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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (11): 8-15    DOI: 10.13523/j.cb.2305027
研究报告     
原核表达及细胞表面展示脯氨酸羟化酶改性鱼明胶
黄明珠1,2,3,沈祺昌2,3,秦春燕2,3,徐阳3,魏怡然3,陈雪岚1,2,3,*()
1 江西师范大学 国家淡水鱼加工技术研发专业中心 南昌 330022
2 江西师范大学生命科学学院 南昌 330022
3 江西师范大学健康学院 南昌 330022
Fish Gelatin Modification Using Prokaryotic Expression and Cell Surface Display Proline Hydroxylases
HUANG Ming-zhu1,2,3,SHEN Qi-chang2,3,QIN Chun-yan2,3,XU Yang3,WEI Yi-ran3,CHEN Xue-lan1,2,3,*()
1 National R&D Center of Freshwater Fish Processing, Jiangxi Normal University, Nanchang 330022, China
2 School of Life Science, Jiangxi Normal University, Nanchang 330022, China
3 School of Health, Jiangxi Normal University, Nanchang 330022, China
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摘要：

鱼明胶次优的凝胶特性限制了其商业应用,酶催化改性鱼明胶有着绿色环保、安全高效等巨大优势,然而,目前报道的明胶改性酶较少,且多为蛋白质共价交联酶,易使明胶形成热不可逆凝胶。克隆出了两种增加胶原蛋白的非共价作用的脯氨酸羟化酶,对其进行原核表达。纯化后,分别对鱼明胶进行催化,结果表明两种酶具有改善鱼明胶胶强度和质构特性的效果。研究了钝齿棒杆菌的表面展示技术,将两种酶分别展示在钝齿棒杆菌表面,制备了一种以钝齿棒杆菌为载体的固定化酶体系,研究两种固定化酶体系对鱼明胶的改性效果。丰富了鱼明胶的催化酶体系,为鱼明胶的催化改性提供新思路。
关键词： 表面展示技术;  鱼明胶;  凝胶强度;  质构分析    
Abstract:

The poor gel properties of fish gelatin limit its commercial application. Enzyme-catalyzed modification of fish gelatin has great advantages such as environmental friendliness, safety and high efficiency. However, there are few gelatin-modified enzymes reported at present, and most of them are covalent cross-linking enzymes, which easily make gelatin form thermal irreversible gel. In this study, two proline hydroxylases that increase the non-covalent action of collagen were cloned and expressed in prokaryotic cells. After purification, the fish gelatin was catalyzed respectively using two proline hydroxylases. The results show that the two enzymes have the effect of improving the gel strength and texture characteristics of fish gelatin. In addition, the surface display technology of Corynebacterium crenatum was studied. The two enzymes were displayed on the surface of C. crenatum, and an immobilized enzyme system with C. crenatum as the carrier was prepared for studying the modification effect of two immobilized enzyme systems on fish gelatin. This study has enriched the catalytic enzyme system of fish gel and provided a new idea for the catalytic modification of fish gelatin.
Key words: Surface display technology    Fish gelatin    Gel strength    Texture analysis
收稿日期: 2023-05-19 出版日期: 2023-12-01
ZTFLH:  Q814  
通讯作者: *陈雪岚     E-mail: xuelanchen162@163.com
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黄明珠
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魏怡然
陈雪岚

引用本文:

黄明珠, 沈祺昌, 秦春燕, 徐阳, 魏怡然, 陈雪岚. 原核表达及细胞表面展示脯氨酸羟化酶改性鱼明胶[J]. 中国生物工程杂志, 2023, 43(11): 8-15.

HUANG Ming-zhu, SHEN Qi-chang, QIN Chun-yan, XU Yang, WEI Yi-ran, CHEN Xue-lan. Fish Gelatin Modification Using Prokaryotic Expression and Cell Surface Display Proline Hydroxylases. China Biotechnology, 2023, 43(11): 8-15.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2305027    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I11/8

