Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.     

Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris

One of the reasons for limited heterologous protein secretion in Pichia pastoris is the suboptimal folding conditions inside the cell. The Hsp70 and Hsp40 chaperone families in the cytoplasm or the ER regulate the folding and secretion of heterologous proteins. Here, we have studied the effect of chaperones Ydj1p, Ssa1p, Sec63p and Kar2p on the secretory expression of Candida antarctica lipase B (CalB) protein. Expression of CalB in P. pastoris resulted in the induction of Kar2p secretion into the medium surpassing the retrieval capacity of the cell. Individual overexpression of Ydj1p, Ssa1p and Sec63p in recombinant P. pastoris increased CalB expression level by 1.6-, 1.4- and 1.4-fold respectively compared to the control strain harboring only the CalB gene. However, overexpression of Kar2p had a negative effect on the expression of CalB. Moreover, Western blot analysis indicated accumulation and secretion of Kar2p in the ER, Golgi and extracellular medium in the chaperone coexpression strains. When expressed in combinations such as Ydj1p–Ssa1p, Ydj1p–Sec63p, Kar2p–Ssa1p, Kar2p–Sec63p, the expression level of CalB was increased by 2.5-, 1.5-, 1.5- and 1.5-fold respectively. Contrastingly, the Kar2p–Ydj1p combination resulted in decreased CalB secretion in the supernatant. From these results, we conclude that overexpression of Kar2p is not required for the secretion of CalB. Also, our work confirmed the synergistic effect of Ssa1p and Ydj1p chaperones in the expression of CalB.     

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the α-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins α-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with α-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with α-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using α-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with α-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.     

南极假丝酵母脂肪酶B基因在大肠杆菌中的表达和发酵优化

旨在利用大肠杆菌实现南极假丝酵母脂肪酶B(CALB)基因的高效可溶性表达,并降低生产成本.构建带有不同信号肽的CALB基因表达质粒,转化至不同大肠杆菌宿主中,在摇瓶中进行基础培养基、诱导条件、培养基组成成分和进程曲线的优化.结果显示,带有PelB信号肽的重组菌pET25b-CALB-1/Rosetta(DE3)在20℃...     

CALA在毕赤酵母中的表达、纯化及酶学特性研究

将南极假丝酵母脂肪酶A(cala)基因克隆至组成型表达载体pGAPZαA中,电激转入X-33,获得高效表达的CALA酵母工程菌株.发酵液上清经超滤浓缩、硫酸铵沉淀和阴离子交换层析等步骤,获得纯化的重组CALA,其比酶活达384.90 U/mg.该酶最适温度为70℃,最适pH值为8.0.经50℃保温2 h,仍含有60%水解酶活力;在pH7.0和8.0溶液中比较稳定.经DMSO处理1 h,仍保持90%的活性;非离子型表面活性剂能提高CALA的酶活,金属离子在不同程度上抑制CALA的酶活.     

Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis

Increasing the expression yield of active Candida antarctica lipase B (CAL-B) in Escherichia coli was achieved by using a codon-optimized synthetic gene and by mutagenesis to introduce hydrophilic residues on the surface of CAL-B. Five residues (four leucines and one isoleucine) on the surface of CAL-B were selected and changed with aspartate after codon optimization. While the codon-optimized synthetic gene of CAL-B did not increase the expression yield, the mutation increased the activity of the enzyme three-fold (3.3 mg/l of culture) compared to the wild type. The mutant enzyme had similar hydrolytic activity toward hydrolysis of p-nitrophenyl acetate or p-nitrophenyl butyrate and enantioselectivity toward hydrolysis of (R, S)-1-phenylethyl acetate compared to the wild-type enzyme. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

