Characterisation of mutagenised acid-resistant alpha-amylase expressed in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> WB600

Based on the original thermostable alpha-amylase gene from Bacillus licheniformis, two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134→Arg and Ser320→Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0 to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein.     

Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained.     

短短小芽孢杆菌大肠杆菌穿梭分泌表达载体的构建

应用PCR技术从具有分泌蛋白能力强且没有胞外蛋白酶活性的短短小芽孢杆菌50中分离出细胞壁蛋白基因的多启动子和信号肽编码序列,利用它与质粒pUB110和pKF3-起构建成穿梭分泌表达载体pBKE50,将α0淀粉酶基因引入该载体转化短短小芽孢杆菌50后,发现α－淀粉酶可以活性形式分泌表达,此工作为下一步建立短短小芽孢杆菌高效分泌表达系统奠定了基础。     

Cloning of L-amino acid deaminase gene from Proteus vulgaris

The L-amino acid degrading enzyme gene from Proteus vulgaris was cloned and the nucleotide sequence of the enzyme gene was clarified. An open reading frame of 1,413 bp starting at an ATG methionine codon was found, which encodes a protein of 471 amino acid residues, the calculated molecular weight of which is 51,518. The amino acid sequence of P. vulgaris was 58.6% identical with the L-amino acid deaminase of P. mirabilis. A significantly conserved sequence was found around the FAD-binding sequence of flavo-proteins. The partially purified wild and recombinant enzymes had the same substrate specificity for L-amino acids to form the respective keto-acids, however not for D-amino acids.     

Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.     

Improvement of cloned alpha-amylase gene expression in fed-batch culture of recombinant Saccharomyces cerevisiae by regulating both glucose and ethanol concentrations using a fuzzy controller

The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B-12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the alpha-amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. The increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy ontroller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed-batch culture. The synthesis of alpha-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory alpha-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory alpha-amylase activities of cells harboring plasmids pNA3 and pNA7 in fed-batch culture were 175 and 395 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled. (c) 1994 John Wiley & Sons, Inc.     

Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.     

Liposome-assisted selective polycondensation of alpha-amino acids and peptides

The main question of this paper is whether and to what extend lipid bilayers can aid in the polycondensation of amino acids and peptides. This means in particular how such bilayers can favor the selection of certain sequences out of a large number of theoretical possible ones. In a first series of experiments we started from a library of Trp-containing dipeptides of the type Trp-X where X is an amino acid residue; and we could show that, when adding this mixture to the POPC liposomes containing a hydrophobic quinoline condensing agent (EEDQ), only the hydrophobic Trp-Trp dipeptide is selected out by the liposomes and transformed into a longer oligomer. Trp-oligomers up to 29 monomers long (water insoluble) could be obtained by using the matrix support of liposomes. Mixed POPC/DDAB liposomes (positive charge) were used to produce co-oligopeptides that contain Trp and Glu residues in the same sequence. Arg/Trp and His/Trp containing sequences were obtained in presence of negatively charged liposomes (mixed POPC/DOPA-liposomes). The polycondensation of racemic NCA-amino acids has been studied to clarify if homochiral sequences are produced preferentially in presence or absence of liposomes. LC-MS and isotope labeling of the L-amino acid, participating in the polymerization reaction achieved this on the level of a direct product analysis. So the individual stereoisomer distribution up to a polymerization degree of 10 (in the case of Trp) could be determined. The data for Trp and other amino acids (Leu, Ile) and amino acid mixtures (Trp/Leu, Trp/Ile, Leu/Ile and Trp/Leu/Ile) show that homochiral sequences are produced preferentially if compared with a random (Bernoulli) distribution.     

Existence of beta-methylnorleucine in recombinant hirudin produced by Escherichia coli.

A gene encoding for hirudin, a potent thrombin inhibitor, was expressed in Escherichia coli, which is the most widely used host. When the recombinant hirudin analog, CX-397, was overproduced by E. coli (600 mg l(-1)) in the absence of nutrient amino acids in the culture medium, the presence of two derivatives in the final product was observed with extremely increased retention times on reverse-phase high-performance liquid chromatography. Each derivative was due to methylation of an isoleucine residue at Ile29 or Ile59 in the CX-397. The structure was deducible as beta-methylnorleucine (beta MeNle; (2S,3S)-2-amino-3-methylhexanoic acid). The modification pathway of beta MeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain. Additionally, beta MeNle is synthesized by mutants of Serratia marcescens that belong to the same family, Enterobacteriaceae, as E. coli (J. Antibiot. 34 (1981a) 1278). These findings suggest that the lack of nutrient amino acids in the culture medium leads to the synthesis of beta MeNle in E. coli, which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein.     

