用氢氧化钾和过氧化氢从甘蔗渣中提取木聚糖的条件优化及甘蔗渣木聚糖酶解产低聚木糖的分析

甘蔗渣含有约40%纤维素和20%半纤维素,是生物炼制生产高附加值生化产品的良好原料。本研究通过响应面分析实验优化了氢氧化钾和过氧化氢处理甘蔗渣从中提取木聚糖的工艺参数:氢氧化钾浓度、过氧化氢浓度和处理温度,以甘蔗渣木聚糖提取率为考察指标,获得处理甘蔗渣的最优条件:10.0%KOH,2.36%H_2O_2,50℃,处理时间6 h,甘蔗渣木聚糖提取率达到78%。用重组内切木聚糖酶水解提取的甘蔗渣木聚糖生产低聚木糖,主要包括木二糖和木三糖,在底物浓度36 g/L、反应温度36℃、p H 5.0、木聚糖酶添加量800 U/g的条件下,水解反应24 h,木聚糖产生低聚木糖的转化率为43%。     

耐热木聚糖酶研究进展

β1,4内切木聚糖酶(EC.3218)能够以内切方式作用于木聚糖主链产生不同长度的木寡糖和少量的木糖,因此是木聚糖降解酶系中最关键的酶。木聚糖酶具有很大的工业应用潜力和价值,由于许多工业应用木聚糖酶的单元操作都是在高温下进行的,寻求耐热木聚糖酶作为催化剂是非常重要的。重点介绍了耐热木聚糖酶的特性、分泌表达和结构区域的研究进展。     

嗜极性木聚糖酶

木聚糖酶内切水解木聚糖主链的1,4-β-D-糖苷键,木聚糖是植物细胞壁中一种主要的多糖。自然界中木聚糖是多种糖类的复合体,这就使得木聚糖酶呈现多态性和多域性,由此需将繁多的木聚糖酶进行归类。木聚糖酶的催化反应属于双置换机制。在已研究的真菌或细菌性木聚糖酶中,大多数在温和的条件下表现出最佳活性,但有很多在极端环境下生长的生物体,为了适应极端环境而产生嗜极性的酶,其中嗜酸的、嗜碱的、嗜热的木聚糖酶,现在已有广泛的研究。对嗜极性木聚糖酶的研究进展作了论述。     

Bacillus pumilus阿拉伯呋喃糖苷酶的重组表达及水解木聚糖的研究

从短小芽孢杆菌中克隆阿拉伯呋喃糖苷酶基因xyn43并重组表达,有利于将该酶分离纯化后应用于其他半纤维素多糖的水解。该研究利用E.coli BL21表达系统对实验室克隆到的短小芽孢杆菌的α-L-阿拉伯呋喃糖苷酶基因xyn43进行重组表达并分析其酶学性质,将重组α-L-阿拉伯呋喃糖苷酶Xyn43和来源于棒曲霉突变菌株的商业木聚糖酶联合作用于燕麦木聚糖。结果表明:以燕麦木聚糖为底物,重组α-L-阿拉伯呋喃糖苷酶Xyn43的最适温度为50℃,最适p H为6.0。该酶在p H 5.0~10.0和45~55℃下较稳定。与木聚糖酶单独作用相比,重组Xyn43酶与商业木聚糖酶同时加入以及先用木聚糖酶水解后加入Xyn43酶,水解产物中的还原糖含量分别增加了16%和20%,木糖含量增加了35%和48%。该结果研究结果表明重组Xyn43酶能够和商业木聚糖酶协同降解燕麦木聚糖,提高水解效率,产生更多的木寡糖,阿拉伯糖和木糖。     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株Bacilluspumilus WL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为26.0kD ,pI值9.5 ,以燕麦木聚糖为底物时的表观Km 值为16.6mg mL ,Vmax值为12.63μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7.2至8.0之间,是耐碱性木聚糖酶;最适作用温度为45℃～55℃,在37℃、45℃以下时该酶热稳定性均较好;50℃保温时,该酶活力的半衰期大约为2h ,在超过50℃的环境下,该酶的热稳定较差,55℃和60℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL-11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL-11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL-11主要合成内切型木聚糖酶A,同时发酵液中不含木糖苷酶,适合用来酶法制备低聚木糖。     

