Prebleaching of kraft pulp with full-length and truncated forms of a thermostable modular xylanase from Rhodothermus marinus

Full-length and truncated forms of a modular thermostable xylanase (EC 3.2.1.8., glycoside hydrolase family 10) were used in bleaching sequences of hardwood and softwood kraft pulps. Enzymatic treatment led to brightness gains of all pulps but the result depended on the pulp source. The presence of the additional domains in the full-length enzyme (including carbohydrate-binding modules) did not improve the bleaching process. No significant change in viscosity was seen after enzyme treatments indicating an unaffected pulp fibre length.     

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.     

Evidence for substrate binding of a recombinant thermostable xylanase originating from Rhodothermus marinus

The xyn1 encoded 5 domain xylanase from the thermophilic bacterium Rhodothermus marinus binds specifically to xylan, β-glucan and amorphous but not crystalline cellulose. Our results show that the binding is mediated by the full length xylanase, but not by the catalytic domain only. Based on similarities concerning both predicted secondary structure and binding specificity found with one cellulose binding domain of CenC from Cellulomonas fimi, we suggest that the binding is mediated by the two N-terminally repeated domains.     

极端耐热木聚糖酶基因在大肠杆菌和毕赤酵母中的高效表达

以海栖热袍菌 (Thermotoga maritima) MSB8菌株基因组DNA为模板,通过PCR扩增出木聚糖酶（XylanaseB）基因, 将此基因克隆至大肠杆菌表达载体pET_28a（+）和毕赤酵母表达载体pPIC9K,并分别转化大肠杆菌 BL21和毕赤酵母GS115。该木聚糖酶在大肠杆菌细胞中表达量高, 但不能分泌; 而在毕赤酵母细胞的表达产物可分泌至胞外。酶学性质分析表明,此酶分子量约为40kD,其最适反应温度为90℃, 最适反应pH值为6.65,且在碱性条件下稳定,具有重要的工业应用前景。     

Immobilization of a Thermostable Cellobiose 2-Epimerase from Rhodothermus marinus JCM9785 and Continuous Production of Epilactose

Previous study has shown that a crude manganese peroxidase (MnP) preparation from the fungus could bleach oxygen-alkaline treated hardwood kraft pulp (OKP) with manganese, glucose, and glucose oxidase. Using purified MnP instead of the crude one also did OKP bleaching with Tween 20. We conclude that MnP is important in this fungal bleaching system.     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

利用游离型表达质粒强化毕赤酵母表达木聚糖酶

巴斯德毕赤酵母是用途广泛的蛋白表达系统。目前用于毕赤酵母的质粒主要以整合型质粒为主,很少见到游离的质粒用于外源基因的表达。文中通过将来源于酵母自身的自主复制序列连接到酵母整合型表达载体pGAP中构成自主复制的游离型表达载体pGAPZαA-PARS,将该载体用于表达木聚糖酶基因。转化毕赤酵母后同传统的整合型表达菌株相比,以甘油为碳源时最高酶活达到343 U/mL,比整合型表达提高了45.9%。同时游离载体表达重组酶比活相对整合表达提高了81.2%。为了节约发酵成本,进一步研究了分别以甘油、葡萄糖、蔗糖、混合碳源(蔗糖︰甘油=1︰2)等不同碳源下游离型重组菌株的表达水平。发现甘油表达水平最高,蔗糖最低,但是以工业葡萄糖为碳源时产酶成本最低。由于pGAP载体不需要以甲醇为碳源,因而文中所构建的游离载体pGAPZαA-PARS极大促进了毕赤酵母在食品行业中的应用。同时,游离型载体可大幅度提高表达水平,为进一步研究提高GAP启动子的高效表达奠定了基础。     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Cloning and expression of a thermostable alpha-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris

The first gene (alpha-gal1) encoding an extracellular alpha-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The alpha-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal alpha-galactosidases belonging to glycosyl hydrolase family 27. The alpha-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant alpha-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at 70 degrees C, pH 4.5, and lost no activity over 10 days at 50 degrees C. alpha-Gal1 followed Michaelis-Menten kinetics (Vmax of 240.3 micronM/min/mg, Km of 0.294 mM) and was inhibited competitively by galactose (Km obs of 0.57 mM, Ki of 2.77 mM). The recombinant T. emersonii alpha-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the alpha-galactosidic linkage. Owing to its substrate preference and noteworthy stability, alpha-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.     

