Berrin J G, Williamson G, Puigserver A, Chaix J C, McLauchlan W R, Juge N
Laboratoire de Biochimie et Biologie de la Nutrition, Faculté des Sciences, St Jérôme, Avenue Escadrille Normandie-Niemen, Marseilles Cedex 20, F-13397, France.
Protein Expr Purif. 2000 Jun;19(1):179-87. doi: 10.1006/prep.2000.1229.
Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris. The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P. pastoris AOX1 promoter. Secretion yields up to 60 mg/liter were obtained in synthetic medium. The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A. niger with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the S. cerevisiae signal peptide was correctly processed in P. pastoris. The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5. Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P. pastoris and the absence of glycosylation. The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A. niger. Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C. The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes. P. pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form. A. niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P. pastoris.
在毕赤酵母中实现了重组黑曲霉11家族1,4-β-木聚糖酶的高效生产。将与酿酒酵母转化酶信号肽融合的编码XylA的cDNA置于毕赤酵母AOX1启动子的控制下。在合成培养基中获得了高达60毫克/升的分泌产量。使用一步纯化方案将重组XylA纯化至同质，发现其在大小、pI和免疫反应性方面与在黑曲霉中过表达的酶相同。重组蛋白的N端序列分析表明，酿酒酵母信号肽在毕赤酵母中被正确加工。纯化后的蛋白分子量为19,893道尔顿，与计算质量高度一致，在等电聚焦上呈现为一条单一的条带，pI值约为3.5。电喷雾电离质谱证实了毕赤酵母产生的一种主要同工型的存在以及糖基化的缺失。以桦木木聚糖为底物，对重组酶的比活性、pH谱、动力学参数和热稳定性进行了进一步表征，并与从黑曲霉中纯化的木聚糖酶进行了比较。两种酶的最适pH均为3.5，在50℃时活性最高。酶活性遵循正常的米氏动力学，两种酶的K(m)和V(max)值相似。毕赤酵母能高产重组木聚糖酶，且易于以单一形式获得。黑曲霉木聚糖酶是第一种由毕赤酵母高效分泌并正确加工的微生物木聚糖酶。
