三株产几丁质酶菌株的筛选、产酶条件优化及水解虾壳的研究

目的:筛选鉴定产几丁质酶的菌株并优化其发酵条件，最终应用于虾壳降解研究。方法:以盐城市滩涂海泥为样品，利用平板筛选水解几丁质的菌株，运用生物信息学方法鉴定菌株，通过单因素优化其发酵条件，并将筛选得到的菌株和优化后的发酵条件用于虾壳发酵。结果:鉴定得到三株显著降解胶体几丁质的菌，分别是发光杆菌（Photobacterium sp. LYM-1）、需钠弧菌（Vibrio sp. WM-1）和希瓦氏菌（Shewanella sp. ZXY-1）；优化发酵条件:发光杆菌的碳源为几丁质10 g/L，氮源为NH₄Cl 2.0 g/L，接种量为3%，发酵液pH为6.5，温度为32 ℃，发酵1 d酶活最高为15.37±0.55 U/mL，是优化前的4.37倍；需钠弧菌的碳源为几丁质10 g/L，氮源为NH₄Cl 2.0 g/L，接种量为3%，发酵液pH为7.5，温度为22 ℃，发酵2 d酶活最高为40.82±6.03 U/mL，是优化前的1.60倍；希瓦氏菌的碳源为几丁质10 g/L，氮源为(NH₄)₂SO₄ 2.0 g/L，接种量为3%，发酵液pH为6.5，温度为22 ℃，发酵1 d酶活最高为25.64±3.29 U/mL，是优化前的2.47倍；三株菌均能利用虾壳产几丁质酶，但利用效率均低于几丁质，酶活力分别为10.25±0.95、32.16±2.25和21.81±4.27 U/mL。结论:本研究从盐碱地筛选得到三株产几丁质酶的菌株，优化后酶活力均得到提高，且均能利用虾壳产几丁质酶，为发酵虾壳制备几丁质酶提供新的菌株来源。

Screening of Three Chitinase-producing Strains,Optimization of Their Chitinase-producing Conditions and Application in the Hydrolysis of Shrimp Shells

Objective: This study aimed to screen and identify chitinase-producing strains and optimize the fermentation conditions of the target bacteria, which were eventually applied to the degradation fermentation of shrimp shells. Methods: The chitin degradation bacteria were screened on chitin hydrolysis zone taking marine mud from beach in Yancheng as samples, identified via bioinformatics technology, and analyzed in the form of phylogenetic tree. Fermentation conditions for the target strains were modified depending on the single-factor experiment. Results: Photobacterium sp. LYM-1, Vibrio sp. WM-1, and Shewanella sp. ZXY-1 were isolated and identified via 16S rDNA sequence analysis. The optimal fermentation conditions were determined as follows: For Photobacterium sp. LYM-1, chitin (10 g/L) as the carbon source, NH₄Cl (2.0 g/L) as the nitrogen source, the inoculated quantified of 3%, initial medium pH6.5, and 32 ℃ for 1 d, in which the highest chitinase activity in the supernatant reached 15.37±0.55 U/mL, achieving 4.37 times higher than that before optimization. For Vibrio sp. WM-1, chitin (10 g/L) as the carbon source, NH₄Cl (2.0 g/L) as the nitrogen source, the inoculated quantified of 3%, initial medium pH7.5, and 22 ℃ for 2 d, in which the highest chitinase activity reached 40.82±6.03 U/mL, achieving 1.60 times higher than that before optimization. For Shewanella sp. ZXY-1, chitin (10 g/L) as the carbon source, (NH₄)₂SO₄ (2.0 g/L) as the nitrogen source, the inoculated quantified of 3%, initial medium pH6.5, and 22 ℃ for 1 d, which contributed the highest chitinase activity to reaching 25.64±3.29 U/mL, achieving 2.47 times higher than that before optimization. All three strains in this study could realize chitinase production under optimized conditions using shrimp shell powder as the sole substrate. The chitinase activities of Photobacterium sp. LYM-1, Vibrio sp. WM-1, and Shewanella sp. ZXY-1 on shrimp shell powders were 10.25±0.95, 32.16±2.25 and 21.81±4.27 U/mL, respectively, which was lower than those on chitin powder. Conclusion: Three chitinase-producing strains were successfully screened from saline-alkali soil, of which the enzyme activity was elevated after optimization. All of them could produce chitinase with the shrimp shell utilized as the substrate, serving as potential strains for chitinase production from the fermentation of shrimp shell.