黄曲霉果胶酶的酶学特性及其在白玉兰落叶水解中的初步应用

Enzymatic characteristics and primary application in hydrolysis of baiyulan leaving leaves of the pectinase produced by an Aspergillus flavus strain

关键词:  果胶酶  酶学性质  黄曲霉  菌株分离和鉴定  落叶水解

Key words:  pectinase  enzyme characteristics  Aspergillus flavus  strain isolation and identification  leaving leaf hydrolysis

摘要:

农残物还田可以改善土壤性质，然而微生物的活性太低拖慢了腐熟进程，为了加快进程，本论文探究了外来果胶酶对落叶的水解作用。本研究采用菌株分离筛选的方法获取土壤中产果胶酶的丝状真菌、用形态学和分子生物学的方法鉴定菌株的种属、用同类文献报道中所用方法测定各项酶学性质、用模拟自然界中的水解环境直接将果胶酶应用于落叶水解，初步衡量了一株菌株所产果胶酶在白玉兰落叶的水解中的作用。从土壤中分离得到一株产果胶酶的菌株TPF2-1，其在液态培养基中培养5 d后，培养液中的果胶酶活力为0.63±0.02 IU/mL。通过观察其形态学特征和分析基因组内转录间隔区序列将其鉴定为黄曲霉。黄曲霉TPF2-1所产的果胶酶在以聚半乳糖醛酸为底物时，最适作用pH值为4.5，最适作用温度为50℃。该果胶酶在35℃保温1 h后酶活力无损失，在pH4.0-7.0下保温（25℃）24 h后仍存活大于90%酶活力。该果胶酶的酶活力不受Na2EDTA影响，说明其发挥酶活力时不需金属离子。在受试金属离子中，1、2和5 mM的Co2+、Cu2+、K+、Li+、Mg2+、Na+、Zn2+均对该果胶酶的酶活力不表现出明显抑制作用，Ca2+、Fe2+和Fe3+仅在5 mM时表现出抑制作用。在进行树叶水解试验时，加入该果胶酶可以明显促进细胞内还原性物质的释放，可使玉兰花落叶水解体系中的还原性物质的量提升至水解前的3.3倍。本论文是首次报道黄曲霉粗果胶酶的多项酶学性质，亦是首次报道用果胶酶对白玉兰花落叶进行酶解，结论为黄曲霉果胶酶在树叶还田方面有潜在的应用价值。

Abstract:

Agricultural residue as fertilizer can enhance the characteristics of soil, but the low fermentation rate of micro-organism draw out the process. Thus, the effect of extra added pectinase on the hydrolysis of leaving leaf was studied. This study used the method of strain screening to obtain pectinase producing strain from soil sample, used the method of analysis of morphological character and molecular data to identify the genus and species of the strain, used the method reported by similar references to determine enzymatic characteristics of the produced crude pectinase, and used the method of directly hydrolysis of leaving leaf, the effect of pectinase produced by a strain in the hydrolysis of baiyulan leaving leaf was evaluated. In this study, a fungal strain TPF2-1 producing pectinase activity of 0.63±0.02 IU/mL in the culture broth after 5-days liquid cultivation was isolated from soil sample. And it was identified as Aspergillus flavus by observation of morphology character and analysis of the genomic internal transcribed spacer sequence. The pectinase produced by the strain showed an optimal pH of 4.5 and optimal temperature of 50°C when using polygalacturonic acid as substrate. It was stable at 35°C after one-hour-incubations, and it remained more than 90% residual activity after incubation at pH 4.0-7.0 for 24 hours (at 25oC). The enzyme activity of A. flavus pectinase did not affected by Na2EDTA, indicating that the enzyme activity did not require metal ions. Among the tested metal ions, Co2+, Cu2+, K+, Li+, Mg2+, Na+ and Zn2+ at 1, 2 and 5 mM did not inhibit the enzyme activity, and only Ca2+, Fe2+ and Fe3+ inhibited the enzyme activity at 5 mM. In the lab-scale trail experiment of leaves hydrolysis, the addition of the pectinase obviously increased the releasing of inter-cellular reducing material of baiyulan leaves. Especially, the concentration of reducing material in baiyulan leaving leaf hydrolysis system was increased to about 3.3 folds. Our study is the first report on the enzyme characteristics of A. flavus crude pectinase, and is the first report on the hydrolysis of baiyulan tree leaving leaf, and the result obtained indicated that the TPF2-1 pectinase has the potential application in the fields of leaving leaf for agricultural fertilization.