The decolorization process was efficiently promoted when methylsyringate was present, with more than 90 % of color removal occurring in 3 h at pH 7.0 or 9.0. These unusual properties indicated a high potential of the novel CotA-laccase for industrial applications.

[Antimicrobial effects of qingkailing injection extract and combination therapy of qingkailing injection and antibiotics on bacteria carrying blaNDM-1 resistance gene].

OBJECTIVE: To research the bacteriostatic effects of Qingkailing Injection Extract (QKLIE) and combination therapy of Qingkailing Injection (QKLI) and antibiotics on bacteria carrying New Delhi metallo-3-lactamase 1 (NDM-1) blaNDM-1 resistance gene, and to determine their minimal inhibitory concentrations (MIC). METHODS: The antimicrobial experiments of QKLIE (Radix Isatidis, baicalin, gardenia, honeysuckle) and combination therapy of QKLI and antibiotics were performed by using the agar dilution method and K-B method. The MIC was determined from each extract. RESULTS: There were different degrees of inhibitory effects on resistant bacteria carrying blaNDM-1 by extracts from main components of QKLI. Of them, the inhibitory effect of baicalin was the best and the MIC of the resistant bacteria was 0.015 g/mL to WD, 0.020 g/mL to WX, 0. 005 g/mL to WJ, and more than 0.020 g/mL to pGEX-4T-NDM-1/DH5alpha (GST-NDM-1), respectively. The MIC value of each extract was sequenced from high to low as baicalin, honeysuckle, gardenia, and Radix Isatidis. Furthermore, combination therapy of QKLI and antibiotics greatly enhanced the antimicrobial activity of each antibiotics when used alone, showing very obvious antibacterial effects on multidrug resistant bacteria carrying blaNDM-1 gene. Of them, the optimal effects were obtained when combined with penicillins (penicillin G, mezlocillin, piperacillin/ tazobactam, ampicillin/sulbactam), with the antibacterial effects improved by 10 folds. The antibacterial effects of other kinds of antibiotics were improved to some extent. Conclusions QKLIE and combination therapy of QKLI and antibiotics showed better bacteriostatic effects on resistant bacteria carrying blaNDM-1 gene. This study provided theoretical bases for drug development, medication and treatment for super-resistant bacteria carrying blaNDM-1.

Production, characterization, cloning and sequence analysis of a monofunctional catalase from Serratia marcescens SYBC08.

A monofunctional catalase from Serratia marcescens SYBC08 produced by liquid state fermentation in 7 liter fermenter was isolated and purified by ammonium sulfate precipitation (ASP), ion exchange chromatography (IEC), and gel filtration (GF) and characterized. Its sequence was analyzed by LC-MS/MS technique and gene cloning. The highest catalase production (20,289 U · ml(-1)) was achieved after incubation for 40 h. The purified catalase had an estimated molecular mass of 230 kDa, consisting of four identical subunits of 58 kDa. High specific activity of the catalase (199,584 U · mg(-1) protein) was 3.44 times higher than that of Halomonas sp. Sk1 catalase (57,900 U · mg(-1) protein). The enzyme without peroxidase activity was found to be an atypical electronic spectrum of monofunctional catalase. The apparent K(m) and V(max) were 78 mM and 188, 212 per µM H(2) O(2) µM heme(-1) s(-1), respectivly. The enzyme displayed a broad pH activity range (pH 5.0-11.0), with optimal pH range of 7.0-9.0: It was most active at 20 °C and had 78% activity at 0 °C. Its thermo stability was slightly higher compared to that of commercial catalase from bovine liver. LC-MS/MS analysis confirmed that the deduced amino acid sequence of cloning gene was the catalase sequence from Serratia marcescens SYBC08. The sequence was compared with that of 23 related catalases. Although most of active site residues, NADPH-binding residues, proximal residues of the heme, distal residues of the heme and residues interacting with a water molecule in the enzyme were well conserved in 23 related catalases, weakly conserved residues were found. Its sequence was closely related with that of catalases from pathogenic bacterium in the family Enterobacteriaceae. This result imply that the enzyme with high specific activity plays a significant role in preventing those microorganisms of the family Enterobacteriaceae against hydrogen peroxide resulted in cellular damage. Calalase yield by Serratia marcescens SYBC08 has potential industrial application in scavenging hydrogen peroxide.

Isolation and identification of a new hypocrellin A-producing strain Shiraia sp. SUPER-H168.

A new hypocrellin A-producing strain, Shiraia sp. SUPER-H168, was isolated from tissues of bamboo, Brachystachyum densiflorum. The morphology of this strain was characterized with a light microscope and a scanning electronic microscope. The mycelia, conidia, pycnidia of fungus were observed. Small pycnidia (10-20 microm in length) full of small conidia appeared on the mycelia, which were first reported in this study. The 18S rDNA region of this strain was amplified and sequenced. Then a neighbor-joining tree of 18S rDNA was constructed. According to the result of analysis, the strain SUPER-H168 was proved to belong to the genus Shiraia. Hypocrellin A was produced by solid-state fermentation, extracted by acetone, isolated by preparative RP-HPLC, and identified by RP-HPLC, ESI-MS and ultraviolet-visible absorbing scanning with diode array detection. The HA production could reach 2.02 mg/g dry solid substrate.