A strategy to enhance and modify fatty acid synthesis in Corynebacterium glutamicum and Escherichia coli: overexpression of acyl-CoA thioesterases.

BACKGROUND: Fatty acid (FA) is an important platform compound for the further synthesis of high-value biofuels and oleochemicals, but chemical synthesis of FA has many limitations. One way to meet the future demand for FA could be to use microbial cell factories for FA biosynthesis. RESULTS: Thioesterase (TE; TesA, TesB, and TE9) of Corynebacterium glutamicum (CG) can potentially improve FA biosynthesis, and tesA, tesB, and te9 were overexpressed in C. glutamicum and Escherichia coli (EC), respectively, in this study. The results showed that the total fatty acid (TFA) production of CGtesB and ECtesB significantly increased to 180.52 mg/g dry cell weight (DCW) and 123.52 mg/g DCW, respectively (P < 0.05). Overexpression strains CG and EC could increase the production of C16:0, C18:1(t), C18:2, C20:1, C16:1, C18:0, and C18:1(c) (P < 0.05), respectively, and the changes of long-chain FA resulted in the enhancement of TFA production. The enzymatic properties of TesA, TesB, and TE9 in vitro were determined: they were specific for long-, broad and short-chain substrates, respectively; the optimal temperature was 30.0 °C and the optimal acid-base (pH) were 8.0, 8.0, and 9.0, respectively; they were inhibited by Fe2+, Cu2+, Zn2+, Mg2+, and K+. CONCLUSION: Overexpression TE enhances and modifies FA biosynthesis with multiple productive applications, and the enzyme properties provided useful clues for optimizing FA synthesis.

Production of ß-glucosidase by Rhodotorula oryzicola and use of enzyme for hydrolysis of sugarcane bagasse delignified.

Bioethanol is obtained by hydrolysis of sugarcane bagasse by cellulases. Commercial cellulases are expensive and have a low concentration of ß-glucosidase (EC 3.2.1.21), which decrease hydrolysis efficiency. The present work aims to produce supernatant rich in ß-glucosidase (BGL) using the yeast Rhodotorula oryzicola and apply it in the hydrolysis of delignified sugarcane bagasse. Yeast fermented in a modified YPD (Yeast Peptone Dextrose) medium with 0.5% (w/v) cellobiose and 1.0% (w/v) glucose produced BGL with a specific activity of 1.44 ± 0.013 U/mg. Partial purification of BGL by acetone showed a specific activity of 3.48 U/mg. The optimum pH and temperature were 6.02 and 65 °C, respectively. BGL partially purified (BGLppR.oryzicola) by acetone showed tolerance to glucose, with a relative activity of 82.89 ± 0.11%. The activity increased with the addition of iron sulfate and zinc sulfate and decreased with manganese sulfate. BGL partially purified was thermal stable, with a relative activity of 85.59% after 60 min at 90 °C. BGL partially purified applied in the hydrolysis of sugarcane bagasse delignified with 3% (w/w) NaOH + 6% (w/w) Na2SO3 showed a conversion rate of 72.46 ± 1.60%. The results showed that BGL partially purified is a glucose tolerant cellulase of low-cost, promising the application of bioethanol production.

Molecular characterization and determination of the biochemical properties of cathepsin L of Trichinella spiralis.

Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis-host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 µM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.

Aliivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical.

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.

Molecular mechanism of high-production tannase of Aspergillus carbonarius NCUF M8 after ARTP mutagenesis: revealed by RNA-seq and molecular docking.

BACKGROUND: Tannase is an enzyme produced by microbial fermentation and is widely used in the food industry; however, the molecular mechanism of tannase production by Aspergillus has not yet been studied. This study was conducted to reveal the differences in Aspergillus carbonarius tannase enzymatic characterization, secondary structures and molecular mechanisms after treatment of the strain with atmospheric and room temperature plasma (ARTP). RESULTS: The results showed that the specific activity of tannase was improved by ARTP treatment, and it showed higher thermostability and tolerance to metal ions and additives. The enzymatic characterization and molecular docking results indicated that tannase had a higher affinity and catalytic rate with tannic acid as a substrate after ARTP treatment. In addition, the docking results indicated that Aspergillus tannases may catalyze tannic acid by forming two hydrogen-bonding networks with neighboring residues. RNA-seq analysis indicated that changes in steroid biosynthesis, glutathione metabolism, glycerolipid metabolism, oxidative phosphorylation pathway and mitogen-activated protein kinase signaling pathways might be crucial reasons for the high production of tannase. CONCLUSION: ARTP enhanced the yield and properties of A. carbonarius tannase by changing the enzyme structure and cell metabolism. This study provides a theoretical basis for elucidating the molecular mechanism underlying high production of Aspergillus tannases. © 2022 Society of Chemical Industry.