Discovery and Investigation of Misincorporation of Serine at Asparagine Positions in Recombinant Proteins Expressed in Chinese Hamster Ovary Cells

Misincorporation of amino acids in proteins expressed in Escherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA^Asn rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (tm) for poly(A).poly(U) and poly(I).poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of tm at low concentrations (approximately 1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala greater than Gly greater than Ser greater than Asn greater than Pro greater than Met, Val greater than Ile, Leu for poly(A).poly(U) and Ala, Gly, Ser greater than Asn greater than Pro greater than Val greater than Ile, Met, Leu for poly(I).poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.     

Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: growth and lysine production

Corynebacterium glutamicum, well known for the industrial production of amino acids, grows aerobically on a variety of mono- and disaccharides and on alcohols and organic acids as single or combined sources of carbon and energy. Members of the genera Corynebacterium and Brevibacterium were here tested for their ability to use the homopolysaccharide starch as a substrate for growth. None of the 24 type strains tested showed growth on or degradation of this substrate, indicating that none of the strains synthesized and secreted starch-degrading enzymes. Introducing the Streptomyces griseus amy gene on an expression vector into the lysine-producer C. glutamicum DM1730, we constructed a C. glutamicum strain synthesizing and secreting alpha-amylase into the culture broth. Although some high-molecular-weight degradation products remained in the culture broth, this recombinant strain effectively used soluble starch as carbon and energy substrate for growth and also for lysine production. Thus, employment of our construct allows avoidance of the cost-intensive enzymatic hydrolysis of the starch, which commercially is used as a substrate in industrial amino acid fermentations.     

GC constituents and relative codon expressed amino acid composition in cyanobacterial phycobiliproteins

The genomic as well as structural relationship of phycobiliproteins (PBPs) in different cyanobacterial species are determined by nucleotides as well as amino acid composition. The genomic GC constituents influence the amino acid variability and codon usage of particular subunit of PBPs. We have analyzed 11 cyanobacterial species to explore the variation of amino acids and causal relationship between GC constituents and codon usage. The study at the first, second and third levels of GC content showed relatively more amino acid variability on the levels of G3 + C3 position in comparison to the first and second positions. The amino acid encoded GC rich level including G rich and C rich or both correlate the codon variability and amino acid availability. The fluctuation in amino acids such as Arg, Ala, His, Asp, Gly, Leu and Glu in α and β subunits was observed at G1C1 position; however, fluctuation in other amino acids such as Ser, Thr, Cys and Trp was observed at G2C2 position. The coding selection pressure of amino acids such as Ala, Thr, Tyr, Asp, Gly, Ile, Leu, Asn, and Ser in α and β subunits of PBPs was more elaborated at G3C3 position. In this study, we observed that each subunit of PBPs is codon specific for particular amino acid. These results suggest that genomic constraint linked with GC constituents selects the codon for particular amino acids and furthermore, the codon level study may be a novel approach to explore many problems associated with genomics and proteomics of cyanobacteria.     

Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.     

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.     

Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids

To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.     

重组水蛭素的纯化和鉴定

本文报导了化学合成的水蛭素基因在酵母细胞中得到表达,井能分泌水蛭素到胞外。将该菌株培养物的上清液经硫酸铵沉淀和Sephadex G-50过滤后,用DEAE-SephadexA-25进行阴离子交换层析,进而用HPLC反相层析,得到表达产物重组水蛭素。经SDS-PAGE,氨基酸序列分析,抗凝血酶活力分析及血浆滴定实验等方法鉴定,证明该基因表达产物与天然水蛭素HV_2相同。     

Cloning, characterization, and inactivation of the Bacillus brevis lon gene.

A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized. The cloned gene (B. brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E. coli protease La, the lon gene product. Fifty-two percent of the amino acid residues of the two polypeptides were identical. The ATP-binding sequences found in E. coli protease La were highly conserved. The promoter of the B. brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E. coli heat shock promoters. The B. brevis lon gene was inactivated by insertion of the neomycin resistance gene. A mutant B. brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin.