木聚糖酶法制备水溶性玉米膳食纤维的工艺研究

利用木聚糖酶对脂肪酶、淀粉酶、蛋白酶预处理后的玉米种皮进行酶解,制备水溶性膳食纤维,并对所得膳食纤维进行气相色谱分析。木聚糖酶酶解最佳工艺条件为:酶解时间40min、加酶量1．0ml/g、酶解温度45℃、pH4．8;酶法得到的可溶性膳食纤维的成分主要有木糖、阿拉伯糖、半乳糖和葡萄糖。     

双酶改性制备玉米皮水溶性膳食纤维的工艺研究

以玉米皮超声提取天然水溶性膳食纤维后的副产物——不溶性玉米皮渣为试材,应用木聚糖酶和纤维素酶组合酶解制备水溶性膳食纤维,采用单因素和正交试验组合研究确立一套由水不溶性膳食纤维改性制备水溶性膳食纤维制备工艺。结果表明,最佳工艺参数为纤维素酶添加量40mg/g 底物、木聚糖酶添加量40mg/g 底物、料液比1:14(g/ml)、酶解时间90min,水溶性膳食纤维得率为5.96%。     

微紫青霉酸性木聚糖酶xynA基因的克隆与序列分析

基于酸性木聚糖酶在饲料及酿酒行业良好的应用前景,通过基因组步移的方法克隆得到微紫青霉酸性木聚糖酶的全长基因xynA,然后采取重叠延伸PCR技术进行内含子的切除获得xynA的cDNA序列,并对其进行了生物信息学分析。序列分析结果显示xynA基因全长720 bp,内含子63 bp,cDNA全长657 bp。推测该木聚糖酶编码信号肽28个氨基酸,成熟肽190个氨基酸;预测该蛋白为分子质量20.61 kD、等电点7.0的亲水性蛋白,且分子内不含二硫键。与其他真菌来源的GH11族耐酸性木聚糖酶进行序列比对,结果显示该酶的相应位置具有特征天冬氨酸残基Asp,且具有糖苷水解酶11族的保守区域特征以及典型的"右手半握"状结构,重组木聚糖酶基因xynA能够在大肠杆菌中成功表达,比酶活力达220.5 U/mg。     

大肠杆菌高密度发酵产木聚糖酶发酵条件优化

该试验对木聚糖酶工业大生产条件下的接种量、pH值、培养温度、溶氧、诱导时间、比生长速率进行了单因素优化试验。在此单因素试验基础上,选取对酶活影响较大的比生长速率、溶氧和pH 3个因素进行了正交试验优化。结果表明,优化后的发酵条件为接种量10%,发酵过程中溶氧值30%,生长期控制pH值为7.0,生长期培养温度37 ℃,生长期比生长速率为0.12 h-1,诱导期pH值为6.8,诱导期培养温度为35 ℃,诱导期比生长速率为0.14 h-1,在对数生长中期（OD600 nm=30）时流加诱导培养基。优化后,放罐木聚糖酶活力最高可达149 000 IU/mL。该研究提升了大生产条件下的木聚糖酶活力,为木聚糖酶的工业化生产提供了指导。     

木聚糖酶产生菌Gh-5培养基配方及发酵条件的优化

研究培养基组分与发酵工艺条件对试验菌株Gh-5产木聚糖酶的发酵影响,并对木聚糖酶的酶学性质进行初步研究。结果表明,该菌最适发酵产酶培养基组分为甘露糖15 g/L,氯化铵10 g/L,ZnSO4 0.3 g/L,KH2PO4 0.5 g/L;最适发酵条件为温度37 ℃;pH值为8.0;接种量14 %;发酵培养生长周期36 h。木聚糖酶产生菌株Gh-5发酵优化后的酶活力为114.64 U/mL,较优化前38.02 U/mL提高了201.53%。木聚糖酶酶学性质研究结果表明,木聚糖酶酶活最适pH值为8.0;最适温度为65 ℃;Zn2+对木聚糖酶酶活有较好促进作用。     

Production,Characterization and Application of a Xylanase from Streptomyces sp. AOA40 in Fruit Juice and Bakery Industries

Streptomyces sp. AOA40, which produces halotolerant and thermotolerant xylanase, was isolated from Mersin soil. Various carbon sources were tested for xylanase production with selected fermentation medium. The best carbon source was selected as corn stover. The effect of corn stover concentration and particle size, composition of fermentation medium, fermentation condition such as initial pH and agitation rate on xylanase production was determined. After production, xylanase was partially purified with ion-exchange chromatography and gel filtration chromatography for characterization of xylanase and application in fruit juice and dough improvement. The optimum pH for the activity of xylanase occurred at pH 6.0 in phosphate buffer, while the optimum temperature was 60°C. The relative xylanase activity in the pH ranges of 4–9 remained between 59.93 and 54.43% of the activity at pH 6.0 (100.00%). The xylanase activity showed a half-life of 172 min at 70°C, which was reduced to 75 min at 80°C. The enzyme was highly inhibited by 10–100 mM of Hg⁺², EDTA, Mg⁺², SDS and 100 mM Cu⁺². Clarity of fruit juices increased after enzymatic treatment of apple (17.85%), grape (17.19%) and orange juice (18.36%) with partially purified xylanase and also reducing sugar concentrations of these fruit juices were improved by 17.21, 16.79 and 19.57%, respectively. Also, dough volume was raised 17.06% with using partially purified Streptomyces sp. AOA40 xylanase in bread making.     

