基于加权KNN的高通量红外光谱法溯源分析青霉素G酰基转移酶菌属分类  

Traceability Analysis of Penicillin G Acylase Genus Classification Using High-Throughput Infrared Spectroscopy Based on the Weighted KNN

作　　者：王琰 张培培 赵瑜 WANG Yan;ZHANG Pei-pei;ZHAO Yu(National Institutes for Food and Drug Control,Chemical Drug Identification Institute,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院化学药品检定所,北京102629

出　　处：《光谱学与光谱分析》2025年第4期1008-1014,共7页Spectroscopy and Spectral Analysis

基　　金：国家药品标准制修订研究课题(2021Y4,2023Y12,2023Y15)资助。

摘　　要：β-内酰胺类抗生素是临床中一类重要的抗感染治疗药物,青霉素G酰基转移酶(PGA)是新型酶法工艺生产β-内酰胺类抗生素的关键技术,不同菌属来源的PGA具有不同的蛋白序列、热稳定性及立体选择性,这些性质会产生不同催化反应活性,对抗生素合成和生产至关重要。红外光谱可对蛋白类物质进行结构表征,基于蛋白质组学的质谱法可从肽段水平对不同蛋白类物质进行鉴定,由于其复杂精密性使得操作更为简便的红外光谱法为PGA蛋白快速鉴定提供了一种有力分析工具。本研究采用超滤法和干燥制膜方法对PGA样本进行前处理,达到纯化PGA原液、去除基质干扰目的,同时又能克服溶液浓度低、信号弱的难题增强红外信号响应。建立高通量红外光谱法对11批不同菌属来源的PGA进行分析,结果显示不同菌属PGA红外光谱特征区(1700~1500 cm^(-1))存在相同蛋白经典酰胺红外吸收峰,同时在指纹区1200~750 cm^(-1)范围内呈现多个差异化红外吸收峰。选择指纹区差异化吸收峰谱带(830~795、1027~1020、1085~1080 cm^(-1))、采用加权K最邻近(KNN)分类算法优化建立PGA溯源模型,结合蛋白质组学质谱法结果对不同PGA进行菌属来源分类分析。结果显示11批PGA分为两类,第I类结果包括PGA 1、PGA 3、PGA 7、PGA 8,为大肠杆菌来源,第Ⅱ类结果包括PGA 2、PGA 4~6和PGA 9~11,为无色杆菌来源。PGA溯源模型分类结果与蛋白质组学分析结果一致,验证了溯源分类模型的适用性和可行性;采用该溯源分类模型对3批未知菌属PGA蛋白进行考察,验证模型的准确性;最后通过对不同天时间的考察,进一步验证模型的耐用性。研究结果表明,加权KNN高通量红外光谱法可实现对不同菌属来源的PGA快速溯源分类,方法简便、准确耐用,为β-内酰胺类半合成抗生素酶法工艺中催化酶的结构表征和快速分析提供了一种新的检测工具。β-lactam antibiotics(BAs)are an important class of anti-infective drugs in clinical practice.Penicillin G acylase(PGA)is a key technology used in the new enzymatic process to produce of BAs.PGAs derived from different bacterial origins have different protein sequence structures,thermal stability,and stereo-selectivity,which cause different catalytic activity and are crucial for antibiotic synthesis and production.Infrared spectroscopy(IR)can be used to characterize the structure of high molecular weight proteins.Proteomics-based mass spectrometry can identify different protein substances at the peptide level,but its complexity makes it harder to operate.The simple IR method provides a powerful analytical tool for rapidly characterizing PGAs.This article explored the selection of ultrafiltration and drying membrane preparation methods for the pre-treatment of PGA samples.This way could purify PGA raw solutions and remove matrix interference,while it could also overcome the problem of low PGA solution concentration to enhance IR signal response.Besides,a high-through put IR method was optimized and established to analyze 11 batches of PGAs from different sources.All IR spectra of PGAs showed classical IR absorption peaks of amide groups at the characteristic region(1700~1500 cm^(-1)).There were still differential IR absorption peaks within the 1200~750 cm^(-1) fingerprint region.A traceability model was established by selecting differentiated absorption peak spectral bands at fingerprint regions(830~795,1027~1020,1085~1080 cm^(-1)).Based on the analysis of proteomics mass spectrometry,a weighted k-nearest neighbor(KNN)algorithm was employed to analyze different PGAs.It showed that 11 batches of PGAs were divided into two classes:those including PGA 1,PGA 3,PGA 7,and PGA 8 belonged to classⅠand were identified as the proteins fermented from E.coli,while the rest of those—PGA 2,PGA 4~6,and PGA 9~11 belonged to classⅡand were produced from Achromobacter sp.CCM 4824.This result verified the applicability and fea

关 键 词：红外光谱 青霉素G酰基转移酶 菌属分类 

分 类 号：O657.33[理学—分析化学]