产几丁质酶的无色杆菌ZWW8的发酵产酶及酶学性质研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0887

摘要/Abstract

摘要：

从湖北武汉地区虾壳堆积土壤中筛选得到一株分泌胞外几丁质酶菌株ZWW8,通过16S rDNA序列比对发现其属于无色杆菌属;通过碳源、氮源、温度、培养基pH、培养时间的单因素优化实验获得ZWW8菌株的最优发酵条件,该菌株在添加0.5%几丁质胶体,0.5% NH₄Cl,初始pH 7.0的培养基中,于37℃培养2 d后胞外上清酶活力可达到33.9 U/L,是优化前酶活力的8倍;通过检测ZWW8菌株分泌胞外几丁质酶的酶学性质发现,其最适反应pH为5,最适反应温度为50℃。Ni ²⁺和Ca ²⁺能提高几丁质酶活性,而Hg ²⁺、Cu ²⁺和Zn ²⁺能明显抑制酶活。此外,几丁质酶还能在高盐条件维持活性和稳定性,并特异性结合几丁质。通过几丁质亲和层析纯化的胞外几丁质酶,在透析除去尿素后仍然具有活性。ZWW8菌株的几丁质酶具有独特的酶学特性,使其能作为高效稳定的生物催化剂,为实现在工业、农业和医学方面的应用潜能,以及几丁质资源的开发和利用奠定基础。

关键词: 无色杆菌, 几丁质酶, 发酵优化, 酶学性质, 耐盐

Abstract:

A extracellular chitinase-secreting bacterium ZWW8 was isolated from shrimp shell-accumulated soil in Hubei and identified as Achromobacter spp. via 16S rDNA sequence analysis. The fermentation conditions for chitinase production by the strain ZWW8 were optimized using the single-factor experiment,and the optimal fermentation conditions were as follows：0.5% colloidal chitin,0.5% ammonium chloride,initial medium pH 7.0,and 37℃ for 2 d. Under the optimized fermentation conditions,the highest chitinase activity in the supernatant reached 33.9 U/mL,which was 8 times of the activity before optimization. By detecting the enzymatic properties of extracellular chitinase secreted by ZWW8 strain,it was revealed that the optimal temperature of chitinase produced by Achromobacter sp. ZWW8 was 50℃ and the optimal pH was 5. Ni ²⁺and Ca ²⁺ increased chitinase activity,but Hg ²⁺,Cu ²⁺ and Zn ²⁺ significantly inhibited the enzymatic activity. Meanwhile,the chitinase not only exhibited activity and stability at high salt concentrations,but also showed chitin binding capacity. The extracellular chitinase from the fermented broth purified by chitin resin column demonstrated high activity after urea removed by dialysis. The chitinase by Achromobacter sp. ZWW8 possesses excellent enzymatic properties,making it a highly efficient and stable biocatalyst,which lays a foundation for exploring its application potential in industry,agriculture and medicine,as well as the development and utilization of chitin resources.

Key words: Achromobacter, chitinase, fermentation optimization, enzymatic property, salt-tolerance

张瑶心, 王亮节, 郑文, 徐汉琴, 郑恋, 钟静. 产几丁质酶的无色杆菌ZWW8的发酵产酶及酶学性质研究[J]. 生物技术通报, 2021, 37(4): 96-106.

ZHANG Yao-xin, WANG Liang-jie, ZHENG Wen, XU Han-qin, ZHENG Lian, ZHONG Jing. Study on Enzyme Production of a Chitinase-producing Strain Achromobacter sp. ZWW8 by Fermentation and Its Enzymatic Characterization[J]. Biotechnology Bulletin, 2021, 37(4): 96-106.

