利用温控载体构建碱性果胶酯裂解酶工程菌

从筛选出的Bacillus subtilisWSHB04-02菌株中扩增出编码碱性果胶酯裂解酶的结构基因PL,将其插入载体pET22b( )多克隆位点,得到带有前导序列PelB重组质粒pET22b( )PL。以pET22b( )PL为模板扩增出带前导序列的碱性果胶酯裂解酶的结构基因PL,将其插入温控载体pHsh,重组载体在大肠杆菌JM109中得到表达。其表达量与以T7为启动子的重组菌BL21DE3[(pET22b( )PL]相比,表达量相近。SDS-PAGE分析显示表达产物的分子量均为43kDa,同核酸序列测定所推导的值相符。研究表明利用pHsh构建的JM109(pHsh PL)诱导表达好,诱导方式简单廉价,这对该酶的大规模发酵具有重要意义。     

Efficient expression of an alkaline pectate lyase gene from Bacillus subtilis and the characterization of the recombinant protein

The gene encoding a novel alkaline pectate lyase (Apel) from Bacillus subtilis was cloned and expressed in B. subtilis WB600. Apel contained an ORF of 1,260 bp, encoding a signal peptide of 21 amino acids and a mature protein of 399 amino acids with a calculated molecular mass of 45497.9 Da. The mature Apel was structurally related to the enzymes in the polysaccharide lyase family 1. After purification, the recombinant Apel had a specific activity of 445 U mg⁻¹. The enzyme was optimally active at 50°C and pH 9.     

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34 805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50°C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl. Received: June 10, 2000 / Accepted: October 3, 2000     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

Regulatory mutations affecting the synthesis of pectate lyase in Xanthomonas campestris

Four classes of Xanthomonas campestris mutants were identified with respect to pectate lyase. Pectate lyase production in the wild-type and classes I and IIb mutants was partially dependent on the growth-phase whereas in classes IIa and III it was totally dependent. Enzyme activity in some of the mutants was constitutive and resistant to catabolite repression.     

Construction and characterization of a novel maltose inducible expression vector in Bacillus subtilis

A maltose-inducible expression vector in Bacillus subtilis has been developed and characterized. The vector permitted β-galactosidase expression at a high level (maximum activity, 8.16 U/ml) when induced and its expression was markedly repressed by glucose. Using this vector, we successfully expressed the other two genes, bioA and vgb. This thus provided a potential expression system for cloned genes in B. subtilis.     

Purification and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20

A Bacillus subtilis AX20 from soil with ability to produce extracellular alpha-amylases was isolated. The characterization of microorganism was performed by biochemical tests as well as 16S rDNA sequencing. Maximum amylase activity (38 U/ml) was obtained at stationery phase when the culture was grown at 37 degrees C. The enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity of 4133 U/mg. The native protein showed a molecular mass of 149 kDa composed of a homodimer of 78 kDa polypeptide by SDS-PAGE. The optimum pH and temperature of the amylase were 6 and 55 degrees C, respectively. The enzyme was inhibited by Hg(2+), Ag(2+), and Cu(2+) and it did not show an obligate requirement of metal ions. The enzyme was not inhibited by EDTA or EGTA, suggesting that this enzyme is not a metalloenzyme. The end products of corn starch and soluble starch were glucose (70-75%) and maltose (20-25%). Rapid reduction of blue value and the end products suggest an endo mode of action for the amylase. The purified amylase shows interesting properties useful for industrial applications.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

