Exploring a high-urease activity Bacillus cereus for self-healing concrete via induced CaCO3 precipitation.

The structural integrity and esthetic appeal of concrete can be compromised by concrete cracks. Promise has been shown by microbe-induced calcium carbonate precipitation (MICP) as a solution for concrete cracking, with a focus on urease-producing microorganisms in research. Bacillus cereus was isolated from soil and employed for this purpose in this study due to its high urease activity. The strain exhibited strong tolerance for alkaline media and high salt levels, which grew at a pH of 13 and 4% salt concentration. The repair of concrete cracks with this strain was evaluated by assessing the effects of four different thickeners at varying concentrations. The most effective results were achieved with 10 g/L of sodium carboxymethyl cellulose (CMC-Na). The data showed that over 90% repair of cracks was achieved by this system with an initial water penetration time of 30 s. The study also assessed the quantity and sizes of crystals generated during the bacterial mineralization process over time to improve our understanding of the process. KEY POINTS: • MICP using Bacillus cereus shows potential for repairing concrete cracks. • Strain tolerates alkaline media and high salt levels, growing at pH 13 and 4% salt concentration. • Sodium carboxymethyl cellulose (CMC-Na) at 10 g/L achieved over 90% repair of cracks.

IPTG-induced high protein expression for whole-cell biosynthesis of L-phosphinothricin.

BACKGROUND: Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase (E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase (E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT. MAIN METHODS AND MAJOR RESULTS: We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g-1 and 896.23 U g-1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g-1 and 109.70 U g-1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L-1 ·h-1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L-1 ·h-1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production. CONCLUSIONS AND IMPLICATIONS: We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations.

Quercetin alleviates subarachnoid hemorrhage-induced early brain injury via inhibiting ferroptosis in the rat model.

Early brain injury (EBI) refers to a series of pathophysiological brain lesions that occur within 72 hr after subarachnoid hemorrhage (SAH), which is an extremely crucial factor in the poor prognosis of patients. In EBI, ferroptosis has been proven to cause neuronal death. Quercetin (QCT) is effective in deactivating reactive oxygen species (ROS), inhibiting lipid peroxidation, and even chelating iron, but its role in SAH remains unclear. In this study, the mortality rate, severity grade of SAH, brain water content (BWC), blood-brain barrier permeability, and neurological function of the rats were detected. Moreover, mitochondrial morphology in cortical neurons were observed and their sizes were subsequently quantified. The levels of lipid peroxidation on glutathione and malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were determined, whereas the protein expressions of glutathione peroxidase 4 (GPX4), SLC7A11 (xCT), transferrin receptor 1 (TfR1), and ferroportin-1 (FPN1) were analyzed by western immunoblotting. The neurodegeneration involved in EBI was investigated by fluoro-Jade C staining, while iron staining was utilized to measure iron content. Our results showed that inhibition of ferroptosis by QCT could suppress EBI and improve neurological function in SAH rats. QCT increased the expression levels of GPX4, xCT, and FPN1, while downregulated TfR1, and exerted protective effects on neurons as well as alleviated iron accumulation and lipid peroxidation in the cortex of SAH rats. In conclusion, our study revealed that QCT might alleviate the EBI by inhibiting ferroptosis in SAH rats.

Strain Screening and Particle Formation: a Lysinibacillus boronitolerans for Self-Healing Concrete.

Microbial-induced calcite precipitation is a promising technology to solve the problem of cracks in soil concrete. The most intensively investigated microorganisms are urease-producing bacteria. Lysinibacillus that is used as urease-producing bacteria in concrete repair has rarely been reported. In this study, Lysinibacillus boronitolerans with a high urease activity was isolated from soil samples. This strain is salt- and alkali-tolerance, and at pH 13, can grow to ~OD600 2.0 after 24 h. At a salt concentration of 6%, the strain can still grow to ~OD600 1.0 after 24 h. The feasibility of using this strain in self-healing concrete was explored. The data showed that cracks within ~0.6 mm could be repaired naturally with hydration when spores and substrates were added to the concrete in an appropriate proportion. Moreover, the number and morphology of CaCO3 crystals that were produced by bacteria can be influenced by the concrete environment. An efficiency method to elucidate the process of microbial-induced calcium carbonate crystal formation was established with Particle Track G400. This study provides a template for future studies on the theory of mineralization based on microorganisms. IMPORTANCE The formation of calcium carbonate crystals in concrete by urease-producing bacteria is not understood fully. In this study, a Lysinibacillus boronitolerans strain with a high urease activity was isolated and used to analyze the counts and sizes of the crystals and the relationship with time. The data showed that the number of crystal particles increases exponentially in a short period with sufficient substrate, after which the crystals grow, precipitate or break. In concrete, the rate-limiting steps of calcium carbonate crystal accumulation are spore germination and urease production. These results provided data support for the rational design of urease-producing bacteria in concrete repair.