图/表 14

图1 基于16S rDNA序列构建的ZWW8菌株的系统进化树括号中的序号代表菌株的GenBank 登录号;分支点上的数字代表计算1 000 次聚类到一起的几率;标尺刻度代表0.5%的序列

Figure 1 Phylogenetic tree of strain ZWW8 based on 16S rDNA sequences Numbers in parentheses refer to the sequence’ accession number in GenBank. The number at each branch point is the percentage supported by bootstrap. Bar: 0.5% sequence divergence

表1 不同碳源对ZWW8菌株发酵产几丁质酶的影响

Table 1 Effect of different carbon sources on chitinase production by Achromobacter sp. ZWW8

碳源Carbon source	酶活Enzyme activity/（U·L^-1）
葡萄糖 Glucose	0.2±0.8
胶体几丁质 Colloidal chitin	20.0±1.9
粉末几丁质 Chitin powder	4.2±0.3
胶体壳聚糖 Colloidal chitosan	0.0±0.1
水溶性壳聚糖 Soluble chitosan	0.2±0.1
N-乙酰氨基葡萄糖 N-Acetyl-D-glucosamine	0.4±0.6

表2 不同氮源对ZWW8菌株发酵产几丁质酶的影响

Table2 Effect of different nitrogen sources on chitinase production by Achromobacter sp. ZWW8

氮源Nitrogen source	酶活Enzyme activity（U·L^-1）
氯化铵 Ammonium chloride	20.7±1.3
硫酸铵 Ammonium sulfate	17.8±1.5
硝酸钠 Sodium nitrate	6.4±1.9
尿素 Urea	13.3±1.0
蛋白胨 Peptone	12.1±1.8
硝酸铵Ammonium nitrate	19.2±0.2

图2 培养基初始pH对ZWW8菌株产几丁质酶的影响

Fig. 2 Effect of initial medium pH on chitinase production by Achromobacter sp. ZWW8

图3 温度对ZWW8菌株产几丁质酶的影响

Fig.3 Effect of temperatures on chitinase production by Achromobacter sp. ZWW8

图4 发酵时间对ZWW8菌株产几丁质酶的影响

Fig.4 Effect of fermentation time on chitinase production by strain ZWW8

图5 温度对ZWW8菌株几丁质酶活性的影响

Fig.5 Effect of temperature on chitinase activity by strain ZWW8

图6 pH对ZWW8菌株几丁质酶活性的影响

Fig.6 Effect of pH on chitinase activity by strain ZWW8

图7 NaCl浓度对ZWW8菌株几丁质酶活性的影响

Fig.7 Effect of NaCl concentration on chitinase activity by strain ZWW8

表3 金属离子对ZWW8菌株几丁质酶活性的影响

Table 3 Effect of metal ions on chitinase activity by strain ZWW8

金属离子Metal ion	相对酶活Relative activity/%
Hg²⁺	3.1±3.3
Zn²⁺	84.4±8.5
Mg²⁺	96.1±7.1
Ca²⁺	103.9±9.3
Ag⁺	99.2±6.8
Cu²⁺	75.9±0.1
Ni²⁺	114.7±10.3

图8 ZWW8菌株几丁质酶在不同温度热处理后的残余活性

Fig.8 Effect of temperature on residual activity of chitinase by strain ZWW8

图9 几丁质酶在不同NaCl浓度热处理后的残余活性

Fig.9 Effect of NaCl concentration on residual activity of chitinase

图10 NaCl浓度对ZWW8菌株几丁质酶的几丁质结合能力的影响

Fig.10 Effect of NaCl concentration on chitin binding ability of chitinase by strain ZWW8

图11 几丁质亲和层析纯化ZWW8菌株几丁质酶的SDS-PAGE检测图 M：低分子量蛋白Marker;1：粗酶液;2：上样流出液;3、4：洗涤液;5：洗脱液

Fig.11 SDS-PAGE analysis of strain ZWW8 chitinase purified by chitin affinity chromatography M: Low molecular weight marker. 1: Crude chitinase. 2: Effluent. 3,4: Scrub solution, 5: Purified chitinase