[Enzymatic characterization of lignan glucosyltransferase of Isatis indigotica].

The lignan glycosyltransferase UGT236(belonging to the UGT71 B family) from Isatis indigotica can catalyze the production of phloridzin from phloretin in vitro. UGT236 shares high identity with P2'GT from apple. In this study, the recombinant plasmid pET28 a-MBP-UGT236 was transferred into Escherichia coli Rosetta(DE3) cells and induced by isopropyl-ß-D-thiogalactoside(IPTG). The purified UGT236 protein was used for enzymatic characterization with phloretin as substrate. The results showed that UGT236 had the optimal reaction temperature of 40 ℃ and the optimal pH 8(Na_2HPO_4-NaH_2PO_4 system). The UGT236 activity was inhibited by Ni~(2+) and Al~(3+), enhanced by Fe~(2+), Co~(2+), and Mn~(2+), and did not affected by Mg~(2+), Ca~(2+), Li~+, Na~+, or K~+. The K_m, K_(cat), and K_(cat)/K_m of phloretin were 61.03 µmol·L~(-1), 0.01 s~(-1), and 157.11 mol~(-1)·s~(-1)·L, and those of UDPG were 183.6 µmol·L~(-1), 0.01 s~(-1), and 51.91 mol~(-1)·s~(-1)·L, respectively. The possible active sites were predicted by homologous modeling and molecular docking. By mutagenisis and catalytic activity detection, three key active sites, Glu391, His15, and Thr141, were identified, while Phe146 was related to product diversity. In summary, we found that the lignan glycosyltransferase UGT236 from I.indigotica could catalyze the reaction of phloretin into phloridzin. Several key amino acid residues were identified by structure prediction, molecular docking, and site-mutagenesis, which provided a basis for studying the specificity and diversity of phloretin glycoside products. This study can provide a reference for artificially producing glycosyltransferase elements with high efficiency and specific catalysis.

Technological and enzymatic characterization of the yeasts isolated from natural fermentation media of Gemlik olives.

AIM: To determine the technological and enzymatic characteristics of 54 yeast strains belonging to 16 species previously isolated from natural fermentation media of Gemlik olives. The distinguishing feature of these strains, according to their selective technological and enzymatic properties using principal component analysis (PCA), was also intended. METHODS AND RESULTS: The technological properties of yeast strains, growth characteristics at different temperatures, pH and salt concentrations were examined. Besides, yeast strains' abilities to use oleuropein as a sole carbon source, to assimilate citric acid and to produce H2 S were examined and their catalase, pectolytic, proteolytic and killer activities were also tested. All strains could grow between 15 and 28°C which are favourable temperatures for natural olive fermentation and they were able to tolerate high salt concentration and low pH in the brine of natural fermentation media. As a result of enzymatic characterization with API-ZYM test system, all strains have esterase activity, which is an important feature for developing table olive aroma. In this research, ß-glucosidase activity, which contributes to removing bitterness out of olives, was one of the main distinguishing features of yeast strains. Several strains of Candida hellenica, Pichia anomala and Candida pelliculosa species had ß-glucosidase activity. PCA tested yeasts and several strains belonging to C. hellenica (AF84-1), P. anomala (BF1-1, BF46-2) and C. pelliculosa (BF46-3, BF143-2) species have promising technological and enzymatic properties for natural table olive production. CONCLUSION: Five promising strains belonging to C. hellenica, P. anomala and C. pelliculosa species may be suitable adjunct starter cultures with lactic acid bacteria in natural fermentation media of table olive. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has been the first contribution to the enzymatic and technological characterization of yeasts isolated from Gemlik olives in Turkey. Some strains could be proposed as a promising adjunct culture in the production of table olives.

Biochemical characterization of extracellular fructosyltransferase from Aspergillus oryzae IPT-301 immobilized on silica gel for the production of fructooligosaccharides.