[Development and practice of national first-class undergraduate course "bioengineering equipment"].

As a strategic emerging industry of China, the biotechnology industry develops rapidly in recent years, which significantly increased the demand for creative and capable talents. As a core curriculum of bioengineering specialty, biotechnology equipment plays an important role in fostering such talents. To address the problems in biotechnology equipment course teaching such as limited equipment availability, limited engineering practice, and lack of learning motivations, curriculum reform and optimization were performed based on curriculum resource development, virtual reality-physical combined engineering training, and boosting learning motivations. The optimized teaching contents focus on fostering morality, intelligence, and creative practice abilities by connecting new requirements of social development, introducing new progress in biotechnology research, as well as new practices in research and development (R & D). Measures such as teaching methods innovation, assessment and evaluation methods optimization, cutting-edge R & D progress, diverse resources integration, and online-offline combined teaching, were developed to boost the learning motivation and foster the innovation competence of students. By above exploration and practice, the practice and innovation competence of students were significantly enhanced.

Development of a fermentation strategy to enhance the catalytic efficiency of recombinant Escherichia coli for l-2-aminobutyric acid production.

Microbial fermentation for enzyme production and then whole-cell catalysis for l-2-aminobutyric acid (l-ABA) production have huge potential for industrial application, but the catalytic capacities of cells are directly related to the fermentation process. Using a 50 L fermenter, the effects of initial glycerol concentration in the medium and rotating speed on cell catalytic capacity were investigated. Fermentation cells showed the best catalytic activity when the initial glycerol concentration was 12 g/L and the rotating speed was 250 rpm. Furthermore, we studied the difference between glycerol and glycerol mixtures as fed-batch media in pH-stat fed-batch fermentation. Results showed that glycerol had better catalytic activity than the glycerol mixture, and the effect of fed-batch fermentation was better than batch fermentation. Meanwhile, the enzyme activities of leucine dehydrogenase and formate dehydrogenase reached 129.87 U/g DCW and 437.02 U/g DCW, respectively, and the intracellular NAD(H) concentration reached 14.94 µmol/g DCW. Using the optimized fermentation parameters, amplified fermentation was then carried out in a 5000 L fermenter to demonstrate the industrial production of l-ABA by Escherichia coli BL21.

[Research progress in spore-based in-situ restoration technology of concrete with microorganisms].

Concrete is the most widely used modern building material. It is easy to crack under the action of stress, which makes the concrete structure permeable, affecting the durability and integrity of the structure, and thus shortening its service life. Microbial in-situ remediation technology is a low cost, effective and green way for concrete crack repairing. Due to its excellent biocompatibility, service life elongation, economic losses and environmental pollution reduction, microbial in-situ remediation technology has been intensively investigated. Bacillus has attracted much attention because of its excellent biomineralization ability, extremely strong environmental tolerance and long-term survival ability of its spores. In order to promote the research, development and large-scale application of microbial in-situ healing of concrete, the paper reviews the mechanism of spore-based in-situ healing of concrete, the survival of spores exposed in concrete, the influence of spores and external additives on the mechanical properties of concrete, progress in research and development of healing agent as well as healing effects. Moreover, future research focuses such as improving the survival ability of spores in the harsh environment of concrete, reducing the influence of external additives on the mechanical properties of concrete, and strengthening the healing effect of actual field applications are also summarized.

Development of a Combination Fermentation Strategy to Simultaneously Increase Biomass and Enzyme Activity of D-amino Acid Oxidase Expressed in Escherichia coli.