参考文献 28

[1]	Kurita K. Chemistry and application of chitin and chitosan[J]. Polymer Degradation and Stability, 1998,59(1):117-120. doi: 10.1016/S0141-3910(97)00160-2 URL
[2]	欧阳石文, 冯兰香, 赵开军. 几丁质酶的三级结构和催化机制[J]. 生命的化学, 2001,21(2):131-133.
	Ou-Yang SW, Feng LX, Zhao KJ. The tertiary structure and catalytic mechanism of chitinase[J]. Chemistry of Life, 2001,21(2):131-133.
[3]	Bhattacharya D, Nagpure A, Gupta RK. Bacterial chitinases:properties and potential[J]. Critical Reviews in Biotechnology, 2007,27(1):21-28. doi: 10.1080/07388550601168223 URL
[4]	Nagpure A, Bharti , Gupta RK. Chitinases:in agriculture and human healthcare[J]. Critical Reviews in Biotechnology, 2013,34(3):215-232. doi: 10.3109/07388551.2013.790874 URL
[5]	Dahiya N, Tewari R, Hoondal GS. Biotechnological aspects of chitinolytic enzymes:a review[J]. Applied Microbiology & Biotechnology, 2006,71(6):773-782.
[6]	郭玉莲. 微生物几丁质酶及其在植物病害防治中的作用[J]. 中国农学通报, 2005(1):283-285.
	Guo Y. Chitinase and its effects on plant disease prevention[J]. Chinese Agricultural Science Bulletin, 2005(1):283-285.
[7]	黄乾生, 谢晓兰, 陈清西. 几丁质酶的结构特征与功能[J]. 厦门大学学报:自然科学版, 2008,47(z2). 232-235.
	Huang QS, Xie XL, Chen QX. The structure and function of chitinase[J]. Journal of Xiamen University:Natural Science, 2008,47(z2). 232-235 .
[8]	冯金荣, 惠丰立, 文祯中. 真菌几丁质酶及其在植物真菌病害防治中的作用[J]. 河南农业科学, 2006(8):83-86.
	Feng JR, Hui FL, Wen ZZ. Fungal chitinase and its role in the control of plant fungal diseases[J]. Journal of Henan Agricultural Sciences, 2006(8):83-86.
[9]	Sakuda S, Isogai A, Makita T, et al. Structures of allosamidins, novel insect chitinase inhibitors, produced by actinomycetes[J]. Journal of the Agricultural Chemical Society of Japan, 2006,51(12):3251-3259.
[10]	Liaqat F, Eltem R. Chitooligosaccharides and their biological activities:A comprehensive review[J]. Carbohydrate Polymers, 2018,184:243-259. doi: 10.1016/j.carbpol.2017.12.067 URL
[11]	Chigaleĭchik AG, Pirieva DA, Rylkin SS, et al. Biosynjournal of chitinase by Achromobacter liquefaciens[J]. Mikrobiologiia, 1976,45:475. pmid: 12452
[12]	Takashi T, Hideyuki M. Antibacterial, anti-nematode and/or plant-cell activating composition,chitinolytic microorganisms for producing the same:European, EP0401560[P]. 1993-05-04.
[13]	Bholay AD, Bhushan K, Rameez S, et al. Novel chitinolytic potential of Achromobacter denitrificans isolated from fishery waste[J]. IOSR Journal of Pharmacy and Biological Sciences, 2015,10(4):31-36.
[14]	Baker GC, Smith JJ, Cowan DA . Review and re-analysis of domain-specific 16S primers[J]. Journal of Microbiological Methods, 2004,55(3):541-555. doi: 10.1016/j.mimet.2003.08.009 URL
[15]	张军毅, 朱冰川, 徐超, 等. 基于分子标记的宏基因组16S rRNA基因高变区选择策略[J]. 应用生态学报, 2015,26(11):3545-3553.
	Zhang JY, Zhu BC, Xu C, et al. Strategy of selecting 16S rRNA hypervariable regions for matagenome-phylogenetic marker genes based analysis[J]. Chinese Journal of Applied Ecology, 2015,26(11):3545-3553.