Functional expression of Candida antarctica lipase B in Eschericha coli

Candida antarctica lipase B (CalB) is an important catalyst in bio-organic synthesis. To optimize its performance, either the reaction medium is changed or the lipase itself is modified. In the latter case, mutants are generated in Eschericha coli and subsequently expressed in fungal hosts for their characterization. Here we present the functional expression of CalB in the periplasm of E. coli. By step-wise deletion of the CalB signal and propeptide we were able to express and purify two different variants of CalB (mature CalB and CalB with its propeptide). A N-terminal FLAG and a C-terminal His tag were used for the purification. For the substrates para-nitrophenol butyrate (p-NPB), para-nitrophenol laurate (p-NPL) and carboxyfluorescein diacetate (CFDA) the specific activity was shown to be similar to CalB expressed in Aspergillus oryzae. The kinetic constants k(M), v(max) and k(cat) were determined using the substrates p-NPB and p-NPL. Almost identical k(cat)/k(M) values (0.423-0.466 min(-1) microM(-1) for p-NPB and 0.068-0.071 min(-1) microM(-1) for p-NPL) were obtained for the CalB variants from E. coli and A. oryzae. The results clearly show that CalB can be functionally expressed in E. coli and that the attachment of tags does not alter the properties of the lipase.     

Soluble expression of Candida antarctica lipase B in Escherichia coli by fusion with Skp chaperone

Candida antarctica lipase B (CalB) is an industrially versatile enzyme, especially for biodiesel production and organic synthesis. Recombinant expression using the E. coli system has advantages, such as lower costs, easier handling, and higher number of clones that can be screened daily compared to expression using higher organism. But the expression of CalB in E. coli is not feasible because insoluble aggregates are formed and proteolytic degradation is known to occur during expression. In this study, fusion proteins were designed to express soluble CalB in E. coli. The periplasmic chaperone of E. coli, Skp was fused with CalB and this fusion protein showed a high solubility (yielding 82.5 ??g/mL). The fusion protein system can be applied to the rapid expression and evaluation of CalB variants for functional improvement.     

Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli

Lipase (EC 3.1.1.3) is a popular enzyme used as an ingredient in detergents and biocatalyst in many biochemical reactions. Lipase is usually expressed in Escherichia coli as an inactive inclusion body and at a low level. In this study, Candida antarctica lipase B (CalB) was fused with various polycationic amino acid tags and expressed in E. coli in order to increase a soluble expression level. By induction with 1.0 mM IPTG, the authentic and fused CalBs were expressed at 27-56% of total protein. The 10-arginine and 10-lysine tags fused at the C-terminal of CalB significantly increased the solubility of CalB by five- to ninefold, relative to the case of the authentic CalB expressed in a recombinant E. coli Origami 2? (DE3) strain. Among a series of the C-terminal poly-arginine tags, the recombinant CalB combined with the 10-arginine tag (CalB-R10) possessed the highest lipase specific activity of 9.5 ± 0.03 U/mg protein, corresponding to a fourfold enhancement compared with the authentic CalB.     

Expression of functional Candida antarctica lipase B in a cell‐free protein synthesis system derived from Escherichia coli

This article reports the cell‐free expression of functional Lipase B from Candida antarctica (CalB) in an Escherichia coli extract. Although most of the cell‐free synthesized CalB was insoluble under conventional reaction conditions, the combined use of molecular chaperones led to the soluble expression of CalB. In addition, the functional enzyme was generated by applying the optimal redox potential. When examined using p‐nitrophenyl palmitate as a substrate, the specific activity of the cell‐free synthesized CalB was higher than that of the reference protein produced in Pichia pastoris. These results highlight the potential of cell‐free protein synthesis technology as a powerful platform for the rapid expression, screening and analysis of industrially important enzymes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effects of organic solvents on activity and conformation of recombinant Candida antarctica lipase A produced by Pichia pastoris

The effect of various organic solvents on the hydrolytic and esterification activities of lipase A from Candida antarctica (CALA) was investigated. Compared to the control group, the esterification and hydrolytic activities of CALA both increased with treatment of acetonitrile or acetone. However, the catalytic activity decreased after treatment with other organic solvents, especially with ethanol or ethyl acetate. Moreover, with treatment by the same organic solvents, the residual esterification activity of CALA was much higher than the hydrolytic activity for the olive oil. Results of Fourier transform-infrared spectroscopy analysis implied that the catalytic activity variance was mainly attributed to the secondary structure changes of CALA. The acetonitrile treatment could also increase the thermal and pH stability of CALA.     

