Gene cloning and molecular characterization of a thermostable chitosanase from Bacillus cereus TY24.

BACKGROUND: An important conceptual advance in health and the environment has been recognized that enzymes play a key role in the green processing industries. Of particular interest, chitosanase is beneficial for recycling the chitosan resource and producing chitosan oligosaccharides. Also, chitosan gene expression and molecular characterization will promote understanding of the biological function of bacterial chitosanase as well as explore chitosanase for utilizing chitosan resources. RESULTS: A chitosanase-producing bacterium TY24 was isolated and identified as Bacillus cereus. Moreover, the chitosanase gene was cloned and expressed in Escherichia coli. Sequence analysis reveals that the recombinant chitosanase (CHOE) belongs to the glycoside hydrolases 8 family. The purified CHOE has a molecular weight of about 48 kDa and the specific activity of 1150 U/mg. The optimal pH and temperature of CHOE were 5.5 and 65 °C, respectively. The enzyme was observed stable at the pH range of 4.5-7.5 and the temperature range of 30-65 °C. Especially, the half-life of CHOE at 65 °C was 161 min. Additionally, the activity of CHOE was remarkably enhanced in the presence of Mn2+, Cu2+, Mg2+ and K+, beside Ca2+ at 5 mM. Especially, the activity of CHOE was enhanced to more than 120% in the presence of 1% of various surfactants. CHOE exhibited the highest substrate specificity toward colloid chitosan. CONCLUSION: A bacterial chitosanase was cloned from B. cereus and successfully expressed in E. coli (BL21) DE3. The recombinant enzyme displayed good stability under acid pH and high-temperature conditions.

Production of surfactant-stable keratinase from Bacillus cereus YQ15 and its application as detergent additive.

BACKGROUND: With the growing concern for the environment, there are trends that bio-utilization of keratinous waste by keratinases could ease the heavy burden of keratinous waste from the poultry processing and leather industry. Especially surfactant-stable keratinases are beneficial for the detergent industry. Therefore, the production of keratinase by Bacillus cereus YQ15 was improved; the characterization and use of keratinase in detergent were also studied. RESULTS: A novel alkaline keratinase-producing bacterium YQ15 was isolated from feather keratin-rich soil and was identified as Bacillus cereus. Based on the improvement of medium components and culture conditions, the maximum keratinase activity (925 U/mL) was obtained after 36 h of cultivation under conditions of 35 °C and 160 rpm. Moreover, it was observed that the optimal reacting temperature and pH of the keratinase are 60 °C and 10.0, respectively; the activity was severely inhibited by PMSF and EDTA. On the contrary, the keratinase showed remarkable stability in the existence of the various surfactants, including SDS, Tween 20, Tween 60, Tween 80, and Triton X-100. Especially, 5% of Tween 20 and Tween 60 increased the activity by 100% and 60%, respectively. Furtherly, the keratinase revealed high efficiency in removing blood stains. CONCLUSION: The excellent compatibility with commercial detergents and the high washing efficiency of removing blood stains suggested its suitability for potential application as a bio-detergent additive.

Recombinant expression and molecular engineering of the keratinase from Brevibacillus parabrevis for dehairing performance.

Keratinase is capable of distinctive degradation of keratin, which provides an eco-friendly approach for keratin waste management towards sustainable development. In this study, the recombinant keratinase (KERBP) from Brevibacillus parabrevis was successfully expressed in Escherichia coli. The purified KERBP had the specific activity of 6005.3 U/mg. It showed remarkable tolerance to various surfactants and also no collagenolytic activity. However, the moderate thermal stability limited its further application. Thus, protein engineering was further adopted to improve its stability. The variants of T218S, S236C and N181D were constructed by site-directed mutagenesis and combinatorial mutagenesis. Compared with the wild type, the t1/2 at 60 °C for the variants T218S, S236C and N181D were 3.05-, 1.18- and 1-fold increase, respectively. Moreover, the double variants N181D-T218S and N181D-S236C significantly improved thermostability with 5.1 and 2.9 °C increase of T50, and prolonging t1/2 at 60 °C with 4.09 and 1.54-fold, respectively. And the catalytic efficiency of the T218S and N181D-T218S variants was also significantly improved. Furthermore, the keratinase displayed favorable ability to dehair wool from skin within 7 h, which showed potential in leather dehairing. Our work contributes to a further insight into the thermostability of keratinase and offers a promising alternative for industrial leather application.

Rutin-Nickel Complex: Synthesis, Characterization, Antioxidant, DNA Binding, and DNA Cleavage Activities.

The rutin-nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV-visible spectroscopy, IR, mass spectrometry, 1H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.

A novel alkaline surfactant-stable keratinase with superior feather-degrading potential based on library screening strategy.

A novel keratinase was mined and expressed in Escherichia coli BL21 (DE3) via function-driven screening with fosmid library. The catalytic properties of purified keratinase were investigated in detail following enzyme purification. The recombinant keratinase was purified to homogeneity with an estimated molecular weight of 26kDa using nickel affinity chromatography, of which the optimal reaction pH and temperature were 10.0 and 55°C, respectively. It could remain stable at pH 5.0-12.0 and 40-60°C. Metal ions such as Ca2+, Mn2+, Ag+, Na+, Mg2+, Li+, Sn2+ (1mM) displayed positive influence on keratinase, and particularly, Ca2+ exhibited remarkable improvement effect by 2.6 folds. It was strongly inhibited by PMSF as a protease inhibitor. On the contrary, it could be obviously activated by various surfactants, such as Tween 40 and Triton X-114. The recombinant keratinase showed high specificity towards casein, soluble keratin, BSA, and wool. The keratinase could efficiently degrade the feathers, which demonstrated its applicable potential for biodegradation of keratin wastes and regeneration of soluble protein.

Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis CGMCC 10798.

Dehairing is a high pollution process in leather industry. Conventionally, the lime-sulfide mediated chemical process for dehairing would lead to the discharge of pollutants and corrosion of industrial equipment. Concerning these problems, keratinase has become a promising candidate for dehairing process in recent years. In this study, a keratinase-producing bacterium was isolated from sheepfold soil and identified as Brevibacillus parabrevis CGMCC 10798 based on the biochemical characteristics and molecular identification. The keratinase was purified to electrophoretic homogeneity with 17.19% of recovery, 13.18 folds of purification and an estimated molecular weight of 28kDa. The enzyme exhibited high keratinase activity and no collagenase activity. Besides, the keratinase showed optimal activity at 60°C and pH 8.0. The enzyme activity could be significantly increased in the presence of Na+ and Ca2+. And it was inhibited by EDTA, and PMSF, which indicated that the keratinase belongs to serine-metallo protease. The enzyme could remain stable in the presence of surfactants. Especially, 5mM Tween 40 and Triton 100 could improve the activity by 11% and 30%, respectively. Moreover, B. parabrevis keratinase could completely dehair goat wool within 7h, which indicated its application potential in leather industry.

A metallo-keratinase from a newly isolated Acinetobacter sp. R-1 with low collagenase activity and its biotechnological application potential in leather industry.

Microbial keratinase is a well-recognized enzyme that can specifically degrade insoluble keratins. A keratinase-producing bacterium was isolated from a duck ranch soil and identified as Acinetobacter sp. R-1 based on the biochemical characteristics and 16S rDNA gene sequencing. It showed high keratinase activity and low collagenase activity. The keratinase was purified to electrophoretic homogeneity with 6.69% recovery, 2.68-fold purification and an estimated molecular weight of 25 kDa. Additionally, the keratinase showed optimal activity at 50 °C and pH11. Keratinase activity of Acinetobacter sp. significantly increased in the presence of Li(+), Na(+), and Ca(2+), while it was completely inhibited by EDTA, indicating it was a metallo-keratinase. Moreover, the crude keratinase from Acinetobacter sp. R-1 could thoroughly depilate goat skin and simultaneously modify the wool surface, which indicated its applicable potential in leather and textile industries.

Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis.

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

Synthesis, characterization, bioactivities of copper complexes with N-allyl di(picolyl)amine.

Four copper(II) complexes with N-allyl di(picolyl)amine were synthesized and characterized by physico-chemical and spectroscopic methods. The spectrophotometric and fluorescence titration data indicate that the [(Aldpa)Cu(L)](ClO(4))(2) (L=dppz, dione, phen) with conjugated aromatic rings as coordinated ligands can be inserted into the base stacks of DNA more deeply than the [(Aldpa)CuCl(2)]. The copper(II) complexes [(Aldpa)Cu(L)](ClO(4))(2) (L=dppz, dione, phen) can inhibit the proliferation of the four cancer cells (Mcf-7, Eca-109, A549 and HeLa) with IC(50) 0.5-19.2 microM, which is larger than that (23.2-84.3 microM) of [(Aldpa)CuCl(2)], suggesting their inhibiting activities on the four cancer cells are correlated with their DNA-binding properties. However, the selectivity of [(Aldpa)CuCl(2)] to cancer cells is better than that of the other three complexes [(Aldpa)Cu(L)](ClO(4))(2), which indicates the substituents introduced on the secondary amino nitrogen atom of dpa have great contribution to the antitumor activities of these copper(II) complexes.

[Hydrogen peroxide is involved in regulation of tobacco leaf senescence].

