以胶体甲壳素作为产酶诱导物,利用蜡状芽胞杆菌和红球菌共同发酵提高甲壳素脱乙酰酶(chitin deacetylase,CDA)的产量。通过监测2菌株混合发酵时甲壳素酶和CDA酶活力随时间的变化,验证了双菌株协同发酵产脱乙酰酶的作用。在此基础上,采用单因素实验考察两菌株配比、发酵周期、温度、接种量、装液量和摇瓶转速等因素对脱乙酰酶活力的影响,并进一步设计Plackett-Burman(PB)实验、最陡爬坡实验、中心组合设计实验和响应面优化实验,确定适宜2菌株协同发酵的工艺条件。结果显示,2菌株接种量最佳配比为2∶8,最佳产酶培养基组分(g/L):蛋白胨0.25、K2HPO4 0.6、MgSO4 1.5、酵母浸粉3.0、NaCl 8.5、KH2PO4 0.8、胶体甲壳素8.0。优化后菌株产CDA的活力约为优化前的1.96倍,研究结果可为工业化产CDA提供参考。
In order to improve the productivity of chitin deacetylase, colloidal chitin were subjected to synergistic fermentation of Rhodococcus sp. and Bacillus cereus. The effects of culture time, ratio of B. cereus and Rhodococcus sp., fermentation time, temperature, inoculum size, liquid loading and shaker speed on chitin deacetylase activity were verified. Combined with the Plackett-Burman experiment and the response surface method central composite experiment, the optimal ratio of B. cereus and Rhodococcus sp. were 2∶8. The optimal production medium component were as follows: 0.25 g/L peptone, 0.6 g/L K2HPO4, 1.5 g/L MgSO4, 3.0 g/L yeast extract, 8.5 g/L NaCl, 0.8 g/L KH2PO4 and 8.0 g/L colloidal chitin. The activity of chitin deacetylase was improved by 0.96 times. The results provide a reference for industrial production of chitin deacetylase.
[1] SILVA D S,DELEZUK J A M,LA PORTA F A,et al.Comparison of experimental and theoretical data on the structural and electronic characterization of chitin and chitosan[J].Current Physical Chemistry,2016,5(3):206-213.
[2] CRINI G.Historical review on chitin and chitosan biopolymers[J].Environmental Chemistry Letters,2019,17(4):1 623-1 643.
[3] LIN S,QIN Z,CHEN Q M,et al.Efficient immobilization of bacterial GH family 46 chitosanase by carbohydrate-binding module fusion for the controllable preparation of chitooligosaccharides[J].Journal of Agricultural and Food Chemistry,2019,67(24):6 847-6 855.
[4] ARAGUNDE H,BIARNÉS X,PLANAS A.Substrate recognition and specificity of chitin deacetylases and related family 4 carbohydrate esterases[J].International Journal of Molecular Sciences,2018,19(2):412.
[5] BLAIR D E,HEKMAT O,SCHüTTELKOPF A W,et al.Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum[J].Biochemistry,2006,45(31):9 416-9 426.
[6] 华苟根,郭坚华.红球菌属的分类及应用研究进展[J].微生物学通报,2003,30(4):107-111.
HUA G G,GUO J H.The taxonomy and application of Rhodococcus[J].Microbiology,2003,30(4):107-111.
[7] SUN Y Y,ZHANG J Q,WU S J,et al.Statistical optimization for production of chitin deacetylase from Rhodococcus erythropolis HG05[J].Carbohydrate Polymers,2014,102:649-652.
[8] MA Q Y,GAO X Z,BI X Y,et al.Isolation,characterisation,and genome sequencing of Rhodococcus equi:A novel strain producing chitin deacetylase[J].Scientific Reports,2020,10:4329.
[9] 柴金龙,王敏卜,杭加豪,等.产几丁质脱乙酰酶海洋细菌的筛选鉴定及产酶条件优化[J].中国酿造,2019,38(10):89-95.
CHAI J L,WANG M B,HANG J H,et al.Screening,identification and enzyme production conditions optimization of chitin deacetylase-producing marine bacteria[J].China Brewing,2019,38(10):89-95.
[10] LIU J L,JIA Z J,LI S,et al.Identification and characterization of a chitin deacetylase from a metagenomic library of deep-sea sediments of the Arctic Ocean[J].Gene,2016,590(1):79-84.
[11] 陈立功,吴家葳,张庆芳,等.产低温几丁质酶菌株的筛选、鉴定与产酶条件优化[J].食品工业科技,2020,41(23):85-90;98.
CHEN L G,WU J W,ZHANG Q F,et al.Screening and identification of a cold-adapted chitinolytic bacterium strain and optimization of its chitinase fermentation conditions[J].Science and Technology of Food Industry,2020,41(23):85-90;98.
[12] HE Y H,XU J P,WANG S J,et al.Optimization of medium components for production of chitin deacetylase by Bacillus amyloliquefaciens Z7,using response surface methodology[J].Biotechnology & Biotechnological Equipment,2014,28(2):242-247.
[13] LIAQAT F,SÖZER BAHADI R P,ELIBOL M,et al.Optimization of chitosanase production by Bacillus mojavensis EGE-B-5.2i[J].Journal of Basic Microbiology,2018,58(10):836-847.
[14] REN J,LIN W T,SHEN Y J,et al.Optimization of fermentation media for nitrite oxidizing bacteria using sequential statistical design[J].Bioresource Technology,2008,99(17):7 923-7 927.
[15] WANG Z W.Advance in study and application on chitinase produced by microbes[J].Letters in Biotechnology,2006,17(3):439-442.
[16] VIENS P,DUBEAU M P,KIMURA A,et al.Uptake of chitosan-derived D-glucosamine oligosaccharides in Streptomyces coelicolor A3(2)[J].FEMS Microbiology Letters,2015,362(9):fnv048.
[17] WIN N,STEVENS W.Shrimp chitin as substrate for fungal chitin deacetylase[J].Applied Microbiology and Biotechnology,2001,57(3):334-341.
[18] NAQVI S,CORD-LANDWEHR S,SINGH R,et al.A recombinant fungal chitin deacetylase produces fully defined chitosan oligomers with novel patterns of acetylation[J].Applied and Environmental Microbiology,2016,82(22):6 645-6 655.
[19] LIU Z L,GAY L M,TUVENG T R,et al.Structure and function of a broad-specificity chitin deacetylase from Aspergillus nidulans FGSC A4[J].Scientific Reports,2017,7(1):1746.
[20] CHAMBON R,PRADEAU S,FORT S,et al.High yield production of Rhizobium NodB chitin deacetylase and its use for in vitro synthesis of lipo-chitinoligosaccharide precursors[J].Carbohydrate Research,2017,442:25-30.
[21] 刘力睿,潘杰,李猛.微生物几丁质酶的研究进展、应用及展望[J].生物资源,2020,42(5):494-504.
LIU L R,PAN J,LI M.Recent research progress,application and future perspectives on microbial chitinases[J].Biotic Resources,2020,42(5):494-504.
