Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Thermomonospora fusca xylanase A (TfxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. The activity of recombinant T. fusca xylanase A (reTfxA) in culture supernatant was 117.3 ± 2.4 U/mg, which is 3 times higher than that of the native TfxA. The optimal temperature and pH for reTfxA were 60 °C and 6.0, respectively. When treated at 70 °C and pH 6.0 for 2 min, the residual activity of the reTfxA was 70%. The reTfxA was very stable over a wide pH range (5.0–9.0). After incubation over pH 5.0–9.0 at 25 °C for 1 h, all the residual activity of reTfxA was over 80%. The K_m and k_cat values for reTfxA were 2.45 mg/ml and 139 s⁻¹, respectively. HPLC analysis revealed that xylobiose (X2) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reTfxA. Hydrolysis results of xylooligosaccharides showed that reTfxA was an endo-acting xylanase and xylobiose, xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolysed. This is the first report on the expression of reTfxA in yeast and on the determining and quantifying of the hydrolysis products released from xylans and xylooligosaccharides by reTfxA.     

甘蔗叶酶法制取低聚木糖的工艺研究

文中研究了木聚糖酶酶解甘蔗叶高温蒸煮液的工艺条件.结果表明,最优化条件为液固比13:1,酶用量40IU/g干基,酶解温度60℃,pH值6.0,反应时间2.5h.     

烟草秸秆低聚木糖的制备和纯化

为优化烟草秸秆低聚木糖制备参数,采用碱解方法提取木聚糖、酶解法制备低聚木糖以及单因素实验法考察了常见因素对工艺的影响。结果表明,木聚糖提取条件为：2.000 g秸秆粉末（≤100目）浸没于20.00 mL浓度为24% NaOH（m/V）和1% NaBH₄（m/V）碱液中,70 ℃条件下浸提4 h,滤液加3倍乙醇体积用量进行醇沉以及0.2倍乙酸体积用量进行中和。制备低聚木糖的条件为：溶液pH为5.50,温度40 ℃,时间6 h,木聚糖溶液（20 mg/mL）10 mL,木聚糖酶液（0.6%,m/V,4.1 U/mL）20 mL。低聚木糖分离提纯条件为：阳离子树脂柱分离纯化,填充高度18.0 cm、直径为4.5 cm;纯化液用高效液相色谱进行定性定量分析。通过上述方法得到的低聚木糖产品纯度较高,对工业制备低聚木糖工艺优化有一定的参考价值。     

Properties of an Alkaline‐Tolerant,Thermostable Xylanase from Streptomyces chartreusis L1105, Suitable for Xylooligosaccharide Production

Abstract: An extracellular xylanase was purified to homogeneity from a culture of Streptomyces chartreusis L1105 by a 2‐step method of ammonium sulfate precipitation and carboxymethyl sepharose fast‐flow chromatography (CMSFF). The xylanase was purified by 6.86‐fold, with a recovery yield of 31.96%. The purified xylanase appeared as a single protein band on SDS‐PAGE with a molecular mass of about 34.2 kDa. The optimum temperature and pH of the purified xylanase activity were 70 °C and 7.2 respectively. The xylanase was more stable under alkaline conditions and retained more than 80% activity after 30 min incubation at pH 6 to 10. It also showed specific activity towards different xylans. Hydrolysis of oat‐spelt and corn‐cob xylans by the xylanase yielded xylobiose and xylotriose as principle products without the formation of xylose. These properties indicate that the purified xylanase may potentially be useful in biotechnological applications, such as xylooligosaccharide preparation. This is the first report about the purification and characterization of a xylanase from S. chartreusis.     