利用真菌纤维素结合域(CBD)保守性序列进行草菇木聚糖酶cDNA的克隆

木聚糖是植物细胞壁的主要组分,它是木糖以β1 ,4 木糖苷键形成主链,乙酰基,阿拉伯糖基等为附链组成的复合多聚糖.木聚糖酶可以降解木聚糖主链,在木聚糖的生物降解中起着非常重要的作用[1 ] .根据木聚糖酶催化域(catalyticdomain ,CD)氨基酸序列的相似性,木聚糖酶可分为两个家     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株BacilluspumilusWL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为2 6 0kD ,pI值9 5 ,以燕麦木聚糖为底物时的表观Km 值为16 6mg mL ,Vmax值为12 6 3μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7 2至8 0之间,是耐碱性木聚糖酶;最适作用温度为4 5℃～5 5℃,在37℃、4 5℃以下时该酶热稳定性均较好;5 0℃保温时,该酶活力的半衰期大约为2h ,在超过5 0℃的环境下,该酶的热稳定较差,5 5℃和6 0℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL_11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL_11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL_11主要合成内切型木聚     

As(Ⅲ)胁迫对小麦发芽过程中木聚糖酶活性及基因表达动态变化的影响

采用室内水培方法,研究了不同浓度As（Ⅲ）胁迫对小麦发芽过程中籽粒、幼根、幼叶3个部位在培养180h内木聚糖酶活性的动态变化。并以小麦稳定表达的肌动蛋白基因作为对照,利用半定量RT-PCR技术,对小麦木聚糖酶基因的表达进行了研究。结果表明,小麦发芽过程中3个部位木聚糖酶活性平均值大小顺序为籽粒〉幼芽〉幼根。随着As（Ⅲ）浓度升高,籽粒中木聚糖酶活性呈现低促高抑的趋势,幼根和幼叶中木聚糖酶活性呈上升趋势。4个浓度As（Ⅲ）处理后木聚糖酶基因表达强度依次为25mg／L〉5mg／L〉0mg／L〉0．1mg／L。     

厌氧菌群SVY42产酶条件分析及产木聚糖酶菌株的分离鉴定

以美国内华达州大盆地温泉采集样品为材料,富集获得纤维素及半纤维素高效稳定降解厌氧菌群SVY42,以巨菌草、甘蔗渣、废菇筒、羧甲基纤维素钠、滤纸、木聚糖为碳源,分析菌群SVY42产内切葡聚糖酶(CMC酶)、β-葡萄糖苷酶和木聚糖酶的情况。在此基础上,以木聚糖为底物筛选高产木聚糖酶的菌株。菌群SVY42在以巨菌草作为碳源时的β-葡萄糖苷酶活最高为0.23 U/mL,以木聚糖作为碳源时CMC酶活和木聚糖酶活均为最高,分别为0.31 U/mL和0.35 U/mL。从菌群SVY42中筛选得到1株高产木聚糖酶厌氧菌株SVY42-1,该菌在最适温度41℃和pH 8.0条件下,其木聚糖酶活力为0.26 U/mL,对其进行16S rDNA序列系统进化分析,SVY42-1与已知菌株的最高同源性仅为93.81%,初步鉴定属于新属。     

食用菌半纤维素酶系研究进展

半纤维素是一类取之不尽而又亟待开发利用的碳水化合物。对食用菌半纤维素酶系的研究已经积累了诸多资料,主要包括β-1,4-内切木聚糖酶、β-木糖苷酶、α-阿拉伯呋喃糖苷酶、乙酰木聚糖酯酶、α-葡萄糖醛酸酶的研究。主要对半纤维素酶系,尤其是木聚糖酶的生物化学与分子生物学研究进行了简要概括,包括结构、酶学性质、基因的克隆与表达等,并综述了在食用菌生产中半纤维素酶活性的变化,最后展望了食用菌半纤维素酶系今后的主要研究方向。     

