Acidophilic xylanase from Aureobasidium pullulans: efficient expression and secretion in Pichia pastoris and mutational analysis

A yeast-like fungus Aureobasidium pullulans var. melanigenum strain ATCC 20524 produces an extracellular acidophilic endo-1,4-beta-xylanase with an optimum pH of 2.0 [Ohta et al., J. Biosci. Bioeng., 92, 262-270 (2001)]. The xynI cDNA encoding the precursor protein (XynI) was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase I gene promoter. The 34 amino acid prepro-signal peptide of the A. pullulans XynI directed the efficient secretion of 178 mg of active xylanase per liter of the culture medium. The secretion level of the xylanase with its own signal peptide was comparable to that of the mature protein fused to the prepro leader from Saccharomyces cerevisiae alpha-mating factor and twofold higher than that of the mature protein fused to the pre-type signal peptide from P. pastoris acid phosphatase. The N-terminal amino acid sequence and the apparent M(r) of 24 kDa of the secreted recombinant protein indicated the native-like processing of the A. pullulans XynI signal sequence in P. pastoris. The three-dimensional model and mutational analysis of the xynI gene product showed that Asp-73 and Glu-157 residues located at the upper and lower edges of the active site cleft, respectively, play a significant role in its low pH optimum.     

海藻糖合成酶产生菌筛选、鉴定及其产酶特性

以麦芽糖为唯一碳源高盐培养基,经高温培养,从温泉水样及其附近土壤中筛选得到一株菌株T2,经过生物合成途径初步验证,该菌株产海藻糖合酶,能够通过海藻糖合成酶（TreS）途径将麦芽糖转化为海藻糖。菌株T2为革兰氏阳性菌,杆状,有芽孢,经过生物学鉴定,将其初步鉴定为芽孢杆菌属（Bacillus sp.）。对其产海藻糖合酶的酶学性质进行了研究：酶反应最适作用温度为60 ℃,在60 ℃条件下保温100 min仍能保持酶活性80.7%;最适作用pH值为7.0,在pH 6.0～7.5范围内稳定。采用正交试验对其发酵培养基配方进行了优化研究,确定了最佳的培养基组成为牛肉膏3.0 g/L,麦芽糖20.0 g/L,蛋白胨7.5 g/L,无机盐（K2HPO4＋NaH2PO4＋MgSO4·7H2O）3.0 g/L。在此条件下,菌株T2产海藻糖合酶酶活力达到310.6 U/L。     

Cloning and sequencing of the malate synthase gene from Hansenula polymorpha

We have cloned the MAS gene, encoding the microbody matrix enzyme malate synthase (EC 4.1.3.2.) from the methylotrophic yeast Hansenula polymorpha. The gene was isolated by screening of a genomic library with a mixed-sequence probe, based on the partial amino acid sequence of the purified enzyme. The nucleotide sequence of a 2.4-kilobase stretch of DNA covering the MAS gene was determined. The gene contains an open reading frame of 555 amino acids, amounting to a calculated molecular mass of 63,254 for the encoded protein. Comparison of the amino acid sequence with the malate synthase sequences of Escherichia coli, Brassica napus L. and Cucumis sativus L. clearly establishes the homology of all four proteins. Compared to the soluble enzyme from E. coli, the malate synthases from H. polymorpha and both plant species, which are located in the microbodies, have a short carboxy-terminal extension. In the plant malate synthases, the extension is probably involved in routing to the microbodies, since it contains the potential peroxisomal targeting signal, Ser-Arg/Lys-Leu, at the carboxy terminus. The H. polymorpha enzyme terminates with similar amino acids, but their sequence, Ser-Leu-Lys, does not conform to any of the known peroxisomal targeting signals.     

Purification, bacteriolytic activity, and specificity of beta-lytic protease from Lysobacter sp. IB-9374

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.     

