复合酶水解云南白鱼制备生物活性肽类物质

研究了复合蛋白酶对云南土著白鱼水解的工艺条件。考察了酶用量、温度、pH值、反应时间、底物浓度、复合酶比例等因素对白鱼水解的影响。确定了最佳酶解条件为:复合酶用量(木瓜蛋白酶+风味蛋白酶1∶1)16000U/g,底物浓度30%,温度55℃,pH值7.5,时间4h。在此条件下,水解度达34.12%,氮回收率90.05%。该结果为云南白鱼生产功能食品提供科学依据。     

动态瞬时高压作用对膳食纤维酶解速度的影响

 对纤维素酶对动态瞬时高压处理后膳食纤维的酶解速度进行了研究。豆渣膳食纤维（Dietary Fiber,DF）经微射流均质机的瞬时高压作用（Instantaneous High Pressure,IHP）,在不同的压力以及同一压力的不同作用次数下产生不同粒径和密度,它们对应的酶解速度是不同的。纤维素酶水解膳食纤维产生纤维二糖和葡萄糖等还原糖,通过测定水解后还原糖的含量判断不同粒径和密度的膳食纤维的酶解速度。结果表明,在40～90 MPa的压力范围内随压力的增大物料粒径呈显著下降,物料密度随之增大,酶解速度增大,在90 MPa的均质压力下物料粒径达到最小,为202.4 nm;当压力继续增大时,物料因膨化作用,物料粒径呈增大趋势,但其密度开始减小,酶解速度继续增大,在140 MPa的均质压力下物料密度达到最小,为1.027 g/mL,此时酶解后还原糖含量最高,酶解速度最快。压力超过140 MPa后,由于超微颗粒间的团聚,物料的密度和粒径均增大,酶解速度减小。在90 MPa和140 MPa下对分别对物料进行多次的瞬时高压处理,发现随处理次数的增多物料粒径增大,体系密度先减小后增大,酶解速度减小。     

丝胶蛋白酶解物的抗氧化性

采用Fenton反应建立羟基自由基(—OH)模型及邻苯三酚在碱性环境中自氧化产生超氧阴离子自由基(—Oz-)模型,研究了碱性蛋白酶酶解丝胶液在不同酶解时间下的抗氧化能力.结果表明丝胶液在酶解4h,水解度为8.72％时的酶解液的抗氧化能力最强,羟基清除率和氧自由基清除率分别为92.18％,33.76％.     

非共振圆偏振光作用下单层二硫化钼电子结构及其自旋/谷输运性质

基于紧束缚近似下的低能有效哈密顿模型和久保线性响应理论,研究了外部非共振圆偏振光作用下单层二硫化钼(MoS_2)电子结构及其自旋/谷输运性质.研究结果表明:单层MoS_2布里渊区K谷和K′谷附近自旋依赖子带间的能隙随着非共振右旋圆偏振光引起的有效耦合能分别线性增大和先减小后增大,随着非共振左旋圆偏振光引起的有效耦合能分别先减小后增大和线性增大,实现了系统能带结构有趣的半导体-半金属-半导体转变.此外,单层MoS_2在外部非共振圆偏振光作用下,呈现有趣的量子化横向霍尔电导和自旋/谷霍尔电导,自旋极化率在非共振右/左旋圆偏振光有效耦合能±0.79 eV附近达到最大并发生由正到负或由负到正的急剧转变,谷极化率随着非共振圆偏振光有效耦合能先增大后减小并在其绝对值0.79-0.87 eV范围内达到100%.因而,可以利用外部非共振圆偏振光将单层MoS_2调制成自旋/谷以及光电特性优异的新带隙材料.     

