Study of the deactivation of β-galactosidase entrapped in alginate-carrageenan gels

The stability of β-galactosidase entrapped in Ca-alginate–K-κ-carrageenan gels under operation conditions was studied. The thermal deactivation of the immobilised enzyme and the biocatalyst protein loss due to gel swelling were taken into account in the mass balance of the enzymatic reaction rate expression.

Time-temperature effect was the most important factor in the biocatalyst deactivation reaction. However, results showed that the enzyme entrapped in gels was partially lost by gel swelling, which was a source of error in predicting results in continuous processes. The enzyme loss determined in this work showed a non-linear behaviour and it depended on mixing conditions of the reactor.

Values of protein loss were used in the modelling of a fixed-bed reactor with similar flow conditions to reduce the error in predicting the operation conditions to maintain a constant conversion.

For reaction conditions similar to those analysed in this work, the β-galactosidase was well entrapped in alginate-carrageenan matrices.     

Cas9 deactivation with photocleavable guide RNAs

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The olfactory G protein Gαolf possesses a lower GDP-affinity and deactivates more rapidly than Gsαshort: consequences for receptor-coupling and adenylyl cyclase activation

The olfactory G protein G(alphaolf) differs from the short splice variant of G(salpha) (G(salphaS)) in 80 amino acids, but little is known about biochemical differences between G(alphaolf) and G(salphaS). We addressed this question by analyzing fusion proteins of the beta2-adrenoceptor (beta2AR) and G(alphaolf) and G(salphaS), respectively, using Sf9 insect cells as expression system. The fusion ensured defined receptor/G protein stoichiometry and efficient coupling. High-affinity agonist binding studies showed that G(alphaolf) possesses a lower GDP-affinity than G(salphaS) As a result, the agonist-free beta2AR and the beta2AR occupied by partial agonists were more efficient at promoting GDP-dissociation from G(alphaolf) than from G(salphaS) a assessed by guanosine 5'-O-(3-thiotriphosphate) binding, adenylyl cyclase (AC) activity and GTP hydrolysis. Basal AC activity in the absence of GTP was almost sixfold lower in membranes expressing beta2AR-G(alphaolf) than in membranes expressing beta2AR-G(salphaS) at similar levels, reflecting the lower abundance of G(alphaolf-GDP) relative to G(salphaS-GDP). The maximum agonist-stimulated AC activity with beta2AR-G(salphaS) was more than twofold higher than with beta2AR-G(alphaolf), but the relative agonist-stimulation of AC with beta2AR-G(alphaolf) was much greater than with beta2AR-G(salphaS). The difference in maximum AC activity can be explained by more rapid deactivation of G(alphaolf-GTP) by GTP hydrolysis and GTP dissociation relative to G(salphaS-GTP). Taken together, there are biochemical differences between G(alphaolf) and G(salphaS), supporting different roles of these G proteins in vivo.     

Factors influencing hydroxylamine inactivation of photosynthetic water oxidation

The kinetics of Mn release during NH₂OH inactivation of the water oxidizing reaction is largely insensitive to the S-state present during addition of NH₂OH. This appears to reflect reduction by NH₂OH of higher S-states to a common more reduced state (S₀ or S_?1) which alone is susceptible to NH₂OH inactivation. Sequences of saturating flashes with dark intervals in the range 0.2–5 s^?1 effectively prevent NH₂OH inactivation and the associated liberation of manganese. This light-induced protection disappears rapidly when the dark interval is longer than about 5 s. Under continuous illumination, protection against NH₂OH inactivation is maximally effective at intensities in the range 10³–10⁴ erg · cm^?2 · s^?1. This behavior differs from that of NH₂OH-induced Mn release, which is strongly inhibited at all intensities greater than 10³ erg · cm^?2 · s^?1. This indicates that two distinct processes are responsible for inactivation of water oxidation at high and low intensities. Higher S-states appear to be immune to the reaction by which NH₂OH liberates manganese, although the overall process of water oxidation is inactivated by NH₂OH in the presence of intense light. The light-induced protection phenomenon is abolished by 50 μM DCMU, but not by high concentrations of carbonyl cyanide m-chlorophenylhydrazone, which accelerates inactivation reactions of the water-splitting enzyme, Y (an ADRY reagent). The latter compound accelerates both inactivation of water oxidation and manganese extraction in the dark.     

Interactions of the cytotoxic RNase A dimers with the cytosolic ribonuclease inhibitor

Ribonuclease A (RNase A) dimers have been recently found to be endowed with some of the special, i.e., non-catalytic biological activities of RNases, such as antitumor and aspermatogenic activities. These activities have been so far attributed to RNases which can escape the neutralizing action of the cytosolic RNase inhibitor (cRI). However, when the interactions of the two cytotoxic RNase A dimers with cRI were investigated in a quantitative fashion and at the molecular level, the dimers were found to bind cRI with high affinity and to form tight complexes.     

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A_C). Caf1A_C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A_C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A_C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

高效絮凝素毕赤酵母表面展示系统的构建

为了获得高效的脂肪酶毕赤酵母表面展示系统,利用来自酿酒酵母絮凝素蛋白Flo1的N端874个氨基酸残基(FS)和C端的1101个氨基酸残基(FL)作为锚定蛋白分别构建了2套载体系统.带有前肽的米黑根毛霉脂肪酶(ProRML)克隆到构建的2套展示载体中,使米黑根毛霉脂肪酶(RML)分别以N端锚定或C端锚定的方式实现在毕赤酵母细胞表面的展示.利用RMLC端的Flag标签,通过流式细胞术和激光扫描共聚焦显微镜检测2套系统中RML在酵母表面的展示情况.研究发现,N端锚定于酵母表面的展示酶FSR以pNPC为底物时,水解活力达到了105.3U/g,大约为C端锚定的展示酶FLR活力的2倍.同时FSR比FLR具有更宽的温度、pH作用范围和更好的热稳定性.与游离酶和固定化酶相比,展示酶FSR也表现出更为优良的热稳定性.结果提示,基于Flo1N端锚定的展示系统更适合展示活性中心近C端的脂肪酶,推动了展示酶的进一步研究和开发.