The Relationship of Freeze Tolerance with Intracellular Compounds in Baker’s Yeasts

Freeze-tolerant baker’s yeasts are required for the processing of frozen doughs. The present study was carried out to investigate the cell survival rate after frozen storage and the change of fermentability in dough due to frozen storage, and to discuss quantitatively the relationship of freeze tolerance with intracellular trehalose, amino acids, and glycerol, using six types of baker’s yeasts as the test materials. The experimental results showed that the fermentability of yeast cells in frozen dough was strongly correlated with the cell survival rate. The baker’s yeast with a higher level of cell survival rate had a larger increase in the total intracellular compound content after frozen storage, and the cell survival rate increased linearly with increasing total intracellular compound content in frozen yeast cells. Trehalose was a primary compound affecting freeze tolerance, followed by glutamic acid, arginine, proline, asparagic acid, and glycerol. The basic information provided by the present study is useful for exploring the freeze-tolerance mechanisms of baker’s yeast cells, breeding better freeze-tolerant baker’s yeast strains, and developing more effective cryoprotectants.     

Essential Role of the N- and C-terminals of Laccase from Pleurotus florida on the Laccase Activity and Stability

POXA1b is the most thermostable laccase isoenzyme from Pleurotus ostreatus. POXA1b is remarkably stable at alkaline pH (the t_1/2 at pH 10 was 30 days), and its C-terminal affects its catalytic and stability properties. We cloned POXA1c from P. florida, which showed 99 % identity with POXA1b. POXA1c was functionally expressed in Pichia pastoris. The functions of the N and C termini of POXA1c were investigated using site-directed mutagenesis. Compared with POXA1c, the N-terminal R5V site effectively increased the specific activities for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol by 2- and 3.5-fold, respectively. A C-terminal truncated mutant, POXA1c△13, also increased the specific activities for ABTS and guaiacol by 2.3- and 3.4-fold, respectively. A double mutant, POXA1cΔ13-R5V, combined the R5V and △13 effects. The specific activity of this double mutant for ABTS was 1,321 U/mg, which indicated a 4-fold increase compared with the wild type. The role of residue V5 on laccase catalytic properties was also observed for laccases from Trametes versicolor and Rigidoporus lignosus. The specific activities of the V5R of the laccases from T. versicolor and R. lignosus were half of that of the wild type. The pH and thermal stability analysis of POXA1c and its mutants showed that the enzymes were remarkably stable because they showed 63 % residual activity after incubation for 108 h at 30 °C over a pH range of 4.5 to 9.0. Similar results were observed for POXA1cΔ13-R5V. POXA1cΔ13-R5V can be widely used in industrial biotechnology because of its excellent catalytic properties.     

Synthesis of binuclear palladium complexes with a rigid phenylene bridge linker

The complexes [PdX₂Py]₂(di-NHC) (X = Br or Cl) in which di-NHC represents a di-N-heterocyclic carbene, featuring a rigid phenylene spacer between the carbene units, have been prepared from reactions of the corresponding diimidazolium halide salts with PdCl₂ in pyridine. The molecular structures of three of the complexes were determined by X-ray diffraction studies. The influences of different substitutions and of the halide ligand (Br or Cl) on the structure and reactivity of the complexes have been studied. The catalytic activity of the binuclear palladium complexes was tested in the Mizoroki–Heck reaction of styrene with bromobenzene.     

Enzymatic Synthesis of γ-Glutamylmethylamide from Glutamic Acid γ-Methyl Ester and Methylamine Catalyzed by Escherichia coli Having γ-Glutamyltranspeptidase Activity

A new method for the synthesis of γ-glutamylmethylamide is presented. Glutamic acid γ-methyl ester was used as substrate for γ-glutamylmethylamide synthesis catalyzed by Escherichia coli with γ-glutamyltranspeptidase activity. Reaction conditions were optimized by using 300 mM glutamic acid γ-methyl ester and 3,000 mM methylamine at pH 10 and 40 °C. Bioconversion rate of γ-glutamylmethylamide reached 87 % after 10 h. γ-Glutamyltranspeptidase was reversibly inhibited only when glutamic acid γ-methyl ester was above 300 mM.     

A New α-Galactosidase from Thermoacidophilic Alicyclobacillus sp. A4 with Wide Acceptor Specificity for Transglycosylation

An α-galactosidase gene (gal36A4) of glycosyl hydrolase family 36 was identified in the genome of Alicyclobacillus sp. A4. It contains an ORF of 2,187 bp and encodes a polypeptide of 728 amino acids with a calculated molecular mass of 82.6 kDa. Deduced Gal36A4 shows the typical GH36 organization of three domains—the N-terminal β-sheets, the catalytic (β/α)₈-barrels, and the C-terminal antiparallel β-sheet. The gene product was produced in Escherichia coli and showed both hydrolysis and transglycosylation activities. The optimal pH for hydrolysis activity was 6.0, and a stable pH range of 5.0–11.0 was found. The enzyme had a temperature optimum of 60 °C. It is specific for α-1,6-glycosidic linkages and had a K _m value of 1.45 mM toward pNPGal. When using melibiose as both donor and acceptor of galactose, Gal36A4 showed the transfer ratio of 23.25 % at 96 h. With respect to acceptor specificity, all tested monosaccharides, disaccharides, and oligosaccharides except for D-xylose and L-arabinose were good acceptors for transglycosylation. Thus, Gal36A4 may find diverse applications in industrial fields, especially in the food industry.     