纤维素酶水解老化纸张中纤维素物质的酶活力分析

应用3,5-二硝基水杨酸测定法,分析了纤维素酶水解老化纸张中纤维素物质的酶活力影响因素,根据酶活力确定了酶解反应条件。pH=4.8,温度为50℃,酶解时间为60min,加酶量为10kU/mL条件下,经纤维素酶水解后测得的还原糖量呈现一定的趋势,可以此来初步判定纸张纤维的老化程度。     

对甲苯偶氮水杨基荧光酮应用于大豆酶解液中钼的形态分布研究

研究了新试剂对甲苯偶氮水杨基荧光酮(MBASF)与钼(Ⅵ)显色反应,以建立水解液中钼的形态分布测定方法.在盐酸介质中,对MBASF与钼(Ⅵ)发生灵敏的显色反应,形成稳定的红色配合物.配合物表观摩尔吸光系数达1.54 L·mor-1·cm-1,在0～12μg·(25mL)-1范围内钼(Ⅵ)服从比耳定律.除常见金属离子外,蛋白、多肽和氨基酸均有高的允许量.大豆酶解液A经等电点沉淀法除去未水解蛋白后得溶液B,溶液B经D301R树脂吸附后,分别采用pH 3.6和9.0的缓冲液洗脱分离得溶液C和D;A,B,C和D溶液分别用于光度法测定总钼、水解钼、结合态钼(多肽和氨基酸螯合态钼)和游离钼的含量.     

十二烷基苯磺酸钠共振散射光谱法测定木瓜蛋白酶活力

在pH值6.5的磷酸盐缓冲溶液中,十二烷基苯磺酸钠(SDBS)与酪蛋白(Casein)形成缔合物微粒,在470,360,400,420和520 nm产生5个瑞利散射峰.在选定条件下,木瓜蛋白酶(Papain)可水解酪蛋白(Casein),加SDBS可中止酶催化反应并与未反应的酪蛋白底物结合形成缔合物微粒.随着Papain浓度的增大,470 nm处的共振散射峰强度降低.Papain的酶活力在0.048～4.8 USP·mL-1范围内与△I470nm呈现良好的线性关系.其线性回归方程为△ISDBS=1.972c 2.31,相关系数分别为r=0.999 9,检测限为0.020USP·mL-1.该法用于嫩肉粉中木瓜蛋白酶活力测定,结果令人满意.     

绿豆酶解过程中Se的形态分布分析

采用AFS-230E型原子荧光光度计对绿豆中Se的含量及绿豆分步酶解过程中Se的形态分布进行了分析。结果表明:(1)绿豆中Se含量为54.79μg.g-1(干重);(2)通过酶解,绿豆中90%以上的Se被提取出来,而水煮对Se的提取率只有19.26%,其中淀粉酶、蛋白酶和纤维素酶酶解过程对Se的提取率分别是36.64%,55.96%和5.189%,可推断,绿豆中大部分Se被蛋白结合或络合;(3)总酶解产物中Se的有机态分布系数为59.87%,远大于绿豆汤中Se的有机态分布系数(3.64%),其中淀粉酶解产物和蛋白酶解产物中Se的有机态分布系数分别为15.51%和44.36%,纤维素酶解产物中未检测到有机态Se,可见,Se在绿豆纤维素中主要以络合形式存在。回收实验表明,方法回收率为97.8%,RSD为1.1%(n=11)。     

丝氨酸蛋白酶中催化三元组内氢键的作用

为了探究丝氨酸蛋白酶催化效率的来源,本文分别研究了丝氨酸酶催化水解多肽CI2、MCTI-A和六肽(SUB)的过程中催化三元组内的氢键所起的作用. 首先采用QM/MM-MD方法计算了在酶-底物复合物和过渡态下组氨酸和天冬氨酸之间质子转移的自由能曲线. 结果表明低能垒氢键仅在CI2酰化反应的过渡态区域形成,而在MCTI-A和SUB酰化反应中均是正常氢键. 与MCTI-A相比,CI2和SUB体系中氢键强度在过渡态时显著增强,因此相应的酰化反应能垒明显降低. 过渡态区域形成的低能垒氢键显然有助于加速酰化反应,同时研究也表明正常氢键也有可能降低能垒. 氢键降低能垒的关键则在于过渡态下氢键强度的增加程度,而不是其是否生成了低能垒氢键. 本文为研究催化三元组间的氢键在丝氨酸蛋白酶中的作用提供了新思路,并有助于理解丝氨酸蛋白酶中催化三元组的催化机制.     

