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.

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.

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.

Biochemical characterization and molecular modification of a zearalenone hydrolyzing enzyme Zhd11D from Phialophora attinorum.

ZEN lactone hydrolase (ZHD) can hydrolyze zearalenone (ZEN) to less or non-toxic product, providing an environment-friendly way for food or feeds-containing ZENs detoxification. Here, a newly identified ZHD from Phialophora attinorum, annotated as Zhd11D, was characterized to exhibit highest activity against ZEN at pH 8.0 and 35 â„ƒ with a specific activity of 304.7 U/mg, which was far higher than most of the reported ZHDs. A nonspecific protein engineering method was introduced through fusing a segment of amphiphilic short peptide S1 at the N-terminus of Zhd11D, resulting in both improved activity (1.5-fold) and thermostability (2-fold at 40 â„ƒ). Biochemical analysis demonstrated that self-aggregation caused by intermolecular interactions between S1 contributed to the improvement of the enzymatic properties of Zhd11D. Additionally, S1-Zhd11D showed a higher hydrolysis rate of ZEN than Zhd11D in peanut oil.

Characterization of a novel GH26 ß-mannanase from Paenibacillus polymyxa and its application in the production of mannooligosaccharides.

A novel glycoside hydrolase family 26 ß-mannanase gene ppman26a was cloned from Paenibacillus polymyxa KF-1. The full-length enzyme PpMan26A and its truncated products CBM35pp (aa 35-328) and PpMan26A-Δ205 (aa 206-656) were overexpressed in Escherichia coli. PpMan26A hydrolyzed locust bean gum, guar gum, konjac gum and ivory nut mannan, with the highest specific activity toward konjac gum. The Km and kcat values for konjac gum were 2.13 mg/mL and 416.66 s-1, respectively. The oligosaccharides fraction obtained from the hydrolysis of konjac gum by PpMan26A was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF-MS). The degradation products were mainly mannooligosaccharides with a degree of polymerization of 3-8. CBM35pp exerted strong binding activity toward mannans but without ß-mannanase activity. PpMan26A-Δ205, with the deletion of the N-terminal CBM domain, showed lower substrate binding capacity, resulting in reduced enzymatic activity and thermostability. This study complements our understanding of GH26 ß-mannanases and expands the potential industrial application of PpMan26A.

Crosslinking Mechanism on a Novel Bacillus cereus Transglutaminase-Mediated Conjugation of Food Proteins.

Until now, Streptoverticillium mobaraense transglutaminase (TG) is the only commercialized TG, but limited information is known about its selection tendency on crosslinking sites at the protein level, restricting its application in the food industry. Here, four recombinant Bacillus TGs were stable in a broad range of pH (5.0−9.0) and temperatures (<50 °C), exhibiting their maximum activity at 50−60 °C and pH 6.0−7.0. Among them, TG of B. cereus (BCETG) demonstrated the maximal specific activity of 177 U/mg. A structural analysis indicated that the Ala147-Ala156 region in the substrate tunnel of BCETG played a vital role in catalytic activity. Furthermore, bovine serum albumin, as well as nearly all protein ingredients in soy protein isolate and whey protein, could be cross-linked by BCETG, and the internal crosslinking paths of three protein substrates were elucidated. This study demonstrated Bacillus TGs are a candidate for protein crosslinking and provided their crosslinking mechanism at the protein level for applications in food processing.

[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.

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.

Isolation of the Thermostable ß-Glucosidase-Secreting Strain Bacillus altitudinis JYY-02 and Its Application in the Production of Gardenia Blue.

Gardenia blue (GB) is a natural blue pigment widely used in textiles and the pharmaceutical industry. The geniposide in gardenia fruits can be hydrolyzed by ß-glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable ß-glucosidase (EC 3.2.1.21) was isolated from soil and identified as Bacillus altitudinis JYY-02. This strain could potentially be used for GB production from geniposide by fermentation. Optimal fermentation results were achieved at pH 6.5 or 8.0 at 45°C for 45 h with additional sucrose. To obtain a large amount of ß-glucosidase, the whole genome of B. altitudinis JYY-02 was sequenced and annotated; it is 3,727,518 bp long and contains 3,832 genes. The gene encoding ß-glucosidase (bgl) in B. altitudinis JYY-02 was screened from the genome and overexpressed in Escherichia coli BL21(DE3). The recombinant ß-glucosidase was purified by affinity chromatography on a Ni Sepharose 6 fast flow (FF) column. The optimal temperature, pH, and Km values for the recombinant ß-glucosidase were 60°C, pH 5.6, and 0.331 mM, respectively, when p-nitrophenyl-ß-d-glucopyranoside (pNPG) was used as the substrate. The recombinant ß-glucosidase catalyzed the deglycosylation reaction of geniposide, which was then used to produce GB. IMPORTANCE ß-Glucosidases are enzymes capable of hydrolyzing ß-glucosidic linkages present in saccharides and glycosides and have many agricultural and industrial applications. Although they are found in all domains of living organisms, commercial ß-glucosidases are still expensive, limiting their application in industry. In the present study, a thermostable ß-glucosidase-producing strain was obtained for GB production by fermentation, engineered bacteria were constructed for preparing recombinant ß-glucosidase, and a one-step method to purify the recombinant enzyme was established. A large amount of purified ß-glucosidase was easily obtained from the engineered bacteria for industrial applications such as GB 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.