昆明假单胞菌HL22-2海藻糖合酶基因的克隆、表达及酶学性质研究

采用热不对称交错聚合酶链式反应（Tail-PCR）克隆云南磷矿来源昆明假单胞菌（Pseudomonas kunmingensis）HL22-2的海藻糖合酶（TreS）基因HL22-2TreS,将该基因与表达载体pETM3C连接后在大肠杆菌（Escherichia coli）BL21（DE3）pLysS中进行异源表达,通过Ni-NTA柱纯化重组酶HL22-2TreS,并对其酶学特性进行分析。结果表明,HL22-2TreS基因全长3 336 bp,编码1 111个氨基酸,氨基酸序列与Genbank数据库中相关的海藻糖合酶具有极高的相似性。重组酶HL22-2TreS的分子质量约126 kDa,该酶的最适反应温度和pH值分别为40 ℃和7.0,在温度20～50 ℃及pH值6.0～9.0条件下比较稳定,Cu2+、Hg2+、Ba2+及Al3+对海藻糖合酶的活力有强烈的抑制效果。HL22-2TreS基因对麦芽糖和海藻糖的米氏常数（Km）分别为20.6 mmol/L和87.5 mmol/L,对麦芽糖具有更高的亲和性,更容易将麦芽糖转化成海藻糖。     

Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.     

Identification of genes whose expressions are enhanced or reduced in baker's yeast during fed-batch culture process using molasses medium by DNA microarray analysis

Genes whose expression levels are enhanced or reduced during the cultivation process that uses cane molasses in baker's yeast production were identified in this study. The results showed that baker's yeast grown in molasses medium had higher fermentation ability and stress tolerance compared with baker's yeast grown in synthetic medium. Molasses apparently provided not only sugar as a carbon source but also provided functional components that enhanced or reduced expression of genes involved in fermentation ability and stress tolerance. To identify the genes whose expression is enhanced or reduced during cultivation in molasses medium, DNA microarray analysis was then used to compare the gene expression profile of cells grown in molasses with that of cells grown in synthetic medium. To simulate the commercial baker's yeast production process, cells were cultivated using a fed-batch culture system. In molasses medium, genes involved in the synthesis or uptake of vitamins (e.g., biotin, pyridoxine and thiamine) showed enhanced expression, suggesting that vitamin concentrations in molasses medium were lower than those in synthetic medium. Genes involved in formate dehydrogenase and maltose assimilation showed enhanced expression in molasses medium. In contrast, genes involved in iron utilization (e.g., siderophore, iron transporter and ferroxidase) showed enhanced expression in synthetic medium, suggesting that iron starvation occurred. The genes involved in the metabolism of amino acids also showed enhanced expression in synthetic medium. This identification of genes provides information that will help improve the baker's yeast production process.     

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.     

产海藻糖合酶菌株发酵培养基的研究

在已筛选出产海藻糖合酶菌株———恶臭假单胞杆菌的基础上 ,研究了该菌株最佳发酵培养基。恶臭假单胞杆菌H76的最适碳源为麦芽糖和葡萄糖 ,最适氮源为蛋白胨和酵母粉 ,无机盐为MgSO4 ·7H2 O ;该菌株产酶最佳培养基配方为 :麦芽糖 3 % ,葡萄糖 3 % ,Mg SO4 7H2 O 0 2 % ,蛋白胨 2 % ,酵母粉 0 7%。     

Cloning and sequencing of cDNA and genomic DNA encoding serine carboxypeptidase of Fusarium moniliforme that was copurified with phosphatase

A previous study [Yoshida, H. et al., J. Biochem., 140, 813-823 (2006)] revealed that a protein of unknown nature was copurified with PDM phosphatase of Fusarium moniliforme. In this study, the identity of this protein was investigated. The results of homology search for the tryptic peptides derived from the purified preparation of PDM phosphatase strongly suggested that it might be serine carboxypeptidase. In fact, carboxypeptidase activity was demonstrated in the preparation and partial separation of carboxypeptidase from PDM phosphatase was achieved by gel filtration high-performance liquid chromatography. Cloning and sequencing of the full-length cDNA encoding the carboxypeptidase was successfully conducted. The cDNA possessed an open reading frame for a protein of 575 amino acid residues with a molecular mass of 64,650 Da, which was highly homologous to certain fungal serine carboxypeptidases. Comparison of the deduced amino acid sequence with the N-terminal sequence of the separated carboxypeptidase revealed that the mature enzyme starts at serine 56 of the precursor and has a molecular mass of 58,487 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA demonstrated that the gene of carboxypeptidase consists of four exons. A limited number of close homologs of F. moniliforme carboxypeptidase were detected among fungi by homology search and their evolutionary relationship was discussed.     