丝氨酸蛋白酶中催化三元组内氢键的作用（英文）

为了探究丝氨酸蛋白酶催化效率的来源,本文分别研究了丝氨酸酶催化水解多肽CI2、MCTI-A和六肽(SUB)的过程中催化三元组内的氢键所起的作用.首先采用QM/MM-MD方法计算了在酶-底物复合物和过渡态下组氨酸和天冬氨酸之间质子转移的自由能曲线.结果表明低能垒氢键仅在CI2酰化反应的过渡态区域形成,而在MCTIA和SUB酰化反应中均是正常氢键.与MCTI-A相比,CI2和SUB体系中氢键强度在过渡态时显著增强,因此相应的酰化反应能垒明显降低.过渡态区域形成的低能垒氢键显然有助于加速酰化反应,同时研究也表明正常氢键也有可能降低能垒.氢键降低能垒的关键则在于过渡态下氢键强度的增加程度,而不是其是否生成了低能垒氢键.本文为研究催化三元组间的氢键在丝氨酸蛋白酶中的作用提供了新思路,并有助于理解丝氨酸蛋白酶中催化三元组的催化机制.     

Ultrasound-ionic liquid enhanced enzymatic and acid hydrolysis of biomass cellulose

The purpose of this paper was to investigate the effect of ultrasound-ionic liquid (IL) pretreatment on the enzymatic and acid hydrolysis of the sugarcane bagasse and wheat straw. The lignocellulosic biomass was dissociated in ILs ([Bmim]Cl and [Bmim]AOC) aided by ultrasound waves. Sonication was performed at different frequencies (20, 28, 35, 40, and 50 kHz), a power of 100 W, a time of 30 min and a temperature of 80 °C. The changes in the structure and crystallinity of the cellulose were studied by Fourier transform infrared (FT-IR), X-ray diffraction (XRD) and thermal gravimetric analysis (TGA). The amounts of the total reducing sugars, glucose, cellobiose, xylose and arabinose in the hydrolysates were determined. The results of FT-IR, XRD and TGA revealed that the structure of cellulose of both biomass samples remained intact after the pretreatment, but the crystallinity decreased. The enzymatic and acid hydrolysis of the biomass samples pretreated with the ultrasound-IL result in higher yields of the reducing sugars compared with the IL-pretreated sample. Enzymatic hydrolysis of bagasse and wheat straw pretreated with [Bmim]Cl-ultrasound resulted in maximal yields of glucose at 20 kHz (40.32% and 53.17%) and acid hydrolysis resulted in maximal yields of glucose at 40 kHz (33.32% and 48.07%). Enzymatic hydrolysis of bagasse and wheat straw pretreated with [Bmim]OAc-ultrasound show maximal yields of glucose at 28 kHz and acid hydrolysis at 50 kHz. Combination of ultrasound with [Bmim]OAc is more effective than [Bmim]Cl in terms of the yields of reducing sugar.     

Effects of ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks

Based on the enzymatic saccharification of the various pulps in the previous 0.8 l ultrasonic stirred tank reactor, the ultrasound-enhanced saccharification of waste papers such as newspaper, carton paper, office paper etc. was carried out in the same reactor as well as larger scale stirred tank reactors of size 3.2 and 6.4 l. The saccharification of each waste paper was less enhanced in the larger reactor at a given ultrasonic intensity. This could be attributed to the decrease in the ultrasonic intensity per reaction volume, i.e., the specific ultrasonic intensity. Most waste papers were more efficiently hydrolyzed with increasing specific ultrasonic intensities, although newspaper was less efficiently done for a too high specific intensity. Such an adverse effect might be due to the fact that some impurities in the newspaper such as lignin were activated by an intensive ultrasonic irradiation to form a rigid and closed network, which inhibited the access and adsorption of cellulase on to the substrate surface. The previous kinetic model was found to be applicable to analyze and simulate the saccharification of each waste paper in the different ultrasonic reactors. The ultimate conversion of a substrate based on the total sugar concentration estimated for an infinite reaction time could be correlated as a function of the ratio of initial substrate to enzyme concentrations at a fixed specific ultrasonic intensity. Either the apparent rate constant or the ultimate conversion increased and tended to approach a constant with an increase in the specific ultrasonic intensity except for the case of newspaper, while neither the apparent Michaelis constant, product inhibition constant nor glucose formation equilibrium constant was influenced by the specific ultrasonic intensity.     

