Expression and characterization of a thermostable serine protease (TfpA) from Thermomonospora fusca YX in Pichia pastoris

A serine protease produced by Thermomonospora fusca YX (TfpA) is heat-stable (up to 85°C) and has a broad pH activity range and strong resistance to detergents. The objective of this study was to determine if the methylotropic yeast Pichia pastoris could express TfpA extracellularly. A 1.0-kb DNA fragment (tfpA) encoding the pro-peptide and mature protein of TfpA was cloned into expression vectors pPICZA (inducible) and pGAPZA (constitutive) and introduced into P. pastoris by electroporation. Expression of r-TfpA was greater in the inducible system than in the constitutive one, producing 135 U ml^–1 medium supernatant 6 days after methanol induction. The r-TfpA was not glycosylated (21.7 kDa), and had pH and temperature optima of 8.5 and 80°C, respectively, using azocasein as a substrate. In conclusion, P. pastoris can be used as a host to produce extracellular r-TfpA, and expression efficiency may be improved by optimizing fermentation conditions and modifying factors related to protein expression and stability.     

A novel raw starch digesting thermostable α-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch

A new strain of Bacillus sp. I-3, isolated from natural soil samples, showed a high raw starch digesting activity towards potato starch. Upon optimization of various environmental and cultural conditions, the yield of α-amylase reached 642 U/mL. The kinetic characterization of partially purified enzyme exhibited the maximum activity at 70 °C, pH 7.0 and revealed a high thermostability in the presence of 10 mM CaCl₂·2H₂O where it could retain more than 90% residual activity at 70 °C after 3.5 h. At 80, 90 and 100 °C, the enzyme retained 80, 59 and 26% of its maximum activity after 2.5, 0.5 and 0.5 h, respectively. The enzyme preparation had a strong affinity towards raw potato starch granules and was almost completely adsorbed onto it. It also hydrolyzed raw potato starch at a concentration of 12.5% significantly in a short period of time of 12 h.     

Production and properties of alpha-amylase from Penicillium chrysogenum and its application in starch hydrolysis

Fungi were screened for their ability to produce alpha-amylase by a plate culture method. Penicillium chrysogenum showed high enzymatic activity. Alpha-amylase production by P. chrysogenum cultivated in liquid media containing maltose (2%) reached its maximum at 6-8 days, at 30 degrees C, with a level of 155 U ml(-1). Some general properties of the enzyme were investigated. The optimum reaction pH and temperature were 5.0 and 30-40 degrees C, respectively. The enzyme was stable at a pH range from 5.0-6.0 and at 30 degrees C for 20 min and the enzyme's 92.1% activity's was retained at 40 degrees C for 20 min without substrate. Hydrolysis products of the enzyme were maltose, unidefined oligosaccharides, and a trace amount of glucose. Alpha-amylase of P. chrysogenum hydrolysed starches from different sources. The best hydrolysis was determined (98.69%) in soluble starch for 15 minute at 30 degrees C.     

耐热羧肽酶Taq基因在毕赤酵母中的表达

为了获取表达羧肽酶Taq毕赤酵母工程菌,通过密码子优化,依据毕赤酵母密码子偏爱性,在体外合成了栖热水生菌的耐热羧肽酶Taq基因。将该基因克隆到毕赤酵母表达载体p HBM905A上并引入6×His标签,构建了重组质粒p HBM905A-Cpase Taq。将该重组质粒转化毕赤酵母GS115,经1%甲醇诱导表达72 h,酶产量达0.1 mg/m L。对纯化的重组酶进行酶活性分析表明在75℃,p H为7.5时,该酶比酶活性为80 U/mg。本研究首次证明了羧肽酶Taq能在毕赤酵母中有效分泌表达,可以被大量制备,进而为多肽水解为氨基酸奠定工业基础。     

Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from Aspergillus awamori KT-11

Complementary DNAs encoding alpha-amylases (Amyl I, Amyl III) and glucoamylase (GA I) were cloned from Aspergillus awamori KT-11 and their nucleotide sequences were determined. The sequence of Amyl III that was a raw starch digesting alpha-amylase was found to consist of a 1,902 bp open reading frame encoding 634 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. On the other hand, the sequence of Amyl I, which cannot act on raw starch, consisted of a 1,500 bp ORF encoding 499 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. The sequence of GA I consisted of a 1,920 bp ORF that encoded 639 amino acids. The signal peptide was composed of 24 amino acids. The amino acid sequence of Amyl III from the N-terminus to the amino acid number 499 showed 63.3% homology with Amyl I. However, the amino acid sequence from the amino acid number 501 to C-terminus, including the raw-starch-affinity site and the TS region rich in threonine and serine, showed 66.9% homology with GA I.     

Heterologous expression of <Emphasis Type=Italic>Thermobifida fusca</Emphasis> thermostable alpha-amylase in <Emphasis Type=Italic>Yarrowia lipolytica</Emphasis> and its application in boiling stable resistant sago starch preparation

A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP _w of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.     