D-amino acid oxidase (DAAO) is widely used in the industrial preparation of L-amino acids, and cultivating Escherichia coli (E. coli) expressing DAAO for the biosynthesis of L-phosphinothricin (L-PPT) is very attractive. At present, the biomass production of DAAO by fermentation is still limited in large-scale industrial applications because the expression of DAAO during the fermentation process inhibits the growth of host cells, which limits higher cell density. In this study, the factors that inhibit the growth of bacterial cells during a 5-L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous D-alanine (D-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation. Under optimal conditions, the DO-STAT feeding mode with DO controlled at 30% ± 5% and the addition of 27.5 g/L lactose mixed with 2 g/L D-Ala during induction at 28 °C resulted in the production of 26.03 g dry cell weight (DCW)/L biomass and 390.0 U/g DCW specific activity of DAAO; an increase of 78% and 84%, respectively, compared with the initial fermentation conditions. The fermentation strategy was successfully scale-up to a 5000-L fermenter.

Efficient racemization of N-phenylacetyl-D-glufosinate for L-glufosinate production.

Most amino acids contain chiral centres and exist as both D-enantiomer and L-enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L-glufosinate (L-PPT) was obtained by coupling amidase-mediated hydrolysis of N-phenylacetyl-D,L-glufosinate with racemization of N-phenylacetyl-D-glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D-enantiomer. To improve the yield of L-PPT, the racemization reaction conditions were optimized, and through single-factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L-PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L-PPT to 97.02%.

Fermentative production of the unnatural amino acid L-2-aminobutyric acid based on metabolic engineering.

BACKGROUND: L-2-aminobutyric acid (L-ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important pharmaceuticals. To make the biosynthesis of L-ABA environmental friendly and more suitable for the industrial-scale production. We expand the nature metabolic network of Escherichia coli using metabolic engineering approach for the production of L-ABA. RESULTS: In this study, Escherichia coli THR strain with a modified pathway for threonine-hyperproduction was engineered via deletion of the rhtA gene from the chromosome. To redirect carbon flux from 2-ketobutyrate (2-KB) to L-ABA, the ilvIH gene was deleted to block the L-isoleucine pathway. Furthermore, the ilvA gene from Escherichia coli W3110 and the leuDH gene from Thermoactinomyces intermedius were amplified and co-overexpressed. The promoter was altered to regulate the expression strength of ilvA* and leuDH. The final engineered strain E. coli THR ΔrhtAΔilvIH/Gap-ilvA*-Pbs-leuDH was able to produce 9.33 g/L of L-ABA with a yield of 0.19 g/L/h by fed-batch fermentation in a 5 L bioreactor. CONCLUSIONS: This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of L-ABA.

Hydrolytic denitrification and decynidation of acrylonitrile in wastewater with Arthrobacter nitroguajacolicus ZJUTB06-99.

Acrylonitrile (C3H3N) widely used in chemical raw materials has biological toxicity with -CN bond, so it is the key to removal of cyanide from acrylonitrile wastewater. In our previous research and investigation, a strain was identified as Arthrobacter nitroguajacolicus named ZJUTB06-99 and was proved to be capable of degrading acrylonitrile. In this paper, the strain ZJUTB06-99 was domesticated with acrylonitrile-containing medium and its decyanidation and denitrification in simulated acrylonitrile wastewater were studied. The intermediate product of acrylonitrile in degradation process was identified through gas chromatography-mass spectrometer, as well as the biodegradation pathway of acrylonitrile in wastewater was deduced tentatively. The kinetics equation of biodegradation of acrylonitrile was lnC = - 0.1784t + 5.3349, with the degradation half-life of acrylonitrile in wastewater by 3.885 h. The results of this study showed that the optimum levels of temperature, pH and bacteria concentration to attain the maximum biodegradation were obtained as 30 °C, 6 and 100 g/L, respectively. The disadvantages of the biodegradation with this strain and its possible enhanced method to degrade acrylonitrile in wastewater were also discussed.

Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid.

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-type nitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity. Co2+ stimulated nitrilase activity, whereas Cu2+, Zn2+ and Tween 80 showed a strong inhibitory effect. The Vmax and kcat of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the Km was decreased 1.53-fold. The yield of Mut-F168V/T201N/S192F/M191T/F192S with 453.2 mM of IDA reached 71.9% in 5 h when 630 mM iminodiacetonitrile was used as substrate. This study indicated that mutant nitrilase obtained in this study is promising in applications for the upscale production of IDAN.

Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

OBJECTIVE: To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. RESULTS: A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). CONCLUSION: The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

Semi-Rational Engineering of Leucine Dehydrogenase for L-2-Aminobutyric Acid Production.

