产κ-卡拉胶酶菌株的筛选及其发酵条件的优化

Screening and optimization of fermentation conditions by a κ-carrageenase producing bacterium

对κ-卡拉胶酶产生菌进行富集培养,以κ-卡拉胶为唯一碳源的平板初筛,从多种海藻上分离纯化获得32株具有卡拉胶酶活性的菌株,经摇瓶复筛,从亮管藻上分离的HC4菌株酶活力最高,为50.75U/mL。测定了HC4菌株的16S rRNA基因序列1436 bp,在核苷酸序列数据库(NCBI)中进行同源性检索,发现它与Tamlana agarivorans的相似性为98%。通过单因子筛选和正交试验,确定该菌株产酶的最佳培养基组成为:碳源κ-卡拉胶5 g/L;氮源蛋白胨3 g/L和NaNO31 g/L;无机盐NaCl 20 g/L、K2HPO4.3H2O 1 g/L、MgSO4.7H2O 0.5 g/L和CaCl20.1 g/L。最佳培养条件为:摇床转速150 r/min,250 mL三角瓶中发酵培养基的装液量50 mL,接种量4%,发酵培养基起始pH7.5,温度28℃,培养时间28 h。经培养基组成及培养条件优化后,HC4菌株的κ-卡拉胶酶酶活力提高到602.30 U/mL,是优化前的11.87倍。

The screening of a K - carrageenase producing bacterium and optimization of fermentation conditions to yield a higher production of extracellular enzyme were conducted by enrichment culture technique and first - screen- ing by K- carrageenan plate containing K- carrageenan as sole source of carbon and energy. A total of 32 isolates that actively degraded K - carrageenan were screened from various seaweeds. Strain HC4 isolated from Hyalosiphonia caespitosa showed the maximum activity by second - screening of shaking culture. Fragment of 1436 bp sequence of strains HC4 16S rRNA gene was amplified, which showed 98% similarity to Tamlana agarivorans when analyzed at National Center for Biotechnology Information （NCBI） database. The culture conditions for the strain HC4 were standardized for the maximal productivity of the extracellular K - carrageenase. The optimal medium components determined by single factor test and orthogonal test were found as the following： K -carrageenan 5 g/L, peptone 3 g/L, NANO3 1 g/L, NaCl 20 g/L, K2HPO4 -3H2O 1 g/L, MgSO4 .7H2O 0.5 g/L, and CaCl2 0.1 g/L. The optimal culture condition were observed as the following： shaking speed 150 r/min, 50 mL medium in 250 mL Erlenmeyer flask, inoculum volume 4%, initial medium pH 7.5, incubation temperature 28 ℃ and incubation time 28 h. In the optimal conditions, the K - carrageenase activity was found to increase by 10. 87 folds compared with the unoptimized conditions.