Mathematical modeling for prediction of endo-xylanase activity and arabinoxylans concentration during mashing of barley malts for brewing

A mathematical model describing the degradation of arabinoxylans by endo-xylanase during mashing process was developed. Endo-xylanase activities and arabinoxylans concentrations in laboratory scale mashing process at different temperature profiles were measured and then used for identifying the model parameters for Harrington barley malt. The modeling errors range for the final concentration of arabinoxylans in wort was -4% to +11.9%. The model developed was also used for predicting the other three different malts mashing processes in laboratory scale, and the prediction errors ranged from -9.5% to +13.6%. The model prediction accuracy for industrial scale mashing process was lower than that in laboratory scale. The simulation results showed that, a lower concentration of arabinoxylans could be achieved when maintaining the mashing-in at 45 degrees C and prolonging the mashing-in time.     

The effect of milling parameters on starch hydrolysis of milled malt in the brewing process

The effect of milling parameters on the hydrolysis of starch during the mashing process was investigated. Hammer milling was compared against roll milling. Roll gap settings, roll speed, speed differential were also analysed, as well as comparing four- to six-roll milling. The parameter of differential speed was also studied through grist particle size distribution. Employing a 65 °C infusion type mashing process for the wort, the glucose and maltose concentrations of malts milled in different ways were analysed. Results showed that the glucose concentration in the wort after 45 min of mashing, obtained using a hammer mill, was the same as that achieved from roll milling in 60 min. For roller mill gap settings the 0.8 mm gap grist required 60 min of mashing to reach a glucose concentration of 3.46 g l⁻¹, whereas the 0.1 mm gap grist achieved the same level of starch hydrolysis in almost half the time, around 30–35 min of mashing. The results regarding roll speed showed that the 300 and 700 rpm mashes required roughly 50 and 40 min, respectively. Comparable sugar concentrations in the 50 rpm mash were obtained in 60 min. Finally, the comparison between simulated four- and six-roll milling showed the latter yielded higher glucose concentrations.     

A kinetic model of starch hydrolysis by alpha- and beta-amylase during mashing

Kinetics of malt starch hydrolysis by endogeneous alpha- and beta-amylases has been experimentally investigated in laboratory-, pilot- and industrial-scale reactors. The production rates of glucose, maltose, maltotriose and total extract, and the separate alpha- and beta-amylases deactivation rates are measured at varying mashing temperature and different initial starch concentrations and qualities. Based on the experimental results, a model is proposed that takes into account the initial carbohydrates and enzymes dissolution, the starch gelatinization, the separate hydrolytic action of alpha-and beta-amylases on insoluble and soluble starch and dextrins, and the influence of temperature both on enzyme activities and thermal denaturation rate. The model can predict, at the three scales, the final sugars concentrations in the wort for given initial malt concentrations and enzymatic contents, and for a fixed temperature profile during the mashing process.     

The barley protein degradation: Mechanism of protein solubilization during barley mashing with neutral proteinase

Kinetics of protein solubilization during barley mashing with neutral proteinase were studied. By plotting the kinetics data in Foster-Niemann coordinates for barley concentration range of 10-30% linear relationships with high correlation coefficients (r >/= 0. 999) were obtained. The slopes of straight lines were very close to corresponding reciprocal initial insoluble nitrogen concentrations. Barley proteolytic inhibitors affected the ordinate intercept; by their addition the values of ordinate intercept decreased. The data suggest that the modified Foster-Niemann equation can be proposed to interpret kinetics of insoluble barley protein degradation. The proteolytic activity decay was studied as well. The enzyme decay was faster in buffer solution than during barley mashing, but in both cases first-order kinetics can be applied. A mathematical model describing protein solubilization and enzyme decay kinetics were developed. The results of computer simulation were in good agreement with experimental data.     