L-2-aminobutyric acid (L-ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD+ regeneration was developed for L-ABA production on a large scale. Previously, the L-ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method. Recently, an HTS assay was developed by us, which enables researchers to engineer LeuDH in a relatively short period of time. Herein, a semirational engineering was performed on LeuDH to increase the catalytic efficiency of BcLeuDH. Firstly, the structure of wild-type (WT) BcLeuDH was modeled and seven potentially beneficial positions were selected for mutation. Five beneficial variants were then identified from the seven site-saturation mutagenesis (SSM) libraries by HTS and confirmed by rescreening via amino acid analyzer. The "best" variant M5 (WT + Q358N) showed 44.5-fold higher catalytic efficiency (k cat/K M) than BcLeuDH WT, which suggested that BcLeuDH M5 is an attractive candidate for L-ABA production on a large scale. Furthermore, the structure-functional relationship was investigated based on the docking and kinetic results.

A high-throughput screening method for amino acid dehydrogenase.

A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)-calcein complex and amino acid (e.g., l-2-aminobutyric acid, l-ABA). When ABA was introduced to a Cu(II)-calcein solution, it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker l-ABA detection and screening for leucine dehydrogenase mutations.

Improvement and characterization of a hyperthermophilic glucose isomerase from Thermoanaerobacter ethanolicus and its application in production of high fructose corn syrup.

High fructose corn syrup (HFCS) is an alternative of liquid sweetener to sucrose that is isomerized by commercial glucose isomerase (GI). One-step production of 55 % HFCS by thermostable GI has been drawn more and more attentions. In this study, a new hyperthermophilic GI from Thermoanaerobacter ethanolicus CCSD1 (TEGI) was identified by genome mining, and then a 1317 bp fragment encoding the TEGI was synthesized and expressed in Escherichia coli BL21(DE3). To improve the activity of TEGI, two amino acid residues, Trp139 and Val186, around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected for site-directed mutagenesis. The specific activity of mutant TEGI-W139F/V186T was 2.3-fold and the value of k cat/K m was 1.86-fold as compared to the wild type TEGI, respectively. Thermostability of mutant TEGI-W139F/V186T at 90 °C for 24 h showed 1.21-fold extension than that of wild type TEGI. During the isomerization of glucose to fructose, the yield of fructose could maintain above 55.4 % by mutant TEGI-W139F/V186T as compared to 53.8 % by wild type TEGI at 90 °C. This study paved foundation for the production of 55 % HFCS using the thermostable TEGI.

Biosynthesis of nicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150.

In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0-12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50-55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.

Biosynthesis of iminodiacetic acid from iminodiacetonitrile by immobilized recombinant Escherichia coli harboring nitrilase.

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl(2), and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase.

Improvement of 1,3-dihydroxyacetone production from Gluconobacter oxydans by ion beam implantation.

Improvement of dihydroxyacetone (DHA) production by mutagenesis of ion beam implantation and medium optimization using response-surface methodology (RSM) were investigated in this work. More than 1000 mutant strains were selected through a mutagenesis method using N(+) ions implantation with a dose of 60 × (2.6 × 10(13)) ions/cm(2) and energy of 10 keV. Several high-yield mutant strains were showed the potent application for DHA production and the genetically stable mutant strain G. oxydans ZJB09113 was selected for optimization of cultivation condition by RSM. The optimal medium for DHA fermentation is composed (in g/L) of yeast extract 4.88, CaCO(3) 2.00, and glycerol 52.86 mL/L (initial pH 4.89). The maximal DHA concentration of 40.0 g/L was achieved after 24 hr of shaken flask fermentation at 30°C with 150 rpm, and 196.3% increase in DHA production in comparison with unoptimized conditions.

A novel synthesis of iminodiacetic acid: biocatalysis by whole Alcaligenes faecalis ZJB-09133 cells from iminodiacetonitrile.

Iminodiacetic acid (IDA) has been widely used as an important intermediate in the fine chemical industry. In this study, a novel synthesis route of IDA from iminodiacetonitrile by whole microorganisms was investigated. A strain with the capability of producing nitrilase, ZJB-09133, was isolated and identified, and later named Alcaligenes faecalis ZJB-09133. In addition, the detailed biocatalysis of iminodiacetonitrile to produce IDA using ZJB-09133 was investigated. The results showed that the conversion reached 65.3% in Na(2)HPO(4)-NaH(2)PO(4) buffer of pH 8.0 under the following conditions: cells in the amount of 0.075-g DCW/L, 1.5% substrate, conversion time of 8 h, and a reaction temperature of 35°C. To the best of our knowledge, this is the first time that the production of IDA using a biocatalysis method has been reported.