重组毕赤酵母表达华根霉脂肪酶发酵液的絮凝除菌条件研究

在重组毕赤酵母表达华根霉脂肪酶的研究中,目的蛋白的提取收率是关键.由于产物脂肪酶是分泌在发酵液中,常用的方法是加入絮凝剂使茵体沉淀,再通过离心获取上清液中的目的蛋白.本研究改良了传统的絮凝剂配方,确定了絮凝剂的新配方:发酵液中加入2%的氯化钙以及磷酸氢二钠按摩尔比1:1混合液,500 mg/L聚丙烯酰胺,同时调节发酵液...     

Enhanced thermostability of a Rhizopus chinensis lipase by in vivo recombination in Pichia pastoris

ABSTRACT: BACKGROUND: Lipase from Rhizopus chinensis is a versatile biocatalyst for various bioconversions and has been expressed at high-level in Pichia pastoris. However, the use of R. chinensis lipase in industrial applications is restricted by its low thermostability. Directed evolution has been proven to be a powerful and efficient protein engineering tool for improvement of biocatalysts. The present work describes improvement of the thermostability of R. chinensis lipase by directed evolution using P. pastoris as the host. RESULTS: An efficient, fast and highly simplified method was developed to create a mutant gene library in P. pastoris based on in vivo recombination, whose recombination efficiency could reach 2.3 x 105 /mug DNA. The thermostability of r27RCL was improved significantly by two rounds of error-prone PCR and two rounds of DNA shuffling in P. pastoris. The S4-3 variant was found to be the most thermostable lipase, under the conditions tested. Compared with the parent, the optimum temperature of S4-3 was two degrees higher, Tm was 22 degrees higher and half-lives at 60degreesC and 65degreesC were 46- and 23- times longer. Moreover, the catalytic efficiency kcat/Km of S4-3 was comparable to the parent. Stabilizing mutations probably increased thermostability by increasing the hydrophilicity and polarity of the protein surface and creating hydrophobic contacts inside the protein. CONCLUSIONS: P. pastoris was shown to be a valuable cell factory to improve thermostability of enzymes by directed evolution and it also could be used for improving other properties of enzymes. In this study, by using P. pastoris as a host to build mutant pool, we succeeded in obtaining a thermostable variant S4-3 without compromising enzyme activity and making it a highly promising candidate for future applications at high temperatures.     

Pilot scale ceramic membrane microfiltration for Pichia pastoris cells separation in production of Rhizopus chinensis lipase

在重组毕赤酵母生产脂肪酶的提取中,应用并优化了陶瓷膜微滤除菌工艺,确定了最佳条件为膜截留分子量500 kDa、膜操作压力0.3 MPa、温度20℃、湿菌体含量35％,先对发酵液稀释1.5倍后再进行洗滤.40L处理量的小试结果显示,在5h处理时间内,能获得高达92.70％的酶活回收率.560 L处理量的中试放大,酶活回收率为89.91％,耗时5.5h.在膜的清洗与再生中,采用2％ NaClO和2％NaOH在60℃、0.3 MPa膜压力下进行清洗40 min,清水膜通量恢复率为98.14％.陶瓷膜与板框除菌的比较试验发现,两种方法都获得了微生物限量合格的产品和较高的酶活回收率,但陶瓷膜微滤的滤液微生物检出量更低,处理时间较短,动力能耗更低,易与超滤膜耦合提取,废水产生量更少,菌体废渣易于回收,是一种节能减排、清洁环保的新型除菌工艺.     

重组毕赤酵母产华根霉脂肪酶的陶瓷膜微滤除菌工艺研究

在重组毕赤酵母生产脂肪酶的提取中,应用并优化了陶瓷膜微滤除茵工艺,确定了最佳条件为膜截留分子量500kDa、膜操作压力0．3Ⅷa、温度20℃、湿菌体含量35％,先对发酵液稀释1．5倍后再进行洗滤。40L处理量的小试结果显示,在5h处理时间内,能获得高达92．70％的酶活回收率。560L处理量的中试放大,酶活回收率为89．91％,耗时5．5h。在膜的清洗与再生中,采用2％NaC10和2％NaOH在60℃、0．3MPa膜压力下进行清洗40min,清水膜通量恢复率为98．14％。陶瓷膜与板框除菌的比较试验发现,两种方法都获得了微生物限量合格的产品和较高的酶活回收率,但陶瓷膜微滤的滤液微生物检出量更低,处理时间较短,动力能耗更低,易与超滤膜耦合提取,废水产生量更少,菌体废渣易于回收,是一种节能减排、清洁环保的新型除茵工艺。     

Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris

The lipases of the Rhizopus species family are important and versatile enzymes that are mainly used in fat and oil modification due to their strong 1,3-regiospecificity. Inexpensive synthetic medium was used for the production of Rhizopus oryzae lipase in the methylotrophic yeast Pichia pastoris. Methanol accumulation inside the bioreactor has previously been shown to negatively influence the production level. Three different methanol fed-batch strategies for maintaining the methanol concentration within optimal limits have been assayed in high-density cultures. One methanol feeding strategy, which is based on the monitoring of the methanol concentration by gas chromatography, resulted in a 2.5-fold higher productivity compared to an initial cultivation, where the feeding rate was adjusted according to the dissolved oxygen concentration (DO) in the supernatant. Finally, productivity could be further increased by introducing a transition phase that involved the simultaneous feeding of glycerol and methanol followed by a single methanol feed. This optimized strategy resulted in the highest productivity (12888 U l(-1) h(-1)), which is 13.6-fold higher than the DO-based strategy.     

Gene expression in yeast: Pichia pastoris.

Recent studies have shown the versatility and utility of the Pichia pastoris expression system. Improvements in strains have boosted the yield of proteins and peptides to the commercially feasible range. The Pichia pastoris expression system will soon be used to manufacture proteins for human clinical trials.     

Cloning,expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35–37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg²⁺, Mn²⁺, Li⁺, Ca²⁺, Ni²⁺, CHAPS, DTT, and EDTA and inhibited by Hg²⁺, Ag⁺, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.     

Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies

Different cultivation strategies have been compared for the production of Rhizopus oryzae lipase (ROL) from Pichia pastoris. Several drawbacks have been found using a methanol non-limited fed-batch. On the one hand, oxygen limitation appeared at early cell dry weights and, on the other hand, high cell death was observed. A temperature limited fed-batch has been proposed to solve both problems. However, in our case study a methanol non-limited fed-batch results in better productivities. Finally, a lower salt medium were used to overcome cell death problems and a temperature limited fed-batch was applied thereafter to solve oxygen transfer limitations. This combined strategy has resulted in lower productivities when compared to a methanol non-limited fed-batch. However the culture could be longer prolonged and a 1.3-fold purer final product was obtained mainly due to cell death reduction.     

共表达伴侣蛋白对重组毕赤酵母产米根霉(Rhizopus oryzae)脂肪酶发酵过程的影响

[目的]通过共表达伴侣蛋白Erolp和PDI获得米根霉(Rhizopus oryzae)脂肪酶在毕赤酵母中的高效表达.[方法]利用毕赤酵母基因重组菌高密度发酵的方法,在7L发酵罐水平上分析共表达伴侣蛋白菌株(BH128)与非共表达伴侣蛋白菌株(H238)对脂肪酶表达的差异.[结果]在相同条件下,BH128发酵产酶能力高于H238,最高酶活可达到2 338.7U/mL,最大比生长速率达到0.02 h-1,最大产物比形成速率达到944.5 U/(gDCW·h),最大底物比消耗速率也能达到0.15 gmethanol/(gDCW·h),分别是H238的1.7、0.5、4.1和1.3倍,且发酵周期缩短了20h.[结论]毕赤酵母基因重组菌BH128通过共表达伴侣蛋白Ero1p和PDI,提高了米根霉脂肪酶的产量,而且缩短了发酵周期,为工业化生产奠定了基础.     

解脂耶氏酵母脂肪酶LIP4和LIP5在毕赤酵母中的异源表达及酶学性质

【目的】克隆解脂耶氏酵母(Yarrowia lipolytica)脂肪酶LIP4和LIP5的cDNA序列,研究其基因结构,并实现其在毕赤酵母中的功能表达,以探讨其酶学性质。【方法】利用反转录PCR首次扩增LIP4和LIP5的编码基因,用SignalP 3.0分析其基因序列,然后分别构建胞内表达载体pPIC3.5K-Lip4、pPIC3.5K-Lip5和胞外表达载体pPIC9K-Lip4、pPIC9K-Lip5,将其转入毕赤酵母GS115中表达,以NTA树脂纯化酶蛋白,研究其酶学性质。【结果】cDNA序列测序结果显示两者均不含内含子,酶蛋白的氨基酸序列中含有典型脂肪酶的活性三联体结构和五肽保守区;酶学性质研究表明,两者的最适底物均为癸酸(C8)对硝基苯酚酯,最适pH为7.0,最适温度为40℃,但LIP4对pH和温度更敏感;两者均能被Ca2+激活,且LIP5还能为Mg2+激活,但均被Hg2+、乙二胺四乙酸(EDTA)和苯甲基磺酰氟(PMSF)强烈抑制。【结论】首次克隆了解脂耶氏酵母脂肪酶LIP4和LIP5编码基因,实现了其在毕赤酵母中的活性表达,并初步研究了其酶学性质,为上述脂肪酶的应用及进一步深入研究解脂耶氏酵母脂肪酶家族奠定了基础。     