毕赤酵母高效表达策略概述

毕赤酵母表达系统是外源蛋白表达的较为理想的系统,但是并不是所有蛋白都能利用此系统获得高效表达,不同来源的蛋白,其表达水平、生物活性和稳定性均存有明显差别。概述了影响毕赤酵母高效表达的主要因素以及外源蛋白在毕赤酵母中的高效表达策略。     

Genome-scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production

The methylotrophic yeast Pichia pastoris has gained much attention during the last decade as a platform for producing heterologous recombinant proteins of pharmaceutical importance, due to its ability to reproduce post-translational modification similar to higher eukaryotes. With the recent release of the full genome sequence for P. pastoris, in-depth study of its functions has become feasible. Here we present the first reconstruction of the genome-scale metabolic model of the eukaryote P. pastoris type strain DSMZ 70382, PpaMBEL1254, consisting of 1254 metabolic reactions and 1147 metabolites compartmentalized into eight different regions to represent organelles. Additionally, equations describing the production of two heterologous proteins, human serum albumin and human superoxide dismutase, were incorporated. The protein-producing model versions of PpaMBEL1254 were then analyzed to examine the impact on oxygen limitation on protein production.     

Codon-optimized expression and characterization of a pH stable fungal xylanase in Pichia pastoris

Novel xylanase (EC 3.2.1.8) is in great demand due to its industrial significance. In this study, we have developed and characterized a novel xylanase-producing yeast strain. This mature xylanase gene xyn11A consists of 870 base pairs and belongs to GH11 family. The gene sequence was optimized and synthesized, and was then cloned into yeast vector pGAPZαA under the control of the constitutive GAP promoter. SDS-PAGE analysis indicates that Xyn11A is extracellularly expressed as a glycosylated protein in P. pastoris. Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase retained more than 90% of its activity after incubation at 50 °C and 60 °C for up to 1 h. Xyn11A is also stable over a wide range of pH (2.0–11.0). Most metal ions tested such as copper (Cu²⁺) and lead (Pb²⁺) have little inhibitory effects on Xyn11A. It is also resistant to pepsin and proteinase K digestion, retaining 80% and 90% of its activity after digestion at 37 °C for 1 h, respectively. Those superior properties make Xyn11A a robust xylanase with great potential for industrial use. To the best of our knowledge, this is the first report of xylanase from the fungus Corynascus thermophilus.     

Cloning, sequence analysis and heterologous expression in Pichia pastoris of a gene encoding a thermostable cellobiose dehydrogenase from Myriococcum thermophilum

We cloned and expressed a gene encoding a thermostable cellobiose dehydrogenase (CDH) from the thermophilic ascomycete Myriococcum thermophilum. The 2904 bp long open reading frame contained six introns located either close to the 5′- or 3′-end of the ORF. The corresponding cDNA of 2487 bp was cloned into the expression vector pPICZαB to achieve inducible heterologous expression and secretion of the recombinant flavocytochrome in the methylotrophic yeast Pichia pastoris. Transformants were selected on media with normal and 10-fold increased zeocin concentration, and selected clones were tested for inducible extracellular production of the recombinant oxidoreductase. The maximally obtained volumetric activity was 0.25 U/ml in YPM (rich) medium and 2.15 U/ml in production stage (minimal) medium in a fed-batch fermentation. Recombinant CDH was purified in two consecutive chromatographic steps leading to a final specific activity of up to 7.4 U/mg protein at 40 °C. Kinetic properties of the recombinant CDH were characterized and the temperature optimum for the recombinant CDH was determined at 63 °C. Certain properties of the sequence of MtCDH are discussed in context with thermal and proteolytic stability.     

The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase

The solution structure is presented for the second family 4 carbohydrate binding module (CBM4-2) of xylanase 10A from the thermophilic bacterium Rhodothermus marinus. CBM4-2, which binds xylan tightly, has a beta-sandwich structure formed by 11 strands, and contains a prominent cleft. From NMR titrations, it is shown that the cleft is the binding site for xylan, and that the main amino acids interacting with xylan are Asn31, Tyr69, Glu72, Phe110, Arg115, and His146. Key liganding residues are Tyr69 and Phe110, which form stacking interactions with the sugar. It is suggested that the loops on which the rings are displayed can alter their conformation on substrate binding, which may have functional importance. Comparison both with other family 4 cellulose binding modules and with the structurally similar family 22 xylan binding module shows that the key aromatic residues are in similar positions, and that the bottom of the cleft is much more hydrophobic in the cellulose binding modules than the xylan binding proteins. It is concluded that substrate specificity is determined by a combination of ring orientation and the nature of the residues lining the bottom of the binding cleft.     

