ZENG Jing, GUO Jianjun, WANG Tong, et al. Enhanced Extracellular Type III Pullulan Hydrolase Production by Co-expressing Molecular Chaperone in Brevibacillus choshinensis and Fermentation Optimization[J]. Science and Technology of Food Industry, 2024, 45(10): 149−157. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023080133.
Citation: ZENG Jing, GUO Jianjun, WANG Tong, et al. Enhanced Extracellular Type III Pullulan Hydrolase Production by Co-expressing Molecular Chaperone in Brevibacillus choshinensis and Fermentation Optimization[J]. Science and Technology of Food Industry, 2024, 45(10): 149−157. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023080133.

Enhanced Extracellular Type III Pullulan Hydrolase Production by Co-expressing Molecular Chaperone in Brevibacillus choshinensis and Fermentation Optimization

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  • Received Date: August 14, 2023
  • Available Online: March 12, 2024
  • This study aimed to increase the expression and secretion of type III pullulan hydrolase (TK-PUL) by Brevibacillus choshinensis through the co-expression of molecular chaperone proteins and the optimization of fermentation conditions. By constructing multiple recombinant B. choshinensis co-expressing TK-PUL and molecular chaperone proteins, and screening them using extracellular enzyme activity as an indicator, the most favorable molecular chaperone protein and corresponding recombinant B. choshinensis for TK-PUL secretion and expression were determined. On this basis, single factor experiments and response surface methodology were used to optimize the fermentation conditions of recombinant B. choshinensis. Results showed that, the extracellular enzyme activity of the recombinant B. choshinensis strain co-expressing the molecular chaperone protein PrsABa reached 98.79 U/mL, representing a 0.31-fold increase. The optimal medium for this recombinant B. choshinensis was composed of 19.65 g/L of glucose, 21.46 g/L of yeast extract, 12.01 g/L of MgCl2·6H2O, 9.02 g/L of proline, 0.01 g/L of FeSO4·7H2O, 0.01 g/L of MnSO4·4H2O, and 0.001 g/L of ZnSO4·7H2O. Culturing the optimized recombinant B. choshinensis in the above-mentioned optimized medium for 66 hours at 35 °C and an initial pH of 7.0 increased the extracellular TK-PUL activity to 192.68 U/mL, which represented a 1.56-fold increase. Efficient secretion of TK-PUL in B. choshinensis was achieved through the co-expression of molecular chaperone proteins and the optimization of fermentation conditions. This study provides a foundation for exploring the industrial-scale application of TK-PUL.
  • [1]
    NAIK B, KUMAR V, GOYAL S K, et al. Pullulanase:unleashing the power of enzyme with a promising future in the food industry[J]. Frontiers in Bioengineering and Biotechnology,2023,11:1139611. doi: 10.3389/fbioe.2023.1139611
    [2]
    NISHA M, SATYANARAYANA T. Characteristics, protein engineering and applications of microbial thermostable pullulanases and pullulan hydrolases[J]. Applied Microbiology and Biotechnology,2016,100(13):5661−5679. doi: 10.1007/s00253-016-7572-y
    [3]
    WANG X Y, NIE Y, XU Y. Industrially produced pullulanases with thermostability:Discovery, engineering, and heterologous expression[J]. Bioresource Technology, 2019:360-371.
    [4]
    AKASSOU M, GROLEAU D. Advances and challenges in the production of extracellular thermoduric pullulanases by wild-type and recombinant microorganisms:A review[J]. Critical Reviews in Biotechnology,2019,39(3):337−350. doi: 10.1080/07388551.2019.1566202
    [5]
    OKAFOR D C, OFOEDU C E, NWAKAUDU A, et al. Enzymes as additives in starch processing:A short overview[M]. New York:Academic Press, 2019:149-168.
    [6]
    MIAO M, JIANG B, JIN Z Y, et al. Microbial starch-converting enzymes:recent insights and perspectives[J]. Comprehensive Reviews in Food Science and Food Safety,2018,17(5):1238−1260. doi: 10.1111/1541-4337.12381
    [7]
    HAN T, ZENG F, LI Z, et al. Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1[J]. Letters in Applied Microbiology,2013,57(4):336−343. doi: 10.1111/lam.12118
    [8]
    AHMAD N, RASHID N, HAIDER M S, et al. Novel maltotriose-hydrolyzing thermoacidophilic type III pullulan hydrolase from Thermococcus kodakarensis[J]. Applied and Environmental Microbiology,2014,80(3):1108−1115. doi: 10.1128/AEM.03139-13
    [9]
    AHMAD N, RASHID N, HAIDER M S, et al. Single step liquefaction and saccharification of corn starch using an acidophilic, calcium independent and hyperthermophilic pullulanase:US9340778[P]. 2016-05-17[2020-04-08]. http://www.google.com/patents/US20140227744.
    [10]
    TOOR K J, AHMAD N, MUHAMMAD M A, et al. TK-PUL, a pullulan hydrolase type III from Thermococcus kodakarensis, a potential candidate for simultaneous liquefaction and saccharification of starch[J]. Amylase,2020,4(1):45−55. doi: 10.1515/amylase-2020-0004
    [11]
    曾静, 郭建军, 袁林. 嗜热酸性普鲁兰水解酶Ⅲ的高效分泌表达及其酶学性质[J]. 食品工业科技,2020,41(3):98−103,109. [ZENG J, GUO J J, YUAN L. Efficient secretory expression of thermoacidiphilic type III pullulan hydrolase and its enzymatic properties[J]. Science and Technology of Food industry,2020,41(3):98−103,109.]