High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris

Candida parapsilosis has been previously shown to produce a lipase/acyltransferase (EC 3.1.1.3) that preferentially catalyses transfer reactions such as alcoholysis over hydrolysis in the presence of suitable nucleophiles other than water, even in aqueous media (aw > 0.9 ). This enzyme has been shown to belong to a new family of lipases. The present work describes the cloning of the gene coding for this lipase/acyltransferase in the yeast Pichia pastoris and the heterologous high-level expression of the recombinant enzyme. The lipase/acyltransferase gene, in which the sequence encoding the signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was placed under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). A transformed P. pastoris clone, containing five copies of the lipase/acyltransferase gene, was selected for the production of recombinant enzyme. The fed-batch culture supernatant contained 5.8 gl(-1) (weighted) of almost pure recombinant lipase/acyltransferase displaying the same catalytic behavior as the original enzyme.     

Immobilization and stability of a Rhizopus oryzae lipase expressed in Pichia pastoris: comparison between native and recombinant variants

The stability of a soluble extract containing a recombinant lipase from Rhizopus oryzae (Cursive) lipase (rROL) produced by Pichia pastoris (Cursive), as well as that for the commercial extract containing the lipase produced by the native organism (nROL), was investigated. The results showed higher residual activity values of the commercial protein compared with the recombinant one. Moreover, two different kinds of support, the polypropylene powder EP100 and Eupergit®C, were tested to immobilize the enzymes. The residual activity of the immobilizated derivatives was also tested to determine whether their stability was enhanced. The results showed a slight improvement in rROL using both supports but a decrease in nROL using Eupergit®C. The study of the residual activity of soluble and immobilized enzymes was performed by means of a central composite rotatable experiment design. In addition, EP100 adsorption isotherms were determined. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

费希尔曲霉脂肪酶在毕赤酵母中的优化表达及高密度发酵

【背景】脂肪酶广泛应用于纺织、食品、药品、皮革等工业领域,其在微生物中的异源表达研究进一步促进了脂肪酶产品的生产和应用。【目的】实现来源于费希尔曲霉的脂肪酶在毕赤酵母中的高效异源表达,探究其合适的表达及发酵条件,提高产量,降低成本。【方法】对费希尔曲霉的脂肪酶编码基因进行密码子优化后,应用pPIC9k质粒整合到毕赤酵母GS115基因组上,构建高产脂肪酶Lip605的毕赤酵母工程菌;并通过响应面发酵条件优化、筛选最适伴侣蛋白和高密度发酵相结合的方法,综合提高脂肪酶表达量。【结果】确定高产脂肪酶毕赤酵母工程菌的最优摇瓶发酵产酶条件为:甲醇3.103%(体积比),生物素0.4 mg/L,酵母粉11.5 g/L,酵母基础氮源培养基(yeast nitrogen base,YNB) 13.4 g/L,初始pH 6.4,装液量50 mL/250 mL,转速220 r/min,温度24°C,培养时间40 h。优化后的胞外脂肪酶酶活达到72.34 U/mL,较优化前提高了5.8倍;进一步选择12个伴侣蛋白分别与脂肪酶Lip605进行共表达,其中共表达伴侣蛋白Rpl10(pPICZA-RPL10)效果最佳,可使Lip605表达量进一步提高46.8%;在此基础上,经过10 L发酵罐分批补料的高密度发酵,工程菌株发酵142 h,胞外脂肪酶酶活最高达到680 U/mL,蛋白浓度为15.89 g/L。【结论】应用复合策略有效提高了脂肪酶Lip605在毕赤酵母中的发酵产量,为其进一步工业化生产奠定了良好的基础。     

High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase

The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZalphaA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPalpha-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000U/mg protein towards tributyrin. The pure enzyme was characterized and its physicochemical properties were compared to those of the BTL2 lipase, which had previously been expressed in Escherichia coli under the control of its native promoter on pUC18 or under the control of the strong temperature inducible promoter lambdaP(L), yielding 600U/g or 54,000U/g wet cells, respectively. The three proteins showed the same N-terminal sequence and had very similar pH optimum, pH stability, temperature optimum, thermostability, and substrate specificity profiles. Three enzymes were extremely stable in the presence of several organic solvents and detergents.     

