Engineering Leifsonia Alcohol Dehydrogenase for Thermostability and Catalytic Efficiency by Enhancing Subunit Interactions

Leifsonia alcohol dehydrogenase (LnADH) is a promising biocatalyst for the synthesis of chiral alcohols. However, limitations of wild-type LnADH observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve its thermostability and catalytic efficiency by altering the subunit interfaces. Residues T100 and S148 were identified to be significant for thermostability and activity, and the melting temperature (ΔT_m) and catalytic efficiency of the mutant T100R/S148I toward ketone substrates was improved by 18.7 °C and 1.8–5.5-fold. Solving the crystal structures of the wild-type enzyme and T100R/S148L revealed beneficial effects of mutations on stability and catalytic activity. The most robust mutant T100R/S148I is promising for industrial applications and can produce 200 g liter⁻¹ day⁻¹ chiral alcohols at 50 °C by only a 1 : 500 ratio of enzyme to substrate.     

Synthesis of silane-modified polyphosphate esters and its application in transparent flame-retardant coatings

Polyphosphate ester (PPAE) made from poly(phosphoric acid), pentaerythritol, and 1,3-butylene glycol has been synthesized. The PPAE reacted with dimethyldichlorosilane (DMDCS) to improve its thermal stability and flame retardancy. Four silane-modified polyphosphate esters (SiPPAEs) were obtained. FTIR, ³¹P NMR, and ²⁹Si NMR results showed that the Si Cl bond of DMDCS could react with the C OH and P OH bonds of PPAE, producing Si O C and P O Si bond in SiPPAEs. Different transparent coatings were obtained by mixing the PPAE and SiPPAEs with amino resin. The results of TGA showed that the stability of SiPPAEs was higher than that of PPAE. With the increase of silane content, the thermal stability of the coating increased. As the silane content increased within a certain range, the fire resistance and expansion coefficient increased. But too high silane content had adverse effects on the expansion and reduced the overall insulation effect. The digital photos and SEM/EDX microphotographs of coatings after combustion showed that silica segments could enriched in the surface to form a hard protective layer to prevent further degradation of the char layer. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47199.     

保护剂对大肠杆菌重组NAD激酶热稳定性的影响

为了提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,研究了几种糖类(葡萄糖、麦芽糖、蔗糖)、醇类(甘油、乙醇、山梨醇)和表面活性剂类(吐温20和司盘20)对NAD激酶热稳定性的影响,通过正交实验得到了一种复合保护剂,并研究了复合保护剂对NAD激酶热稳定性、最适反应温度、p H及储存稳定性的影响。结果表明:麦芽糖、甘油、乙醇、吐温20均能显著提高NAD激酶热稳定性。复合保护剂配方为w(麦芽糖)=0.6%、φ(甘油)=8%、φ(乙醇)=8%、φ(吐温20)=0.6%。55℃时,添加复合保护剂后,NAD激酶半衰期由1.1 h提高到18.4 h,提高了16.7倍;最适反应温度由35℃提高到50℃;p H稳定范围由6.0~9.0扩大到5.5~9.5;在4℃储存28 d后,酶活保留率提高31%。     

Computational Library Design for Increasing Haloalkane Dehalogenase Stability

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (T_m,app, 72.5 °C) and an over 200‐fold longer half‐life at 60 °C. The most stable LinB variants also displayed increased compatibility with co‐solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild‐type enzyme.     

The Stability of G6PD Is Affected by Mutations with Different Clinical Phenotypes

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The k_cat (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T₅₀ (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP⁺ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP⁺, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP⁺, leading to enzyme instability.     

Thermostability of montmorillonitic clays

Bentonite is one of the most widespread used clays connected with various applications. In the case of foundry technology, bentonite is primarily used as a binder for mold manufacture. Thermal stability of bentonites is a natural property of clay minerals and it depends on the genesis, source and chemical composition of the clay. This property is also closely connected to bentonite structure. According to DTA analysis if only one peak of dehydroxylation is observed（about 600 oC）, the cis- isomerism of bentonite is expected, while two peaks of de-hydroxylation（about 550 and 850 oC） are expected in the trans- one. In this overview, the bentonite structure, the water - bentonite interaction and the swelling behavior of bentonite in connection with the general technological properties of bentonite molding mixture are summarized. Further, various types of methods for determination of bentonite thermostability are discussed, including instrumental analytical methods as well as methods that employ evaluation of various technological properties of bentonite binders and/or bentonite molding mixtures.     

Comparison of Gel-forming Properties of Silver Carp (Hypophthalmichthys molitrix) Surimi Prepared in Different Seasons

ABSTRACT: The gel-forming properties of silver carp surimi made in different seasons were compared. Surimi prepared in winter and spring formed gel at 30°C, while autumn and summer surimi required a higher temperature of 40°C for gel formation. All surimi showed marked disintegration when incubated at 60°C. Ca²⁺-ATPase inactivation rate of myofibrils prepared from 4 surimi samples showed that myofibrils in autumn and summer surimi were much more stable than those in winter and spring surimi by about 10°C. These results demonstrated a close relationship between the gel-forming temperature of surimi and the thermal stability of myofibrils in surimi, namely that autumn and summer surimi containing stable myofibrils required higher temperature than winter and spring surimi for the gel formation.     

Structural stability of myosin rod from silver carp as affected by season

Chymotrypsin digestion and Circular dichroism (CD) spectroscopy were used to study the structural stability of myosin rods prepared from silver carp in summer and winter. Differences in the thermal stability were clearly demonstrated by the chymotryptic digestion patterns and CD data obtained at pH 7.5 in 0.5 M KCl for myosin rods in both seasons, indicating the structural properties differed seasonally. In winter, the myosin rods were more susceptible to be cleaved into short (40 kDa) subfragments when digested at high temperature (30 °C). The major peaks in unfolding profiles showed that transition temperatures for the rods were 35 °C in winter, and 36 and 41 °C in summer. Rod subfragments (S-2 and LMM) were isolated and further analyzed by CD. In both winter and summer, the LMM fragments showed an unfolding transition at around 35.5 °C, but the S-2 fragments showed large seasonal difference; in winter, they showed a major unfolding peak around 35 °C and a minor peak at 40.5 °C, while in summer, they unfolded mostly at 44.5 °C, which was 8 °C higher than that for the LMM fragments. Short (40 kDa) S-2 subfragments isolated from winter rods gave a single unfolding transition at 41 °C. These results suggest that differences in the thermal stability of myosin rods between winter and summer were due to differences in the stability of S-2 region. PRACTICAL APPLICATION: The aim of this study was to detect the differences in the structure thermal stability of myosin rods between summer and winter, which is a very important portion for gel formation. The information will be very useful for producing surimi from freshwater fish species in different seasons.     

荞麦α-淀粉酶抑制剂的初步研究

收集了我国荞麦主要产地和部分国外的甜荞和苦荞样品,研究荞麦粉中α-淀粉酶抑制剂的活性和热稳定性。结果显示,苦荞粉中α-淀粉酶抑制剂平均活性(44.8±3.2U/g)高于甜荞粉(37.8±2.4U/g),品种内的α-淀粉酶抑制剂活性无明显的地区间差异。抑制剂具有高的热不稳定性,在100℃处理20min即完全失活,荞麦种子萌发过程中α-淀粉酶抑制剂活性逐渐丧失。研究表明,荞麦食品中的a-淀粉酶抑制剂无内源性抗营养作用。