毕赤酵母中tRNAPro CCG基因的表达及其作用效果

转运核糖核酸 (tRNA) 是蛋白质合成过程中重要参与成分之一,为了探索稀有密码子对应的tRNA (稀少tRNA) 丰度改变对外源基因表达量的影响,文中构建了毕赤酵母稀少tRNA基因与外源基因共表达体系。首先在GFP基因中添加由4个连续脯氨酸稀有密码子CCG组成的阻遏区,结果显示该GFP基因的表达量明显降低。然后将带有阻遏区的GFP基因和tRNAPro CCG基因顺次连接于pPIC9K载体上,在毕赤酵母GS115中共表达,结果使GFP表达量提高了4.9%;另将带有阻遏区的GFP基因和tRNAPro CCG基因分别连接于pPIC9K和pFLDα载体,在毕赤酵母GS115中共表达,GFP表达量最高提高了12.5%;应用同样方式将tRNAPro CCG基因与NFATc3T-GFP融合基因共表达,其表达量提高了21.3%。可见,tRNAPro CCG在毕赤酵母GS115中确为稀少tRNA,通过共表达tRNAPro CCG基因可显著提高带有连续该密码子的外源基因表达量,并且,文中构建的共表达体系将同样适用于其他稀少tRNA基因的筛选和验证。     

人Tumstatin在毕赤酵母中的表达和活性分析

利用PCR技术从重组质粒pET-3c-tum中扩增人tumstatin的cDNA片段,连入pPICZαA酵母表达载体,获得的重组质粒pPICZα-tum电激法转化毕赤酵母GS115。经表型鉴定、诱导表达筛选,得到可分泌表达人tumstatin的重组酵母转化子,表达蛋白质的相对分子量约30kD,表达量约25mg/L。表达上清经超滤浓缩和离子交换法初步纯化,所得产物具有免疫活性,能够抑制内皮细胞增殖,诱导其发生细胞凋亡,并能抑制鸡胚尿囊膜血管生成。     

Direct screening of libraries of yeast clones for alpha-amylase activity on raw starch hydrolysis

High-throughput screening for high-activity barley alpha-amylase mutants expressed in Saccharomyces cerevisiae is hampered by the interference of reducing agents, particularly the glucose used in yeast growth media. The present investigation employed colorimetric and chemiluminescent detection systems that enable direct and rapid screening of activities on raw starch substrate. Active clones could be separated into two groups, based on high total activity or high specific activity.     

A novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization

This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase.     

Purification and characterization of a thermostable raw starch digesting amylase from a Streptomyces sp. isolated in a milling factory

A raw starch utilizing microbe was isolated from mud in a milling factory. The 16S ribosomal DNA (rDNA) sequencing and morphological properties of the strain indicated that it belongs to the genus Streptomyces. A strongly raw starch digesting amylase was purified from the culture supernatant of the strain by chromatographic procedures. The specific activity of the enzyme was 11.7 U/mg, molecular mass 47 kDa, optimum pH 6.0, and optimum temperature 50 to 60 degrees C. The enzyme showed sufficient activity even at 70 degrees C. It was activated by calcium, cobaltous, and magnesium ions, and inhibited by copper, nickel, zinc, and ferrous ions. It formed maltose mainly from raw and gelatinized starch, and glycogen. No products were formed from glucose, maltose, maltotriose, pullulan, or cyclodextrins (CDs). The enzyme digested raw wheat, rice, and waxy rice starch rapidly, and raw corn, waxy corn, sweet potato, tapioca, and potato starch normally.     

耐高温α—淀粉酶基因在马铃薯中的表达

The thermostable alpha-amylase gene cloned from Bacillus lichemiformis was reconstructed into an expression vector pAMY721M under the CaMV35S promoter. The vector was transferred into A. tumerfaciens ABI. The thermostable alpha-amylase gene was transferred into Solanum tuberosum L. via Agrobacterium mediation according to the revised method of ZHAO Shu Juan et al (1997) and YANG Mei Zhu et al (1992). Shoots were induced and regenerated on MS medium with 2 mg/L ZT, 0.1 mg/L IAA and 100 mg/L kanamycin. Putative transformants were selected with kanamycin and roots induced on MS medium with 0.15 mg/L IAA. PCR analysis and thermostable alpha-amylase activity assay were done to identify the transgenic plantlets. Among them, 102,001, 102,607, and 110,402 were showed to have a higher alpha-amylase activity than untransformed control.     

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris

AIMS: Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. METHODS AND RESULTS: First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4.5-5.0 and 65 degrees C. The enzyme was stable at 60 degrees C, the enzyme activity kept 80% after 60 min incubation at 70 degrees C. The half-life was 40 and 10 min under incubation at 80 and 90 degrees C respectively. CONCLUSIONS: A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.     