米根霉脂肪酶基因pro-ROL和m-ROL在毕赤酵母中的密码子优化、表达和酶学性质的比较分析

米根霉Rhizopus oryzae脂肪酶不仅在众多工业领域中具有良好的应用价值,其典型的分子内伴侣结构(Intramolecular chaperon)也是研究蛋白质翻译后加工和成熟的理想材料.以尼oryzae HU3005为材料,克隆了其脂肪酶前体基因(pro-ROL)和成熟脂肪酶基因(m-ROL),并实现了其在巴斯德毕赤酵母Pichia pastoris GS 115中的分泌表达.酶学性质比较分析表明:m-ROL对中等链长底物(Cl0和C12)具有更高的水解活性,而pro-ROL更倾向于短链底物(C4),且在pH 8.0时活性最高.再者,pro-ROL具有比m-ROL更好的温度稳定性.推测m-ROL和pro-ROL脂肪酶酶学性质的差异可能是由前序列对脂肪酶的折叠和修饰的影响而导致.为提高m-ROL的表达水平,采用重叠延伸PCR技术将基因中8个低频密码子替换为高频密码子.在摇瓶条件下,发酵72 h后,经密码子优化后的m-ROL酶活和蛋白质含量分别达到132.7 U/mL和50.4 U/mL,而初始m-ROL和pro-ROL酶活和蛋白质含量分别仅为28.7 U/mL和14.4 mg/L、29.6 U/mL和14.1 mg/L.     

Impact of NH4+ nitrogen source on the production of Rhizopus oryzae lipase in Pichia pastoris

BackgroundPichia pastoris is a highly successful system for heterologous expression. During the induction stage, the ammonium ion released into the fermentation broth has a deep impact on cell growth and protein expression. The impact of NH₄⁺ concentration on the expression of the Rhizopus oryzae lipase proAROL in P. pastoris was investigated.ResultsThe lipase activity under the optimum NH₄⁺ concentration of 440 mmol/L reached 12,019 U/mL. Increased concentrations of NH₄⁺ in the broth prevented the protease production, resulting in higher specific lipase activity in the supernatant. Furthermore, analysis of carbon metabolism and energy regeneration pattern revealed that under the definite NH₄⁺ concentrations more carbon source (methanol) was consumed with surged AOX activity and then the higher energy and amino acid precursors demand for recombinant protein synthesis is compensated for by the TCA cycle.ConclusionsIn this study, the R. oryzae lipase activity reaches the highest level ever reported under optimized NH₄⁺ concentration and the analysis of the carbon metabolism provides useful information for future optimization of protein production by P. pastoris in a molecular level.     

Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris

To improve the specific production rate of Rhizopus oryzae lipase (ROL) in Pichia pastoris, a protein that triggers the unfolded protein response in P. pastoris, the effect of sorbitol/methanol mixed substrates was tested in batch and fed-batch cultures. Remarkably, a different substrate consumption behaviour was observed depending on the host's phenotype (Mut(+) or Mut(s)) in batch cultures: when the methanol assimilation capacity is genetically reduced (Mut(s) phenotype), both substrates were consumed simultaneously, allowing not only a higher specific growth rate but also higher lipase levels (8.7-fold) compared to those obtained by cells growing on methanol as a sole carbon source in batch culture. This effect was not observed in Mut(+) phenotype, where the two substrates were consumed sequentially and the levels of heterologous product were only slightly higher (1.7-fold). A mixed substrate strategy was also applied to a Mut(s) fed-batch culture at a low methanol concentration set-point (0.5 gl(-1)). This resulted in a 2.2-fold increase in the heterologous protein level achieved, compared with the methanol-only feeding strategy. In addition, sorbitol co-feeding permitted the achievement of higher specific growth rates, and avoided the drastic decrease of the specific production rate observed after the start of the induction phase when methanol was used as sole carbon source This resulted in a significant increase in the overall bioprocess volumetric productivity (2.2-fold) and specific productivity (1.7-fold). Moreover, whereas increased ROL gene dosage in Mut(s) strains have been previously reported to be deleterious for P. pastoris cells growing on methanol, sorbitol co-feeding allowed for sustained cell growth and lipase production.     

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning,expression and characterization

An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP₀₃80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca²⁺ and inhibited by Fe²⁺, Zn²⁺, K⁺, and Ag⁺. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.