Effects of various exogenous hydrogen peroxide (H2O2) concentrations on leaf senescence of tobacco (Nicotiana tabacum L.) were studied. The results showed that 0.1 and 1 mmol/L H2O2 had little effects on the leaf senescence of tobacco, the contents of chlorophyll and soluble protein were decreased in varying degrees by 10, 100 mmol/L H2O2 treatment, the decreased chlorophyll and soluble protein contents were significantly correlated with increased endogenous H2O2 content. SDS-PAGE of soluble protein and localized staining of H2O2 showed consistency with respective content. Chloroplast ultrastructure were observed in the mesophyll cells of tobacco and there was a little change by 10 mmol/L H2O2 treatment, and a remarkable breakdown of granal stacks and disorganized thylakoid membranes were revealed by 100, 200 mmol/L H2O2 treatment. RT-PCR analysis showed the expression level of senescence associated genes SAG12 was low by 10mmol/L H2O2 treatment and increased by 100 mmol/L H2O2 treatment. These results strongly suggested the tobacco leaf senescence was induced by 10 mmol/L H2O2 and accelerated the progression by high concentration (100, 200 mmol/L) H2O2 stress.

[Study on molluscicidal effect of the extracts from sarcotesta of Ginkgo biloba].

OBJECTIVE: To observe the molluscicidal activity and the fish acute toxicity of the molluscicides extracted from Ginkgo biloba sarcotesta by benzinum (EGSB) (with major component of ginkgolic acid), arecoline (ARE) and their combination. METHODS: Oncomelania hupensis snails were immersed in different concentrations of dry powder sarcotesta of Ginkgo biloba (DPGB), extract of Ginkgo biloba sarcotesta by water (EGSW) and EGSB by WHO recommended method for molluscicide screening to observe the molluscicidal activity, and also the inhibiting effect on the snails' climbing-up as well as acute toxicity to Brachydanio rerio. Niclosamide was used as control. RESULTS: The three extractions from Ginkgo biloba all showed molluscicidal activity, with EGSB as the best. Its 24 h LC50 and LC90 were 0.65 mg/L and 5.50 mg/L, and the 48 h LC50 and LC90 were 0.07 mg/L and 0.85 mg/L, respectively. The combination of EGSB and ARE showed better effect than EGSB alone. Its 24 h LC50 and LC90 were 0.26 mg/L and 0.56 mg/L respectively, a sharp decrease by 60% and 90% compared to EGSB (P<0.05). Under the concentration of 2.50 mg/L of EGSB, the rate of snails' climbing-up was 10%, while under the concentration of 0.16 mg/L of the EGSB+ARE combination, the rate was 8%. The inhibition on the snails' climbing-up of the combination was stronger than EGSB. The fish survived for 24 h and 10th respectively at the concentration of 1 x LC90 and 2 x LC90 of EGSB. Under the concentration of 2 x LC90 of the combination, only 50% of the fish died and no fish died at the concentration of 1 x LC90. The toxicity of the combination was lower than EGSB alone. CONCLUSION: EGSB shows an adequate molluscicidal activity and it is worth of further investigation.

[Relation between the decline of photosynthetic function and H2O2 accumulation in leaf life span of tobacco].

Correlations among H2O2 accumulation, chloroplast photosynthetic function decline and H2O2-scavenging enzymes of AsA-GSH cycle in leaf life span of tobacco (Nicotiana tabacum L. cv NC89) were studied. Photosynthetic rate displayed elevated phase,active photosynthesis duration and sharp fall phase while the chlorophyll content displayed elevated phase,relative steady phase and sharp fall phase during leaf life span. The decline of photosynthetic parameters was slow first and then sharp,and the decline of ribulose 1,5-bisphosphate carboxylase (RuBPCase) activity was obviously faster than that of photosynthetic electron transport activity during the decline of photosynthetic function. The decline of photosynthetic function was divided into reversible decline and irreversible decline. Ascorbate peroxidase (APX) and Glutathione reductase (GR) remained higher level in reversible decline stage,while declined fast in irreversible decline stage. H2O2 content was significantly negative correlated with chlorophyll content, photosynthetic rate, RuBPCase activity,APX and GR activity during the decline of photosynthetic function in tobacco leaves. Localized staining of H2O2 stated that the decline of photosynthetic function was closely correlative with H2O2 accumulation. The decline of photosynthetic function in tobacco leaves was faster than the decline of AsA-GSH cycle function.

[QTL analysis for traits associated with photosynthetic functions in rice (Oryza sativa L.)].