    ZENG J, GUO J J, YUAN L. Efficient secretory expression of thermoacidiphilic type III pullulan hydrolase and its enzymatic properties[J]. Science and Technology of Food industry, 2020, 41(3): 98−103,109.
    [12]
    HANAGATA H, MIZUKAMI M, MIYAUCHI A. Efficient expression of antibody fragments with the Brevibacillus expression system[J]. Antibodies,2014,3(2):242−252. doi: 10.3390/antib3020242
    [13]
    MIZUKAMI M, HANAGATA H, MIYAUCHI A. Brevibacillus expression system:Host-vector system for efficient production of secretory proteins[J]. Current Pharmaceutical Biotechnology,2010,11(3):251−258. doi: 10.2174/138920110791112031
    [14]
    YAO D B, ZHANG K, WU J. Available strategies for improved expression of recombinant proteins in Brevibacillus expression system:A review[J]. Critical Reviews in Biotechnology,2021,40(7):1044−1058.
    [15]
    OKAMOTO A, KOSUGI A, KOIZUMI Y, et al. High efficiency transformation of Bacillus brevis by electroporation[J]. Bioscience, Biotechnology, and Biochemistry,1997,61(1):202−203. doi: 10.1271/bbb.61.202
    [16]
    GREEN M R, SAMBROOK J. Molecular cloning:A laboratory manual[M]. New York:Cold Spring Harbor Laboratory Press, 2012:101-200.
    [17]
    LI Z, SU L Q, DUAN X G, et al. Efficient expression of maltohexaose-forming α-amylase from Bacillus stearothermophilus in Brevibacillus choshinensis SP3 and its use in maltose production[J]. Biomed Research International,2017,2017:5479762.
    [18]
    MILLER G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical Chemistry,1959,31(3):426−428. doi: 10.1021/ac60147a030
    [19]
    YANG H Q, QU J F, ZOU W, et al. An overview and future prospects of recombinant protein production in Bacillus subtilis[J]. Applied Microbiology and Biotechnology,2021,105(18):6607−6626. doi: 10.1007/s00253-021-11533-2
    [20]
    ZHANG K, TAN R T, YAO D B, et al. Enhanced production of soluble Pyrococcus furiosus α-amylase in Bacillus subtilis through chaperone co-expression, heat treatment and fermentation optimization[J]. Journal of Microbiology and Biotechnology,2021,31(4):570. doi: 10.4014/jmb.2101.01039
    [21]
    YAO D B, ZHANG K, ZHU X Y, et al. Enhanced extracellular α-amylase production in Brevibacillus choshinensis by optimizing extracellular degradation and folding environment[J]. Journal of Industrial Microbiology and Biotechnology,2022,49(1):kuab061. doi: 10.1093/jimb/kuab061
    [22]
    XU L Y, ZHANG Y Y, DONG Y H, et al. Enhanced extracellular β-mannanase production by overexpressing PrsA lipoprotein in Bacillus subtilis and optimizing culture conditions[J]. Journal of Basic Microbiology,2022,62(7):815−823. doi: 10.1002/jobm.202200080
    [23]
    QUESADA-GANUZA A, ANTELO-VARELA M, MOURITZEN J C, et al. Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase[J]. Microbial Cell Factories,2019,18(1):158. doi: 10.1186/s12934-018-1049-x
    [24]
    CHEN J Q, GAI Y M, FU G, et al. Enhanced extracellular production of α-amylase in Bacillus subtilis by optimization of regulatory elements and over-expression of PrsA lipoprotein[J]. Biotechnology Letters,2015,37:899−906. doi: 10.1007/s10529-014-1755-3
    [25]
    ZOU C, DUAN X G, WU J. Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis[J]. Journal of Industrial Microbiology and Biotechnology,2016,43(4):495−504. doi: 10.1007/s10295-015-1719-1
    [26]
    CHENG Y M, LU M T, YEH C M. Functional expression of recombinant human trefoil factor 1 by Escherichia coli and Brevibacillus choshinensis[J]. BMC Biotechnology,2015,15(1):32. doi: 10.1186/s12896-015-0115-2
    [27]
    ZOU C, DUAN X G, WU J. Magnesium ions increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis[J]. Applied Microbiology and Biotechnology,2016,100:7115−7123. doi: 10.1007/s00253-016-7386-y
    [28]
    MATSUNAGA R, TSUMOTO K. Addition of arginine hydrochloride and proline to the culture medium enhances recombinant protein expression in Brevibacillus choshinensis:The case of RBD of SARS-CoV-2 spike protein and its antibody[J]. Protein Expression and Purification,2022,194:106075. doi: 10.1016/j.pep.2022.106075
    [29]
    MAEHASHI K, MATANO M, SAITO M, et al. Extracellular production of riboflavin-binding protein, a potential bitter inhibitor, by Brevibacillus choshinensis[J]. Protein Expression and Purification,2010,71(1):85−90. doi: 10.1016/j.pep.2009.12.016
    [30]
    LI H P, XU C M, WEN B Y, et al. Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis[J]. Experimental and Therapeutic Medicine,2020,19(3):2149−2154.
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