Cloning and sequencing of kojibiose phosphorylase gene from Thermoanaerobacter brockii ATCC35047

A gene encoding kojibiose phosphorylase was cloned from Thermoanaerobacter brockii ATCC35047. The kojP gene encodes a polypeptide of 775 amino acid residues. The deduced amino acid sequence was homologous to those of trehalose phosphorylase from T. brockii and maltose phosphorylases from Bacillus sp. and Lactobacillus brevis with 35%, 29% and 28% identities, respectively. Kojibiose phosphorylase was efficiently overexpressed in Escherichia coli JM109. The DNA sequence of 3956 bp analyzed in this study contains three open reading frames (ORFs) downstream of kojP. The four ORFs, kojP, kojE, kojF, and kojG, form a gene cluster. The amino acid sequences deduced from kojE and kojF are similar to those of the N-terminal and C-terminal regions of a sugar-binding periplasmic protein from Thermoanaerobacter tengcongensis MB4. Furthermore, the amino acid sequence deduced from kojG is similar to that of a permease of the ABC-type sugar transport systems from T. tengcongensis MB4. Each of three amino acid substitutions, D362N, K614Q and E642Q, caused a complete loss of kojibiose phosphorylase activity. These results suggest that D362, K614 and E642 play an important role in catalysis. Another mutation, D459N, increased K(m) values for kojibiose (7-fold that for the wild type), beta-G1P (11-fold) and glucose (7-fold), whereas K(m) for inorganic phosphate was minimally affected by this mutation, suggesting that D459 may be involved in the binding to saccharides.     

Novel energy metabolism in anaerobic hyperthermophilic archaea: a modified Embden-Meyerhof pathway

Hyperthermophiles, a group of microorganisms whose optimum growth temperatures are above 80 degrees C, have been isolated mainly from marine and continental volcanic environments. They are viewed as potential sources of extraordinarily stable biomolecules with applications in novel industrial processes. Most hyperthermophiles belong to the domain Archaea, the third domain of life, and are considered to be the most ancient of all extant life forms. Recent studies have revealed unusual energy metabolic processes in hyperthermophilic archaea, e.g. a modified Embden-Meyerhof pathway, that have not been observed so far in organisms belonging to the Bacteria and Eucarya domains. Several novel enzymes--ADP-dependent glucokinase, ADP-dependent phosphofruktokinase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase, phosphoenolpyruvate synthase, pyruvate: ferredoxin oxidoreductase, and ADP-forming acetyl-CoA synthetase--have been found to be involved in the modified Embden-Meyerhof pathway of the hyperthermophilic archaeon Pyrococcus furiosus. In addition, a novel regulation site for energy metabolism and a unique mode of ATP regeneration have been postulated to exist in the pathway of P. furiosus. The metabolic design observed in this microorganism might reflect the situation at an early stage of evolution. This review focuses mainly on the unique energy metabolism and related enzymes of P. furiosus that have recently been described.     

Cloning and sequencing of the deacetylase gene from Vibrio alginolyticus H-8

A gene encoding deacetylase DA1 that is specific for N, N'-diacetylchitobiose was cloned using the shot-gun method with pUC118 and sequenced. The open reading frame encoded a protein of 427 amino acids including the signal peptide. The molecular mass of the mature enzyme estimated from the amino acid sequence data was 44.7 kDa, which is approximately similar to that, estimated by SDS-PAGE (48.0 kDa), of the purified enzyme reported previously. The N-terminal amino acid sequence deduced from the cloned deacetylase gene showed partial sequence homology with the Nod B protein from Rhizobium sp. (37% identity) and chitin deacetylase from Mucor rouxii (28%). It contained a domain, which showed homology with a chitin-binding domain of chitinase A from Bacillus circulans (39%).     

Yeast sequencing reports. Molecular cloning and sequencing of the hcs gene,which encodes 3-hydroxy-3-methylglutaryl coenzyme a synthase of Schizosaccharomyces pombe

We have cloned and sequenced the hcs gene, which is thought to encode a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase consisting of 447 amino acids, from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence of the hcs product of S. pombe has homology with the HMG-CoA synthase of rat (47·8%), chicken (49·2%), hamster (47·1%) and human cells (46·9%). One of the hcs genes was replaced with a marker gene in the diploid cell. No viable hcs-disrupted haploid was isolated after tetrad dissection, suggesting that the hcs gene is essential for growth. However the hcs-defective mutant could be grown on a medium containing 5 mg/ml mevalonate. These results strongly support that the hcs gene encodes HMG-CoA synthase and S. pombe contains a single copy of the hcs gene. The sequence of the hcs gene has been entered into the public data libraries under Accession Number U32187.     