The effect of fibrolytic enzymes sprayed onto forages and fed in a total mixed ratio to lactating dairy cows

We investigated the effect of spraying different combinations of fibrolytic enzymes onto forages on their nutritive value for lactating cows. Holstein cows were fed a TMR consisting of 30% corn silage, 15% alfalfa hay, and 55% concentrate (dry matter basis). During a 12-wk treatment period, the forages were treated with no enzymes (control), cellulase D and sultanas B, or cellulase D and xylanase C. Enzymes were diluted in water and sprayed onto the forages while mixing. Both combinations of enzymes supplied similar amounts of fibrolytic activity based on classical enzyme assays conducted at 50 degrees C. Cows fed forages treated with cellulase D and xylanase B tended to produce more 3.5% FCM (+2.5 kg/d) than did cows fed the untreated forages. Dry matter intake, milk production, milk fat, and milk protein were unaffected by treatment. In vitro production of gas from forages treated with enzymes was greater than from untreated forage, but 96-h volatile fatty acid production was not different among treatments. With an alternative enzyme assay based on the depolymerization of dyed substrate at 40 degrees C, activity of xylanase C was greatest at a pH of 6.5 but was substantially reduced as the pH of the assay was decreased. In contrast, xylanase B showed highest activity at pH 5 and enzyme activity was twice that of xylanase B at pH 5.5 and 6. Overall, the results of this study provide more evidence that fibrolytic enzymes can be used to improve milk production in lactating cows.     

Acetylated Xylan Degradation by Glycoside Hydrolase Family 10 and 11 Xylanases from the White-rot Fungus Phanerochaete chrysosporium

Endo-type xylanases are key enzymes in microbial xylanolytic systems, and xylanases belonging to glycoside hydrolase (GH) families 10 or 11 are the major enzymes degrading xylan in nature. These enzymes have typically been characterized using xylan prepared by alkaline extraction, which removes acetyl sidechains from the substrate, and thus the effect of acetyl groups on xylan degradation remains unclear. Here, we compare the ability of GH10 and 11 xylanases, PcXyn10A and PcXyn11B, from the white-rot basidiomycete Phanerochaete chrysosporium to degrade acetylated and deacetylated xylan from various plants. Product quantification revealed that PcXyn10A effectively degraded both acetylated xylan extracted from Arabidopsis thaliana and the deacetylated xylan obtained by alkaline treatment, generating xylooligosaccharides. In contrast, PcXyn11B showed limited activity towards acetyl xylan, but showed significantly increased activity after deacetylation of the xylan. Polysaccharide analysis using carbohydrate gel electrophoresis showed that PcXyn11B generated a broad range of products from native acetylated xylans extracted from birch wood and rice straw, including large residual xylooligosaccharides, while non-acetylated xylan from Japanese cedar was readily degraded into xylooligosaccharides. These results suggest that the degradability of native xylan by GH11 xylanases is highly dependent on the extent of acetyl group substitution. Analysis of 31 fungal genomes in the Carbohydrate-Active enZymes database indicated that the presence of GH11 xylanases is correlated to that of carbohydrate esterase (CE) family 1 acetyl xylan esterases (AXEs), while this is not the case for GH10 xylanases. These findings may imply co-evolution of GH11 xylanases and CE1 AXEs.     

G/11家族木聚糖酶特征序列与其最适温度定量关系的研究

本文研究了G/11家族木聚糖酶特征序列与其最适温度的定量关系,所得逐步回归模型达到极显著(p<0.0001),相关系数为0.965。该模型对文献[5]报道的23种木聚糖酶的预测值与其实测值吻合较好,平均绝对误差和平均绝对百分比误差分别为3.42%和1.84℃。对比文献[5]的两种模型,它优于基于单个氨基酸的模型(2.26℃和8.1%),但略逊于基于二肽的模型(1.22℃和2.27%)。同时,在这两个特征序列中,存在7个对木聚糖酶最适温度影响较大的位点。利用在大肠杆菌中表达的重组木聚糖酶的特征序列和最适温度验证该模型的可靠性,该模型的预测误差仅为0.2%(预测值和实测值分别为50℃和49.9℃)。此模型适用于G/11家族中的所有木聚糖酶且有很好的精度,所以它可为木聚糖酶定向改造其耐高温的活性提供更多的生物信息。