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各位大神：本人最近投了个杂志，评审说摘要要找英语好的润色一下，求大神帮忙。 β-D-1,4-内切木聚糖酶(EC.3.2.1.8)是木聚糖降解酶系中最重要的成员，广泛用于饲料、食品、造纸和生物能源等领域。本研究根据毕赤酵母（Pichia pastoris）对基因密码子偏好性对厌氧真菌Orpinomyces sp. PC-2的β-D-1,4-内切木聚糖酶xynA基因进行密码子优化。通过全基因合成技术合成优化后的β-D-1,4-内切木聚糖酶基因xynAm，并克隆到毕赤酵母表达载体pPIC9K的多克隆位点，构建重组质粒pPIC9K-xynAm，电击转化至毕赤酵母GS115菌株中，以29℃、0.5%甲醇诱导表达，获得重组β-D-1,4-内切木聚糖酶，并对该酶的酶学特性进行了研究。结果表明：在7 L发酵罐中诱导表达96 h后，β-D-1,4-内切木聚糖酶的活性为3515 IU/mL，比活性为2411 IU/mg。酶学性质分析表明：重组β-D-1,4-内切木聚糖酶的最适反应温度为55 ℃，最适反应pH为6.0，在温度为30~45 ℃、pH 4.0~10.6之间该酶具有较好的稳定性。底物特异性分析表明：重组β-D-1,4-内切木聚糖酶可水解低粘度阿拉伯木聚糖、高粘度阿拉伯木聚糖、燕麦木聚糖、桦木木聚糖和可溶性木聚糖，但不降解大麦β-葡聚糖和地衣多糖。10mmol/L的Co2+和K+对该酶的活性略有激活作用，Mn2+、Fe2+和SDS对该酶活性有一定的抑制作用。本研究为进一步工业化应用来源于Orpinomyces sp.PC-2的β-D-1,4-内切木聚糖酶奠定了基础。 Endo-1,4-β- xylanase （EC.3.2.1.8） is the most important member of xylanolytic enzymes. It is widely used in feed, food, papermaking and bioenergy and other fields. In the present research, according to the codon usage frequency of highly expressed genes in Pichia pastoris, a endo-1,4-β-xylanase gene (xynA) derived from anaerobic fungi Orpinomyces sp. PC-2 was optimized, and the modified gene (xynAm) was chemical synthesized and constructed a yeast expression vector pPIC9K-xynAm. Then, the xynAm gene was expressed in Pichia pastoris GS115, and enzymatic properties of the recombinant endo-1,4-β-xylanase were studied. The results showed, in 7L fermenter, the enzyme activity and specific activity of the recombinant endo-1,4-β-xylanase reached the maximum of 3515 IU/mL and 2411 IU/mg after 96 h of induction. Enzymatic properties analysis showed that, the optimum pH and reaction temperature of the purification xylanase was 55 ℃ and 6.0, and relatively more stable at 37~50 ℃ and pH 4.0~10.6. Substrate specificity analysis showed that, the xylanase could hydrolyze low and high viscosity arabinoxylans, oat spelt xylan, birch xylan and 4-O-Methyl-D-glucurono-D-xylan but not barley β-glucan, lichenan and sodium carboxymethyl cellulose. The enzyme activity was slightly activated by 10 mmol/L Co2+ and K+, and inhibited by Mn2+, Fe2+ and SDS. This study lays a foundation for further industrial application of the endo-1,4-β-xylanase from the Orpinomyces sp.PC-2. |
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