Influence of malted barley and exogenous enzymes on the glucose/maltose balance of worts with sorghum or barley as an adjunct

The sugar profile of wort from laboratory malted barley, malted sorghum, unmalted barley and unmalted sorghum grains mashed with commercial enzyme preparations were studied. Similar levels of glucose to maltose (1:7) were observed in wort of malted barley and malted sorghum. Mashing barley or sorghum grains with commercial enzymes changed the glucose to maltose ratio in both worts, with a greater change in wort from sorghum grains. Although hydrolysis with commercial enzymes released more glucose from maltose in sorghum wort, the same treatment retained more maltose in barley wort. Adding malted barley to sorghum grains mashed with commercial enzymes restored the glucose to maltose ratio in sorghum mash. Fermentation of wort produced from all barley malt (ABM) mash and commercial enzyme/barley malt/sorghum adjunct (CEBMSA) mash of similar wort gravity was also studied. ABM and CEBMSA worts exhibited similar glucose to maltose ratios and similar amino acid spectra. However, ABM released more individual amino acids and five times more proline than wort from commercial enzyme/barley malt/sorghum adjunct. ABM produced 27% more glucose and 7% more maltose than CEBMSA. After fermentation, ABM mash produced 9.45% ABV whilst commercial enzyme/barley malt/sorghum adjunct mash produced 9.06% ABV. Restoration of the glucose/maltose ratio in the CEBMSA mash produced wort with a sugar balance required for high gravity brewing. © 2020 The Institute of Brewing & Distilling     

Biochemical and genetic characterization of a D(-)-3-hydroxybutyrate dehydrogenase from Acidovorax sp. strain SA1

D(-)-3-hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) from a poly(D(-)-3-hydroxybutyrate) (PHB) degrading bacterium, Acidovorax sp. SA1, was purified using Toyopearl DEAE-650M, red-Sepharose CL-4B, and Q Sepharose FF. The molecular mass of the enzyme was estimated as 27 kDa by SDS-PAGE and 110 kDa by gel filtration. The gene encoding BDH was cloned and sequenced, and expressed in Escherichia coli. The gene product was purified in two steps with a high yield. The N-terminal amino acid sequence of the enzyme purified from E. coli agreed with that of the purified enzyme from strain SA1. The BDH of strain SA1 had high amino acid sequence homology to that of Ralstonia eutropha H16. The Km values for D(-)-3-hydroxybutyrate and NAD+ in the oxidation reaction were 4.5 x 10(-4) M and 8.9 x 10(-5) M, respectively. The Km values for acetoacetate and NADH in the reduction reaction were 2.4 x 10(-4) M and 2.9 x 10(-5) M, respectively.     

Gene analysis and enzymatic properties of thermostable beta-glycosidase from Pyrococcus kodakaraensis KOD1

A beta-glycosidase with broad substrate specificity was identified from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding beta-glycosidase (Pk-gly) consists of 1449 nucleotides corresponding to a polypeptide of 483 amino acids. The protein showed similarity with other beta-glycosidases from family-1 glycosyl hydrolases, in particular, it showed high identity to beta-mannosidase from P. furiosus (55.7%), beta-glycosidase from Sulfolobus solfataricus (42.7%) and beta-glucosidase from P. furiosus (41.9%). The cloned gene was expressed in Escherichia coli and the recombinant protein was purified. The beta-glycosidase showed optimal activity at pH 6.5 and at an extremely high temperature of 100 degrees C, and had a half-life of 18 h at 90 degrees C. The beta-glycosidase hydrolyzed various pNp-beta-glycopyranosides, with kcat K(m) values in the order of pNp-beta-glucopyranoside = pNp-beta-mannopyranoside > pNp-beta-galactopyranoside > pNp-beta-xylopyranoside. pNp-beta-mannopyranoside was the substrate exhibiting the lowest K(m) value [0.254 mM] with a kcat K(m) ratio comparable to that of pNp-beta-glucopyranoside. This substrate specificity was distinct from previously reported beta-glycosidases. We observed that the region in PK-Gly corresponding to the fifth alpha-helix and beta-strand region of beta-glycosidase from S. solfataricus, which constitutes a large portion of the channel for substrate incorporation, displayed a chimeric structure, with the N-terminal region similar to beta-glycosidases and the C-terminal region similar to beta-mannosidases. An exo-type hydrolytic activity and transglycosylation activity were also observed towards cellooligomers.