Li X L, Ljungdahl L G
Department of Biochemistry, University of Georgia, Athens 30602-7229.
Appl Environ Microbiol. 1994 Sep;60(9):3160-6. doi: 10.1128/aem.60.9.3160-3166.1994.
Aureobasidium pullulans Y-2311-1 growing on xylan secretes four major xylanases with different masses and isoelectric points. Two of these enzymes, named APX-I and APX-II, have been purified previously. Their N-terminal amino acid sequences are identical except that APX-I has Asp and APX-II has Asn at position 7. An 83-bp DNA region was amplified by PCR and used as a probe for the xylanase gene cloning. The longest cDNA (xynA) obtained by cDNA cloning and PCR amplification consisted of 895 bp. A. pullulans xynA had an open reading frame encoding a polypeptide of 221 amino acids with a calculated mass of 23,531 Da and contained a putative 34-amino-acid signal peptide in front of the amino terminus of the mature enzyme. Strong homology was found between the deduced amino acid sequence of XynA and some xylanases from bacterial and fungal sources. It is suggested that A. pullulans XynA belongs to the family G glycanases. Northern (RNA blot) analysis revealed that only one transcript of 900 bases was present in cultures grown in medium containing D-xylose or oat spelt xylan. Transcription was completely repressed in the presence of glucose in the medium. Southern blot analysis indicated that A. pullulans xynA was present as a single copy in the genome. Comparison between the genomic and cDNA sequences revealed that one intron of 59 bp was present in the coding region. The data presented suggest that the highly active xylanases, APX-I and APX-II, secreted by A. pullulans are encoded by the same gene.
在木聚糖上生长的出芽短梗霉Y-2311-1分泌四种分子量和等电点不同的主要木聚糖酶。其中两种酶，命名为APX-I和APX-II，此前已被纯化。它们的N端氨基酸序列相同，只是在第7位APX-I为天冬氨酸，APX-II为天冬酰胺。通过PCR扩增出一个83bp的DNA区域，并用作木聚糖酶基因克隆的探针。通过cDNA克隆和PCR扩增获得的最长cDNA（xynA）由895bp组成。出芽短梗霉xynA有一个开放阅读框，编码一个221个氨基酸的多肽，计算分子量为23,531Da，并且在成熟酶氨基端前含有一个推定的34个氨基酸的信号肽。在XynA的推导氨基酸序列与来自细菌和真菌来源的一些木聚糖酶之间发现了很强的同源性。表明出芽短梗霉XynA属于G家族聚糖酶。Northern（RNA印迹）分析表明，在含有D-木糖或燕麦麸木聚糖的培养基中生长的培养物中仅存在一种900个碱基的转录本。在培养基中存在葡萄糖的情况下转录被完全抑制。Southern印迹分析表明，出芽短梗霉xynA在基因组中以单拷贝形式存在。基因组和cDNA序列之间的比较表明，在编码区存在一个59bp的内含子。所呈现的数据表明，出芽短梗霉分泌的高活性木聚糖酶APX-I和APX-II由同一基因编码。