[16]	Kim SK, Rajapakse N. Enzymatic production and biological activities of chitosan oligosaccharides(COS):A review[J]. Carbohydrate Polymers, 2005,62(4):357-368. doi: 10.1016/j.carbpol.2005.08.012 URL
[17]	Dou J, Xu Q, Tan C, et al. Effects of chitosan oligosaccharides on neutrophils from glycogen-induced peritonitis mice model[J]. Carbohydrate Polymers, 2009,75(1):119-124. doi: 10.1016/j.carbpol.2008.07.005 URL
[18]	Aam BB, Heggset EB, Norberg AL, et al. Production of chitooligosaccharides and their potential applications in medicine[J]. Marine Drugs, 2010,8(5):1482-1517. doi: 10.3390/md8051482 URL
[19]	Hough DW, Danson MJ. Extremozymes[J]. Current Opinion in Chemical Biology, 1999,3(1):39-46. pmid: 10021406
[20]	Karan R, Capes MD, Dassarma S . Function and biotechnology of extremophilic enzymes in low water activity[J]. Aquatic Biosystems, 2012,8(1):1-4. doi: 10.1186/2046-9063-8-1 URL
[21]	Vaidya R, Shah I, Vyas P, et al. Production of chitinase and its optimization from a novel isolate Alcaligenes xylosoxydans:potential in antifungal biocontrol[J]. World Journal of Microbiology & Biotechnology, 2001,17(7):691-696. doi: 10.1023/A:1012927116756 URL
[22]	Vaidya R, Vyas P, Chhatpar HS. Statistical optimization of medium components for the production of chitinase by Alcaligenes xylosoxydans[J]. Enzyme & Microbial Technology, 2003,33(1):92-96.
[23]	Vaidya R, Roy S, Macmil S, et al. Purification and characterization of chitinase from Alcaligenes xylosoxydans[J]. Biotechnology Letters, 2003,25(9):715-717. doi: 10.1023/A:1023406630791 URL
[24]	付星, 闫巧娟, 江正强, 等. 高产几丁质酶巴伦葛兹类芽孢杆菌的筛选和发酵条件优化[J]. 微生物学通报, 2015,42(4):625-633.
	Fu X, Yan QJ, Jiang ZQ, et al. Screening of a high-level chitinase producing strain, Paenibacillus barengoltzii and optimization of its fermentation conditions[J]. Microbiology China, 2015,42(4):625-633.
[25]	Vyas P, Deshpande MV . Chitinase production by Myrothecium verrucaria and its significance for fungal mycelia degradation[J]. J Gen Appl Microbiol, 1989,35(5):343-350. doi: 10.2323/jgam.35.343 URL
[26]	Kavitha A, Vijaylakshmi M. Partial purification and antifungal profile of chitinase produced by Streptomyces tendae TK-VL_333[J]. Annals of Microbiology, 2011,61(3):597-603. doi: 10.1007/s13213-010-0178-1 URL
[27]	Uchiyama T, Katouno F, Nikaidou N, et al. Roles of the exposed aromatic residues in crystalline chitin hydrolysis by chitinase A from Serratia marcescens 2170[J]. Journal of Biological Chemistry, 2001,276(44):41343-41349. doi: 10.1074/jbc.M103610200 URL
[28]	Orikoshi H, Nakayama S, Miyamoto K, et al. Roles of four chitinases(ChiA, ChiB, ChiC, and ChiD)in the chitin degradation system of marine bacterium Alteromonas sp. strain O-7[J]. Applied and Environmental Microbiology, 2005,71(4):1811-1815. doi: 10.1128/AEM.71.4.1811-1815.2005 URL