Shahbaz Umar, Yu Xiaobin
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
J Genet Eng Biotechnol. 2020 Aug 31;18(1):45. doi: 10.1186/s43141-020-00059-1.
Chitin is an important biopolymer next to cellulose, extracted in the present study. The exoskeleton of marine bycatch brachyuran crabs, namely Calappa lophos, Dromia dehaani, Dorippe facchino and also from stomatopod Squilla spp. were used to extract chitin through fermentation methods by employing two bacterial strains such as Pseudomonas aeruginosa, Serratia marcescens. The yield of chitin was 44.24%, 37.45%, 11.56% and 27.24% in C. lophos, D. dehaani, D. facchino and Squilla spp. respectively. FT-IR spectra of the produced chitin exhibit peaks which is more or less coherent to that of standard chitin which is further analysed by Scanning Electron Microscope. The quality of produced chitin was assessed through moisture, protein, ash and lipid content analysis ensured that chitin obtained from trash crustaceans are on par with that of standard chitin.
A total of 10 samples were collected from different areas of Jiangsu China for screening of chitinase-producing bacteria. Based on the clearance zone, two of the best samples were chosen for further study. 16S rRNA sequence analysis showed that this strain belongs to genus Myxococcus and species Myxococcus fulvus. Phylogenetic analysis was performed and it shows strain UM01 is a novel bacterial strain. UM01 isolate shows maximum chitinase production at 35 °C and 8 pH. Among all, these colloidal chitins were found to be the best for chitinase production. Three chitinase-producing genes were identified and sequenced by using degenerative plasmid. UMCda gene (chitin disaccharide deacetylase) was cloned into E. coli DH5a by using PET-28a vector, and antagonistic activity was examined against T. reesei.
To our knowledge, this is the earliest study report to gene cloning and identification of the chitinase gene in Myxococcus fulvus. Chitinase plays a key role in decomposition and utilization of chitin as a raw material. This research indicates that Myxococcus fulvus UM01 strain is a novel myxobacteria strain and can produce large amounts of chitinase within a short time. The UMCda gene cloned into E. coli DH5a showed a promising effect as antifungal activity. In overall findings, the specific strain UM01 has endowed properties of bioconversation of waste chitin and other biological applications.
几丁质是仅次于纤维素的一种重要生物聚合物，在本研究中被提取出来。利用两种细菌菌株，即铜绿假单胞菌和粘质沙雷氏菌，通过发酵方法从海洋兼捕短尾蟹（如丽文寄居蟹、德氏互敬蟹、中华虎头蟹）以及口足类虾蛄属的外骨骼中提取几丁质。丽文寄居蟹、德氏互敬蟹、中华虎头蟹和虾蛄属的几丁质产量分别为44.24%、37.45%、11.56%和27.24%。所制备几丁质的傅里叶变换红外光谱显示出的峰与标准几丁质的峰大致一致，并用扫描电子显微镜进一步分析。通过水分、蛋白质、灰分和脂质含量分析对所制备几丁质的质量进行评估，确保从废弃甲壳类动物中获得的几丁质与标准几丁质相当。
从中国江苏不同地区共采集了10个样本用于筛选产几丁质酶的细菌。根据透明圈，选择了两个最佳样本进行进一步研究。16S rRNA序列分析表明，该菌株属于粘球菌属和富黄粘球菌种。进行了系统发育分析，结果表明UM01菌株是一种新型细菌菌株。UM01分离株在35℃和pH值为8时几丁质酶产量最高。其中，发现这些胶体几丁质最适合用于几丁质酶的生产。利用简并质粒鉴定并测序了三个产几丁质酶基因。通过PET-28a载体将UMCda基因（几丁质二糖脱乙酰酶）克隆到大肠杆菌DH5α中，并检测其对里氏木霉的拮抗活性。
据我们所知，这是最早关于富黄粘球菌几丁质酶基因克隆与鉴定的研究报告。几丁质酶在几丁质作为原材料的分解和利用中起关键作用。本研究表明，富黄粘球菌UM01菌株是一种新型粘细菌菌株，能在短时间内产生大量几丁质酶。克隆到大肠杆菌DH5α中的UMCda基因具有良好的抗真菌活性。总体而言，特定菌株UM01具有将废弃几丁质进行生物转化及其他生物学应用的特性。
