Katsuhisa Saeki

Purification and characterization of an alkaline amylopullulanase with both α-1,4 and α-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378

Abstract The novel alkaline amylopullulanase produced by alkalophilic Bacillus sp. KSM-1378 was purified to an electrophoretically homogeneous state from culture medium. The purified enzyme was a glycoprotein with an apparent molecular mass of about 210 kDa and an isoelectric point of pH 4.8. The N-terminal amino acid sequence was Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and showed no homology to the N-terminal regions of other amylopullulanases reported to date. The enzyme was able to attack specifically the α-1,6 linkages in pullula.n to generate maltotriose as the major end product, as well as the α-1,4 linkages in amylose, amylopectin and glycogen to generate various oligosaccharides. The pH and temperature optima for the pullulanase and α-amylase activities were pH 9.5 and 50°C and pH 8.5 and 50°C respectively. Both activities were strongly inhibited by well characterized inhibitors, such as diethyl pyrocarbonate and N-bromosuccinimide. The pullulanase activity was specifically inactivated by Hg2+ ions, α-cyclodextrin and β-cyclodextrin while the amylase activity was strongly inhibited by EDTA and EGTA, although inhibition could be reversed by Ca2+ ions. It is suggested that the single alkaline amylopullulanase protein has two different active sites, one for the cleavage of α-1,4-linked substrates and one for the cleavage of α-1,6-linked substrates.

Purification and characterization of an alkaline isoamylase from an alkalophilic strain of Bacillus

Summary: Alkaline isoamylase (glycogen 6-glucanohydrolase, EC 3.2.1.68) activity was detected in the culture medium of an alkalophilic strain of Bacillus sp., designated KSM-3309, which was isolated from a soil sample. This novel enzyme was purified to homogeneity from the culture filtrate by precipitation with ammonium sulphate, chromatography on DEAE-cellulose and DEAE-Bio-Gel A, and gel filtration on Sephacryl S-200. The purified enzyme had a pH optimum of approximately 9.0, and displayed maximum catalytic activity at 55 °C. The enzyme had a molecular mass of 65 kDa, as determined by both SDS-polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200. The isoelectric point was 4.2. This enzyme cleaved the branching points of both amylopectin and glycogen, and incubation of the enzyme with these glucans caused large increases in coloration of the iodine reagent. Amylose, pullulan and maltose were practically insensitive to the enzyme. The enzyme activity was inhibited by Hg2+ ions and by N-bromosuccinimide, but the thiol inhibitors iodoacetate, 4-chloromercuribenzoate and N-ethylmaleimide had either no effect or a slightly inhibitory effect. β-Cyclodextrin, an inhibitor of pullulanase, was not inhibitory.

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

Abstract 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.

Nucleotide and deduced amino acid sequences of a new subtilisin from an alkaliphilic Bacillus isolate.

The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.

Alkaliphilic Bacillus sp. strain KSM-LD1 contains a record number of subtilisin-like serine proteases genes

The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337–340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.

Crystallization and preliminary X-ray diffraction studies of a novel alkaline serine protease (KP-43) from alkaliphilic Bacillus sp. strain KSM-KP43.

A novel alkaline serine protease (KP-43) which belongs to a new class of the subtilisin superfamily was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 43.50 (2), b = 110.4 (1), c = 168.9 (1) A. The crystals diffract X-rays beyond 1.9 A resolution using Cu Kalpha radiation from a rotating-anode generator and are suitable for high-resolution crystal structure analysis.

A single mutation within a Ca2 + binding loop increases proteolytic activity, thermal stability, and surfactant stability

We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca(2+) ion. This residue lies 15-20Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate-enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca(2+) and affects the packing of the Ca(2+) binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.