Mechanistic investigations in ultrasound-assisted xylitol fermentation

This study has investigated ultrasound-assisted xylitol production through fermentation of dilute acid (pentose-rich) hydrolysate of sugarcane bagasse using free cells of Candida tropicalis. Sonication of fermentation mixture at optimum conditions was carried out in ultrasound bath (37 kHz and 10% duty cycle). Time profiles of substrate and product in control (mechanical shaking) and test (mechanical shaking + sonication) fermentations were fitted to kinetic model using Genetic Algorithm (GA) optimization. Max. xylitol yield of 0.56 g/g and 0.61 g/g of xylose was achieved in control and test fermentations, respectively. The biomass yield also increased marginally (∼17%) with sonication. However, kinetics of fermentation increased drastically (2.5×) with sonication with 2× rise in xylose uptake and utilization by the cells. With comparative analysis of kinetic parameters in control and test experiments, this result was attributed to enhanced permeability of cell membrane that allowed faster diffusion of nutrients, substrates and products across cell membrane, higher enzyme-substrate affinity, dilution of toxic components and reduced inhibition of intracellular enzymes by substrate.     

Hydrodynamic cavitation-assisted continuous pre-treatment of sugarcane bagasse for ethanol production: Effects of geometric parameters of the cavitation device

For biotechnological conversion of lignocellulosic biomass, a pre-treatment step is required before enzymatic hydrolysis of carbohydrate fractions of the material, which is required to produce fermentable sugars for generation of ethanol or other products in a biorefinery. The most of the reported pre-treatment technologies are in batch operation mode, presenting some disadvantages as dead times in the process. In this context, hydrodynamic cavitation (HC)-assisted alkaline hydrogen peroxide (AHP) pre-treatment in continuous process was proposed for pre-treatment of sugarcane bagasse (SCB). The system was designed with a main reactor containing two devices to generate cavitation by passing liquid medium through orifice plates. For SCB pretreated in continuous process, 52.79, 34.31, 22.13 and 15.81 g of total reducing sugars (TRS) per 100 g of SCB were released in samples pretreated using orifice plates with a number of holes of 24 (d = 0.45 mm), 16 (d = 0.65 mm), 12 (d = 0.8 mm) and 8 (d = 1 mm), respectively. Computational Fluid Dynamics (CFD) tools showed that 0.94 of vapor phase volume fraction and 0.19 of cavitation number were achieved at 31 m/s of throat velocity and upstream pressure of 350,000 Pa. By using pretreated SCB, 28.44 g of ethanol/L (84.31% of yield respect to theoretical value) was produced by immobilized Scheffersomyces stipitis NRRL-Y7124 in a simultaneous hydrolysis and fermentation process at high solid loading (16% S/L). Thus, HC-assisted process was proved as a promising technology for valorization of lignocellulosic biomass.     

The promoted hydrolysis effect of cellulase with ultrasound treatment is reflected on the sonicated rather than native brown rice

Brown rice is nutritionally superior to polished white rice, as it maintains a large content of external bran that involves a series of bioactive compounds. However, the presence of bran also restricts water diffusion and results in adverse quality of brown rice. In this work, ultrasound conditions were optimized for cellulase to improve its hydrolysis effect on rice bran, and combinations of enzymatic and ultrasound treatment in different manners were conducted on brown rice, to improve the textural attributes. The results showed significant improvements in the catalytic activity and efficiency of cellulase after ultrasonication at the optimal intensity of 1.67 W cm⁻³ and duration of 30 min, with the conformational variation of cellulase observed from the fluorescence spectra and circular dichroism (CD). Despite the enhanced activity of ultrasonicated cellulase, it leaded to a similar rice surface morphology and a comparable amount of released glucose, and equivalent textural parameters of brown rice treated by native cellulase. However, for the pre-sonicated brown rice, the ultrasonicated cellulase showed a significantly higher hydrolysis capacity than the untreated enzyme, suggesting the important influence of ruptured bran surface on amplifying the hydrolysis effect of cellulase. Compared to the successive ultrasound stimulation on both cellulase and brown rice, ultrasound-assisted cellulase treatment on brown rice produced less glucose from rice bran, but induced similar textural properties of brown rice, possibly resulting from the simultaneously promoting effect of ultrasonication on cellulase and water diffusion. Ultimately, this study highlighted that the mild rice surface rupture is a crucial factor to display the promoted hydrolysis effect of ultrasonicated cellulase on brown rice. Ultrasound-assisted cellulase treatment potentially provides an effective strategy to improve the edible quality of brown rice.     