引物名称 序列 目的
pgsA-s ATTAATTAAGCTTGCATGCCTATGAAAAAAGAACTGAGCTTTCATG 扩增pgsA ORF
pgsA-x TCCAGTGAAAAGTTCTTCTCGTTTATGCATTTTAGATTTTAGTTTATCGCTATGATCAA 扩增pgsA ORF
pgsA/gfp-s TTGATCATAGCGATAAACTAAAATCTAAAATGCATAAACGAGAAGAACTTTTCACTGGA 通过搭桥PCR连接pgsA和gfp
gfp-x CTGAATTCGAGCTCGGTACCCTTATTATTTGTAGAGCTCATCCATGCCATGT 扩增gfp ORF
ncgl1337-s ATTAATTAAGCTTGCATGCCTATGGCTCAGCGAAAACTGGCCTCTGTG 扩增ncgl1337跨膜区
ncgl1337-x CCAGTGAAAAGTTCTTCTCGTTTATGCATAGCCACACCACCACTTGAGGTGAGTGC 扩增ncgl1337跨膜区
ncgl1337/gfp-s GCACTCACCTCAAGTGGTGGTGTGGCTATGCATAAACGAGAAGAACTTTTCACTGG 通过搭桥PCR连接ncgl1337和gfp
proH-s ATTAATTAAGCTTGCATGCCTATGGATCTTTCCGTTCTCA 扩增proH ORF
proH-x TCCAGTGAAAAGTTCTTCTCGTTTATGCATGGAAGAGAGCTTATCCAGGTT 扩增proH ORF
proH/gfp-s AACCTGGATAAGCTCTCTTCCATGCATAAACGAGAAGAACTTTTCACTGGA 通过搭桥PCR连接proH和gfp
pbp4h/pgsA-s TTGATGTTGATCATAGCGATAAACTAAAATCTAAAATGACAAATAAATTTATTTCGTACAATA 扩增pbp4h ORF,通过搭桥PCR连接pgsA和pbp4h
pbp4h-x CTGAATTCGAGCTCGGTACCTTATTTAACAGCACGGATCCATTGGTTCGCG 扩增pbp4h ORF
pbp4h-s ATTAATTAAGCTTGCATGCCTATGCATCATCATCATCATCATACAAATAAATTTATTTCG
TACAATA
bap4h/pgsA -s TTGATGTTGATCATAGCGATAAACTAAAATCTAAAATGACAAACAACAATCAAATAGG
TGA		 扩增pbp4h ORF,通过搭桥PCR连接pgsA和pap4h
bap4h-x CTGAATTCGAGCTCGGTACCTTACTTATAAGTACCTCTTCTCACCCAC 扩增bap4h ORF
bap4h-s ATTAATTAAGCTTGCATGCCTATGCATCATCATCATCATCATACAAACAACAATCAAAT
AGGTGA
表1  本研究使用的引物
图1  蛋白质SDS-PAGE图
酶 Vmax /[μmol/ (mL·min) Km /(μmol/mL) Kcat /s-1 Kcat/Km /[mL/(μmol·s)]
Pbp4h 2.46 952.51 352.40 0.37
Bap4h 3.70 1650.11 473.05 0.29
PgsA-Pbp4h 1.63 359.73 - -
PgsA-Bap4h 1.45 292.84 - -
表2  酶动力参数
图2  酶催化对凝胶强度的影响
样品 对照组 Bap4h Pbp4h
硬度/g 553.11±8.48a 570.12±5.94b 579.75±9.06b
黏附性/(g.s) -23.72±0.71a -20.90±0.55c -22.52±0.37b
弹性 0.97±0.02a 0.94±0.03a 0.94±0.01a
黏结性 0.78±0.01a 0.79±0.04a 0.83±0.02b
胶黏性/g 467.45±15.00a 504.52±17.10b 492.66±13.02a
咀嚼性/g 385.91±5.70a 425.28±9.48b 416.16±3.88b
弹力 0.85±0.12a 0.88±0.03b 0.82±0.11a
表3  酶催化对质构特性的影响
图3  构建钝齿棒杆菌表面展示系统
图4  酶催化对凝胶强度的影响
样品 对照组 Bap4h Pbp4h
硬度/g 553.51±7.12a 574.56±5.70b 576.33±7.74b
黏附性/(g.s) -23.63±0.76a -20.64±0.29a -21.95±0.72b
弹性 0.94±0.02a 0.96±0.01a 0.95±0.02a
黏结性 0.77±0.02a 0.81±0.02b 0.8±0.05a
胶黏性/g 483.74±12.05a 499.13±9.97a 497.22±14.52a
咀嚼性/g 404.17±5.46a 425.7±6.67b 428.33±7.05b
弹力 0.84±0.11a 0.87±0.04a 0.85±0.02a
表4  酶催化对质构特性的影响
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