Mutation of cysteine residues increases heterologous expression of peach expansin in the methylotrophic yeast Pichia pastoris

The study of Carbohydrate-Active enZymes (CAZymes) associated with plant cell wall metabolism is important for elucidating the developmental mechanisms of plants and also for the utilization of plants as a biomass resource. The use of recombinant proteins is common in this context, but heterologous expression of plant proteins is particularly difficult, in part because the presence of many cysteine residues promotes denaturation, aggregation and/or protein misfolding. In this study, we evaluated two phenotypes of methylotrophic yeast Pichia pastoris as expression hosts for expansin from peach (Prunus persica (L.) Batsch, PpEXP1), which is one of the most challenging targets for heterologous expression. cDNAs encoding wild-type expansin (PpEXP1_WT) and a mutant in which all cysteine residues were replaced with serine (PpEXP1_CS) were each inserted into expression vectors, and the protein expression levels were compared. The total amount of secreted protein in PpEXP1_WT culture was approximately twice that of PpEXP1_CS. However, the amounts of recombinant expansin were 0.58 and 4.3 mg l⁻¹, corresponding to 0.18% and 2.37% of total expressed protein, respectively. This 13-fold increase in production of the mutant in P. pastoris indicates that the replacement of cysteine residues stabilizes recombinant PpEXP1.     

Modification of a gene encoding hybrid xylanase and its expression in Pichia pastoris

To obtain the protein expression of a hybrid xylanase in yeast, the gene encoding it was modified according to the codon bias of Pichia pastoris and expressed extracellularly in this yeast as an active xylanase, MBtx, exhibited a molecular mass of approximately 35 kDa on SDS–PAGE. The pH behavior of MBtx in terms of both activity and stability was similar to that of Btx, original gene product in Escherichia coli, while a certain difference was observed in optimal temperature for activity and in thermal stability. HPLC analysis revealed the xylan in wheat could be hydrolyzed by MBtx and the major hydrolysis product was xylotriose. These results showed codon usage played a key role in regulating the expression of the hybrid xylanase in P. pastoris and the recombinant hybrid xylanase, MBtx, produced by P. pastoris could be potentially useful in feed industry.     

甘油相控制策略对重组毕赤酵母表达瑞替普酶(reteplase)的影响

研究了甘油补料策略对毕赤酵母表达reteplase(rPA)的发酵过程中细胞的生长和rPA表达的影响。通过将对甘油补料速率由10g/L.h-1增大到20g/L.h-1,细胞比生长速率,甘油的比消耗速率,甘油得率等都得到极大提高。而且,诱导期细胞生长,甲醇消耗和rPA的生成速率都增加,rPA的最大表达量从140.2mg/L增加到199.5mg/L。此另外,甘油补料时间也是影响rPA表达的重要因素,甘油补料时间短,细胞密度小,表达rPA少,甘油补料时间长,细胞密度增大,rPA的表达速率也增加,但是到诱导后期,细胞死亡率增大,蛋白酶释放增加,rPA的降解增强。     

青霉菌聚半乳糖醛酸酶基因在毕赤酵母中的表达及酶学性质研究

筛选得到一株能分解果胶的青霉菌(Penicillium sp.),使用简并引物PCR和TAIL-PCR方法从该菌中克隆了一个聚半乳糖醛酸酶基因pgp1.pgp1基因全长1 225 bp,包含2个内含子,其cDNA全长1 104 bp,编码367个氨基酸和一个终止密码子,前18个氨基酸为信号肽序列.将pgp1基因连接pPIC9载体,在巴斯德毕赤酵母表达系统中进行了异源表达.在3L发酵罐水平,培养基中聚半乳糖醛酸酶活力达到700 U/mL.酶学性质测定表明,重组酶蛋白PGP1的最适pH为5.0,在pH4.0 -6.0下处理1h后,剩余酶活力超过90％;最适温度为38℃,以聚半乳糖醛酸为底物,PGP1的Km=(1.172±0.169)mg/mL,Vmax=(0.061±0.002) mg/min/mL.     

Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris

The human interferon alpha2b (hIFN-alpha2b) is the most widely used member of IFNalpha family, and it exerts many biological actions including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. Herein, the cDNA coding for hIFN-alpha2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-alpha2b has been achieved. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-alpha2b, a 18.8 kDa protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using Source Q ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 298 mg of the protein was obtained in high purity from 1l of the supernatant and its identity to hIFN-alpha2b was confirmed by NH(2)-terminal amino acid sequence analysis. The bioassay of the recombinant protein gave a specific activity of 1.9 x 10(9)IU/mg. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hIFN-alpha2b for both research and industrial purpose.