酶法降解蔗渣制备低聚木糖酶解特性的研究

响应面优化酶解条件:加酶量0.82%,粗木聚糖浓度8.4%,温度46℃,水解时间4.5h;低聚木糖得率43.55%;薄层分析初步确定有木二糖、木三塘、木糖;红外光谱分析产物具有低聚木糖结构特征;液相色谱确定产物以木二糖为主,含量达85%。     

酶解花生壳制备低聚木糖的研究

以花生壳为原料,在固液比1∶20,H_2SO_4浓度为1.0%,120℃条件下处理30min,木聚糖提取率为40.1%。利用木聚糖酶降解木聚糖制备低聚木糖,确立了酶解工艺条件:50℃,pH4.8,加酶量2%(相对固体花生壳原料).搅拌速度80r/min,反应时间24h。在上述反应条件下,低聚木糖得率为81.2%。     

高温蒸煮法与微波法处理玉米芯制备低聚木糖比较

通过对高温蒸煮法和微波法处理玉米芯制备低聚木糖的比较，碍出：高温蒸煮法产物复杂，玉米芯经稀酸浸泡后再高温蒸煮，木聚糖提取率和水解液中还原糖含量均较高，但产物主要是单糖；微波法产物较单一，玉米芯经稀碱液浸泡后再微波处理，木聚糖提取率和水解液中还原糖含量均高，且其主要成分是木二糖，糠醛含量少。比较2种预处理方法，微波处理玉米芯制备低聚木糖更理想。     

酶水解爆破秸秆制备低聚木糖

研究了木聚糖酶水解爆破秸秆制备低聚木糖的工艺,得到如下结论:当爆破秸秆与水质量比为1∶7.5、pH6.0、黑曲霉木聚糖酶添加量为198U/g(干基)、53℃、酶解12h时,可获得较好的酶解效果,酶解液总糖含量达到49.80mg/mL,还原糖含量达到17.03mg/mL、木聚糖水解率达到63.77%(对原料木聚糖)、木聚糖平均聚合度降至3.10;酶解产物中低聚糖主要为木二糖和木三糖,低聚木糖含量达到50.80%(对固形物)。     

功能性甜味剂低聚木糖的研发

选用富含木聚糖的甘蔗渣为原料,利用木聚糖酶对其进行选择性降解制备功能性食品添加剂低聚木糖,并对其性质进行研究。结果表明:木聚糖酶对原料甘蔗渣进行酶解,底物专一性强,产品纯度高,外观质量为乳白色或淡黄色粉末,易溶于水,甜味纯正,风味宜人。产品具有良好的pH稳定性、热稳定性和优良的保存性能,可广泛应用于保健品、食品、饮品领域,特别适用于各种“文明病”患者、婴幼儿、老年人和亚健康人群,社会和经济效益明显。     

Characterization of Enzyme Released Antioxidant Phenolic Acids and Xylooligosaccharides from Different Graminaceae or Poaceae Members

Xylooligosacchrides (XOS) and phenolic acids were simultaneously produced from hemicelluloses using crude enzyme mixture synthesized by a novel Bacillus subtilis KCX006. The strain synthesized XOS-forming endo-xylanase and de-branching α-L-arabinofuranosidase and esterase but not β-xylosidase. This enzyme mixture can improve the yield of XOS and phenolic acids from xylan due to the absence of hydrolysis of XOS by β-xylosidase and release of phenolic acids by esterase. Hence, the enzyme mixture was tested for simultaneous production of XOS and phenolic acids from xylan (28–35%)-rich Graminaceae or Poaceae plant biomass, such as wheat bran, sugarcane bagasse, bamboo, and rise husk. Hemicellulose fractions of the biomasses were extracted by alkaline treatment and hydrolyzed using crude xylanase mixture. The profiles of XOS and phenolic acids formed were analyzed by HPLC. The lyophilized hydrolytic products were further analysed by ¹H NMR to identify the substitutions in XOS. The observed profile of XOS and phenolic acids varied with the biomass sources. Maximum amounts of XOS (665.2 mg/g dwt) and phenolic acids (89.69 μg/g dwt) were produced from hemicellulose A fractions of sugarcane bagasse and bamboo bagasse, respectively. HPLC and ¹H NMR analysis of XOS revealed the formation of free- and arabino-XOS. Phenolic acids consisted of hydroxycinnamic and hydroxybenzoic acids and the antioxidant activity correlated well with hydroxycinnamic acids content. The crude enzyme of B. subtilis is useful to produce mixture of XOS and phenolic acids from biomass.