OBJECTIVE: Extracellular fructosyltransferase (FTase, E.C.2.4.1.9) from Aspergillus oryzae IPT-301 was immobilized on silica gel by adsorption and biochemically characterized aiming at its application in the transfructosylation reaction of sucrose for the production of fructooligossaccarides (FOS). RESULTS: The transfructosylation activity (AT) was maximized by the experimental design in function of the reaction pHs and temperatures. The AT of the immobilized enzyme showed the kinetics behavior described by the Hill model. The immobilized FTase showed reuse capacity for six consecutive reaction cycles and higher pH and thermal stability than the soluble enzyme. CONCLUSION: These results suggest a high potential of application of silica gel as support for FTase immobilization aiming at FOS production.

Engineering Xaa-Pro dipeptidyl aminopeptidase for specific cleavage of glucagon and glucagon-like peptide 1 from fusion proteins.

N-terminal extensions ("tags") have proven valuable for producing peptides using high throughput recombinant expression technologies. However, the applicability is hampered by the limited options for specific and efficient proteases to release the fully native sequence without additional amino acids in the N-terminal. Here we describe the Escherichia coli (E. coli) expression, purification and characterization of engineered variants of Xaa-Pro dipeptidyl aminopeptidase (Xaa-Pro-DAP) derived from Lactococcus lactis for cleavage of Gly-Pro dipeptide extension in the N-terminal of glucagon and glucagon-like peptide 1 (GLP-1(7-37)). By single amino acid substitution in the Xaa-Pro-DAP protease, significantly higher product yields were achieved. The combination of HRV14 3C protease and engineered Xaa-Pro-DAP is suggested for obtaining native N-terminal of peptides.

Expression, homology modeling and enzymatic characterization of a new ß-mannanase belonging to glycoside hydrolase family 1 from Enterobacter aerogenes B19.

BACKGROUND: ß-mannanase can hydrolyze ß-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, ß-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. ß-mannanase is widespread in various organisms, however, microorganisms are the main source of ß-mannanases. Microbial ß-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial ß-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce ß-mannanase, which shows more advantages than traditional microbial fermentation in various aspects. RESULTS: A ß-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The ß-mannanase activity was activated by low concentration of Co2+, Mn2+, Zn2+, Ba2+ and Ca2+. Man1E showed the highest affinity for Locust bean gum (LBG). The Km and Vmax values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 µmol/(mL min), respectively. CONCLUSIONS: A new type of ß-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported ß-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.

Cloning and characterization of the ß-xylosidase from Dictyoglomus turgidum for high efficient biotransformation of 10-deacetyl-7-xylosltaxol.

With the aim of finding an extracellular biocatalyst that can efficiently remove the C-7 xylose group from 10-deacetyl-7-xylosltaxol, a Dictyoglomus turgidum ß-xylosidase was cloned and expressed in Escherichia coli BL21 (DE3). The molecular mass of purified Dt-Xyl3 was approximately 84 kDa. The recombinant Dt-Xyl3 was most active at pH 5.0 and 75 °C, retaining 88% activity at 65 °C for 1 h, and displaying excellent stability over pH 4.0-7.5 for 24 h. In terms of kinetic parameters, the Km and Vmax values for pNPX were 0.8316 mM and 5.0178 µmol/mL·min, respectively. Moreover, Dt-Xyl3 was activated by Mn2+ and Ba2+ and inhibited by Cu2+, Ni+ and Al3+. In particular, it displayed high tolerance to salts with 60.8% activity in 20% (w/v) NaCl. Ethanol and methanol at 5-15% showed little effect on the enzymatic activity. Dt-Xyl3 demonstrated multifunctional activities followed by pNPX, pNPAraf and pNPG and had a high selectivity for cleaving the outer xylose moieties of 10-deacetyl-7-xylosltaxol with Kcat/Km 110.87 s-1/mM, which produced 10-deacetyl-taxol to semi-synthesize paclitaxel. Under the optimized conditions (60 °C, pH 4.5, enzyme dosage of 0.5 U/mL), 1 g of 10-deacetyl-7-xylosltaxol was transformed to its corresponding aglycone 10-deacetyl-taxol within 30 min, with a molar conversion of 98%. This is the first report that Dictyoglomus turgidum can produce extracellular GH3 ß-xylosidase with highly specific activity for 10-deacetyl-7-xylosltaxol biotransformation, thus leading to the application of ß-xylosidase Dt-Xyl3 as a biocatalyst in biopharmaceutics.

