Abstract：
Millions of tons of citrus peel waste are produced every year as a byproduct of the juice industry. Citrus peel is rich in pectin and xyloglucan, but while the pectin is extracted for use in the food industry, the xyloglucan is currently not valorized. To target hydrolytic degradation of citrus peel xyloglucan into oligosaccharides, we have used bioinformatics to identify three glycoside hydrolase 12 (GH12) endoxyloglucanases (EC 3.2.1.151) from the citrus fruit pathogens Penicillium italicum GL-Gan1 and Penicillium digitatum Pd1 and characterized them on xyloglucan obtained by alkaline extraction from citrus peel. The enzymes displayed pH-temperature optima of pH 4.6-5.3 and 35-37°C. PdGH12 from P. digitatum and PiGH12A from P. italicum share 84% sequence identity and displayed similar kinetics, although kcat was highest for PdGH12. In contrast, PiGH12B from P. italicum, which has the otherwise conserved Trp in subsite -4 replaced with a Tyr, displayed a 3 times higher KM and a 4 times lower kcat/KM than PiGH12A, but was the most thermostable enzyme of the three Penicillium-derived endoxyloglucanases. The benchmark enzyme AnGH12 from Aspergillus nidulans was more thermally stable and had a higher pH-temperature optimum than the enzymes from Penicillum spp. The difference in structure of the xyloglucan oligosaccharides extracted from citrus peel xyloglucan and tamarind xyloglucan by the new endoxyloglucanases was determined by LC-MS. The inclusion of citrus peel xyloglucan demonstrated that the endoxyloglucanases liberated fucosylated xyloglucan oligomers, implying that these enzymes have the potential to upgrade citrus peel residues to produce oligomers useful as intermediates or bioactive compounds.
摘要：
作为果汁行业的副产品，每年产生数百万吨的柑橘皮废物。柑橘皮富含果胶和木葡聚糖，但是当果胶被提取用于食品工业时，木葡聚糖目前没有增值。以柑橘皮木葡聚糖水解降解为寡糖为目标，我们已经使用生物信息学从柑橘类水果病原体中鉴定了三种糖苷水解酶12（GH12）内木葡聚糖酶（EC3.2.1.151）。意大利青霉GL-Gan1和指状青霉Pd1，并对它们进行了表征。从柑橘皮中通过碱提取获得的木葡聚糖。酶表现出pH4.6-5.3和35-37°C的最佳pH-温度。PdGH12与Pdigitum的PiGH12A具有84%的序列同一性，并表现出相似的动力学。尽管Kcat对PdGH12最高。相比之下,PiGH12B来自P.italicum,子站点-4中的其他保守Trp替换为Tyr，显示比PiGH12A高3倍的KM和低4倍的kcat/KM，但是是三种青霉衍生的内切木葡聚糖酶中最热稳定的酶。来自构巢曲霉的基准酶AnGH12比来自青霉的酶具有更高的热稳定性，并且具有更高的最适pH-温度。通过LC-MS确定了通过新的内切木葡聚糖酶从柑橘皮木葡聚糖和罗望子木葡聚糖中提取的木葡聚糖寡糖的结构差异。柑橘皮木葡聚糖的包含表明，内木葡聚糖酶释放岩藻糖基化木葡聚糖低聚物，暗示这些酶具有升级柑橘皮残留物以产生可用作中间体或生物活性化合物的低聚物的潜力。