Synergism of sweeping frequency ultrasound and deep eutectic solvents pretreatment for fractionation of sugarcane bagasse and enhancing enzymatic hydrolysis

Sugarcane bagasse (SCB) is an abundant agricultural waste in China and the conversion of the waste into plethora of useful resources is very vital. To achieve this, fractionation of the waste is highly important in the biomass biorefinery. The present study aims at investigating the synergistic role of deep eutectic solvents (DES) with sweeping frequency ultrasound (SFUS) and fixed frequency ultrasound (FFUS) in the fractionation of SCB to enhance the enzymatic saccharification process. Therefore, the effects of ultrasound (US) and DES conditions on the pretreatment efficiency were investigated. Under optimum SCB pretreatment conditions, FFUS (40 kHz, 60 min) + DES (choline chloride (ChCl)-lactic acid (LA), 120 °C, 3 h) and SFUS (40 kHz, 60 min) + DES (ChCl-LA, 120 °C, 3 h), the lignin removal rates were 80.13 and 85.62%, respectively. The hemicellulose removal rates were 78.08 and 90.46%, respectively; and the contents of glucose, xylose and arabinose in the liquid fractions after FFUS + DES pretreatment were 7.07, 17.95 and 3.01%, respectively. However, the yield of glucose, xylose, and cellobiose after enzymatic hydrolysis of the SFUS + DES pretreated SCB were 86.76, 38.68, and 20.76%. Analytical studies revealed that the SFUS + DES pretreatment can effectively destroy the ultrastructure of SCB and reduce the crystallinity of cellulose. Furthermore, the mechanism of pretreatment with SFUS + DES was proposed, which confirmed the excellent performance of SFUS + DES. Thus, the application of SFUS + DES pretreatment was able to improve the removal of lignin and hemicellulose from SCBs.     

云南盘鮈酶水解条件研究

采用动物蛋白水解酶对云南盘鮈进行酶水解,通过正交实验法确定了酶水解条件。结果表明,酶水解的最适条件为:温度50℃,pH7.0,时间3h,加酶量0.5%,固液比1∶2.5。在该条件下盘鮈肉蛋白的水解度为32.85%。氨基酸分析表明,游离氨基酸含量36.47mg/mL,必需氨基酸14.42mg/mL,占39.54%。尤其是亮氨酸、缬氨酸、苏氨酸、赖氨酸含量丰富,为低值鱼深加工研究提供理论依据。     

Effect of ultrasound and enzymatic pre-treatment on yield and properties of banana juice

Effect of ultrasound and enzymatic pre-treatments with cellulase and pectinase on yield and properties of banana juice were investigated. A two-level full factorial design was employed. The factors selected were ultrasonication time (0 and 30 min), cellulase concentration (0 and 0.2%) and pectinase concentration (0 and 0.2%). The responses studied were yield, viscosity, clarity, total soluble solids (TSS) and pH. It was observed that pectinase was more effective in increasing the yield of juice compared to cellulase. Ultrasonic pre-treatment alone did not significantly increase the yield of juice. When ultrasound was combined with pre-treatment with both the enzymes maximum yield of 89.40% was obtained compared to 47.30% in the control. The viscosity of the juice decreased with addition of enzymes and with application of ultrasound. The clarity of the juice was not affected by cellulase treatment, but improved with pectinase treatment. Ultrasonication alone was found to be more effective than pectinase or cellulase treatment in improving the clarity of the juice. The TSS increased with enzymatic treatment, ultrasonication and their combination. pH was not affected by treatment type, but was found to be lower for the treated juices. Significant correlations were observed between the various responses.