胡麻籽油咪唑啉缓蚀剂的制备及其对A30钢的缓蚀效果

以胡麻籽油、羟乙基乙二胺为原料合成咪唑啉中间体,用苄基氯进行改性,得到阳离子咪唑啉衍生物。利用FT IR对合成产物进行了表征,用静态失重法、电化学极化曲线和FESEM对其缓蚀性能进行了评价,并考察了不同缓蚀剂浓度、腐蚀浸泡时间对缓蚀效果的影响,探讨了其在A30钢表面的吸附行为。结果表明,合成的缓蚀剂在盐酸体系中对A30钢有较好的缓蚀性能,在浓度为100mg/L时对低碳钢的缓蚀效率可达87%,并且其产物为阳离子型缓蚀剂,吸附满足Langmuir等温吸附方程。最后采用量子化学方法对其缓蚀剂的缓蚀机理进行了分析。     

Fe~(3+)诱导全氟羧酸的光化学降解:pH值及链长的影响

以全氟辛酸(PFOA)为代表的全氟化合物是环境水体中新出现的一类持久性有机污染物,Fe3+的存在促进了其在254 nm紫外光下的有效降解.在此基础上,主要考察了溶液初始pH值对Fe3+诱导PFOA光化学降解的影响,并以全氟丁酸(PFBA)、全氟戊酸(PFPeA)、全氟己酸(PFHxA)和全氟庚酸(PFHpA)为对象,研究了Fe3+诱导短链全氟羧酸(PFCAs)的降解,通过对降解中间产物的分析,进而推断了其降解机理.结果表明,强酸性条件有利于PFOA的降解,弱酸性或中性反应条件下,PFOA的降解和脱氟均受到明显地抑制,进一步证实PFOA的降解主要是溶解性铁作用的结果,此时Fe(OH)2+则是铁(III)-羟基配合物的主要分配形态.Fe3+诱导PFCAs的降解表明:当碳原子数大于5,长链的PFCAs更易于降解,但对于碳原子数小于6的PFCAs,其降解没有明显的规律.降解中间产物主要是链更短的PFCAs,由此推断,PFCAs的降解遵循逐级降解的规律.     

An E1-Catalyzed Chemoenzymatic Strategy to Isopeptide-N-Ethylated Deubiquitylase-Resistant Ubiquitin Probes

Triazole-based deubiquitylase (DUB)-resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain-specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB-resistant Ub probes is reported based on isopeptide-N-ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one-pot, ubiquitin-activating enzyme (E1)-catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi-milligram scale. Proteomic studies using label-free quantitative (LFQ) MS indicated that the isopeptide-N-ethylated Ub probes may complement the triazole-based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.     

Extending the Sensitivity of CEST NMR Spectroscopy to Micro-to-Millisecond Dynamics in Nucleic Acids Using High-Power Radio-Frequency Fields

Biomolecules undergo motions on the micro-to-millisecond timescale to adopt low-populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr–Purcell–Meiboom–Gill (CPMG), chemical exchange saturation transfer (CEST), and R_1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R_1ρ experiment are more broadly sensitive to motions on the micro-to-millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high-power radio-frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro-to-millisecond timescale. Given the ease of implementing high-power fields in CEST, this should make it easier to characterize micro-to-millisecond dynamics in biomolecules.     

Palladium-Catalyzed Asymmetric [8+2] Dipolar Cycloadditions of Vinyl Carbamates and Photogenerated Ketenes

Higher-order cycloadditions, particularly [8+2] cycloadditions, are a straightforward and efficient strategy for constructing significant medium-sized architectures. Typically, configuration-restrained conjugated systems are utilized as 8π-components for higher-order concerted cycloadditions. However, for this reason, 10-membered monocyclic skeletons have never been constructed via catalytic asymmetric [8+2] cycloaddition with high peri- and stereoselectivity. Here, we accomplished an enantioselective [8+2] dipolar cycloaddition via the merger of visible-light activation and asymmetric palladium catalysis. This protocol provides a new route to 10-membered monocyclic architectures bearing chiral quaternary stereocenters with high chemo-, peri-, and enantioselectivity. The success of this strategy relied on the facile in situ generation of Pd-containing 1,8-dipoles and their enantioselective trapping by ketene dipolarophiles, which were formed in situ via a photo-Wolff rearrangement.