Saccharomyces yeast growth on beet molasses effects of substrate concentration on alcohol toxicity

Summary Growth of a baker's yeast strain has been studied on beet molasses wort to determine the influence of molasses quality and concentration on alcohol toxicity with regard to yeast specific growth rate. A quantitative relation, based on Levenspiel's model has been found useful to describe the growth of our strain. The results show that ethanol toxicity can be directly linked to initial concentration of beet molasses in wort. The proposed model shows that in industrial distilleries, the batch-process may be more suitable than the fed-batch process.     

Cloning and expression of ferulic acid esterase gene and its effect on wort filterability

Objectives

To optimize the expression of type A ferulic acid esterase (FaeA) from Aspergillus niger in Pichia pastoris X-33 using codon optimization.

Results

Recombinant FaeA was purified from the fermentation broth, with the maximum specific activity of 48.4 ± 0.1 U mg^?1. Adding it during mashing process for beer brewing raised the filtration rate by 14.5% while the turbidity and viscosity declined by 22 and 6.9%, respectively. Addition of FaeA increased the concentrations of free ferulic acid (FA) and arabinoxylan (AX) in the wort, while the polymeric arabinoxylans content declined significantly.

Conclusions

Recombinant FaeA was capable to prevent the oxidative gelation of PAX formation by breaking the cross-linking of FA among AX chains and improve the filtration performance of wort.

     

啤酒生产酵母全循环新工艺的研究

以改善浅色啤酒质量,降低生产成本,提高经济效益的目的,提出一种新颖的浅色啤酒的酿造方法。采用煮－浸法糖化工工艺及５０％麦芽和５０％大米作为啤酒酿造的原料及辅料。在糖化过程中添加啤酒酵母提取物作为补充氮源,不仅使所酿造的成品啤酒色泽浅,口味淡爽,纯正,泡沫洁白细腻,持久挂杯,而且具有较显著的经济效益和社会效益。     

The biodiversity of predominant lactic acid bacteria in dolo and pito wort for the production of sorghum beer

AIM: To quantify and identify the predominant lactic acid bacteria (LAB) in dolo and pito wort processing, and to examine their biodiversity at strain level. MATERIALS AND RESULTS: The processing of dolo and pito wort was studied at four production sites in Burkina Faso and Ghana. The succession of dominant micro-organisms, pH and titratable acidity were determined from sorghum malt through mashing and acidification to final wort. In the sorghum malt and during mashing, the LAB counts were 5.7-7.5 log CFU g(-1). Similar levels of yeasts and gram-negative, catalase-positive bacteria were observed. These levels decreased to 3.7-4.5 log CFU g(-1) and相似文献   

Changes in the yeast metabolism at very high-gravity wort fermentation

The rate of ethanol production increased with increasing wort gravity up to the initial wort concentration of 24%, reaching the maximum ethanol concentration of 6.2%, but its attenuation reached only 49%. The intracellular trehalose accumulation was proportional to the inital wort gravity, at 24 or 30% wort fermentation increased 3 or 4.5 times, respectively, compared to 12% wort fermentation. Trehalose accumulation began after exhaustion of glucose, ceased after uptake of approximately 65% reducing saccharides, despite of increasing ethanol or remaining saccharide concentration in the environment.     

Industrialization of a Noncooking System for Alcoholic Fermentation from Grains

Alcoholic fermentation from grains with a noncooking system was successfully carried out for the first time on an industrial scale. The results were compared with those with a conventional high-temperature cooking system and a low-temperature cooking one and it was found that:

(1) The fermentation efficiency is equal or superior to that of the high-temperature cooking system.

(2) Mashing at a concentration high enough to obtain an average 14.2% final alcohol concentration can be very easily done on an industrial scale.

(3) The need for heavy fuel oil for the mashing process is eliminated.

(4) The noncooking system allows much energy saving in industrial production of alcohol from starchy materials.