Biotransformation of phytoestrogens from soy in enzymatically characterized liver microsomes and primary hepatocytes of Atlantic salmon.

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.

Directed evolution of Aspergillus oryzae lipase for the efficient resolution of (R,S)-ethyl-2-(4-hydroxyphenoxy) propanoate.

Aspergillus oryzae lipase (AOL) is a potential biocatalyst for industrial application. In this study, a mutant lipase AOL-3F38N/V230R was screened through two rounds of directed evolution, resulting in a fourfold increase in lipase activity, and threefold in catalytic efficiency (kcat/Km), while maintaining its excellent stereoselectivity. AOL-3F38N/V230R enzyme activity was maximum at pH 7.5 and also at 40 °C. And compared with wild-type AOL-3, AOL-3F38N/V230R preferentially hydrolyzed the fatty acid ethyl ester carbon chain length from C4 to C6-C10. In the same catalytic reaction conditions, the conversion of (R,S)-ethyl-2-(4-hydroxyphenoxy) propanoate ((R,S)-EHPP) by AOL-3F38N/V230R can be increased 169.7% compared to the original enzyme. The e.e.s of (R,S)-EHPP achieved 99.4% and conversion about 50.2% with E value being 829.0. Therefore, AOL-3F38N/V230R was a potential biocatalyst for obtaining key chiral compounds for aryloxyphenoxy propionate (APP) herbicides.

Functional Characterization of Carbohydrate-Binding Modules in a New Alginate Lyase, TsAly7B, from Thalassomonas sp. LD5.

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.

Characterization of a novel arabinose-tolerant α-L-arabinofuranosidase with high ginsenoside Rc to ginsenoside Rd bioconversion productivity.

AIMS: (i) To investigate the enzymatic characterization of α-L-arabinofuranosidase from Thermotoga thermarum DSM5069. (ii) To evaluate the performance of its excellent properties on converting ginsenoside Rc to ginsenoside Rd. METHODS AND RESULTS: The thermostable α-L-arabinofuranosidase (Tt-Afs) gene from T. thermarum DSM5069 was cloned and overexpressed. Recombinant Tt-Afs was purified, and its molecular weight was approx. 55 kDa. Its optimal activity was at pH 5·0 and 95°C. It has high selectivity for cleaving the outer arabinofuranosyl moieties at the C-20 carbon of ginsenoside Rc and its sugar-tolerance makes Tt-Afs a promising candidate for the production of ginsenoside Rd. In a reaction at 85°C and pH 5·0, 25 g l(-1) of ginsenoside Rc was transformed into 21·8 g l(-1) of Rd within 60 min, with a corresponding molar conversion of 99·4% and a high ginsenoside Rd productivity of 21800 mg l(-1) h(-1). CONCLUSIONS: We have successfully cloned and overexpressed the novel α-l-arabinofuranosidase from T. thermarum DSM5069. The high ginsenoside Rd productivity and detailed characterization of recombinant Tt-Afs was provided. SIGNIFICANCE AND IMPACT OF THE STUDY: The result shows a high productivity on the bioconversion from high concentration ginsenoside Rc to ginsenoside Rd, which also give rise to a potential commercial enzyme application.

Purification and partial characterization of intact and truncated chitinase from Bacillus thuringiensis HZP7 expressed in Escherichia coli.

OBJECTIVE: To ascertain the effect of chitin-binding domain (ChBD) and fibronectin type III domain (FN3) on the characterization of the intact chitinase from Bacillus thuringiensis. RESULTS: An intact chitinase gene (chi74) from B. thuringiensis HZP7 and its truncated genes (chi54, chi63 and chi66) were expressed in Escherichia coli BL21. The expression products were analyzed after purification. All chitinases were active from pH 4-7.5 and from 20 to 80 °C with identical optimal: pH 5.5 and 60 °C. The activity of colloid chitin degradation for Chi74 was the highest, followed by Chi66, Chi63 and Chi54. Ag(+) reduced the activity of Chi74, Chi54, Chi63 and Chi66, but Mg(2+) enhanced them. The effect of Ag(+) and Mg(2+) was more significant on the activity of Chi54 than on the activities of Chi63, Chi66 and Chi74. CONCLUSION: ChBDChi74 and FN3Chi74 domains play a role in exerting enzymatic activity and can improve the stability of chitinase.

