Screening Of High Pectinase-producing Strain And Enzymatic Purification

Pectinase is widely used in various industries, which can be used for degradation of pectic substance in plant tissues. Aging flue-cured tobacco generally contains 10~15% pectin, high pectin content will generate toxic and harmful substances during tobacco burning, and has negative effect on tobacco smoking quality. Pectin in tobacco can be degraded by pectinase, which are produced by various microorganisms. At present, vast majority of the research working on fungus with higher pectinase activity, only few studies on bacteria. Nevertheless, bacteria with high pectinase activity have gradually attracted much attention due to its unique fast growth rate, enzymatic diversity, short enzyme production period and diverse sources. In this study, a strain with high pectinase activity was isolated from aging flue-cured tobacco, then the fermentation conditions were optimized, finally the pectinase was purified and its enzymatic characteristic was partial investigated. The main results are as follows:1. Screening and identification of pectinase-producing strain: 32 strains with hydrolysis circle were selected using Congo red stain. The bacteria(named PB-1) with highest pectinase activity was finally screened by determining pectinase activity, and preliminarily identified as Bacillus subtilis based on the morphological characteristics and 16 S rDNA(99% homology).2. Optimization of PB-1 fermentation conditions: The culture media for production of pectinase and cultural conditions of Bacillus subtilis PB-1 was optimized by single factor experiments, Plackett-Burman design and central composite design. The experimental results showed that the three factors(peptone, pH and pectin) significantly influence the pectinase activity. The optimized culture medium consisted of soluble starch 15 g/L, pectin 12 g/L, peptone 20 g/L, K2HPO4 1 g/L, MnCl2 0.1 g/L, FeSO4 0.01 g/L, MgSO4 0.5 g/L, with initial pH of 9.0 and inoculation size of 8% of the medium, at 37 ℃ and 150 r/min for 28 hours. The pectinase enzyme activity reached up to 20.2 U/mL under the optimal fermentation conditions, which was 9.7-fold of the initial enzyme activity.3. Purification and enzymatic characteristics of the pectinase: The crude pectinase was obtained by ammonium sulfate fractionation precipitation and dialysis from the fermentation broth. After lyophilization, the crude enzyme was subjected to QXL ion exchange column and Sephadex G-75 gel filtration column. A single protein band of 44.3 kDa was obtained by SDS-PAGE, which indicated that the pectinase reached electrophoresis purity. The peptide mass fingerprint determined by MALDI-TOF MS/MS showed that the enzyme was pectate lyase. The enzymatic properties were also studied, which showed that the optimum temperature and reaction pH was 50 ℃ and 9.0, respectively. Temperature stability range below 50 ℃, and pH stability range from 5.0~11.0 indicated that the pectinase has a wide pH tolerance range.