毕赤酵母展示表达南极假丝酵母脂肪酶B

将南极假丝脂肪酶B(CALB)基因N端和C端,分别与酿酒酵母絮凝蛋白(Flo1p)絮凝结构域序列的N端(FS)和C端(FL)融合,构建成脂肪酶毕赤酵母表面展示载体KFS和KFL,并转化毕赤酵母GS115后获得重组子KFS-CALB和KFL-CALB。免疫荧光检测证实脂肪酶已展示于毕赤酵母细胞表面。甲醇诱导120 h后展示酶活性分别达到286 U/g干细胞和182 U/g干细胞。酶的热稳定性较游离酶有较大提高,50℃孵育4 h后KFS-CALB菌株的残留酶活力仍保持初始酶活力70%以上;KFL-CALB在50℃孵育2 h后的酶活力也达到初始酶活力50%,远远高于游离态的CALB,其在50℃孵育0.5 h后仅残留18%的初始酶活力。     

Double Candida antarctica lipase B co-display on Pichia pastoris cell surface based on a self-processing foot-and-mouth disease virus 2A peptide

To develop a high efficiency Candida antarctica lipase B (CALB) yeast display system, we linked two CALB genes fused with Sacchromyces cerevisiae cell wall protein genes, the Sed1 and the 3′-terminal half of Sag1, separately by a 2A peptide of foot-and-mouth disease virus (FMDV) in a single open reading frame. The CALB copy number of recombinant strain KCSe2ACSa that harbored the ORF was identified, and the quantity of CALB displayed on the cell surface and the enzyme activity of the strain were measured. The results showed that the fusion of multiple genes linked by 2A peptide was translated into two independent proteins displayed on the cell surface of stain KCSe2ACSa. Judging from the data of immunolabeling assay, stain KCSe2ACSa displayed 94?% CALB-Sed1p compared with stain KCSe1 that harbored a single copy CALB-Sed1 and 64?% CALB-Sag1p compared with stain KCSa that harbored a single copy CALB-Sag1 on its surface. Besides, strain KCSe2ACSa possessed 170?% hydrolytic activity and 155?% synthetic activity compared with strain KCSe1 as well as 144?% hydrolytic activity and 121?% synthetic activity compared with strain KCSa. Strain KCSe2ACSa even owned 124?% hydrolytic activity compared with strain KCSe2 that harbored two copies CALB-Sed1. The heterogeneous glycosylphosphatidylinositol-anchored proteins co-displaying yeast system mediated by FMDV 2A peptide was shown to be an effective method for improving the efficiency of enzyme-displaying yeast biocatalysts.     

Biomimetic affinity purification of Candida antarctica lipase B

Candida antarctica lipase B (CalB) is one of the most widely used biocatalysts in organic synthesis. The traditional method for purification of CalB is a multi-step, high cost and low recovery procedure. Biomimetic affinity purification had high efficiency purification. We selected 298 ligand columns from a 700-member library of synthetic ligands to screen Pichia pastoris protein extract. Of the 298, three columns (named as A9-14, A9-10, and A11-33) had one-step purification effect, and A9-14 of these affinity ligands, had both high purification and recovery. The one-step recovery of CalB reached 73% and the purification reached 91% upon purification. The active groups of A9-14 were cyclohexylamine and propenylamine. Furthermore, both A9-14 and A9-10 had the same R1 active group of cyclohexylamine which might act the main binding role for CalB. The synthetic ligand A9-14 had a binding capacity of 0.4 mg/mL and had no negative effects on its hydrolytic activity. Unlike a natural affinity ligand, this synthetic ligand is highly stable to resist 1M NaOH, and thus has great potential for industrial scale production of CalB.     

Expression of chondroitin-4- O -sulfotransferase in Escherichia coli and Pichia pastoris

Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.     

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.     

Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris

Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria. However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme. We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols. When expressed as a fusion protein in E. coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins. The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P. pastoris. To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated. Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme. Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme. This supports previous observations that the enzyme in vivo may be stabilized by additional interactions.