L-苹果酸产生菌筛选及高产突变株诱变选育

THE SCREENING OF L-MALIC ACID PRODUCING STRAINS AND THE SELECTION OF L-MALIC ACID HIGH-YIELD MUTANTS

通过广泛收集和分离,获得根霉属(Rhizopus)、曲霉属(Aspergillus)及裂褶菌属(Schizophyllum)等属菌株897株。产酸指示平板上的变色圈测定结果表明,它们中间628株为产酸菌。通过纸层析对产酸菌发酵液酸谱的分析,获得129株L-苹果酸产生菌,经进一步测定发酵液中L-苹果酸的含量,筛选出以葡萄糖为原料,摇瓶发酵140小时,L-苹果酸产率48.37g/L,对糖转化率48.37×10^(-2)的菌株LM02。经初步鉴定,这一菌株为曲霉(Asper-gillus sp.)以LM02作为出发株,采用亚硝基胍、自然污染细菌、甲基磺酸乙酯及紫外线进行诱变处理,选育出葡萄糖为原料,L-苹果酸产率较高的突变株N1-14、N1-14′、NE1412、NU1416及NU1419。其中N1-14′的L-苹果酸产量最高,比出发株提高46.2×10^(-2)。N1-14′的菌丝生长速度快,产孢能力强,摇瓶发酵葡萄糖140小时,平均L-苹果酸产率为72.53g/L,对糖转化率53.74×10^(-2)。全发酵液经薄层层析测定,不含黄曲霉毒素。发酵产物分离提纯后,得到白色粉末状结晶,经纸层析、质谱及红外光谱测定,证明为L-苹果酸。

Through collection and isolation,897 strains belonging to the genera of Rhizopus,Aspergillus and Schizophyllum etc.were obtained.The acid producing indicative medium indicated that there were 628 acid producing strains among 897 ones,and 129 L-malic acid producing strains were selected from 628 acid producing strains by the analysis of paper chromatography.After the quantitative determination of L-malic acid contents in the fermentation liquid,the strain LM02 was found to be the best one for production of L-malic acid from glucose.The yield was 48.37 g/L and the conversion rate of glucose to L-malic acid was 48.37 ×10^(-2) after flask fermentation for 140 hours.According to the morphological cha- racters,LM02 was tentatively referred to Atspergillus sp. The strain Aspergillus sp.LM02 was chosen as the start strain for mutagenic treatments with nitrosoguanidine,natural contamination bacteria,methyl methane sulfonate and ultraviolet irradiation.After the mutagenic treatments mutants were selected in the medium with glucose as sole carbon sources. The mutant N1-14' showed good growth character.The average L-malic acid yield and conversion rate of glucose to L-malic acid were 72.53 g/L and 53.74× 10^(-2) respectively with a fermentation time of 140hr.Aflatoxin was not detected from culture broth by thin layer chromatography.After isolation and purification white crystal was identified to be L-malic acid by the analysis of paper chromato- graphy,mass spectrum and infrared absorption spectrum.