热稳定高比活纤维素酶在Pichia pastoris中的表达研究

Bacillus amyoliquefaciens DL-3纤维素酶具有热稳定高比活多功能的酶学特性,本文根据该酶的氨基酸序列合成了其编码基因(cel),构建了pPIC9K-cel表达载体,并用P.pastoris进行了表达。工程菌株三角瓶发酵酶活性达0.50 U/mL,酶解滤纸的产物为低聚糖,表明人工合成的热稳定高比活纤维素酶基因在P.pastoris中可以正常表达、加工及分泌,重组酶的分子量由天然酶的53 kDa增加至68 kDa,糖基化严重。     

人纤溶蛋白酶原K5在毕赤酵母中的表达及其生物活性鉴定

化学合成人纤溶蛋白酶原K5 (pK5 )的编码基因并克隆到毕赤氏酵母表达系统的分泌型载体pPIC9K上 ,将重组质粒经BglⅡ单酶切后电转化PichiapastorisGS115菌株 ,筛选出对G4 18有高抗性和在MM培养基上生长缓慢的转化子。经摇瓶发酵和甲醇诱导后 ,用 15 %SDS PAGE检测发酵上清液 ,表明有重组蛋白pK5的高表达。经CM-Sepherose离子交换柱和Superdex 75分子筛层析两步分离纯化 ,获得了纯度达到 98%的rpK5。用MTT方法检测的结果表明 ,纯化的rpK5可显著地抑制人血管内皮细胞的生长     

Mutational Bias and Gene Expression Level Shape Codon Usage in Thermobifida fusca YX

In this study major factors shaping codon and amino acid usage variation in Thermobifida fusca YX are reported. It is a major degrader of plant cell walls. It produces spores that can be allergenic and has been associated with a condition called farmers lung. For comparison, two other closely related Actinobacteria, S. coelicolor and N. farcinica were considered. Correspondence analysis on RSCU (Relative Synonymous Codon Usage) showed significant correlation between the major trend of codon usage variation and gene expression level assessed by the "Codon Adaptation Index" (CAI) values. The result was further confirmed from distribution of genes along the first axis. In addition, N_{c} (effective number of codons) plot, SCUO (synonymous codon usage order) plot and correlation analyses showed that base composition and mutational bias have a dominant role in codon usage variation. Furthermore, gene expression level, hydrophobicity and aromaticity have played a significant role in the source of variations for amino acid usage. In addition, codon preference for genes at higher expression level was found to be similar among three different genera. Notably, 14 codons optimally used by Thermobifida fusca YX and its comparative study with S. coelicolor and N. farcinica might provide some useful information for their further study of molecular evolution and genetic engineering.     

一种新型巴斯德毕赤酵母表达载体的构建及甘露聚糖酶的表达

目的:构建以带自身启动子的蔗糖转化酶基因(suc2)为选择标记的载体,用于外源基因在巴斯德毕赤酵母中的正确分泌表达。方法:根据已发表的蔗糖转化酶基因序列设计并合成1对引物,应用PCR技术,以啤酒酵母INVSC1总DNA为模板,扩增出包含自身启动子和终止区序列的suc2基因。将该基因与毕赤酵母表达载体pPIC9K连接,构建了以suc2为选择标记的表达载体pPIC12K。将甘露聚糖酶基因man克隆入载体pPIC12K,用PEG/LiCl法转化毕赤酵母GS115菌株。以蔗糖为惟一碳源筛选转化子,利用底物平板检测筛选到的转化子中man基因的表达,并对重组表达菌株进行连续传代实验。结果:部分转化子周围产生明显的水解圈,证明甘露聚糖酶已经得到分泌表达;对重组表达菌株的连续传代实验证实了该表达载体具有良好的遗传稳定性。结论:以带自身启动子的suc2基因为选择标记的表达载体构建成功,并且这个新型表达载体能够对外源基因进行稳定有效的分泌表达。     

Expression of lignocellulolytic enzymes in Pichia pastoris

ABSTRACT: BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes even gram quantities of enzyme per liter were obtained by fed-batch cultivation. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly see the importance of gene optimization and strain characterization for successfully improving secretion levels. We also give a basic guideline for understanding the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.     

重组瑞替普酶在毕赤酵母中的表达

【目的】瑞替普酶(重组组织纤溶酶原激活物,rt PA)被认为是第三代安全有效的溶栓剂,以p PIC9K为载体,以3种不同表型的毕赤酵母(Pichia pastoris)为宿主,探索适合rt PA分泌型表达的最佳体系。【方法】以质粒p ET28a-rt PA为模板,设计特异性引物,PCR扩增目的基因rt PA,插入分泌型表达载体p PIC9K中,获得重组表达质粒p PIC9K-rt PA。重组质粒经限制性内切酶Sal I线性化后,电击转化至3种不同表型的P.pastoris(GS115、SMD1168、KM71)中进行组成型表达;重组表达体系进行甲醇诱导表达,对产物进行Western blot鉴定,并采用纤维蛋白平板溶圈法测定其活性。【结果】重组蛋白分子量约为43 k D;rt PA-GS115和rt PA-KM71均在39 k D处有特异性条带,且前者在32 k D处有轻微降解条带,而后者并无此现象;rt PA-SMD1168无降解现象,且rt PA-SMD1168比活性较rt PA-GS115高27%;rt PA-KM71表达量和活性均为最低。【结论】从重组蛋白生物活性出发,P.pastoris SD1168可作为rt PA的最佳表达体系,在控制宿主蛋白酶活性、减少产物降解的前提下,P.pastoris GS115也是rt PA表达的优选体系。