A mapping population of 81 F11 lines (Recombinant Inbred Lines, RILs), derived from a cross between a japonica variety Kinmaze and an indica variety DV85 by single-seed descent method, was used to detect quantitative trait loci (QTL) for traits associated with photosynthetic functions. Total leaf nitrogen content (TLN), chlorophyll a/b ratio (Chl. a:b) and chlorophyll content (Chl) were measured in leaves of rice (Oryza sativa L.) at the 7th day after heading. A total of six putative QTLs were detected with percentage of variance explained (PVE) ranging between 11.2% -29.6%, and LOD of QTLs 2.66-4.81. Of those putative QTLs, three for TLN were detected on chromosomes 1,2 and 11, with PVE of 17.3%, 15.3% and 13.7%, respectively;Two controlling Chl. a:b on chromosomes 3 and 4, PVE of 13.8% and 29.6%, one for Chl on chromosome 1, PVE of 11.2%. Four of those detected QTLs were newly reported in this study. Interestingly, the QTL controlling chlorophyll content,namely qCC-1 reported here,was detected in the region of the RFLP marker C122 on chromosome 1, where harbored NADH-glutamate synthase structure gene according to a previous study. Because the biosynthesis of chlorophyll begins with glutamate, qCC-1 would play a vital role in photosynthetic functions. Whereas,no QTL controlling chlorophyll content were detected at the 30th day after heading, suggesting that the effect of the QTL controlling chlorophyll content decreased during leaf senescence.

Interaction of hydrogen peroxide with ribulose-1,5-bisphosphate carboxylase/oxygenase from rice.

The properties of rice-derived ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in different concentrations of hydrogen peroxide (H2O2) solutions have been studied. The results indicate that at low H2O2 concentrations (0.2-10 mM), the properties of rubisco (e.g., carboxylase activities, structure, and susceptibility to heat denaturation) change slightly. However, at higher H2O2 concentrations (10-200 mM), rubisco undergoes an unfolding process, including the loss of secondary and tertiary structure, forming extended hydrophobic interface, and leading to cross-links between large subunits. High concentrations of H2O2 can also result in an increase in susceptibility of rubisco to heat denaturation. Further pre-treatments with or without reductive reagents to rubisco show that the disulfide bonds in rubisco help to protect the enzyme from damage by H2O2 as well as other reactive oxygen species.

QTL and epistatic interaction underlying leaf chlorophyll and H2O2 content variation in rice (Oryza sativa L.).

It is meaningful to study the genetics of the traits associated with photosynthesis such as leaf chlorophyll and H2O2 contents for high yield breeding in rice. A mapping population of 98 BC1F9 lines (backcross Inbred Lines, BIL), derived from a backcross of Nipponbare (japonica)/Kasalath(indica)//Nipponbare by the single-seed-descent methods, was employed to map quantitative trait loci (QTL) underlying such rice traits as leaf chlorophyll and H2O2 contents. Five and two QTLs were detected for leaf Chlorophyll and H2O2 content variation, respectively. Among the QTLs detected, q-Chl1 on chromosome 1 accounted for 22% variation for leaf chlorophyll content variation. The alleles from Nipponbare increased stature at the locus; On the other hand, q-H2O2(1) which associated with H2O2 content was also located on the same region as q-Chl1. The alleles from Nipponbare decreased stature at this locus. The 2 traits have the 2 QTL sharing the same chromosome locations, which was located between C86 and C813 on chromosome 1. Epistasis analysis showed QTL with interaction distributed on chromosome 2, 6, 11 and 12. However, no QTL interacted with q-Chl1 or q-H2O2(1) was detected. It can be deduced that alleles from Nipponbare at the region between C86 and C813 on chromosome 1 increases leaf chlorophyll content and decreases H2O2 content at the mean time with significant additive effect but little epistasis.

Expression of Choline Oxidase Gene (codA) Enhances Salt Tolerance of the Tobacco.

The codA gene for choline oxidase, which converts choline into betaine. This enzyme, cloned from a soil bacterium Arthrobactor globiformis, has been transferred and expressed in tobacco by the Agrobatcterium-method transformation through the binary plasmid pGAH/codA. The pGAH/codA carried with Km(R) and Hyg(R),and coding sequence for a transit peptide from Rubisco small subunit gene (rbcS) was inserted between 35 S promoter and codA, so that the COD could be introduced into the chloroplast by this transit peptide. The transformed plants were screened on the medium containing the Km and Hyg. PCR, Western and gold immunolocalization tests showed that the codA gene has integrated into the tobacco DNA genome and its protein was expressed and the mature peptide has gone into the chloroplasts by the transit peptide. The results of salt-tolerance measuring for transgenic plants showed that the transgenic plants were more tolerance to the salt than the control plants. The young transgenic plants (1.0--1.5 cm) could survive at 400 mmol/L NaCl MS medium for more than 30 days. From them the higher tolerance plant T4-400 was obtained, which could grow at the 300 mmol/L NaCl MS medium well. The transgenic plants (6--8cm) could grow normally at the 400 mmol/L NaCl MS medium while wild plants failed to do so. So the transferred plant with codA enhanced its tolerance to the salt stress.