Biochemical and Structural Properties of a High-Temperature-Active Laccase from Bacillus pumilus and Its Application in the Decolorization of Food Dyes.

A novel laccase gene isolated from Bacillus pumilus TCCC 11568 was expressed, and the recombinant laccase (rLAC) displayed maximal activity at 80 °C and at pH 6.0 against ABTS. rLAC maintained its structural integrity at a high temperature (355 K) compared to its tertiary structure at a low temperature (325 K), except for some minor adjustments of certain loops. However, those adjustments were presumed to be responsible for the formation of a more open access aisle that facilitated the binding of ABTS in the active site, resulting in a shorter distance between the catalytic residue and the elevated binding energy. Additionally, rLAC showed good thermostability (≤70 °C) and pH stability over a wide range (3.0-10.0), and displayed high efficiency in decolorizing azo dyes that are applicable to the food industry. This work will improve our knowledge on the relationship of structure-function for thermophilic laccase, and provide a candidate for dye effluent treatment in the food industry.

Excavation, expression, and functional analysis of a novel zearalenone-degrading enzyme.

Zearalenone (ZEN) is a toxic secondary metabolite of Fusarium sp. commonly found in wheat, corn, and other crops. In addition to economic losses, ZEN can seriously endanger the health of both humans and livestock, thus presenting an urgent need for ZEN-detoxifying enzymes that function in the extreme heat or pH conditions of industrial fermenters. Here, we identify and characterize the activity of the ZEN-degrading enzyme from Exophiala spinifera, ZHD_LD, which shares 60.15% amino acid identity and a conserved catalytic triad with the well-characterized ZEN-detoxifying protein ZHD101 from Clonostachys rosea. Biochemical activity and stability assays indicated that purified recombinant ZHD_LD exhibited high activity against ZEN with optimal reaction conditions of 50 ℃ and pH 7.0-10.0. Structural modeling of the ZHD_LD active site and comparison with ZHD101 revealed its likely mechanism of ZEN degradation. This research provides an industrially valuable candidate enzyme for ZEN detoxification in food and livestock feed.

Enzymatic Characterization of Purified ß-Glucosidase from Non-Saccharomyces Yeasts and Application on Chardonnay Aging.

The application of ß-glucosidase from non-Saccharomyces yeasts to improve wine aroma has been widely explored. However, few enzymes are active under the severe conditions of wine aging (high ethanol concentration, low temperature, and low pH). Therefore, the application of ß-glucosidase in wine aging needs further research. In this study, the ß-glucosidases Mg-ßgl and Hu-ßgl extracted from Meyerozyma guilliermondii NM218 and Hanseniaspora uvarum BF345 were purified and used in young Chardonnay wines aged for 50 days. The enzyme activity of the two enzymes was measured. The effects of the two enzymes and a commercial ß-glucosidase (An-ßgl) on the volatile composition and sensory quality of the wine were also determined. The results showed that Mg-ßgl and Hu-ßgl had high specific activity of 1.95 U/mg and 2.11 U/mg, respectively, maintaining the activity of 70-80% at 20 °C, pH of 3.0-4.0, and 15% ethanol, corresponding to wine aging conditions. Analysis of volatiles with GC-MS showed a 65-70% increase in total terpenoids and new detection of C13-norisoprenoids when the wines were treated with the three ß-glucosidases. In addition, wines treated with Mg-ßgl and Hu-ßgl had more hexanol, phenylethanol, ethyl octanoate, ethyl heptanoate, and ethyl caprate than wines treated without and with An-ßgl. In sensory analysis, the judges showed a greater preference for Hu-ßgl-treated wines, to which they attributed pleasant sweet, floral, honey, pomelo, and banana aromas. The results of this study not only offer a way to improve flavor complexity in wine but also provide a reference for the use of other edible sources of ß-glucosidase in wine aging.

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.

Screening and characterization of a GH78 α-l-rhamnosidase from Aspergillus terreus and its application in the bioconversion of icariin to icaritin with recombinant ß-glucosidase.

In this study, a GH78 α-L-rhamnosidase AtRha from Aspergillus terreus CCF3059 was screened and expressed in Pichia pastoris KM71H. The maximum enzyme activity of AtRha was 1000 U/mL after 12 days. AtRha was most active at 65 °C and pH 6.5, displaying excellent thermal stability and pH stability. The kinetic parameters Km, Vmax, kcat and kcat/Km values for pNPR were 0.481 mM, 659 µmol/min·mg, 1065 s-1 and 2214 s-1mM-1, respectively. AtRha could be inhibited by Fe2+, Hg2+ and Cu2+. Moreover, it displayed good tolerance to organic reagents with 52.6% activity in 15%(w/v) methanol. AtRha can hydrolyze icariin containing the α-1 rhamnoside linkage. Furthermore, AtRha and ß-glucosidase TthBg3 showed excellent selectivity to cleave the rhamnose at the 3rd position and the glucosyl at the C-7 group of icariin, which established an effective and green method to produce the more pharmacological active icaritin. In addition, the optimal enzyme addition schemes and the reaction conditions were screened and optimized. After a two-stage transformation under optimized conditions, 0.5 g/L of icariin was transformed into 0.25 g/L of icaritin, with a corresponding molar conversion rate of 91.2%. Our findings provide a new, specific and cost-effective method for the production of icaritin in the industry.

