中国麻业科学
中國痳業科學
중국마업과학
PLANT FIBER SCIENCES IN CHINA
2015年
2期
87-94
,共8页
张懿翔%丁若篧%陈婷%郁崇文%张兴群
張懿翔%丁若篧%陳婷%鬱崇文%張興群
장의상%정약착%진정%욱숭문%장흥군
生物脱胶%果胶酶%枯草芽孢杆菌%vgb 基因%同源重组
生物脫膠%果膠酶%枯草芽孢桿菌%vgb 基因%同源重組
생물탈효%과효매%고초아포간균%vgb 기인%동원중조
bio -degumming%Pectinase%Bacillus subtilis%gene vgb%homologous recombination
【目的】将透明颤菌血红蛋白(VHb)基因(vgb)在枯草芽孢杆菌中进行整合表达,以改善枯草芽孢杆菌的呼吸强度,通过高密度培养提高果胶酶的表达量,最终明显改善脱胶效果。【方法】构建含有 vgb 基因、amyE 基因以及启动子 P43的 pSK -vgb (+)质粒,通过同源重组的方法,将 vgb 基因整合到枯草芽孢杆菌 A5的染色体上,得到稳定表达透明颤菌血红蛋白的枯草芽孢杆菌 A5-vgb (+)。分别采用 PCR 和 CO 差光谱分析法检测 vgb 基因的整合状态与血红蛋白的稳定表达,并通过发酵摇瓶试验研究 VHb 对菌株果胶酶产量的影响。将构建好的 A5-vgb (+)菌株进行脱胶试验,通过 XRD 结晶度与电镜扫描分析鉴定脱胶效果。【结果】PCR 与 CO 差光谱检测表明,vgb 基因成功整合在 A5菌株中,且正确表达的 VHb 具有生物学活性,在150 rpm,37℃,pH =8.0,16小时发酵的条件下,vgb 基因的表达促使 A5-vgb (+)菌株的果胶酶产量较不含 vgb 基因的菌株提升了29.5%。XRD 与电镜扫描结果表明,构建的 A5-vgb (+)菌株的脱胶效果相较于不含 vgb 的菌株,在结晶度上提高了0.55%,表面更光滑。【结论】将 vgb 基因整合至枯草芽孢杆菌 A5菌株中,可提高 A5菌株的脱胶效果。
【目的】將透明顫菌血紅蛋白(VHb)基因(vgb)在枯草芽孢桿菌中進行整閤錶達,以改善枯草芽孢桿菌的呼吸彊度,通過高密度培養提高果膠酶的錶達量,最終明顯改善脫膠效果。【方法】構建含有 vgb 基因、amyE 基因以及啟動子 P43的 pSK -vgb (+)質粒,通過同源重組的方法,將 vgb 基因整閤到枯草芽孢桿菌 A5的染色體上,得到穩定錶達透明顫菌血紅蛋白的枯草芽孢桿菌 A5-vgb (+)。分彆採用 PCR 和 CO 差光譜分析法檢測 vgb 基因的整閤狀態與血紅蛋白的穩定錶達,併通過髮酵搖瓶試驗研究 VHb 對菌株果膠酶產量的影響。將構建好的 A5-vgb (+)菌株進行脫膠試驗,通過 XRD 結晶度與電鏡掃描分析鑒定脫膠效果。【結果】PCR 與 CO 差光譜檢測錶明,vgb 基因成功整閤在 A5菌株中,且正確錶達的 VHb 具有生物學活性,在150 rpm,37℃,pH =8.0,16小時髮酵的條件下,vgb 基因的錶達促使 A5-vgb (+)菌株的果膠酶產量較不含 vgb 基因的菌株提升瞭29.5%。XRD 與電鏡掃描結果錶明,構建的 A5-vgb (+)菌株的脫膠效果相較于不含 vgb 的菌株,在結晶度上提高瞭0.55%,錶麵更光滑。【結論】將 vgb 基因整閤至枯草芽孢桿菌 A5菌株中,可提高 A5菌株的脫膠效果。
【목적】장투명전균혈홍단백(VHb)기인(vgb)재고초아포간균중진행정합표체,이개선고초아포간균적호흡강도,통과고밀도배양제고과효매적표체량,최종명현개선탈효효과。【방법】구건함유 vgb 기인、amyE 기인이급계동자 P43적 pSK -vgb (+)질립,통과동원중조적방법,장 vgb 기인정합도고초아포간균 A5적염색체상,득도은정표체투명전균혈홍단백적고초아포간균 A5-vgb (+)。분별채용 PCR 화 CO 차광보분석법검측 vgb 기인적정합상태여혈홍단백적은정표체,병통과발효요병시험연구 VHb 대균주과효매산량적영향。장구건호적 A5-vgb (+)균주진행탈효시험,통과 XRD 결정도여전경소묘분석감정탈효효과。【결과】PCR 여 CO 차광보검측표명,vgb 기인성공정합재 A5균주중,차정학표체적 VHb 구유생물학활성,재150 rpm,37℃,pH =8.0,16소시발효적조건하,vgb 기인적표체촉사 A5-vgb (+)균주적과효매산량교불함 vgb 기인적균주제승료29.5%。XRD 여전경소묘결과표명,구건적 A5-vgb (+)균주적탈효효과상교우불함 vgb 적균주,재결정도상제고료0.55%,표면경광활。【결론】장 vgb 기인정합지고초아포간균 A5균주중,가제고 A5균주적탈효효과。
The gene Vitroscilla Hemoglobin was integrated and expressed in Bacillus subtilis to en-hance the effect of degumming by improving pectinase production.Promoter p43 and gene amyE,pSK -vgb (+)were integrated into expression vector.Through the method of homologous recombination, gene vgb was integrated into Bacillus subtilis A5,and then got a vgb integrated expression strain named Bacillus subtilis A5 -vgb (+).The result was identified by PCR analysis and CO -spectral analysis.A5 -vgb (+)strain was applied in flax degumming,shake flask experiment was used to study the func-tion of protein VHb in production of Pectinase.X -ray diffraction (XRD)and canning electron micro-scope (SEM)were uesd to analysis the effect of degumming.PCR analysis and CO -spectral analysis showed that the gene vgb was succeed in being integrated into Bacillus subtilis A5 and protein VHb had bioactivity.The results of shake flask experiment showed that the expression of gene vgb could improve Pectinase production,and the activity of Pectinase was 29.5% higher than that of the control under 150 rpm,37℃,pH =8.0,16 hours .The data of XRD and SEM showed that being compared with the strain without gene vgb,the degumming effect of A5 -vgb (+)strain improved 0.55% in crystallinity, and had a smoother surface.To some extent,the VHb could enhance the degumming effect of Bacillus subtilis A5.