Identification and characterization of a novel citrate synthase from Streptomyces diastaticus No. 7 strain M1033.

Citrate synthase (CS) is a key enzyme of the tricarboxylic acid cycle and is widely distributed among prokaryotes and eukaryotes. Here, we report for the first time, the cloning, expression, and characterization of a novel CS from Streptomyces diastaticus No. 7 strain M1033 (SdCS). Gel filtration chromatography and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analyses indicate that SdCS forms homodimers with a molecular mass of approximately 100.0 kDa. The predicted amino acid sequence of SdCS is highly similar to those of bacterial homodimeric type I CSs. The pH and temperature optima for SdCS activity were 8.0 and 35 °C, respectively. The half-life (t1/2 ) of SdCS was 10 Min at 50 °C and was increased to 210 Min in the presence of oxaloacetate. The kinetic parameters of SdCS (kcat = 262.8 and 230.7 s(-1); Km = 58.4 and 11.2 µM for acetyl-CoA and oxaloacetate, respectively) were comparable to those of dimeric CSs isolated from Gram-positive bacteria and eukaryotes. Moreover, SdCS activity was inhibited by ATP and ADP and stimulated by AMP. These findings provide a foundation for further investigations on the three-dimensional structure and mechanism of catalysis of SdCS.

Identification of a Novel Chitinase from Bacillus paralicheniformis: Gene Mining, Sequence Analysis, and Enzymatic Characterization.

In this study, a novel strain for degrading chitin was identified as Bacillus paralicheniformis HL37, and the key chitinase CH1 was firstly mined through recombinant expression in Bacillus amyloliquefaciens HZ12. Subsequently, the sequence composition and catalytic mechanism of CH1 protein were analyzed. The molecular docking indicated that the triplet of Asp526, Asp528, and Glu530 was a catalytic active center. The enzymatic properties analysis revealed that the optimal reaction temperature and pH was 65 °C and 6.0, respectively. Especially, the chitinase activity showed no significant change below 55 °C and it could maintain over 60% activity after exposure to 85 °C for 30 min. Moreover, the optimal host strain and signal peptide were obtained to enhance the expression of chitinase CH1 significantly. As far as we know, it was the first time finding the highly efficient chitin-degrading enzymes in B. paralicheniformis, and detailed explanations were provided on the catalytic mechanism and enzymatic properties on CH1.

Biochemical Characterization of a Marine Pseudoalteromonas citrea-Derived Fatty Acyl-AMP Ligase That Exhibits N-Acyl Amino Acid Synthetic Activity.

Activation of fatty acids as acyl-adenylates by fatty acid-AMP ligase (FAAL) is a well-established process contributing to the formation of various functional natural products. Enzymatic characterization of FAALs is pivotal for unraveling both the catalytic mechanism and its role in specific biosynthetic pathways. In this study, we recombinantly expressed and characterized a novel FAAL derived from marine Pseudoalteromonas citrea (PcFAAL). PcFAAL was a cold-adapted neutral enzyme, demonstrating optimal activity at 30 °C and pH 7.5. Notably, its specific activity relied on the presence of Mg2+; however, higher concentrations exceeding 10 mM resulted in inhibition of enzyme activity. Various organic solvents, especially water-immiscible organic solvents, demonstrated an activating effect on the activity of PcFAAL on various fatty acids. The specific activity exhibited a remarkable 50-fold increase under 4% (v/v) n-hexane compared to the aqueous system. PcFAAL displayed a broad spectrum of fatty acid substrate selectivity, with the highest specific activity for octanoic acid (C8:0), and the catalytic efficiency (kcat/Km) for octanoic acid was determined to be 1.8 nM-1·min-1. Furthermore, the enzyme demonstrated biocatalytic promiscuity in producing a class of N-acyl amino acid natural products, as verified by LC-ESI MS. Results indicated that the PcFAAL exhibits promiscuity towards 10 different kinds of amino acids and further demonstrated their potential value in the biosynthesis of corresponding functional N-acyl amino acids.