Enzymatic characterization of D-lactate dehydrogenase and application in alanine aminotransferase activity assay kit.

D-lactate dehydrogenase (D-LDH) is widely used for the clinical detection of alanine aminotransferase (ALT) activity. It is a key enzyme in ALT detection kits, and its enzymatic properties directly determine sensitivity and accuracy of such kits. In this study, D-lactate dehydrogenase (WP_011543503, ldLDH) coding sequence derived from Lactobacillus delbrueckii was obtained from the NCBI database by gene mining. LdLDH was expressed and purified in Escherichia coli, and its enzyme activity, kinetic parameters, optimum temperature, and pH were characterized. Furthermore, stabilizers, including sugars, polyols, amino acids, certain salts, proteins, and polymers, were screened to improve stability of ldLDH during freeze-drying and storage. Finally, a kit based on ldLDH was tested to determine whether the enzyme had potential clinical applications. The results showed that ldLDH had a specific activity of 1,864 U/mg, Km value of 1.34 mM, optimal reaction temperature of 55°C, and an optimal pH between 7.0 and 7.5. When sucrose or asparagine was used as a stabilizer, freeze-dried ldLDH remained stable at 37°C for > 2 months without significant loss of enzymatic activity. These results indicated that ldLDH possesses high activity and stability. Test results using the ALT assay kit prepared with ldLDH were consistent with those of commercial kits, with a relative deviation <5%. These results indicated that ldLDH met the primary requirements for ALT assays, laying a foundation for the development of new ALT kits with potential clinical applications.

Biochemical Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. HS Isolated in the Odiel Marshlands.

Alpha-amylases are a large family of α,1-4-endo-glycosyl hydrolases distributed in all kingdoms of life. The need for poly-extremotolerant amylases encouraged their search in extreme environments, where archaea become ideal candidates to provide new enzymes that are able to work in the harsh conditions demanded in many industrial applications. In this study, a collection of haloarchaea isolated from Odiel saltern ponds in the southwest of Spain was screened for their amylase activity. The strain that exhibited the highest activity was selected and identified as Haloarcula sp. HS. We demonstrated the existence in both, cellular and extracellular extracts of the new strain, of functional α-amylase activities, which showed to be moderately thermotolerant (optimum around 60 °C), extremely halotolerant (optimum over 25% NaCl), and calcium-dependent. The tryptic digestion followed by HPLC-MS/MS analysis of the partially purified cellular and extracellular extracts allowed to identify the sequence of three alpha-amylases, which despite sharing a low sequence identity, exhibited high three-dimensional structure homology, conserving the typical domains and most of the key consensus residues of α-amylases. Moreover, we proved the potential of the extracellular α-amylase from Haloarcula sp. HS to treat bakery wastes under high salinity conditions.

Expression and Characterization of a Thermostable Carrageenase From an Antarctic Polaribacter sp. NJDZ03 Strain.

The complete genome of Polaribacter sp. NJDZ03, which was isolated from the surface of Antarctic macroalgae, was analyzed by next-generation sequencing, and a putative carrageenase gene Car3206 was obtained. Car3206 was cloned and expressed in Escherichia coli BL21(DE3). After purification by Ni-NTA chromatography, the recombinant Car3206 protein was characterized and the antioxidant activity of the degraded product was investigated. The results showed that the recombinant plasmid pet-30a-car3206 was highly efficiently expressed in E. coli BL21(DE3). The purified recombinant Car3206 showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 45 kDa. The optimum temperature of the recombinant Car3206 was 55°C, and it maintain 60-94% of its initial activity for 4-12 h at 55°C. It also kept almost 70% of the initial activity at 30°C, and more than 40% of the initial activity at 10°C. These results show that recombinant Car3206 had good low temperature resistance and thermal stability properties. The optimum pH of recombinant Car3206 was 7.0. Car3206 was activated by Na+, K+, and Ca2+, but was significantly inhibited by Cu2+ and Cr2+. Thin-layer chromatographic analysis indicated that Car3206 degraded carrageenan generating disaccharides as the only products. The antioxidant capacity of the degraded disaccharides in vitro was investigated and the results showed that different concentrations of the disaccharides had similar scavenging effects as vitamin C on O 2 • - , •OH, and DPPH•. To our knowledge, this is the first report about details of the biochemical characteristics of a carrageenase isolated from an Antarctic Polaribacter strain. The unique characteristics of Car3206, including its low temperature resistance, thermal stability, and product unity, suggest that this enzyme may be an interesting candidate for industrial processes.

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.