麻类脱胶高效菌株果胶裂解酶基因克隆与表达

【目的】克隆麻类脱胶高效菌株Dickeya sp.DCE-01的果胶裂解酶基因并进行原核表达,对表达产物进行纯化和酶学性质研究。【方法】根据该菌株全基因组序列预测的果胶裂解酶基因Q59419设计引物,PCR扩增后将该基因连接到pEASY-E1和pACYCDuet-1载体上,导入E.coli BL21(DE3)进行表达。选择酶活力高的阳性克隆子进行大量诱导表达后,采用超滤和Sephadex G-100凝胶层析两步法纯化出果胶裂解酶,研究其酶学性质。【结果】克隆到果胶裂解酶基因pel(GenBank登录号:JX964997),其序列全长1 128 bp,编码375个氨基酸。pACYCDuet-1-pel-BL表达胞外果胶裂解酶活力最高,发酵液粗酶活达298.8 IU/mL。其最适反应温度为50°C,最适pH为9.0;保温1 h,酶活稳定温度≤45°C,稳定pH为9.0?10.0。酶催化作用依赖于Ca2+,其最适作用浓度为2 mmol/L;Zn2+、Ca2+和NH4+促进酶活力,Fe3+和Pb2+严重抑制酶活力;聚半乳糖醛酸钠为该酶的最适底物。【结论】从麻类脱胶高效菌株中发掘到碱性果胶裂解酶基因,其表达产物在生物质加工过程中具有重要工业化应用前景。     

Expression,purification and characterization of pectate lyase A from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml⁻¹ medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺ and Fe³⁺ ions stimulated the pectate lyase activity, but Cu²⁺ and Zn²⁺ inhibited it. The recombinant PelA had a V _max of 77 μmol min⁻¹ mg⁻¹ and an apparent K _m of 0.50 mg ml⁻¹ for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.     

Purification and characterization of pectate lyase from banana (Musa acuminata) fruits

Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.     

The mmgA gene from Bacillus subtilis encodes a degradative acetoacetyl-CoA thiolase

Early in sporulation, the mother cell compartment of Bacillus subtilis transcribes the mother cell metabolic gene (mmg) operon. The gene mmgA was assigned by other workers using sequence homology as an acetyl-CoA acetyltransferase [E.C. 2.3.1.9]. The gene was overexpressed in Escherichia coli, and the protein was purified by Ni²⁺-affinity chromatography. However, the expected MmgA-catalyzed biosynthesis of acetoacetyl-CoA from acetyl-CoA was undetectable by a standard UV assay, HPLC, and mass spectrometry. These methods indicated a preference for the reverse degradative thiolytic reaction, with a k _cat of 80 s⁻¹, and a K _m of 70 and 50 μM for CoA and acetoacetyl-CoA, respectively.     

Large-scale degumming of ramie fibre using a newly isolated <Emphasis Type="Italic">Bacillus pumilus</Emphasis> DKS1 with high pectate lyase activity

A combined (enzymatic and chemical) process using a Bacillus pumilus strain (DKS1), isolated from the soil, was used to degum ramie bast fibres. After 24 h of incubation with the isolated pectinolytic strain using a low-cost medium, the weight loss of the ramie fibre was found to be 25% under small scale. High activity of pectate lyase was detected in the culture supernatants; 400 kg of ramie fibres was degummed with 24% weight loss in large-scale degumming under field conditions. No cellulase activity was found. Microbial intervention followed by mild (0.1%) alkali treatment showed high percentage of weight loss from the ramie fibre. Bacterial degumming followed by chemical treatment resulted in an increase of single fibre tenacity (cN/tex) by more than 20.81% as compared to non-degummed (decorticated) fibre samples. Scanning electron micrographs (SEM) and fluorescence microscope showed that after Bacillus pumilus DKS1 treatment the surface of the decorticated ramie fibre becomes very smooth. These results indicate the process provides an economical and eco-friendly method for the small scale as well as large-scale degumming of decorticated ramie fibre. This study has great relevance to the textile as well as paper industry.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.     

Design of an expression vector and its application to heterologous protein expression in Bacillus subtilis

An expression vector, pUBEX, was constructed for extracellular production of heterologous proteins in Bacillus subtilis using a polyhistidine tag on the C-terminal sequence, providing an efficient and easy purification of the protein. A CII protein, a member of Bowman–Birk protease inhibitors, which was expressed as an inactive protein in Escherichia coli, was successfully expressed in Bacillus subtilis using the pUBEX vector and was purified to 6.4 mg l^–1 by the immobilized metal affinity chromatography.