两种汉逊酵母细胞高压匀浆破碎方式的比较

Comparison of two high⁃pressure homogenization methods for disrupting Hansenula polymorpha cells

目的比较分步和循环2种高压匀浆破碎方式对汉逊酵母细胞的破碎效果,以期为今后汉逊酵母细胞工业化规模生产破碎方式的选择提供实验依据。方法采用分步和循环2种高压匀浆破碎方式对3批汉逊酵母细胞浓缩发酵液进行破碎,取破碎前后样品,置光学显微镜下观察细胞形态,检测宿主蛋白含量、细胞破碎率、蛋白释出度、释出蛋白粒径分布等指标,并进行比较。结果分步和循环破碎2种高压匀浆破碎方式破碎3批汉逊酵母细胞浓缩发酵液后,完整细胞数量减少,细胞碎片增多,但细胞形态未见明显差异;宿主蛋白含量(平均灰度值分别为115.259和109.125)、细胞破碎率(均值分别为60.29%和58.96%)、蛋白释出度(均值分别为39.55%和37.28%)、释出蛋白粒径分布(分步方式破碎后释出蛋白粒径平均分布在90.41和525.07 nm处,分别占样品蛋白含量的31.37%和68.63%;循环方式破碎后释出蛋白粒径平均分布在109.11和547.37 nm处,分别占样品蛋白含量的29.93%和70.07%)的差异均无统计学意义(F分别为1.054、1.159、4.245、8.875,t分别为1.0360、0.5041、0.6050、0.1397,P均<0.05)。结论循环和分步2种高压匀浆破碎方式对汉逊酵母细胞的破碎效果相当,但循环破碎方式更简便,且可避免污染风险,更适用于工业化规模生产汉逊酵母细胞的破碎。

Objective To compare the disrupting effects of Hansenula polymorpha cells by two kinds of high-pressure homogenization methods,step-by-step disrupting and cyclic disrupting,so as to provide experimental basis for the selection of disrupting methods in the industrial large-scale production of Hansenula polymorpha cells in the future.Methods Three batches of concentrated fermentation broth of Hansenula polymorpha cells were disrupted by two high-pressure homogenization methods:step-by-step disrupting and cyclic disrupting.The samples before and after disrupting were taken,and the cell morphology was observed under light microscope.The host protein content,cell disruption rate,protein release rate and particle size distribution of released protein were detected and compared.Results After disrupting three batches of concentrated fermentation broth of Hansenula polymorpha cells by step-by-step disrupting and cyclic disrupting,the number of intact cells decreased and cell fragments increased,while there was no obvious difference in cell morphology.In addition,there was no significant difference in host protein content(average gray value of 115.259 and 109.125 respectively),cell disruption rate(average value of 60.29%and 58.96%respectively),protein release rate(average value of 39.55%and 37.28%respectively),and released protein particle size distribution(average size of 90.41 and 525.07 nm after step-by-step disrupting,accounting for 31.37%and 68.63%of sample protein content;average size of 109.11 and 547.37 nm after cyclic disrupting,accounting for 29.93%and 70.07%of sample protein content,respectively.F=1.054,1.159,4.245 and 8.875,t=1.0360,0.5041,0.6050 and 0.1397,respectively,each P<0.05).Conclusion Cyclic and step-by-step high-pressure homogenization disrupting methods have the equivalent disrupting effect on Hansenula polymorpha cells,while cyclic disrupting is simpler and can avoid pollution risks,which is more suitable for the disrupting of Hansenula polymorpha cells for industrial large-scale production.